Brenner and Rector's The Kidney, 8th ed.

CHAPTER 5. Transport of Inorganic Solutes: Sodium, Chloride, Potassium, Magnesium, Calcium, and Phosphate

David B. Mount   Alan S.L. Yu



Sodium and Chloride Transport, 156



Proximal Tubule, 156



Loop of Henle and Thick Ascending Limb, 166



Distal Convoluted Tubule, Connecting Tubule, and Collecting Duct, 172



Potassium Transport, 180



Proximal Tubule, 180



The Loop of Henle and Medullary K+ Recycling, 181



K+ Secretion by the Distal Convoluted Tubule, Connecting Tubule, and Cortical Collecting Duct, 181



K+ Reabsorption by the Collecting Duct, 182



Regulation of Distal K+ Transport, 183



Calcium Transport, 185



Calcium Homeostasis, 185



Renal Handling of Calcium, 186



Regulation of Renal Calcium Handling, 190



Magnesium Transport, 192



Magnesium Homeostasis, 192



Renal Magnesium Handling, 193



Regulation of Renal Magnesium Handling, 195



Phosphate Transport, 196



Phosphate Homeostasis, 196



Renal Handling of Phosphate, 197



Regulation of Renal Phosphate Handling, 200


Sodium (Na+) is the principal osmole in extracellular fluid; as such, the total body content of Na+ and Cl-, its primary anion, determine the extracellular fluid volume. Renal excretion or retention of salt (Na+-Cl-) is thus the major determinant of the extracellular fluid volume, such that genetic loss-in-function or gains-in-function in renal Na+-Cl- transport can be associated with relative hypotension or hypertension, respectively. On a quantitative level, at a glomerular filtration rate of 180 liters/day and serum Na+ of ≈140 μM, the kidney filters some 25,200 millimoles per day of Na+; this is equivalent to ≈1.5 kilograms of salt, which would occupy roughly ten times the extracellular space.[1] Minute changes in renal Na+-Cl- excretion can thus have profound effects on the extracellular fluid volume; furthermore, 99.6% of this filtered Na+-Cl- must be reabsorbed to excrete 100 millimoles per day. Energetically, this renal absorption of Na+ consumes one molecule of ATP per 5 molecules of Na+.[1] This is gratifyingly economical, given that the absorption of Na+-Cl- is driven by basolateral Na+/K+-ATPase, which has a stoichiometry of three molecules of transported Na+ per molecule of ATP. This estimate reflects a net expenditure, however, because the cost of transepithelial Na+-Cl- transport varies considerably along the nephron, from a predominance of passive transport by thin ascending limbs to the purely active transport mediated by the aldosterone-sensitive distal nephron (distal convoluted tubule, connecting tubule, and collecting duct). The bulk of filtered Na+-Cl- transport is reabsorbed by the proximal tubule and thick ascending limb ( Fig. 5-1 ), nephron segments which utilize their own peculiar combinations of paracellular and transcellular Na+-Cl- transport; whereas the proximal tubule can theoretically absorb as much as 9 Na+ molecules for each hydrolyzed ATP,[1] paracellular Na+ transport by the thick ascending limb doubles the efficiency of transepithelial Na+-Cl- transport (6 Na+ per ATP).[2] Finally, the “fine-tuning” of renal Na+-Cl- absorp-tion occurs at full cost[1] (3 Na+ per ATP) in the aldosterone-sensitive distal nephron, while affording the generation of considerable transepithelial gradients.



FIGURE 5-1  Percentage reabsorption of filtered Na+-Cl- along the euvolemic nephron. ALH, thin ascending limb of the loop of Henle; CCD, cortical collecting duct: DCT, distal convoluted tubule; DLH, descending thin limb of the loop of Henle; PCT, proximal convoluted tubule; PST, proximal straight tubule; TAL, thick ascending limb; IMCD, inner medullary collecting duct; OMCD, outer medullary collecting duct.



The nephron thus constitutes a serial arrangement of tubule segments with considerable heterogeneity in the physiological consequences, mechanisms, and regulation of transepithelial Na+-Cl- transport. These issues will be reviewed in this section, in anatomical order, with an emphasis on particularly recent developments.

Proximal Tubule

A primary function of the renal proximal tubule is the near-isosomotic reabsorption of two thirds to three quarters of the glomerular ultrafiltrate. This encompasses the reabsorption of approximately 60% of filtered Na+-Cl- (see Fig. 5-1 ), such that this nephron segment plays a critical role in the maintenance of extracellular fluid volume. Although all segments of the proximal tubule share the ability to transport a variety of inorganic and organic solutes, there are considerable differences in the transport characteristics and capacity of early, mid, and late segments of the proximal tubule. There is thus is a gradual reduction in the volume of transported fluid and solutes as one proceeds along the proximal nephron. This corresponds to distinct ultrastructural characteristics in the tubular epithelium, moving from the S1 segment (early proximal convoluted tubule), to the S2 segment (late proximal convoluted tubule and beginning of the proximal straight tubule), and the S3 segment (remainder of the proximal straight tubule) ( Fig. 5-2 ). Cells of the S1 segment are thus characterized by a tall brush border, with extensive lateral invaginations of the basolateral membrane.[3] Numerous elongated mitochondria are located in lateral cell processes, with a proximity to the plasma membrane that is characteristic of epithelial cells involved in active transport. Ultrastructure of the S2 segment is similar, albeit with a shorter brush border, fewer lateral invaginations, and less prominent mitochondria. In epithelial cells of the S3 segment, lateral cell processes and invaginations are essentially absent, with small mitochondria that are randomly distributed within the cell.[3] The extensive brush border of proximal tubular cells serves to amplify the apical cell surface that is available for reabsorption; again, this amplification is axially distributed, increasing apical area 36-fold in S1 and 15-fold in S3.[4] At the functional level, there is a rapid drop in the absorption of bicarbonate and Cl- after the first millimeter of perfused proximal tubule, consistent with a much greater reabsorptive capacity in S1 segments.[5]



FIGURE 5-2  Schematic representation of the distribution of S1, S2, and S3 segments in the proximal tubules of superficial (SF) and juxtamedullary (JM) nephrons.  (Redrawn from Woodhall PB, Tisher CC, Simonton CA, Robinson RR: Relationship between para-aminohippurate secretion and cellular morphology in rabbit proximal tubules. J Clin Invest 61:1320–1329, 1978.)




There is also considerable axial heterogeneity in the quantitative capacity of the proximal nephron for organic solutes such as glucose and amino acids, with predominant reabsorption of these substrates in S1 segments.[6] The Na+-dependent reabsorption of glucose, amino acids, and other solutes in S1 segments results in a transepithelial potential difference (PD) that is initially lumen-negative, due to electrogenic removal of Na+ from the lumen[7] ( Fig. 5-3 ). This is classically considered the first phase of volume reabsorption by the proximal tubule. [8] [9] The lumen-negative PD serves to drive both paracellular Cl- absorption and a “backleak” of Na+ from the peritubular space to the lumen. Paracellular Cl- absorption in this setting accomplishes the net transepithelial absorption of a solute such as glucose, along with equal amounts of Na+ and Cl-; in contrast, backleak of Na+ leads only to reabsorption of the organic solute, with no net transepithelial transport of Na+ or Cl-. The amount of Cl- reabsorption that is driven by this lumen-negative PD thus depends on the relative permeability of the paracellular pathway to Na+ and Cl-. There appears to be considerable heterogeneity in the relative paracellular permeability to Na+ and Cl-; for example, whereas superficial proximal convoluted tubules and proximal straight tubules in the rabbit are Cl--selective, juxtamedullary proximal tubules in this species are reportedly Na+-selective. [10] [11] Regardless, the component of paracellular Cl- transport that is driven by this lumen-negative PD is restricted to the very early proximal tubule.



FIGURE 5-3  Reabsorption of solutes along the proximal tubule, in relation to transepithelial potential difference (PD). TF/P represents to ratio of tubule fluid to plasma concentration. OSM, osmolality.  (From Rector FC, Jr: Sodium, bicarbonate, and chloride absorption by the proximal tubule. Am J Physiol 244: F461–71, 1983.)




The second phase of volume reabsorption by the proximal tubule is dominated by Na+-Cl- reabsorption, via both paracellular and transcellular pathways.[9] In addition to the Na+-dependent reabsorption of organic solutes, the early proximal tubule has a much higher capacity for HCO3- absorption,[6] via the coupling of apical Na+-H+ exchange, carbonic anhydrase, and basolateral Na+-HCO3- cotransport. As the luminal concentrations of HCO3- and other solutes begin to drop, the concentration of Na+-Cl- rises to a value greater than that of the peritubular space.[8] This is accompanied by a reversal of the lumen-negative PD, to a lumen-positive value generated by passive Cl-diffusion[12] (see Fig. 5-3 ). This lumen-positive PD serves to drive paracellular Na+ transport, whereas the chemical gradient between the lumen and peritubular space provides the driving force for paracellular reabsorption of Cl-. This passive, paracellular pathway is thought to mediate ≈40% of transepithelial Na+-Cl- reabsorption by the mid-to-late proximal tubule.[11] Of note, however, there may be heterogeneity in the relative importance of this paracellular pathway, with evidence that active (i.e., transcellular) reabsorption predominates in proximal convoluted tubules from juxtamedullary versus superficial nephrons.[13] Regardless, the combination of both passive and active transport of Na+-Cl- explains how the proximal tubule is able to reabsorb ≈60% of filtered Na+-Cl- despite Na+/K+-ATPase activity that is considerably lower than that of distal segments of the nephron[14] ( Fig. 5-4 ).



FIGURE 5-4  Distribution of Na+/K+-ATPase activity along the nephron. CAL, cortical thick ascending limb; CCT, cortical collecting duct; DCT, distal convoluted tubule; MAL, medullary thick ascending limb; MCT, medullary collecting duct; PCT, proximal convoluted tubule; PR, pars recta; TAL, thin ascending limb of the loop of Henle; TDL, descending thin limb of the loop of Henle.  (From Katz AI, Doucet A, Morel F: Na-K-ATPase activity along the rabbit, rat, and mouse nephron. Am J Physiol 237:F114–120, 1979.)




The transcellular component of Na+-Cl- reabsorption initially emerged from studies of the effect of cyanide, ouabain, luminal anion transport inhibitors, cooling, and luminal/peritubular K+ removal.[9] For example, the luminal addition of SITS, an inhibitor of anion transporters, reduces volume reabsorption of proximal convoluted tubules perfused with a high Cl-, low HCO3- solution that mimics the luminal composition of the late proximal tubule; this occurs in the absence of an effect on carbonic anhydrase.[15] This transcellular component of Na+-Cl- reabsorption is clearly electroneutral. For example, in the absence of anion gradients across the perfused proximal tubule there is no change in transepithelial PD after the inhibition of active transport by ouabain, despite a marked reduction in volume reabsorption.[16] Transcellular Na+-Cl- reabsorption is accomplished by the coupling of luminal Na+-H+exchange or Na+-SO42- cotransport with a heterogeneous population of anion exchangers, as reviewed later.

Paracellular Na+-Cl- Transport

A number of factors serve to optimize the conditions for paracellular Na+-Cl- transport by the mid-to-late proximal tubule. First, the proximal tubule is a low-resistance, “leaky” epithelium,[11] with tight junctions that are highly permeable to both Na+ and Cl-.[10] Second, these tight junctions are preferentially permeable to Cl- over HCO3-,[17] a feature that helps generate the lumen-positive PD in the mid-to-late proximal tubule. Third, the increase in luminal Na+-Cl- concentrations in the mid-to-late proximal tubule generates the electrical and chemical driving forces for paracellular transport. Diffusion of Cl- thus generates a lumen-positive PD,[12] which drives paracellular Na+ transport; the chemical gradient between the lumen and peritubular space provides the driving force for paracellular reabsorption of Cl-. This rise in luminal Na+-Cl- is the direct result of the robust reabsorption of HCO3- and other solutes by the early S1 segment, [6] [8] combined with the iso-osmotic reabsorption of filtered water.[18]

A highly permeable paracellular pathway is a consistent feature of epithelia that function in the near-isosmolar reabsorption of Na+-Cl-, including small intestine, proximal tubule, and gallbladder. Morphologically, the apical tight junction of proximal tubular cells and other “leaky” epithelia is considerably less complex than that of “tight” epithelia. Freeze-fracture microscopy thus reveals that the tight junction of proximal tubular cells is comparatively shallow, with as few as one junctional strand ( Fig. 5-5 ); in contrast, high-resistance epithelia have deeper tight junctions with a complex, extensive network of junctional strands.[19] At the functional level, tight junctions of epithelia function as charge- and size-selective “paracellular tight-junction channels”,[20] physiological characteristics that are thought to be conferred by integral membrane proteins that cluster together at the tight junction[21]; changes in the expression of these proteins can have marked effects on permeability, without affecting the number of junctional strands.[22] In particular, the charge[23] and size[24] selectivity of tight junctions appears to be conferred in large part by the claudins, a large (>20) gene family of tetraspan transmembrane proteins. The repertoire of claudins expressed by proximal tubular epithelial cells may thus determine the high paracellular permeability of this nephron segment. At a minimum, proximal tubular cells co-express claudin-2, -10, and -11. [25] [26] The robust expression of claudin-2 in proximal tubule is of particular interest because this claudin can dramatically decrease the resistance of transfected epithelial cells.[22] Consistent with this cellular phenotype, targeted deletion of claudin-2 in knockout mice generates a “tight” epithelium in the proximal tubule, with a reduction in Na+-Cl- reabsorption.[27]



FIGURE 5-5  Freeze fracture electron microscopy of tight junctions in mouse proximal and distal nephron. A, Proximal convoluted tubule, a “leaky” epithelium; the tight junction contains only one junctional strand, seen as a groove in the fracture face (arrows). B, Distal convoluted tubule, a “tight” epithelium. The tight junction is deeper and contains several anastamosing strands, seen as grooves in the fracture face.  (From Claude P, Goodenough DA: Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J Cell Biol 58:390–400, 1973.)




The reabsorption of HCO3- and other solutes from the glomerular ultrafiltrate would be expected to generate an osmotic gradient across the epithelium, resulting in a hypotonic lumen. This appears to be the case, although the absolute difference in osmolality between lumen and peri-tubular space has been a source of considerable controversy.[18] Another controversial issue has been the relative importance of paracellular versus transcellular water transport from this hypotonic lumen. These issues have both been elegantly addressed through characterization of knockout mice with a targeted deletion of Aquaporin-1, a water channel protein expressed at the apical and basolateral membranes of the proximal tubule. Mice deficient in Aquaporin-1 have an 80% reduction in water permeability in perfused S2 segments, with a 50% reduction in transepithelial fluid transport.[28] Aquaporin-1 deficiency also results in a marked increase in luminal hypotonicity, providing definitive proof that near-isosmotic reabsorption by the proximal tubule requires transepithelial water transport via Aquaporin-1.[18] The residual water transport in the proximal tubules of Aquaporin-1 knockout mice is mediated in part by Aquaporin-7.[29] Alternative pathways for water reabsorption may include “co-transport” of H2O via the multiple Na+-dependent solute transporters in the early proximal tubule[30]; this novel hypothesis is, however, a source of considerable controversy.[31] A related issue is the relative importance of diffusional versus convective transport of Na+-Cl-, also known as “solvent drag”, across the paracellular tight junction[11]; convective transport of Na+-Cl- with water would seem to play a lesser role than diffusion, given the evidence that the transcellular pathway is the dominant transepithelial pathway for water in the proximal tubule. [18] [28] [29]

Transcellular Na+-Cl- transport

Apical Mechanisms

Apical Na+-H+ exchange plays a critical role in both transcellular and paracellular reabsorption of Na+-Cl- by the proximal tubule. In addition to providing an entry site in the transcellular transport of Na+, Na+-H+ exchange plays a dominant role in the robust absorption of HCO3- by the early proximal tubule[32]; this absorption of HCO3- serves to increase the luminal concentration of Cl-, which in turn increases the driving forces for the passive paracellular transport of both Na+ and Cl-. Increases in luminal Cl- also help drive the apical uptake of Cl- during transcellular transport. Not surprisingly, there is a considerable reduction in fluid transport of perfused proximal tubules exposed to concentrations of amilo-ride that are sufficient to inhibit proximal tubular Na+-H+ exchange.[33]

Na+-H+ exchange is predominantly mediated by the NHE proteins, encoded by the nine members of the SLC9 gene family; NHE3 in particular plays an important role in proximal tubular physiology. The NH3 protein is expressed at the apical membrane of S1, S2, and S3 segments.[34] The apical membrane of the proximal tubule also expresses alternative Na+-dependent H+ transporters,[35] including NHE8.[36] Regardless, the primacy of NHE3 in proximal Na+-Cl- reabsorption is illustrated by the renal phenotype of NHE3-null knock-out mice, which have a 62% reduction in proximal fluid absorption[37] and a 54% reduction in baseline chloride absorption.[38]

Much as amiloride and other inhibitors of Na+-H+ exchange revealed an important role for this transporter in transepithelial salt transport by the proximal tubule,[33] evidence for the involvement of an apical anion exchanger first came from the use of anion transport inhibitors; DIDS, furosemide, and SITS all reduce fluid absorption from the lumen of PT segments perfused with solutions containing Na+-Cl-. [15] [33] In the simplest arrangement for the coupling of Na+-H+ exchange to Cl- exchange, Cl- would be exchanged with the OH- ion during Na+-Cl- transport ( Fig. 5-6 ). Evidence for such a Cl--OH- exchanger was reported by a number of groups in the early 1980's, using membrane vesicles isolated from the proximal tubule (reviewed in Ref 39). These findings could not however be replicated in similar studies from other groups. [39] [40] Moreover, experimental evidence was provided for the existence of a dominant Cl--formate exchange activity in brush border vesicles, in the absence of significant Cl--OH- exchange.[40] It was postulated that recycling of formate by the back-diffusion of formic acid would sustain the net transport of Na+-Cl- across the apical membrane. Vesicle formate transport stimulated by a pH gradient (H+-formate cotransport or formate-OH- exchange) is saturable, consistent with a carrier-mediated process rather than diffusion of formic acid across the apical membrane of the proximal tubule.[41] Transport studies using brush border vesicles have also detected the presence of Cl--oxalate exchange mechanisms in the apical membrane of the PT,[42] in addition to SO42--oxalate exchange.[43] Based on differences in the affinities and inhibitor sensitivity of the Cl--oxalate and Cl--formate exchange activities, it was suggested that there are two separate apical exchangers in the proximal nephron, a Cl--formate exchanger and a Cl--formate/oxalate exchanger capable of transporting both formate and oxalate (see Fig. 5-6 ).



FIGURE 5-6  Transepithelial Na+-Cl- transport in the proximal tubule. A, In the simplest scheme, Cl- enters the apical membrane via a Cl--OH- exchanger, coupled to Na+ entry via NHE-3. B, Alternative apical anion exchange activities that couple to Na+-H+ exchange and Na+-SO42- cotransport; see text for details.



The physiological relevance of apical Cl-formate and Cl-oxalate exchange has been addressed by perfusing individual proximal tubule segments with solutions containing Na+-Cl- and either formate or oxalate. Both formate and oxalate significantly increased fluid transport under these conditions, in rabbit, rat, and mouse proximal tubule.[38] This increase in fluid transport was inhibited by DIDS, suggesting involvement of the DIDS-sensitive anion exchanger(s) detected in brush border vesicle studies. A similar mechanism for Na+-Cl- transport in the distal convoluted tubule (DCT) has also been detected, independent of thiazide-sensitive Na+-Cl- cotransport.[44] Further experiments indicated that the oxalate- and formate-dependent anion transporters in the PT are coupled to distinct Na+ entry pathways, to Na+-SO42- cotransport and Na+-H+ exchange, respectively.[45] The coupling of Cl--oxalate transport to Na+-SO42- cotransport requires the additional presence of SO42--oxalate exchange, which has been demonstrated in brush border membrane vesicle studies.[43] The obligatory role for NHE3 in formate stimulated Cl-transport was illustrated using NHE3-null mice, in which the formate effect is abolished[38]; of note, oxalate stimulation of Cl- transport is preserved in the NHE3-null mice. Finally, tubular perfusion data from superficial and juxtamedullary proximal convoluted tubules suggest that there is heterogeneity in the dominant mode of anion exchange along the PT, such that Cl--formate exchange is absent in juxtamedul-lary PCTs, in which Cl--OH- exchange may instead be dominant.[46]

The molecular identity of the apical anion exchanger(s) involved in transepithelial Na+-Cl- by the proximal tubule has been the object of more than two decades of investigation. A key breakthrough was the observation that the SLC26A4 anion exchanger, also known as pendrin, is capable of Cl--formate exchange when expressed in Xenopus laevis oocytes.[47] However, expression of SLC26A4 in the proximal tubule is minimal or absent in several species, and murine Slc26a4 is quite clearly not involved in formate-stimulated Na+-Cl- transport in this nephron segment.[48] There is however robust expression of SLC26A4 in distal type B intercalated cells[49]; the role of this exchanger in Cl- transport by the distal nephron is reviewed elsewhere in this chapter (see Na+-Cl- transport in the CNT and CCD; Cl- transport). Regardless, this data for SLC26A4 led to the identification and characterization of SLC26A6, a widely expressed member of the SLC26 family that is expressed at the apical membrane of proximal tubular cells. Murine Slc26a6, when expressed in Xenopus oocytes, mediates the multiple modes of anion exchange that have been implicated in transepithelial Na+-Cl- by the proximal tubule, including Cl--formate exchange, Cl--OH- exchange, Cl--SO42-, and SO42--oxalate exchange.[50] However, tubule perfusion experiments in mice deficient in Slc26a6 do not reveal a reduction in baseline Cl- or fluid transport, indicative of considerable heterogeneity in apical Cl- transport by the proximal tubule.[51] Candidates for the residual Cl- transport in Slc26a6-deficient mice include Slc26a7, which is expressed at the apical membrane of proximal tubule[52]; however, this member of the SLC26 family appears to function as a Cl- channel rather than as an exchanger.[53] It does however appear that Slc26a6 is the dominant Cl--oxalate exchanger of the proximal brush border. The usual increase in tubular fluid transport induced by oxalate is thus abolished in Slc26a6-knockout mice,[51] with an attendant loss of Cl--oxalate exchange in brush border membrane vesicles.[54]

Somewhat surprisingly, Slc26a6 mediates electrogenic Cl--OH- and Cl--HCO3- exchange,[50] and most if not all the members of this family are electrogenic in at least one mode of anion transport.[55] This begs the question of how the electroneutrality of transcellular Na+-Cl- transport is preserved. Notably, however, the stoichiometry and electrophysiology of Cl--base exchange differ for individual members of the family; for example, Slc26a6 exchanges one Cl- for two HCO3- anions, whereas SLC26A3 exchanges two Cl- anions for one HCO3- anion.[55] Co-expression of two or more electrogenic SLC26 exchangers in the same membrane may thus yield a net electroneutrality of apical Cl- exchange. Alternatively, apical K+ channels in the proximal tubule may function to stabilize membrane potential during Na+-Cl- absorption.[56]

Another puzzle is why Cl--formate exchange preferentially couples to Na+-H+ exchange mediated by NH3 38 (see Fig. 5-6 ), without evident coupling of Cl--oxalate exchange to Na+-H+ exchange or Cl--formate exchange to Na+-SO42- cotransport; it is evident that Slc26a6 is capable of mediating SO42--formate exchange,[50] which would be necessary to support coupling between Na+-SO42- cotransport and formate. Scaffolding proteins may serve to cluster these different transporters together in separate “micro-domains”, leading to preferential coupling. Notably, whereas both Slc26a6 and NHE have been reported to bind to the scaffolding protein PDZK1, distribution of Slc26a6 is selectively impaired in PDZK1 knockout mice.[57] Petrovic and colleagues[58] have also reported a novel activation of proximal Na+-H+ exchange by luminal formate, suggesting a direct effect of formate per se on NHE3; this may in part explain the preferential coupling of Cl--formate exchange to NHE3.

Basolateral Mechanisms

As in other absorptive epithelia, basolateral Na+/K+-ATPase activity establishes the Na+ gradient for transcellular Na+-Cl- transport by the proximal tubule and provides a major exit pathway for Na+. To preserve the electroneutrality of transcellular Na+-Cl- transport[16] this exit of Na+ across the basolateral membrane must be balanced by an equal exit of Cl-. Several exit pathways for Cl- have been identified in proximal tubular cells, including K+-Cl- cotransport, Cl- channels, and various modalities of Cl--HCO3- exchange (see Fig. 5-6 ).

Several lines of evidence support the existence of a swelling-activated basolateral K+-Cl- cotransporter (KCC) in the proximal tubule.[59] The KCC proteins are encoded by four members of the cation-chloride cotransporter gene family; KCC1, KCC3, and KCC4 are all expressed in kidney. In particular, there is very heavy co-expression of KCC3 and KCC4 at the basolateral membrane of the proximal tubule, from S1 to S3.[60] At the functional level, basolateral membrane vesicles from renal cortex reportedly contain K+-Cl- cotransport activity.[59] The use of ion-sensitive microelectrodes, combined with luminal charge injection and manipulation of bath K+ and Cl-, suggest the presence of an electroneutral K+-Cl- cotransporter at the basolateral membrane proximal straight tubules. Increases or decreases in basolateral K+ increase or decrease intracellular Cl- activity, respectively, with reciprocal effects of basolateral Cl- on K+ activity; these data are consistent with coupled K+-Cl- transport. [61] [62] Notably, a 1 μM concentration of furosemide, sufficient to inhibit all four of the KCCs, does not inhibit this K+-Cl-cotransport under baseline conditions.[61] However, only 10% of baseline K+ efflux in the proximal tubule is mediated by furosemide-sensitive K+-Cl- cotransport, which is likely quiescent in the absence of cell swelling. Thus the activation of apical Na+-glucose transport in proximal tubular cells strongly activates a barium-resistant (Ba2+) K+ efflux pathway that is 75% inhibited by 1 μM furosemide.[63] In addition, volume regulatory decrease (VRD) in Ba2+-blocked proximal tubules swollen by hypotonic conditions is blocked by 1 μM furosemide.[59] Cell swelling in response to apical Na+ absorption[64] is postulated to activate a volume-sensitive basolateral K+-Cl-cotransporter, which participates in transepithelial absorption of Na+-Cl-. Notably, targeted deletion of KCC3 and KCC4 in the respective knockout mice reduces VRD in the proximal tubule.[65] Furthermore, perfused proximal tubules from KCC3-deficient mice have a considerable reduction in transepithelial fluid transport,[66] suggesting an important role for basolateral K+-Cl- cotransport in transcellular Na+-Cl- reabsorption.

The basolateral chloride conductance of mammalian proximal tubular cells is relatively low, suggesting a lesser role for Cl- channels in transepithelial Na+-Cl- transport. Basolateral anion substitutions have minimal effect on the membrane potential, despite considerable effects on intracellular Cl- activity,[67] nor for that matter do changes in basolateral membrane potential affect intracellular Cl-. [61] [62] However, as with basolateral K+-Cl- cotransport, basolateral Cl- channels in the proximal tubule may be relatively inactive in the absence of cell swelling. Cell swelling thus activates both K+ and Cl- channels at the basolateral membranes of proximal tubular cells. [68] [69] [70] Seki and colleagues[71] have reported the presence of a basolateral Cl- channel within S3 segments of the rabbit nephron, wherein they did not seen affect of the KCC inhibitor H74 on intracellular Cl- activity. The molecular identity of these and other basolateral Cl- channels in the proximal nephron is not known with certainty, although S3 segments have been shown to exclusively express mRNA for the swelling-activated CLC-2 Cl- channel[72]; the role of this channel in transcellular Na+-Cl- reabsorption is not known.

Finally, there is functional evidence for both Na+-dependent and Na+-independent Cl--HCO3- exchange at the basolateral membrane of proximal tubular cells. [10] [67] [73] The impact of Na+-independent Cl--HCO3- exchange on basolateral exit is thought to be minimal.[67] For one, this exchanger is expected to mediate Cl- entry under physiological conditions.[73] Second, there is only a modest difference between the rate of decrease in intracellular Cl-activity between the combined removal of Na+ and Cl- versus Cl- and HCO3- removal, suggesting that pure Cl--HCO3- exchange does not contribute significantly to Cl- exit. In contrast, there is a 75% decrease rate of decrease in intracellular Cl- activity after the removal of basolateral Na+.[67] The Na+-dependent Cl--HCO3- exchanger may thus play a considerable role in basolateral Cl- exit, with recycled exit of Na+ and HCO3- via the basolateral Na+-HCO3- cotransporter NBC1 (see Fig. 5-6 ). Molecular candidates for this Na+-dependent Cl--HCO3- exchanger have emerged from the human, squid, and Drosophila genomes[74]; however, immunolocalization in mammalian proximal tubule has not as yet been reported.

Regulation of Proximal Tubular Na+-Cl- Transport

Glomerulotubular Balance

A fundamental property of the kidney is the phenomenon of glomerulotubular balance, wherein changes in glomerular filtration rate (GFR) are balanced by equivalent changes in tubular reabsorption, thus maintaining a constant fractional reabsorption of fluid and Na+-Cl- ( Fig. 5-7 ). Although the distal nephron is capable of adjusting reabsorption in response to changes in tubular flow,[75] the impact of GFR on Na+-Cl- reabsorption by the proximal tubule is particularly pronounced ( Fig. 5-8 ). Glomerulotubular balance is indepen-dent of direct neurohumoral control, and thought to be mediated by the additive effects of luminal and peri-tubular factors.[76]



FIGURE 5-7  Glomerulotubular balance; fractional water absorption by the proximal tubule does not change as a function of single nephron GFR.  (From Schnermann J, Wahl M, Liebau G, Fischbach H: Balance between tubular flow rate and net fluid reabsorption in the proximal convolution of the rat kidney. I. Dependency of reabsorptive net fluid flux upon proximal tubular surface area at spontaneous variations of filtration rate. Pflugers Arch 304:90–103, 1968.)






FIGURE 5-8  Glomerulotubular balance; linear increase in absolute fluid reabsorption by the late proximal tubule as a functional of single nephron GFR (SNGFR).  (From Spitzer A, Brandis M: Functional and morphologic maturation of the superficial nephrons. Relationship to total kidney function. J Clin Invest 53:279–287, 1974.)




At the luminal side, changes in GFR increase the filtered load of HCO3-, glucose, and other solutes, increasing their reabsorption by the load-responsive proximal tubule[6] and thus preserving a constant fractional reabsorption. Changes in tubular flow rate have additional stimulatory effects on luminal transport, in both the proximal and distal nephron.[75] In the proximal tubule, increases in tubular perfusion clearly increase the rate of both Na+ and HCO3- absorption, due to increases in luminal Na+-H+ exchange.[75] Increases in GFR during volume expansion are also accompanied by a modest increase in the capacity of Na+-H+ exchange, as measured in brush-border membrane vesicles, with the opposite effect in volume contraction.[75]

Notably, influential experiments from almost four decades ago, performed in rabbit proximal tubules, failed to demonstrate a significant effect of tubular flow on fluid absorption.[77] This issue has been revisited by Du and co-workers, who recently reported a considerable flow-dependence of fluid and HCO3- transport in perfused murine proximal tubules [76] [78] ( Fig. 5-9 ). These data were analyzed using a mathematical model that estimated microvillus torque as a function of tubular flow[78]; accounting for increases in tubular diameter, which reduce torque, there is a linear relationship between calculated torque and both fluid and HCO3- absorption. [76] [78] Consistent with an effect of torque rather than flow per se, increasing viscosity of the perfusate by the addition of dextran increases the effect on fluid transport; the extra viscosity increases the hydrodynamic effect of flow and thus increases torque. The mathematical analysis of Du and assoicates provide an excellent explanation of the discrepancy between their results and those of Burg and co-workers.[77] Whereas Burg and colleagues performed their experiments in rabbit,[77] the more recent report utilized mice [76] [78]; other studies that had found an effect of flow utilized perfusion of rat proximal tubules, presumably more similar to mouse than rabbit.[75] Increased flow has a considerably greater effect on tubular diameter in rabbit proximal tubule, thus reducing the increase in torque. Mathematical analysis of the rabbit data[77] thus predicts a 43% increase in torque, due to a 41% increase in tubule diameter at a threefold increase in flow; this corresponds to the statistically insignificant 36% increase in volume reabsorption reported by Burg and colleagues (Table 2 in Ref 77 ).



FIGURE 5-9  Glomerulotubular balance; flow dependent increases in fluid (Jv) and HCO3- (JHCO3) absorption by perfused mouse proximal tubules. Absorption increases when bath albumin concentration increases from 2.5 g/dl to 5 g/dl.  (From Du Z, Yan Q, Duan Y, et al: Axial flow modulates proximal tubule NHE3 and H-ATPase activities by changing microvillus bending moments. Am J Physiol Renal Physiol 290:F289–96, 2006.)




Pharmacological inhibition reveals that tubular flow activates proximal HCO3- reabsorption mediated by both NHE3 and apical H+-ATPase.[76] The flow-dependent increase in proximal fluid and HCO3- reabsorption is also attenuated in NHE3-deficient knockout mice. [76] [78] Inhibition of the actin cytoskeleton with cytochalasin-D reduces the effect of flow on fluid and HCO3- transport, suggesting that flow-dependent movement of microvilli serves to activate NHE3 and H+-ATPase via their linkage to the cytoskeleton (see Fig. 5-13 for NHE3).



FIGURE 5-13  The scaffolding protein NHERF (NHE Regulatory Factor) links the Na+-H+ exchanger NHE3 to the cytoskeleton and signaling proteins. NHERF binds to ezrin, which in turn links to protein kinase A (PKA) and the actin cytoskeleton. NHERF also binds to the SGK-1 protein kinase, which activates NHE-3. PDZ, PSD95, Discs large (Drosophila), and ZO-1 domain; SGK-1, serum and glucocorticoid-induced kinase-1.  (Redrawn from Weinman EJ, Cunningham R, Shenolikar S: NHERF and regulation of the renal sodium-hydrogen exchanger NHE3. Pflugers Arch 450:137–144, 2005.)




Peritubular factors also play an important, additive role in glomerulotubular balance. Specifically, increases in GFR result in an increase in filtration fraction and an attendant increase in postglomerular protein and peritubular oncotic pressure. It has long been appreciated that changes in peritubular protein concentration have important effects on proximal tubular Na+-Cl- reabsorption[79]; these effects are also seen in combined capillary and tubular perfusion experiments (reviewed in Ref 76). Peritubular protein also has an effect in isolated perfused proximal tubule segments, where the effect of hydrostatic pressure is abolished.[76] Increases in peritubular protein concentration have an additive effect on flow-dependent activation of proximal fluid and HCO3- absorption (see Fig. 5-9 ). The effect of peritubular protein on HCO3- absorption, which is a predominantly transcellular phenomenon,[17] suggests that changes in peritubular oncotic pressure do not affect transport via the paracellular pathway. However, the mechanism of the stimulatory effect of peritubular protein on transcellular transport is still not completely clear.[76]

Neurohumoral Influences

Fluid and Na+-Cl- reabsorption by the proximal tubule is affected by a number of hormones and neurotransmitters. The major hormonal influences on renal Na+-Cl- transport are shown in Figure 5-10 . Renal sympathetic tone exerts a particularly important stimulatory influence, as does angiotensin II (AII); dopamine is a major inhibitor of proximal tubular Na+-Cl- reabsorption.



FIGURE 5-10  Neurohumoral influences on Na+-Cl- absorption by the proximal tubule, thick ascending limb, and collecting duct. Factors that stimulate (➙) and inhibit (000035   ) sodium reabsorption are as follows: ANGII, angiotensin II (low and high referring to pico- and micromolar concentrations); adr, adrenergic agonists; AVP, arginine vasopressin; PTH, parathyroid hormone; GC, glucocorticoids; MC, mineralocorticoids; PGE2, prostaglandin E2; ET, endothelin; ANP/Urod, atrial natriuretic peptide and urodilatin; PAF, platelet-activating factor; BK, bradykinin. PCT, proximal convoluted tubule; PST, proximal straight tubule; MTAL, medullary thick ascending limb of loop of Henle; CTAL, cortical thick ascending limb; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct.  (Redrawn from Feraille E, Doucet A: Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81:345–418, 2001.)




Unilateral denervation of the rat kidney causes a marked natriuresis and a 40% reduction in proximal Na+-Cl- reabsorption, without effects on single nephron GFR or on the contralateral innervated kidney.[80] In contrast, low-frequency electrical stimulation of renal sympathetic nerves reduces proximal tubular fluid absorption, with a 32% drop in natriuresis and no change in GFR.[81] Basolateral epinephrine and/or nor-epinephrine stimulate proximal Na+-Cl- reabsorption via both α- and β-adrenergic receptors. Several lines of evidence suggest that α1-adrenergic receptors exert a stimulatory effect on proximal Na+-Cl- transport, via activation of basolateral Na+/K+-ATPase and apical Na+-H+ exchange; the role of α2-adrenergic receptors is more controversial.[82] Ligand-dependent recruitment of the scaffolding protein NHERF-1 by β2-adrenergic receptors resorts in direct activation of apical NHE3,[83]bypassing the otherwise negative effect of downstream cyclic AMP (cAMP—see later).

Angiotensin II (ANGII) has potent, complex effects on proximal Na+-Cl- reabsorption. Several issues unique to ANGII deserve emphasis. First, it has been appreciated for three decades that this hormone has a biphasic effect on the proximal tubule[84]; stimulation of Na+-Cl- reabsorption occurs at low doses (10-12 to 10-10 M), whereas concentrations greater than 10-7 M are inhibitory ( Fig. 5-11 ). Further complexity arises from the presence of AT1 receptors for ANGII at both luminal and basolateral membranes in the proximal tubule.[85] ANGII application to either the luminal or peritubular side of perfused tubules has a similar biphasic effect on fluid transport, albeit with more potent effects at the luminal side.[86] Experiments using both receptor antagonists and knockout mice have indicated that the stimulatory and inhibitory effects of ANGII are both mediated via AT1 receptors, due to signaling at both the luminal and basolateral membrane.[87] Finally, ANGII is also synthesized and secreted by the proximal tubule, exerting a potent autocrine effect on proximal tubular Na+-Cl- reabsorption.[88] Proximal tubular cells thus express mRNA for angiotensinogen, renin, and angiotensin-converting enzyme,[82] allowing for autocrine generation of ANGII. Indeed, luminal concentrations of ANGII can be 100-1000-fold higher than circulating levels of the hormone.[82] Proximal tubular and systemic synthesis of ANGII may be subject to different control. In particular, Thomson and co-workers have recently demonstrated that proximal tubular ANGII is increased considerably after high-salt diet, with a preserved inhibitory effect of losartan on proximal fluid reabsorption.[89] These authors have argued that the increase in proximal tubular ANGII after a high-salt diet contributes to a more stable distal salt delivery.[89]



FIGURE 5-11  The biphasic effect of angiotensin II (ANGII) on proximal tubular Na+-Cl- absorption. The steady-state Na+ concentration gradient (DCNa) is plotted as a function of peritubular ANGII concentration; low concentrations activate Na+-Cl- absorption by the proximal tubule, whereas higher concentrations inhibit.  (From Harris PJ, Navar LG: Tubular transport responses to angiotensin. Am J Physiol 248:F621–630, 1985.)




The proximal tubule is also a target for natriuretic hormones; in particular, dopamine synthesized in the proximal tubule has negative autocrine effects on proximal Na+-Cl- reabsorption.[82] Proximal tubular cells have the requisite enzymatic machinery for the synthesis of dopamine, using L-dopa reabsorbed from the glomerular ultrafiltrate. Dopamine synthesis by proximal tubular cells and release into the tubular lumen is increased after volume expansion or high-salt diet, resulting in a considerable natriuresis. [90] [91] Luminal dopamine antagonizes the stimulatory effect of epinephrine on volume absorption in perfused proximal convoluted tubules,[92] consistent with an autocrine effect of dopamine released into the tubular lumen.[90] Dopamine primarily exerts its natriuretic effect via D1-like dopamine receptors (D1 and D5 in human); as is the case for the AT1 receptors for ANGII,[85] D1 receptors are expressed at both the apical and luminal membranes of proximal tubule.[93] Targeted deletion of the D1A[94] and D5 receptors[95] in mice leads to hypertension, by mechanisms that include reduced proximal tubular natriuresis.[94]

The natriuretic effect of dopamine in the proximal tubule is modulated by atrial natriuretic peptide (ANP), which inhibits apical Na+-H+ exchange via a dopamine-dependent mechanism.[96] ANP appears to induce recruitment of the D1 dopamine receptor to the plasma membrane of proximal tubular cells, thus sensitizing the tubule to the effect of dopamine.[97] The inhibitory effect of ANP on basolateral Na+/K+-ATPase occurs via a D1-dependent mechanism, with a synergistic inhibition of Na+/K+-ATPase by the two hormones.[97] Furthermore, dopamine and D1 receptors appear to play critical permissive roles in the in vivo natriuretic effect of ANP. [98] [99]

Finally, there is considerable crosstalk between the major anti-natriuretic and natriuretic influences on the proximal tubule. For example, ANP inhibits ANGII dependent stimulation of proximal tubular fluid absorption,[100]presumably via the dopamine-dependent mechanisms discussed earlier.[96] Dopamine also decreases the expression of AT1 receptors for ANGII in cultured proximal tubular cells.[101] Furthermore, the provision of L-dopa in the drinking water of rats decreases AT1 receptor expression in the proximal tubule, suggesting that dopamine synthesis in the proximal tubule “resets” the sensitivity to ANGII.[101] ANGII signaling through AT1 receptors decreases expression of the D5 dopamine receptor, whereas renal cortical expression of AT1 receptors is in turn increased in knockout mice deficient in the D5 receptor.[102] Similar interactions have been found between proximal tubular AT1 receptors and the D2-like D3 receptor.[103]

Regulation of Proximal Tubule Transporters

The apical Na+-H+ exchanger NHE3 and the basolateral Na+/K+-ATPase are primary targets for signaling pathways elicited by the various anti-natriuretic and natriuretic stimuli discussed earlier; NHE3 mediates the rate-limiting step in transepithelial Na+-Cl- absorption,[78] and as such is perhaps the dominant target for regulatory pathways. NHE3 is regulated by the combined effects of direct phosphorylation and interaction with scaffolding proteins, which primarily regulate transport via changes in trafficking of the exchanger protein to and from the brush border membrane (see Fig. 5-2 ). [34] [104] [105] Increases in cyclic AMP (cAMP) have a profound inhibitory effect on apical Na+-H+ exchange in the proximal tubule. Intracellular cAMP is increased in response to dopamine signaling via D1-like receptors and/or PTH-dependent signaling via the PTH receptor, whereas ANGII-dependent activation of NHE3 is associated with a reduction in cAMP.[106] PTH is a potent inhibitor of NHE3, presumably so as to promote distal delivery of Na+-HCO3- and an attendant stimulation of distal calcium reabsorption.[105] The activation of protein kinase A (PKA) by increased cAMP results in direct phosphorylation of NHE3; although several sites in NHE3 are phosphorylated by PKA, the phosphorylation of serine 552 (S552) and 605 (S605) been specifically implicated in the inhibitory effect of cAMP on Na+-H+ exchange.[107] “Phospho-specific” antibodies that specifically recognize the phosphorylated forms of S552 and S605 were recently utilized to demonstrate dopamine-dependent increases in the phosphorylation of both these serines.[108] Moreover, immunostaining of rat kidney revealed that S552-phosphorylated NHE3 localizes at the coated pit region of the coated pit region of the brush border membrane,[108] where the oligomerized inactive form of NHE3 predominates.[109] The cAMP-stimulated phosphorylation of NHE3 by PKA thus results in a redistribution of the transporter from the microvillar membrane to an inactive, sub-microvillar population ( Fig. 5-12 ).



FIGURE 5-12  The effect of dopamine on trafficking of the Na+-H+ exchanger NHE3 in the proximal tubule. Microdissected proximal convoluted tubules were perfused for 30 minutes with 10-5 mol/L dopamine (DA) in the lumen or the bath, as noted, inducing a retraction of immunoreactive NHE3 protein from the apical membrane.  (From Bacic D, Kaissling B, McLeroy P, et al: Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal proximal tubule. Kidney Int 64:2133–2141, 2003.)




The regulation of NHE3 by cAMP also requires the participation of homologous scaffolding proteins that contain protein-protein interaction motifs known as PDZ domains (named for the PSD95, Discs large (Drosophila), and ZO-1 proteins in which these domains were first discovered) ( Fig. 5-13 ). The first of these proteins, NHE Regulatory Factor-1 (NHERF-1), was purified as a cellular factor required for the inhibition of NHE3 by PKA.[104] NHERF-2 was in turn cloned by yeast two-hybrid screens as a protein that interacts with the C-terminus of NHE3; NHERF-1 and NHERF-2 have very similar effects on the regulation of NHE3 in cultured cells. The related protein PDZK1 interacts with NHE3 and a number of other epithelial transporters, and is required for expression of the anion exchanger Slc26a6 at brush border membranes of the proximal tubule.[57]

NHERF-1 and NHERF-2 are both expressed in human and mouse proximal tubule cells; NHERF-1 colocalizes with NHE3 in microvilli of the brush border, whereas NHERF-2 is predominantly expressed at the base of microvilli in the vesicle-rich domain.[104] The NHERFs assemble a multi-protein signaling complex in association with NHE3 and other epithelial transporters and channels. In addition to NHE3 they bind to the actin-associated protein ezrin, thus linking NHE3 to the cytoskeleton[104]; this linkage to the cytoskeleton may be particularly important for the mechanical activation of NHE3 by microvillar bending, as has been implicated in glomerulotubular balance (see earlier). [76] [78] Ezrin also functions as an anchoring protein for PKA, bringing PKA into close proximity with NHE3 and facilitating its phosphorylation (see Fig. 5-13 ).[104] Analysis of knockout mice for NHERF-1 has revealed that it is not required for baseline activity of NHE3; as expected, however, it is required for cAMP-dependent regulation of the exchanger by PTH.[104] One longstanding paradox has been that β-adrenergic receptors, which increase cAMP in the proximal tubule, cause an activation of apical Na+-H+ exchange.[82] This has been resolved by the observation that the first PDZ domain of NHERF-1 interacts with the β2-adrenergic receptor in an agonist-dependent fashion; this interaction serves to disrupt the interaction between the second PDZ domain and NHE3, resulting in a stimulation of the exchanger despite the catecholamine-dependent increase in cAMP.[104]

As discussed earlier, at concentrations greater than 10-7 M (see Fig. 5-11 ) ANGII has an inhibitory effect on proximal tubular Na+-Cl- absorption.[84] This inhibition is dependent on the activation of brush border phospholipase A2, which results in the liberation of arachidonic acid.[86] Metabolism of arachidonic acid by cytochrome P450 mono-oxygenases in turn generates 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrioenoic acids (EETs), compounds that inhibit NHE3 and the basolateral Na+/K+-ATPase. [82] [110] EETs and 20-HETE have also been implicated in the reduction in proximal Na+-Cl- absorption that occurs during pressure natriuresis, inhibiting Na+/K+-ATPase and retracting NHE3 from the brush border membrane.[111]

Anti-natriuretic stimuli such as ANGII acutely increase expression of NHE3 at the apical membrane, at least in part by inhibiting the generation of cAMP.[106] “Low-dose” ANGII (10-10 M) also increases exocytic insertion of NHE3 into the plasma membrane, via a mechanism that is dependent on phosphatidylinositol 3-kinase (PI 3-kinase).[112] Treatment of rats with captopril thus results in a retraction of NHE3 and associated proteins from the brush border of proximal tubule cells.[113] Glucocorticoids also increase NHE3 activity, due to both transcriptional induction of the NHE3 gene and an acute stimulation of exocytosis of the exchanger to the plasma membrane.[114]Glucorticoid-dependent exocytosis of NHE3 appears to require NHERF-2, which acts in this context as a scaffolding protein for the glucocorticoid-induced serine-threonine kinase SGK1 (see also Regulation of Na+-Cl- transport in the CNT and CCD; aldosterone).[115] The acute effect of dexamethasone has thus been shown to require direct phosphorylation of serine 663 in the NHE3 protein by SGK1.[116]

Finally, many of the natriuretic and anti-natriuretic pathways that influence NHE3 have parallel effects on the basolateral Na+/K+-ATPase (see Ref 82 for a detailed review). The molecular mechanisms underlying inhibition of Na+/K+-ATPase by dopamine have been particularly well characterized. Inhibition by dopamine is associated with removal of active Na+/K+-ATPase units from the basolateral membrane,[117] analogous somewhat to the effect on NHE3 expression at the apical membrane. This inhibitory effect is primarily mediated by protein kinase C (PKC), which directly phosphorylates the α1 subunit of Na+/K+-ATPase, the predominant a subunit in the kidney.[82] The effect of dopamine requires phosphorylation of serine 18 of the α1 subunit by PKC; this phosphorylation event does not affect enzymatic activity of the Na+/K+-ATPase, but rather induces a conformational change that enhances the binding of PI 3-kinase to an adjacent proline-rich domain. The PI-3 kinase recruited by this phosphorylated α1 subunit then stimulates the dyamin-dependent endocytosis of the Na+/K+-ATPase complex via clathrin-coated pits.[117]

Loop of Henle and Thick Ascending Limb

The loop of Henle encompasses the thin descending limb, the thin ascending limb, and the thick ascending limb (TAL). The descending and ascending thin limbs function in passive absorption of water[118] and Na+-Cl-, [119] [120] [121] respectively, whereas the TAL reabsorbs ≈30% of filtered Na+-Cl- via active transport. There is considerable cellular and functional heterogeneity along the entire length of the loop of Henle, with consequences for the transport of water, Na+-Cl-, and other solutes. The thin descending limb begins in the outer medulla after an abrupt transition from S3 segments of the proximal tubule, marking the boundary between the outer and inner stripes of the outer medulla. Thin descending limbs end at a hairpin turn at the end of the loop of Henle. Short-looped nephrons that originate from superficial and midcortical nephrons have a short descending limb within the inner stripe of the outer medulla; close to the hairpin turn of the loop these tubules merge abruptly into the TAL (see also later). Long-looped nephrons originating from juxtamedullary glomeruli have a long ascending thin limb that then merges with the TAL. The TALs of long-looped nephrons begin at the boundary between the inner and outer medulla, whereas the TALs of short-looped nephrons may be entirely cortical. The ratio of medullary to cortical TAL for a given nephron is a function of the depth of its origin, such that superficial nephrons are primarily composed of cortical TALs whereas juxtamedullary nephrons primarily possess medullary TALs.

Transport Characteristics of the Descending Thin Limb

It has long been appreciated that the osmolality of tubular fluid increases progressively between the corticomedullary junction and the papillary tip, due to either active secretion of solutes or passive absorption of water along the descending thin limb.[122] Subsequent reports revealed a very high water permeability of perfused outer medullary thin descending limbs, in the absence of significant permeability to Na+-Cl-.[123] Notably, however, the permeability properties of descending thin limbs vary as a function of depth in the inner medulla and inclusion in short-versus long-looped nephrons. [124] [125] Descending thin limbs from short-looped nephrons contain “type I” cells—very flat, endothelial-like cells with intermediate-depth tight junctions suggesting a relative tight epithelium (reviewed in 124, 125). The epithelium of descending limbs from long-looped nephrons is initially more complex, with taller type II cells possessing more elaborate apical microvilli and more prominent mitochondria. In the lower medullary portion of long-looped nephrons these cells change into a type III morphology, endothelial-like cells similar to the type I cells from short-looped nephrons.[124] The permeability properties appear to change as a function of cell type, with a progressive axial drop in water permeability of long-looped descending limbs; the water permeability of descending thin limbs in the middle part of the inner medulla is thus ≈42% that of outer medullary thin descending limbs.[126] Furthermore, the distal 20% of descending thin limbs have a very low water permeability.[126] These changes in water permeability along the descending thin limb are accompanied by a progressive increase in Na+-Cl- permeability, although the ionic permeability remains considerably less than that of the ascending thin limb.[125]

Consistent with a primary role in passive water and solute absorption, Na+/K+-ATPase activity in the descending thin limb is almost undetectable,[14] suggesting that these cells do not actively transport Na+-Cl-; those ion transport pathways that have been identified in descending thin limb cells are thought to primarily contribute to cellular volume regulation.[127] In contrast to the relative lack of Na+-Cl- transport, transcellular water reabsorption by the thin descending limb is a critical component of the renal countercurrent concentrating mechanism. [118] [123] Consistent with this role, epithelial cells of the descending thin limbs express very high levels of the Aquaporin-1 water channel, at both apical and basolateral membranes.[128] The expression is highest in type II cells of descending thin limbs in the outer medulla,[128] which have the highest Aquaporin-1 content of all the tubule segments along the nephron.[129] Aquaporin-1 is also expressed in type I cells of short-looped nephrons[128]; notably, however, Aquaporin-1-expressing cells in descending limbs from short-looped nephrons extend into segments that do not express Aquaporin-1, just prior to the juncture with thick ascending limbs.[128] In addition, the terminal sections of deep descending limbs of long-looped nephrons, which do not exhibit appreciable water permeability,[126] do not express Aquaporin-1.[130] The analysis of knockout mice with targeted deletion of Aquaporin-1 has dramatically proven the primary role of water absorption, as opposed to solute secretion, in the progressive increase in osmolality along the descending thin limb.[122] Homozygous Aquaporin-1 knockout mice thus have a marked reduction in water permeability of perfused descending thin limbs, resulting in a vasopressin-resistant concentrating defect.[118]

Na+-Cl- Transport by the Thin Ascending Limb

Fluid entering the thin ascending limb has a very high concentration of Na+-Cl-, due to osmotic equilibration by the water-permeable descending limbs. The passive reabsorption of this delivered Na+-Cl- by the thin ascending limb is a critical component of the passive equilibration model of the renal countercurrent multiplication system. [119] [120] Consistent with this role, the permeability properties of the thin ascending limb are dramatically different from those of the descending thin limb, with a much higher permeability to Na+-Cl-125 and vanishingly-low water permeability.[131] Passive Na+-Cl- reabsorption by thin ascending limbs occurs via a combination of paracellular Na+transport [121] [132] [133] and transcellular Cl- transport. [134] [135] [136] The inhibition of paracellular conductance by protamine thus selectively inhibits Na+ transport across perfused thin ascending limbs, consistent with paracellular transport of Na+.[132] As in the descending limb, thin ascending limbs have a modest Na+/K+-ATPase activity (see Fig. 5-4 ); however, the active transport of Na+ across thin ascending limbs for only an estimated 2% of Na+reabsorption by this nephron segment.[137] Anion transport inhibitors[134] and chloride channel blockers[135] reduce Cl- permeability of the thin ascending limb, consistent with passive transcellular Cl- transport. Direct measurement of the membrane potential of impaled hamster thin ascending limbs has also yielded evidence for apical and basolateral Cl- channel activity.[136] This transepithelial transport of Cl-, but not Na+, is activated by vasopressin, with a pharmacology that is consistent with direct activation of thin ascending limb Cl- channels.[138]

Both apical and basolateral Cl- transport in the thin ascending limb appears to be mediated by the CLC-K1 Cl- channel, in co-operation with the Barttin subunit (see also Na+-Cl- transport in the thick ascending limb; basolateral mechanisms). Immunofluorescence[139] and in situ hybridization[140] indicate a selective expression of CLC-K1 in thin ascending limbs, although single-tubule RT-PCR studies have suggested additional expression in the thick ascending limb, distal convoluted tubule, and cortical collecting duct.[141] Notably, immunofluorescence and immunogold labeling indicate that CLC-K1 is expressed exclusively at both the apical and basolateral membrane of thin ascending limbs,[139] such that both the luminal and basolateral Cl- channels of this nephron segment[136] are encoded by the same gene. Homozygous knockout mice with a targeted deletion of CLC-K1 have a vasopressin-resistant nephrogenic diabetes insipidus,[142] reminiscent of the phenotype of Aquaporin-1 knockout mice.[118] Given that CLC-K1 is potentially expressed in the thick ascending limb (TAL),[141] dysfunction of this nephron segment might also contribute to the renal phenotype of CLC-K1 knockout mice; however, the closely homologous channel CLC-K2 (CLC-NKB) is clearly expressed in TAL,[141] where it can likely substitute for CLC-K1. Furthermore, loss-of-function mutations in CLC-NKB are an important cause of Bartter syndrome,[143] indicating that CLC-K2, rather than CLC-K1, is critical for transport function of the TAL.

Detailed characterization of CLC-K1 knockout mice has revealed a selective impairment in Cl- transport by the thin ascending limb.[121] Whereas Cl- absorption is profoundly reduced, Na+ absorption by thin ascending limbs is not significantly impaired ( Fig. 5-14 ). The diffusion voltage induced by a transepithelial Na+-Cl- gradient is reversed by the absence of CLC-K1, from +15.5 mV in homozygous wild-type controls (+/+) to -7.6 mV in homozygous knockout mice (-/-). This change in diffusion voltage is due to the dominance of paracellular Na+ transport in the CLC-K1 deficient -/- mice, leading to a lumen-negative potential; this corresponds to a marked reduction in the relative permeability of Cl- to that of Na+ (PCl/PNa), from 4.02 to 0.63 (see Fig. 5-14 ). The inhibition of paracellular transport by protamine has a comparable effect on the diffusion voltage in -/- mice versus +/- and +/+ mice that have been treated with NPPB to inhibit CLC-K1; the respective diffusion voltages are 7.9 mV (-/- plus protamine), 8.6 mV (+/- plus protamine and NPPB), and 9.8 (+/+ plus protamine and NPPB). Therefore, the paracellular Na+conductance is unimpaired and essentially the same in CLC-K1 mice, when compared to littermate controls. This study thus provides elegant proof for the relative independence of paracellular and transcellular conductances for Na+and Cl-, respectively, in thin ascending limbs.



FIGURE 5-14  Role of the CLC-K1 chloride channel in Na+ and Cl- transport by thin ascending limbs. Homozygous knockout mice (CLC-K1-/-) are compared to their littermate controls (CLC-K1+/+). A, Efflux coefficients for 36Cl- and 22Na+ in the thin ascending limbs; Cl-absorption is essentially abolished in the knockout mice, whereas there is no significant effect of CLC-K1 deficiency on Na+ transport. B, The diffusion voltage induced by a transepithelial Na+-Cl- gradient is reversed by the absence of CLC-K1, from +15.5 mV in controls to -7.6 mV in homozygous knockout mice. This change in diffusion voltage is due to the dominance of paracellular Na+ transport in the CLC-K1 deficient -/- mice, leading to a lumen-negative potential; this corresponds to a marked reduction in the relative permeability of Cl- to that of Na+ (PCl/PNa), from 4.02 to 0.63.  (From Liu W, Morimoto T, Kondo Y, et al: Analysis of NaCl transport in thin ascending limb of the loop of Henle in CLC-K1 null mice. Am J Physiol Renal Physiol 282:F451–457, 2002.)




Finally, CLC-K1 associates with “Barttin”, a novel accessory subunit identified via positional cloning of the gene for Bartter syndrome with sensorineural deafness[144] (see also Na+-Cl- transport in thick ascending limb: basolateral mechanisms). Barttin is expressed with CLC-K1 in thin ascending limbs, in addition to TAL, distal convoluted tubule, and α-intercalated cells. [141] [144] Rat CLC-K1 is unique among the CLC-K orthologs and paralogs (CLC-K1/2 in rodent, CLC-NKB/NKA in humans) in that it can generate Cl- channel activity in the absence of co-expression with Barttin [139] [145]; however, its human ortholog CLC-NKA is non-functional in the absence of Barttin.[144] Regardless, Barttin co-immunoprecipitates with CLC-K1,[141] and increases expression of the channel protein at the cell membrane. [141] [145] This “chaperone” function seems to involve the transmembrane core of Barttin, whereas domains within the cytoplasmic carboxy terminus modulate channel properties (open probability and unitary conductance).[145]

Thick Ascending Limb

Apical Na+-Cl- Transport

The thick ascending limb (TAL) reabsorbs ≈30% of filtered Na+-Cl- (see Fig. 5-1 ). In addition to an important role in the defense of the extracellular fluid volume, Na+-Cl- reabsorption by the water-impermeable TAL is a critical component of the renal countercurrent multiplication system. The separation of Na+-Cl- and water in the TAL is thus responsible for the capacity of the kidney to either dilute or concentrate the urine. In collaboration with the countercurrent mechanism, Na+-Cl- reabsorption by the thin and thick ascending limb increases medullary tonicity, facilitating water absorption by the collecting duct.

The TAL begins abruptly after the thin ascending limb of long-looped nephrons and after the Aquaporin-negative segment of short-limbed nephrons.[128] The TAL extends into the renal cortex, where it meets its parent glomerulus at the vascular pole; the plaque of cells at this junction form the macula densa, which function as the tubular sensor for both tubuloglomerular feedback and tubular regulation of renin release by the juxtaglomerular apparatus. Cells in the medullary TAL are 7 μM to 8 μM in height, with extensive invaginations of the basolateral plasma membrane and interdigitations between adjacent cells.[3] As in the proximal tubule, these lateral cell processes contain numerous elongated mitochondria, perpendicular to the basement membrane. Cells in the cortical TAL are considerably shorter, 2 μM in height at the end of the cortical TAL in rabbit, with less mitochondria and a simpler basolateral membrane.[3] Macula densa cells also lack the lateral cell processes and interdigitations that are characteristic of medullar TAL cells.[3] However, scanning electron microscopy has revealed that the TAL of both rat[146] and hamster[147] contains two morphological subtypes, a rough-surfaced cell type (R cells) with prominent apical microvilli and a smooth-surfaced cell type (S cells) with an abundance of sub-apical vesicles. [3] [148] In the hamster TAL, cells can also be separated into those with high apical and low basolateral K+ conductance and a weak basolateral Cl- conductance (LBC cells), versus a second population with low apical and high basolateral K+ conductance, combined with high basolateral Cl- conductance (HBC). [136] [147] The relative frequency of the morphological and functional subtypes in the cortical and medullary TAL suggests that HBC cells correspond to S cells and LBC cells to R cells.[147]

Morphological heterogeneity notwithstanding, the cells of the medullary TAL, cortical TAL, and macula densa share the same basic transport mechanisms ( Fig. 5-15 ). Na+-Cl- reabsorption by the TAL is thus a secondarily active process, driven by the favorable electrochemical gradient for Na+ established by the basolateral Na+/K+-ATPase. [149] [150] Na+, K+, and Cl- are co-transported across by the apical membrane by an electroneutral Na+-K+-2Cl-cotransporter; this transporter generally requires the simultaneous presence of all three ions, such that the transport of Na+ and Cl- across the epithelium is mutually co-dependent and dependent on the luminal presence of K+. [151] [152] [153] Of note, under certain circumstances apical Na+-Cl- transport in the TAL appears to be K+-independent; this issue is reviewed below (see Regulation of Na+-Cl- transport in the TAL). Regardless, this transporter is universally sensitive to furosemide, which has been known for more than three decades to inhibit transepithelial Cl- transport by the TAL.[154] Apical Na+-K+-2Cl- cotransport is mediated by the cation-chloride cotransporter NKCC2, encoded by the SLC12A1 gene.[155] Functional expression of NKCC2 in Xenopus laevis oocytes yields Cl-- and Na+-dependent uptake of 86Rb+ (a radioactive substitute for K+) and Cl-- and K+-dependent uptake of 22Na+. [155] [156] [157] As expected, NKCC2 is sensitive to micromolar concentrations of furosemide, bumetanide, and other loop diuretics.[155]



FIGURE 5-15  Transepithelial Na+-Cl- transport pathways in the thick ascending limb (TAL). NKCC2, Na+-K+-2Cl- cotransporter-2; ROMK, renal outer medullary K+ channel; CLC-NKB, human Cl- channel; Barttin, Cl- channel subunit; KCC4, K+-Cl- cotransporter-4.



Immunofluorescence indicates expression of NKCC2 protein along the entire length of the TAL.[155] In particular, immunoelectron microscopy reveals expression in both rough (R—see earlier) and smooth (S) cells of the TAL (also see earlier).[148] NKCC2 expression in subapical vesicles is particularly prominent in smooth cells,[148] suggesting a role for vesicular trafficking in the regulation of NKCC2 (see Regulation of Na+-Cl- transport in the TAL). NKCC2 is also expressed in macula densa cells,[148] which have been shown to possess apical Na+-K+-2Cl- cotransport activity.[158] This latter observation is of considerable significance, given the role of the macula densa in tubuloglomerular feedback and renal renin secretion; luminal loop diuretics block both tubuloglomerular feedback[159] and the suppression of renin release by luminal Cl-.[160]

Alternative splicing of exon 4 of the SLC12A1 gene yields NKCC2 proteins that differ within transmembrane domain 2 and the adjacent intracellular loop. There are thus three different variants of exon 4, denoted “A”, “B”, and “F”; the variable inclusion of these cassette exons yields the NKCC2-A, NKCC2-B, and NKCC2-F proteins. [155] [157] Kinetic characterization reveals that these isoforms differ dramatically in ion affinities. [155] [157] In particular, NKCC2-F has a very low affinity for Cl- (Km of 113 μM) and NKCC2-B has a very high affinity (Km of 8.9 μM); NKCC2-A has an intermediate affinity for Cl- (Km of 44.7 μM).[157] These isoforms differ in axial distribution along the tubule, with the F cassette expressed in inner stripe of the outer medulla, the A cassette in outer stripe, and the B cassette in cortical TAL.[161] There is thus an axial distribution of the anion affinity of NKCC2 along the TAL, from a low-affinity, high-capacity transporter (NKCC2-F) to a high-affinity, low-capacity transporter (NKCC2-B). Although technically compromised by the considerable homology between the 3′ end of these 96 base-pair exons, in situ hybridization has suggested that rabbit macula densa exclusively expresses the NKCC2-B isoform.[162] Notably, however, selective knockout of the B cassette exon 4 does not eliminate NKCC2 expression in the murine macula densa, which also seems to express NKCC2-A by in situ hybridization.[163] These NKCC2-B knockout mice do however have a shift in the sensitivity of both tubuloglomerular feedback and tubular regulation of renin release.[163]

It should be mentioned in this context that the Na+-H+ exchanger NHE3 functions as an alternative mechanism for apical Na+ absorption by the TAL. There is also evidence in mouse cortical TAL for Na+-Cl- transport via parallel Na+-H+ and Cl--HCO3- exchange,[164] although the role of this mechanism in transepithelial Na+-Cl- transport seems less prominent than in the proximal tubule. Indeed, apical Na+-H+ exchange mediated by NHE3 appears to function primarily in HCO3- absorption by the TAL.[165] There is thus a considerable upregulation of both apical Na+-H+ exchange and NHE3 protein in the TAL of acidotic animals,[166] paired with an induction of AE2, a basolateral Cl--HCO3- exchanger.[167]

Apical K+ Channels

Microperfused TALs develop a lumen-positive potential difference (PD) during perfusion with Na+-Cl-. [168] [169] This lumen-negative PD plays a critical role in physiology of the TAL, driving the paracellular transport of Na+, Ca2+, and Mg2+ (see Fig. 5-15 ). Originally attributed to electrogenic Cl- transport,[169] the lumen-positive, transepithelial PD in the TAL is generated by the combination of apical K+ channels and basolateral Cl- channels. [149] [150] [170] The conductivity of the apical membrane of TAL cells is predominantly, if not exclusively, K+ selective. Luminal recycling of K+ via Na+-K+-2Cl- cotransport and apical K+ channels, along with basolateral depolarization due to Cl- exit through Cl- channels, results in the lumen-negative transepithelial PD. [149] [150]

Several lines of evidence indicate that apical K+ channels are required for transepithelial Na+-Cl- transport by the TAL. [149] [150] First, the removal of K+ from luminal perfusate results in a marked decrease in Na+-Cl-reabsorption by the TAL, as measured by short circuit current; the residual Na+-Cl- transport in the absence of luminal K+ is sustained by the exit of K+ via apical K+ channels, since the combination of K+ removal and a luminal K+ channel inhibitor (barium) almost abolishes the short-circuit current.[151] Apical K+ channels are thus required for continued functioning of NKCC2, the apical Na+-K+-2Cl- cotransporter; the low luminal concentration of K+in this nephron segment would otherwise become limiting for transepithelial Na+-Cl- transport. Second, the net transport of K+ across perfused TAL is <10% that of Na+ and Cl-171; ≈90% of the K+ transported by NKCC2 is recycled across the apical membrane via K+ channels, resulting in minimal net K+ absorption by the TAL.[150] Third, the intracellular K+ activity of perfused TAL cells is ≈15 mV to 20 mV above equilibrium, due to furosemide-sensitive entry of K+ via NKCC2.[170] Given an estimated apical K+ conductivity of ≈12 m/cm2, this intracellular K+ activity yields a calculated K+ current of ≈200 mA/cm2; this corresponds quantitatively to the uptake of K+ by the apical Na+-K+-2Cl- cotransporter.[149] Finally, the observation that Bartter syndrome can be caused by mutations in ROMK[172] provides genetic proof for the importance of K+ channels in Na+-Cl- absorption by the TAL (see later).

Three types of apical K+ channels have been identified in the TAL, a 30 pS channel, a 70 pS channel, and a high-conductance, calcium-activated maxi K+ channel [173] [174] [175] (see Fig. 5-15 ). The higher open probability and greater density of the 30 pS and 70 pS channels, versus the maxi K+ channel, suggest that these are the primary route for K+ recycling across the apical membrane; the 70 pS channel in turn appears to mediate ≈80% of the apical K+conductance of TAL cells.[176] The low conductance 30 pS channel shares several electrophysiological and regulatory characteristics with ROMK, the cardinal inward-rectifying K+ channel that was initially cloned from renal outer medulla.[177] ROMK protein has been identified at the apical membrane of medullary TAL, cortical TAL, and macula densa.[178] Furthermore, the 30 pS channel is absent from the apical membrane of mice with homozygous deletion of the gene encoding ROMK.[179] Notably, not all cells in the TAL are labeled with ROMK antibody, suggesting that ROMK might be absent in the co-called HBC cells with high basolateral Cl- conductance and low apical/high basolateral K+ conductance (see also earlier). [136] [147] HBC cells are thought to correspond to the smooth-surfaced morphological subtype of TAL cells (S cells)[147]; however, distribution of ROMK protein by immunoelectron microscopy has not as yet been published.

ROMK clearly plays a critical role in Na+-Cl- absorption by the TAL, given that loss-of-function mutations in this gene are associated with Bartter syndrome.[172] The role of ROMK in Bartter syndrome was initially discordant with the data suggesting that the 70 pS K+ channel is the dominant conductance at the apical membrane of TAL cells[176]; heterologous expression of the ROMK protein in Xenopus oocytes had yielded a channel with a conductance of ≈30 pS,[177] suggesting that the 70 pS channel was distinct from ROMK. This paradox has been resolved by the observation that the 70 pS channel is absent from the TAL of ROMK knockout mice, indicating that ROMK proteins form a subunit of the 70 pS channel.[180] ROMK activity in the TAL is clearly modulated by association with other proteins, such that co-association with other subunits to generate the 70 pS channel is perfectly compatible with the known physiology of this protein. ROMK thus associates with scaffolding proteins NHERF-1 and NHERF-2 (see Proximal tubule, neurohumoral influences), via the C-terminal PDZ-binding motif of ROMK; NHERF-2 is co-expressed with ROMK in the TAL.[181] The association of ROMK with NHERFs serves to bring ROMK into closer proximity to the cystic fibrosis transmembrane regulator protein (CFTR).[181] This ROMK-CFTR interaction is in turn required for the native ATP and glybenclamide sensitivity of apical K+ channels in the TAL.[182]

Paracellular Transport

Microperfused TALs perfused with Na+-Cl- develop a lumen-positive transepithelial potential difference (PD) [168] [169] generated by the combination of apical K+ secretion and basolateral Cl- efflux. [149] [150] [170] This lumen-positive PD plays a critical role in the paracellular reabsorption of Na+, Ca2+, and Mg2+ by the TAL (see Fig. 5-15 ). In the transepithelial transport of Na+, the stoichiometry of NKCC2 (1Na+:1K+:2Cl-) is such that other mechanisms are necessary to balance the exit of Cl- at the basolateral membrane; consistent with this requirement, data from mouse TAL indicate that ≈50% of transepithelial Na+ transport occurs via the paracellular pathway. [2] [183] For example, the ratio of net Cl- transepithelial absorption to net Na+ absorption through the paracellular pathway is 2.4 +/- 0.3 in microperfused mouse medullary TAL segments,[183] the expected ratio if 50% of Na+transport occurs via the paracellular pathway. In the absence of vasopressin, apical Na+-Cl- cotransport is not K+-dependent (see Regulation of Na+-Cl- transport in the TAL), reducing the lumen-positive PD; switching to K+-dependent Na+-K+-2Cl- cotransport in the presence of vasopressin results in a doubling of Na+-Cl- reabsorption, without an effect on oxygen consumption.[2] Therefore, the combination of a cation-permeable paracellular pathway and an “active transport” lumen-positive PD,[149] generated indirectly by the basolateral Na+/K+-ATPase,[184] results in a doubling of active Na+-Cl- transport for a given level of oxygen consumption.[2]

Unlike the proximal tubule,[12] the voltage-positive PD in the TAL is generated almost entirely by transcellular transport, rather than diffusion across the lateral tight junction. In vasopressin-stimulated mouse TAL segments, with a lumen-positive PD of 10 mV, the maximal increase in Na+-Cl- in the lateral interspace is ≈10 μM.[183] Tight junctions in the TAL are cation-selective, with PNa/PCl ratios of 2 to 5. [149] [183] Notably, however, PNa/PCl ratios can be highly variable in individual tubules, ranging from 2 to 5 in a single study of perfused mouse TAL.[183] Regardless, assuming a PNa/PCl ratio of ≈3, the maximal dilution potential in the mouse TAL is between 0.7 mV to 1.1 mV, consistent with a dominant effect of transcellular processes on the lumen-positive PD.[183]

The reported transepithelial resistance in the TAL is between 10 and 50 W-cm2; although this resistance is higher than that of the proximal tubule, the TAL is not considered a “tight” epithelium. [149] [184] Notably, however, water permeability of the TAL is extremely low, <1% that of the proximal tubule.[149] These “hybrid” characteristics[184]—relatively low resistance and very low water permeability—allow the TAL to generate and sustain Na+-Cl-gradients of up to 120 μM.[149] Not unexpectedly, given its lack of water permeability, the TAL does not express aquaporin water channels; as in the proximal tubule, the particular repertoire of claudins expressed in the TAL determines the resistance and ion-selectivity of this nephron segment. Mouse TAL segments co-express claudin-3, -10, -11, -16, and -19. [26] [185] [186] Of particular significance, mutations in human claudin-16 (paracellin-1)[185] and claudin-19[186] are associated with hereditary hypomagnesemia, suggesting that these claudins are particularly critical for the cation-selectivity of TAL tight junctions. Heterologous expression of claudin-16 (paracellin-1) in the anion-selective LLC-PK1 cell line markedly increases Na+ permeability, without affecting Cl- permeability; this yields a marked increase in the PNa/PCl ratio ( Fig. 5-16 ).[187] LLC-PK1 cells expressing claudin-16 also have increased permeability to other monovalent cations. There is however only a modest increase in Mg2+ permeability, suggesting that claudin-16 does not form a Mg2+-specific pathway in the tight junction; rather, it may serve to increase the overall cation selectivity of the tight junction. Notably, no such effects on PNa/PCl ratio or Mg2+ permeability were seen in cation-selective MDCK-II cells[187]; Kausalya and colleagues also report minimal effect of claudin-16 on PNa/PCl ratio in MCDK-C7 cells, although they did detect a modest increase in Mg2+ permeability.[188] Regardless, the functional[187] and genetic[185] data suggest that claudin-16 expression is critical for the cation-selectivity of tight junctions in the TAL.



FIGURE 5-16  The effect of claudin-16 (paracellin-1) overexpression in LLC-PK1 cells. A, Effects of paracellin-1 on the permeability of Na+ and Cl- in LLC-PK1 cells. B, Ratio of PNa to PCl and diffusion potential (bottom) across a LLC-PK1 cell monolayer. C, Transepithelial resistance across an LLC-PK1 cell monolayer over a period of 12 days in cells expressing paracellin-1 and control cells. D, Summary of the effects of paracellin-1 upon permeability of various cations in LLC-PK1 cells.  (From Hou J, Paul DL, Goodenough DA: Paracellin-1 and the modulation of ion selectivity of tight junctions. J Cell Sci 118:5109–5118, 2005.)




Basolateral Mechanisms

The basolateral Na+/K+-ATPase is the primary exit pathway for Na+ at the basolateral membrane of TAL cells. The Na+ gradient generated by Na+/K+-ATPase activity is also thought to drive the apical entry of Na+, K+, and Cl-via NKCC2, the furosemide-sensitive Na+-K+-2Cl- cotransporter.[150] Inhibition of Na+/K+-ATPase with ouabain thus collapses the lumen-positive PD and abolishes transepithelial Na+-Cl- transport in the TAL. [168] [169] [184]Basolateral exit of Cl- from TAL cells is primarily but not exclusively[189] electrogenic, mediated by Cl- channel activity. [149] [150] Reductions in basolateral Cl- depolarize the basolateral membrane, whereas increases in intracellular Cl- induced by luminal furosemide have a hyperpolarizing effect.[189] Intracellular Cl- activity during transepithelial Na+-Cl- transport is above its electrochemical equilibrium,[190] with an intracellular-negative voltage of -40 to -70 mV that drives basolateral Cl- exit. [149] [150] [189]

There has been considerable recent progress in the molecular physiology of basolateral Cl- channels in the TAL. At least two CLC channels, CLC-K1 and CLC-K2 (CLC-NKA and CLC-NKB in humans), are co-expressed in this nephron segment. [141] [144] However, an increasing body of evidence indicates that the dominant Cl- channel in the TAL is encoded by CLC-K2. First, CLC-K1 is heavily expressed at both apical and basolateral membranes of the thin ascending limb,[139] and the phenotype of the corresponding knockout mouse is consistent with primary dysfunction of thin ascending limbs, rather than the TAL [121] [142] (see Na+-Cl- transport in the thin ascending limb). Second, loss-of-function mutations in CLC-NKB are associated with Bartter syndrome,[143] genetic evidence for a dominant role of this channel in Na+-Cl- transport in the TAL. More recently, a very common T481S polymorphism in human CLC-NKB was shown to increase channel activity by a factor of 20[191]; preliminary data indicate an association with hypertension,[192] suggesting that this gain-of-function in CLC-NKB increases Na+-Cl- transport by the TAL and/or other segments of the distal nephron. Finally, CLC-K2 protein is heavily expressed at the basolateral membrane of the mouse TAL, with additional expression in the DCT, CNT, and α-intercalated cells.[193]

A key advance was the characterization of the “Barttin” subunit of CLC-K channels, which is co-expressed with CLC-K1 and CLC-K2 in several nephron segments, including TAL (see also Na+-Cl- transport in the thin ascending limb). [141] [144] Unlike rat CLC-K1, the rat CLC-K2, human CLC-NKA, and human CLC-NKB paralogs are not functional in the absence of Barttin co-expression. [144] [145] CLC-NKB co-expressed with Barttin is highly selective for Cl-, with a permeability series of Cl->>Br-=NO3->I-. [141] [144] [191] CLC-NKB/Barttin channels are activated by increases in extracellular Ca2+ and are pH-sensitive, with activation at alkaline extracellular pH and marked inhibition at acidic pH.[144] CLC-NKA/Barttin channels have similar pH and calcium sensitivities, but exhibit higher permeability to Br.[144] Strikingly, despite the considerable homology between the CLC-NKA/NKB proteins, these channels also differ considerably in pharmacological sensitivity to various Cl- channel blockers, potential lead compounds for the development of paralog-specific inhibitors.[194]

Correlation between functional characteristics of CLC-K proteins with native Cl- channels in TAL has been problematic. In particular, a wide variation in single channel conductance has been reported for basolateral Cl- channels in this nephron segment (reviewed in Ref 195). This is perhaps due to the use of collagenase and other conditions for the preparation of tubule fragments and/or basolateral vesicles, manipulations that potentially affect channel characteristics.[195] There may also be considerable molecular heterogeneity of Cl- channels in the TAL, although the genetic evidence would seem to suggest a functional dominance of CLC-NKB.[143] Notably, single channel conductance has not been reported for CLC-NKB/Barttin channels, due to the difficulty in expressing the channel in heterologous systems; this complicates the comparison of CLC-NKB/Barttin to native Cl- channels. Single channel conductance has however been reported for the V166E mutant of rat CLC-K1, which alters gating of the channel without expected effects on single channel amplitude; co-expression with Barttin increases the single channel conductance of V166E CLC-K1 from ≈7 pS to ≈20 pS.[145] Therefore, part of the reported variability in native single channel conductance may reflect heterogeneity in the interaction between CLC-NKB and/or CLC-NKA with Barttin. Regardless, a recent study using whole-cell recording techniques suggests that CLC-K2 (CLC-NKB in humans) is the dominant Cl- channel in TAL and other segments of the rat distal nephron.[195] Like CLC-NKB/Barttin[141] [144] [191] this native channel is highly Cl--selective, with considerably weaker conductance for Br- and I-195; CLC-NKA/Barttin channels exhibit higher permeability to Br-.[144] This renal channel is also inhibited by acidic extracellular pH,[195] but seems to lack the activation by alkaline pH seen in CLC-NKB/Barttin-expressing cells.[144]

Electroneutral K+-Cl- cotransport has also been implicated in transepithelial Na+-Cl- transport in the TAL (see Figure 5-15 ), functioning in K+-dependent Cl- exit at the basolateral membrane.[189] The K+-Cl- cotransporter KCC4 is thus expressed at the basolateral membrane of medullary and cortical TAL, in addition to macula densa. [196] [197] There is also functional evidence for K+-Cl- cotransport at the basolateral membrane of this section of the nephron. First, TAL cells contain a Cl--dependent NH4+ transport mechanism that is sensitive to 1.5 μM furosemide and 10 μM barium (Ba2+).[198] NH4+ ions have the same ionic radius as K+ and are transported by KCC4 and other K+-Cl- cotransporters[199]; KCC4 is also sensitive to Ba2+ and millimolar furosemide[200], consistent with the pharmacology of NH4+-Cl- cotransport in the TAL.[198] Second, to account for the effects on transmembrane potential difference of basolateral Ba2+ and/or increased K+, it has been suggested that the basolateral membrane of TAL contains a Ba2+-sensitive K+-Cl- transporter [189] [201]; this is also consistent with the known expression of Ba2+-sensitive[200] KCC4 at the basolateral membrane. [196] [197] Third, increases in basolateral K+ cause Cl--dependent cell swelling in Amphiuma early distal tubule, an anolog of the mammalian TAL; in Amphiuma LBC cells with low basolateral conductance, analogous to mammalian LBC cells [136] [147] (see Na+-Cl- transport in the TAL: Apical Na+-Cl- transport), this cell swelling was not accompanied by changes in basolateral membrane voltage or resistance,[202] consistent with K+-Cl- transport.

There is thus considerable evidence for basolateral K+-Cl- cotransport in the TAL, mediated by KCC4. [196] [197] However, direct confirmation of a role for basolateral K+-Cl- cotransport in transepithelial transepithelial Na+-Cl-transport is lacking. Indeed, KCC4-deficient mice do not have a prominent defect in function of the TAL, but exibit instead a renal tubular acidosis.[197] The renal tubular acidosis in these mice has been attributed to defects in acid extrusion by H+-ATPase in α-intercalated cells[197]; however, this phenotype is conceivably due to reduction in medullary NH4+ reabsorption by the TAL,[203] due to the loss of basolateral NH4+ exit mediated by KCC4.[199]

Finally, there is also evidence for the existence of Ba2+-sensitive K+ channel activity at the basolateral membrane of TAL, [204] [205] [206] providing an alternative exit pathway for K+ to that mediated by KCC4. These channels may function in transepithelial transport of K+, which is however only <10% that of Na+ and Cl- transport by the TAL.[171] Basolateral K+ channels may also attenuate the increases in intracellular K+ that are generated by the basolateral Na+/K+-ATPase, thus maintaining transepithelial Na+-Cl- transport. [204] [205] [206] In addition, basolateral K+ channel activity may help stabilize the basolateral membrane potential above the equilibrium potential for Cl-,[206] thus maintaining a continuous driving force for Cl- exit via CLC-NKB/Barttin Cl- channels.

Regulation of Na+-Cl- Transport by the Thick Ascending Limb

Activating Influences

Transepithelial Na+-Cl- transport by the TAL is regulated by a complex blend of competing neurohumoral influences. In particular, increases in intracellular cAMP tonically stimulate ion transport in the TAL; the list of stimulatory hormones and mediators that increase cAMP in this nephron segment includes vasopressin, PTH, glucagon, calcitonin, and β-adrenergic activation (see Fig. 5-10 ). These overlapping cAMP-dependent stimuli are thought to result in maximal baseline stimulation of transepithelial Na+-Cl- transport.[82] For example, characterization of the in vivo effect of these hormones requires the prior simultaneous suppression or absence of circulating vasopressin, PTH, calcitonin, and glucagon.[82] This baseline activation is in turn modulated by a number of negative influences; most prominently PGE2 and extracellular Ca2+ (see Fig. 5-10 ).

Vasopressin is perhaps the most extensively studied positive modulator of transepithelial Na+-Cl- transport in the TAL. The TAL expresses V2 vasopressin receptors at both the mRNA and protein level, and micro-dissected TALs respond to the hormone with an increase in intracellular cAMP.[207] Vasopressin activates apical Na+-K+-2Cl- cotransport within minutes in perfused mouse TAL segments, and also exerts longer term influence on NKCC2 expression and function. The acute activation of apical Na+-K+-2Cl- cotransport is achieved at least in part by the stimulated exocytosis of NKCC2 proteins, from subapical vesicles to the plasma membrane.[208] This trafficking-dependent activation is abrogated by treatment of perfused tubules with tetanus toxin, which cleaves the vesicle-associated membrane proteins VAMP-2 and VAMP-3.[208] Activation of NKCC2 is also associated with the phosphorylation of a cluster of N-terminal threonines in the transporter protein; treatment of rats with the V2 agonist dDAVP induces phosphorylation of these residues in vivo, as measured with a potent phospho-specific antibody.[208] These threonine residues are thought to be substrates for the SPAK and OSR1 kinases, recently identified by Delpire and colleagues as key regulatory kinases for NKCC1 and other cation-chloride cotransporters.[209] SPAK and OSR1 are in turn activated by upstream WNK (With No Lysine (K) kinases) (see also Regulation of Na+-Cl- transport in the DCT), such that SPAK or OSR1 require co-expression with WNK4 to fully activate NKCC1.[209] WNK kinases can however influence transport when co-expressed alone in Xenopus oocytes with cation-chloride cotransporters, in the absence of exogenous SPAK/OSR1, reflective perhaps of the activation of endogenous Xenopus laevis orthologs of SPAK and/or OSR1. Regardless, co-expression with WNK3 in Xenopus oocytes results in activatory phosphorylation of the N-terminal threonines in NKCC2 that are phosphorylated in TAL cells after treatment with dDAVP. [208] [210] The WNK3 protein is also expressed in TAL cells,[210] although the link(s) between activation of the V2 receptor and this particular kinase are as yet uncharacterized.

Vasopressin has also been shown to alter the stoichiometry of furosemide-sensitive apical Cl- transport in the TAL, from a K+-independent Na+-Cl- mode to the classical Na+-K+-2Cl- cotransport stoichiometry.[2] In the absence of vasopressin, 22Na+ uptake by mouse medullary TAL cells is not dependent on the presence of extracellular K+, whereas the addition of the hormone induces a switch to K+-dependent 22Na+ uptake. Underscoring the metabolic advantages of paracellular Na+ transport, which is critically dependent on the apical entry of K+ via Na+-K+-2Cl- cotransport (see earlier), vasopressin accomplishes a doubling of transepithelial Na+-Cl- transport without affecting 22Na+ uptake (an indicator of transcellular Na+-Cl- transport); this doubling in transepithelial absorption occurs without an increase in O2 consumption,[2] highlighting the energy efficiency of ion transport by the TAL. The mechanism of this shift in the apparent stoichiometry of NKCC2 is not completely clear. However, splice variants of mouse NKCC2 with a novel, shorter C-terminus have been found to confer sensitivity to cAMP when co-expressed with full-length NKCC2.[211] Notably, these shorter splice variants appear to encode furosemide-sensitive, K+-independent Na+-Cl- cotransporters when expressed alone in Xenopus oocytes.[212] The in vivo relevance of these phenomena is not clear, however, nor is it known whether similar splice variants exist in species other than mouse.

In addition to its acute effects on NKCC2, the apical Na+-K+-2Cl- cotransporter, vasopressin increases transepithelial Na+-Cl- transport by activating apical K+ channels and basolateral Cl- channels in the TAL. [82] [207] Details have yet to emerge of the regulation of the basolateral CLC-NKB/Barttin Cl- channel complex by vasopressin, cAMP, and related pathways. However, the apical K+ channel ROMK is directly phosphorylated by protein kinase A on three serine residues (S25, S200, and S294 in the ROMK2 isoform). Phosphorylation of at least two of these three serines is required for detectable K+ channel activity in Xenopus oocytes; mutation of all three serines to alanine abolishes phosphorylation and transport activity, and all three serines are required for full channel activity.[213] These three phospho-acceptor sites have distinct effects on ROMK activity and expression.[214] Phosphorylation of the N-terminal S25 residue appears to regulate trafficking of the channel to the cell membrane, without affects on channel gating; this serine is also a substrate for the SGK1 kinase, which activates the channel via an increase in expression at the membrane.[214] In contrast, phosphorylation of the two C-terminal serines modulates open channel probability, via effects on pH-dependent gating[215] and on activation by the binding of phosphatidyl 4,5-biphosphate (PIP2) to the C-terminal domain of the channel.[216]

Vasopressin also has considerable long-term effects on transepithelial Na+-Cl- transport by the TAL. Sustained increases in circulating vasopressin result in marked hypertrophy of medullary TAL cells, accompanied by a doubling in baseline active Na+-Cl- transport.[207] Water restriction or treatment with dDAVP also results in an increase in abundance of the NKCC2 protein in rat TAL cells. Consistent with a direct effect of vasopressin-dependent signaling, expression of NKCC2 is reduced in mice with a heterozygous deletion of the Gs stimulatory G protein, through which the V2 receptor activates cAMP generation.[207] Increases in cAMP are thought to directly induce transcription of the SLC12A1 gene that encodes NKCC2, given the presence of a cAMP-response element in the 5′ promoter. [207] [208] Abrogation of the tonic negative effect of PGE2 on cAMP generation with indomethacin also results in a considerable increase in abundance of the NKCC2 protein.[207] Finally, in addition to these effects on NKCC2 expression, water restriction or dDAVP treatment increases abundance of the ROMK protein at the apical membrane of TAL cells.[217]

Inhibitory Influences

The tonic stimulation of transepithelial Na+-Cl- transport by cAMP-generating hormones (e.g., vasopressin, PTH) is modulated by a number of negative neurohumoral influences (see Fig. 5-10 and Ref 82). In particular, extracellular Ca2+ and PGE2 exert dramatic inhibitory effects on ion transport by this and other segments of the distal nephron, through a plethora of synergistic mechanisms. Both extracellular Ca2+ and PGE2 activate the Giinhibitory G protein in TAL cells, opposing the stimulatory, Gs-dependent effects of vasopressin on intracellular levels of cAMP. [218] [219] Extracellular Ca2+ exerts its effect through the calcium-sensing receptor (CaSR), which is heavily expressed at the basolateral membrane of TAL cells [219] [220]; PGE2 primarily signals through EP3 prostaglandin receptors.[82] The increases in intracellular Ca2+ due to the activation of the CaSR and other receptors directly inhibits cAMP generation by a Ca2+-inhibitable adenylate cyclase that is expressed in the TAL, accompanied by an increase in phosphodiesterase-dependent degradation of cAMP [219] [221] ( Fig. 5-17 ).



FIGURE 5-17  Inhibitory effects of the calcium-sensing receptor (CaSR) on transepithelial Na+-Cl- transport in the TAL. A, Activation of the basolateral CaSR inhibits the generation of cyclic AMP (cAMP) in response to vasopressin and other hormones (see text for details). B, Stimulation of phospholipase A2 by the CaSR leads to liberation of arachidonic acid, which is in turn metabolized by P450 w-hydroxylase to 20-HETE (20-hydroxyeicosatetraenoic acid), or by cyclooxygenase-2 (COX-2) to PGE2. 20-HETE is a potent natriuretic factor, inhibiting apical Na+-K+-2Cl-cotransport, apical K+ channels, and the basolateral Na+/K+-ATPase. Activation of the CaSR also induces TNFa expression in the TAL, which activates COX-2 and thus generation of PGE2, leading to additional inhibition of Na+-Cl- transport.  (Redrawn from Hebert SC: Calcium and salinity sensing by the thick ascending limb: A journey from mammals to fish and back again. Kidney Int Suppl: S28–33, 2004.)




Activation of the CaSR and other receptors in the TAL also results in the downstream generation of arachidonic acid metabolites with potent negative effects on Na+-Cl- transport (see Fig. 5-17 ). Extracellular Ca2+ thus activates phospholipase A2 in TAL cells, leading to the liberation of arachidonic acid. This arachidonic acid is in turn metabolized by P450 w-hydroxylase to 20-HETE (20-hydroxyeicosatetraenoic acid), or by cyclooxygenase-2 (COX-2) to PGE2; P450 w-hydroxylation generally predominates in response to activation of the CaSR in TAL.[219] 20-HETE has very potent negative effects on apical Na+-K+-2Cl- cotransport, apical K+ channels, and the basolateral Na+/K+-ATPase. [82] [219] PLA2-dependent generation of 20-HETE also underlies in part the negative effect of bradykinin and angiotensin-II on Na+-Cl- transport. [82] [219] Activation of the CaSR also induces TNFa expression in the TAL, which activates COX-2 and thus generation of PGE2 (see Fig. 5-17 ); this PGE2 in turn results in additional inhibition of Na+-Cl- transport.[219]

The relative importance of the CaSR in the regulation of Na+-Cl- transport by the TAL is dramatically illustrated by the phenotype of a handful of patients with gain-of-function mutations in this receptor. In addition to suppressed PTH and hypocalcemia, the usual phenotype caused by gain-of-function mutations in the CaSR (autosomal dominant hypoaparathyroidism), these patients manifest a hypokalemic alkalosis, polyuria, and increases in circulating renin and aldosterone. [222] [223] Therefore, the persistent inhibition of Na+-Cl- transport in the TAL by these over-active mutants of the CaSR causes a rare subtype of Bartter syndrome, type V in the genetic classification of this disease.[219]

Distal Convoluted Tubule, Connecting Tubule, and Collecting Duct

The distal nephron that extends beyond the thick ascending limb is the final arbiter of urinary Na+-Cl- excretion, and a critical target for both natriuretic and anti-natriuretic stimuli. The understanding of the cellular organization and molecular phenotype of the distal nephron continues to evolve, and merits a brief review in this context. The distal convoluted tubule (DCT) begins at a variable distance after the macula densa, with an abrupt transition between NKCC2-positive cortical TAL cells and DCT cells that express the thiazide-sensitive Na+-Cl- cotransporter NCC. Considerable progress has been made in the phenotypic classification of cell types in the DCT and adjacent nephron segments, based on the expression of an expanding list of transport proteins and other markers[224] ( Fig. 5-18 ). This analysis has revealed considerable differences in the organization of the DCT, connecting segment (CNT), and cortical collecting duct (CCD) in rodent, rabbit, and human kidneys. In general, rabbit kidneys are unique in the axial demarcation of DCT, CNT, and CCD segments, at both a molecular and morphological level; the organization of the DCT to CCD is considerably more complex in other species, with boundaries that are much less absolute.[224] Notably, however, the overall repertoire of transport proteins expressed does not vary between these species; what differs is the specific cellular and molecular organization of this segment of the nephron.



FIGURE 5-18  Schematic representation of the segmentation of the mouse distal nephron and of the distribution and abundance of Na+-, Ca2+- and Mg2+-transporting proteins. ENaC, epithelial Na+ channel; NCC, thiazide-sensitive Na+-Cl- cotransporter; CB, calbindin D28K; PV, paravalbumin. TRPV5 and TRPV6, apical Ca2+ entry channels; NCX1, Na+-Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; CBP-D28k, calbindin D28k; TRPM6, apical Mg2+ entry channel.  Data compiled from Refs 225, 522, 661.


The early DCT (DCT1) of mouse kidney expresses NCC and a specific marker, parvalbumin, which also distinguishes the DCT1 from the adjacent cortical TAL[225] (see Fig. 5-18 ). Mouse DCT2 cells co-express NCC with proteins involved in transcellular Ca2+ transport, including the apical calcium channel ECaC1 (TRPV5), the cytosolic calcium-binding protein calbindin D28K, and the basolateral Na+-Ca2+ exchanger NCX1.[225] NCC is co-expressed with the amiloride-sensitive Na+ channel (ENaC) in the late DCT2 of mouse, with robust expression of ENaC continuing in the downstream CNT and CCD.[225] In contrast, rabbit kidney does not have a DCT1 or DCT2, and exhibits abrupt transitions between NCC- and ENaC-positive DCT and CNT segments, respectively.[224] Human kidneys that have been studied thus far exhibit expression of calbindin D28K all along the DCT and CNT, extending into the CCD; however, the intensity of expression varies at these sites. Approximately 30% of cells in the distal convolution of human kidney express NCC, with 70% expressing ENaC (CNT cells); ENaC and NCC overlap in expression at the end of the human DCT segment. Finally, cells of the early CNT of human kidneys express ENaC in the absence of Aquaporin-2, the apical vasopressin-sensitive water channel.[224]

Although primarily contiguous with the DCT, CNT cells share several traits with principal cells of the CCD, including apical expression of ENaC and ROMK, the K+ secretory channel; the capacity for Na+-Cl- reabsorption and K+secretion in this nephron segment is as much as 10 times higher than that of the CCD[226] (see also Na+-Cl- transport in the CNT and CCD; Apical Na+ transport). Intercalated cells are the minority cell type within the distal nephron, emerging within the DCT and CNT and extending into the early inner medullary collecting duct (IMCD).[227] Three subtypes of intercalated cells have been defined, based on differences in the subcellular distribution of the H+-ATPase and the presence or absence of the basolateral AE1 Cl--HCO3- exchanger. Type A intercalated cells extrude protons via an apical H+-ATPase in series with basolateral AE1; type B intercalated cells secrete HCO3- and OH- via an apical anion exchanger (SLC26A4 or pendrin) in series with basolateral H+-ATPase.[227] In rodents, the most prevalent subtype of intercalated cells in the CNT is the non-A, non-B intercalated cell, which possess an apical Cl--HCO3- exchanger (SLC26A4 or pendrin) along with apical H+-ATPase.[227] Although intercalated cells play a dominant role in acid-base homeostasis, Cl- transport by type B intercalated cells performs an increasingly appreciated role in distal nephron Na+-Cl- transport (see Na+-Cl- transport in the CNT and CCD; Cl- transport).

The outer medullary collecting duct encompasses two separate subsegments, corresponding to the outer and stripes of the inner medulla, OMCDo and OMCDi, respectively. OMCDo and OMCDi contain principal cells with apical amiloride-sensitive Na+ channels (ENaC)[228]; however, the primary role of this nephron segment is renal acidification, with a particular dominance of Type A intercalated cells in OMCDi.[3] The OMCD also plays a critical role in K+ reabsorption, via the activity of apical H+/K+-ATPase pumps. [229] [230] [231]

Finally, the inner medullary collecting duct begins at the boundary between the outer and inner medulla, and extends to the tip of the papilla. The IMCD is arbitrarily separated into three equal zones, denoted IMCD1, IMCD2, and IMCD3; at the functional level, an early IMCD (IMCDi) and a terminal portion (IMCDt) can be appreciated.[3] The IMCD plays particularly prominent roles in vasopressin-sensitive water and urea transport.[3] The early IMCD contains both principal cells and intercalated cells; all three subsegments (IMCD1-3) express apical ENaC protein, albeit considerably weaker expression than in the CNT and CCD.[232] The roles of the IMCD and OMCD in Na+-Cl- homeostasis have been more elusive than that of the CNT and CCD; however, to the extent that ENaC is expressed in the IMCD and OMCD, homologous mechanisms are expected to function in Na+-Cl- reabsorption by CNT, CCD, OMCD, and IMCD segments.

Distal Convoluted Tubule

Mechanisms of Na+-Cl- Transport in the Distal Convoluted Tubule

Earlier micropuncture studies that did not distinguish between early and late DCT indicate that this nephron segment reabsorbs ≈10% of filtered Na+-Cl-. [233] [234] The apical absorption of Na+ and Cl- by the DCT is mutually dependent; ion substitution does not affect transepithelial voltage, suggesting electroneutral transport.[235] The absorption of Na+ by perfused DCT segments is also inhibited by chlorothiazide, localized proof that this nephron segment is the target for thiazide diuretics.[236] Similar thiazide-sensitive Na+-Cl- cotransport exists in the urinary blander of winter flounder, the species in which the thiazide-sensitive Na+-Cl- cotransporter (NCC) was first identified by expression cloning.[237] Functional characterization of rat NCC indicates very high affinities for both Na+ and Cl- (Michaelis-Menten constants of 7.6 ± 1.6 and 6.3 ± 1.1 μM, respectively)[238]; equally high affinities had previously been obtained by Velazquez and co-workers in perfused rat DCT.[235] The measured Hill coefficients of rat NCC are ≈1 for each ion, consistent with electroneutral co-transport.[238]

NCC expression is the defining characteristic of the DCT[224] (see Figs. 5-18 and 5-19 [18] [19]). There is also evidence for expression of this transporter in osteoblasts, peripheral blood mononuclear cells, and intestinal epithelium[239]; however, kidney is the dominant expression site.[155] Loss-of-function mutations in the SLC12A2 gene encoding human NCC cause Gitelman syndrome, familial hypokalemic alkalosis with hypomagnesemia, and hypocalciuria (see also Chapter 15 ). Mice with homozygous deletion of the Slc12a2 gene encoding NCC exhibit marked morphological defects in the early DCT, [240] [241] with both a reduction in the absolute number of DCT cells and changes in ultrastructural appearance. Similarly, thiazide treatment promotes marked apoptosis of the DCT,[242] suggesting that thiazide-sensitive Na+-Cl- cotransport plays an important role in modulating growth and regression of this nephron segment (see also Regulation of Na+-Cl- transport in the DCT).



FIGURE 5-19  Transport pathways for Na+-Cl- and K+ in (A) DCT cells and (B) principal cells of the CNT and CCD. ENaC, epithelial Na+ channel; NCC, thiazide-sensitive Na+-Cl- cotransporter; ROMK, renal outer medullary K+ channel; KCC4, K+-Cl- cotransporter-4; NHE-2, Na+-H+ exchanger-2.



Co-expression of NCC and the amiloride-sensitive Na+ channel (ENaC) occurs in the late DCT and CNT segments of many species, either in the same cells or in adjacent cells in the same tubule.[224] Notably, ENaC is the primary Na+ transport pathway of CNT and CCD cells, rather than DCT. There is however evidence for other Na+ and Cl- entry pathways in DCT cells. In particular, the Na+-H+ exchanger NHE2 is co-expressed with NCC at the apical membrane of rat DCT cells.[243] As in the proximal tubule, perfusion of distal convoluted tubule with formate and oxalate stimulates DIDS-sensitive Na+-Cl- transport that is distinct from the thiazide-sensitive transport mediated by NCC.[44] Therefore, a parallel arrangement of Na+-H+ exchange and Cl--anion exchangers may play an important role in electroneutral Na+-Cl- absorption by the DCT (see Fig. 5-19 ). Of note, the anion exchanger SLC26A6 is evidently expressed in the human distal nephron, including perhaps in DCT cells[244]; NHE2[243] and SLC26A6 are thus candidates mechanisms for this alternative pathway of DCT Na+-Cl- absorption.

At the basolateral membrane, as in other nephron segments, Na+ exits via Na+/K+-ATPase; bearing in mind the considerable caveats in morphological identification of the DCT,[224] this nephron segment appears to have the highest Na+/K+-ATPase activity of the entire nephron[14] (see Fig. 5-4 ). Basolateral membranes of DCT cells in both rabbit[245] and mouse[196] express the K+-Cl- cotransporter KCC4, a potential exit pathway for Cl-. However, several lines of evidence indicate that Cl- primarily exits DCT cells via basolateral Cl- channels. First, the basolateral membrane of rabbit DCT contains Cl- channel activity, with functional characteristics that are similar to those of CLC-K2. [195] [246] Second, CLC-K2 protein is expressed at the basolateral membrane of DCT and CNT cells[193]; mRNA for CLC-K1 can also be detected by RT-PCR of microdissected DCT segments.[141] Third, loss-of-function mutations in CLC-NKB, the human ortholog of CLC-K2, typically cause Bartter syndrome (dysfunction of the TAL)[143]; however, in some of these patients, mutations in CLC-NKB lead to more of a Gitelman syndrome phenotype, consistent with loss-of-function of DCT segments.[247]

Regulation of Na+-Cl- Transport in the Distal Convoluted Tubule

Considerable hypertrophy of the DCT occurs in response to chronic increases in delivery of Na+-Cl- to the DCT, typically induced by furosemide treatment with dietary Na+-Cl- supplementation. [224] [233] These morphological changes are reportedly independent of changes in aldosterone or glucocorticoid, suggesting that increased Na+-Cl- entry via NCC promotes hypertrophy of the DCT[224]; this is the inverse of the hypomorphic changes seen in NCC-deficiency [240] [241] or thiazide treatment.[224] Notably, however, changes in aldosterone do have dramatic effects on both the morphology of the DCT[248] and expression of NCC. [224] [249] [250] The DCT is thus an aldosterone-sensitive epithelium, expressing both mineralocorticoid receptor and the 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) enzyme that confers specificity for aldosterone over glucocorticoids.[224] Mice with a targeted deletion of 11β-HSD2, with activation of the mineralocorticoid receptor by circulating gluocorticoid, exhibit massive hypertrophy of what appear to be DCT cells[248]; this suggests an important role for mineralocorticoid activity in shaping this nephron segment. Furthermore, NCC expression is dramatically increased by treatment of normal rats with fludrocortisone or aldosterone[249]; adrenalectomized rats also show an increase in NCC expression after rescue with aldosterone, and treatment with spironolactone reduces expression of NCC in salt-restricted rats.[250]

Considerable insight into the role of NCC in the pathobiology of the DCT has recently emerged from the study of the WNK4 kinase.[251] WNK1 and WNK4 were initially identified as causative genes for pseudohypopaldosteronism type II (PHA-II) (also known as Gordon syndrome or “hereditary hypertension with hyperkalemia”). PHA-II is in every respect the “mirror image” of Gitelman syndrome, encompassing hypertension, hyperkalemia, hyperchloremic metabolic acidosis, suppressed PRA and aldosterone, and hypercalciuria.[252] Furthermore, PHA-II behaves like a gain-of-function in NCC and/or the DCT, in that treatment with thiazides typically results in resolution of the entire syndrome.[252] Intronic mutations in WNK1 have been detected in patients with PHA-II, leading to increased abundance of WNK1 mRNA in patient leukocytes; WNK4-associated disease is due to clustered point mutations in an acidic-rich, conserved region of the protein.[253] The WNK1 and WNK4 proteins are co-expressed within the distal nephron, in both DCT and CCD cells; whereas WNK1 localizes to the cytoplasm and basolateral membrane, WNK4 protein is found at the apical tight junctions.[253]

Consistent with the physiological gain-of-function in NCC associated with PHA-II,[252] WNK4 co-expression with NCC in Xenopus oocytes inhibits transport, and both kinase-dead and disease-associated mutations abolish the effect. [254] [255] WNK1 in turn has no effect on NCC, but abrogates the inhibitory effect of WNK4.[256] WNK4 reportedly interacts directly with the NCC protein[254]; however, the WNK kinases appear to exert their effect on NCC and other cation-chloride cotransporters via the phosphorylation and activation of the SPAK and OSRI serine/threonine kinases, which in turn phosphorylate the transporter proteins. [209] [257] [258] Notably, however, the in vivo relevance of these cell culture experiments is not entirely clear, particularly because the WNK1 and WNK4 kinases appear to regulate a number of other transport pathways in the distal nephron. [259] [260] [261]

To develop in vivo models relevant to both PHA-II and the physiological role of WNK4 in the distal nephron, Lalioti and colleagues generated two strains of BAC-transgenic mice that overexpress wild-type WNK4 (TgWnk4WT) or a PHA-II mutant of WNK4 (TgWnk4PHAII, bearing a Q562E mutation associated with the disease).[251] Consistent with the inhibitory effect of WNK4 on NCC, [254] [255] the blood pressure of TgWnk4WT is less than that of wild-type littermate controls; in contrast, TgWnk4PHAII mice are hypertensive. The biochemical phenotype of TgWnk4PHAII is also similar to that of PHA-II (i.e., hyperkalemia, acidosis, and hypercalciuria, with a suppressed expression of renal renin). TgWnk4PHAII mice also exhibit marked hyperplasia of the DCT, compared to a relative hypoplasia in the TgWnk4WT mice; morphology and phenotype of the CCD was not particularly affected. Of particular significance, the DCT hyperplasia of TgWnk4PHAII mice was completely suppressed on an NCC-deficient background, generated by mating TgWnk4PHAII mice with NCC knockout mice. [240] [241] Therefore, the DCT is the primary target for PHA-II associated mutations in WNK4. In addition, as suggested by prior studies [224] [240] [241] changes in Na+-Cl- entry via NCC can evidently modulate hyperplasia or regression of the DCT.[251] Furthermore, the results obtained with transgenic mice[251] provide a dramatic validation of the selective use of Xenopus oocytes for the analysis of regulatory interactions with ion transport proteins such as NCC. [254] [255] [256]

Connecting Tubules and Cortical Collecting Duct

Apical Na+ Transport

The apical membrane of CNT cells and principal cells contain prominent Na+ and K+ conductances, [226] [262] without a measurable apical conductance for Cl-.[195] The entry of Na+ occurs via the highly selective epithelial Na+channel (ENaC), which is sensitive to micromolar concentrations of amiloride (see Fig. 5-19 ).[263] This selective absorption of positive charge generates a lumen-negative potential difference (PD), the magnitude of which varies considerably as a function of mineralocorticoid status and other factors (see also Regulation of Na+-Cl- transport in the CNT and CCD). This lumen-negative PD serves to drive the following critical processes: (1) K+ secretion via apical K+ channels; (2) paracellular Cl- transport through the adjacent tight junctions; or (3) electrogenic H+ secretion via adjacent Type A intercalated cells.

ENaC is a heteromeric channel complex formed by the assembly of separate, homologous subunits, denoted α-, β-, and γ-ENaC.[264] These channel subunits share a common structure, with intracellular N- and C-terminal domains, two transmembrane segments, and a large glycosylated extracellular loop.[265] Xenopus oocytes expressing α-ENaC alone have detectable Na+ channel activity ( Fig. 5-20 ), which facilitated the initial identification of this subunit by expression cloning; functional complementation of this modest activity was then utilized to clone the other two subunits by expression cloning.[264] Full channel activity requires the co-expression of all three subunits, which causes a dramatic increase in expression of the channel complex at the plasma membrane[266] (see Fig. 5-20 ). The subunit stoichiometry has been a source of considerable controversy, with some reports favoring a tetramer with ratios of two α-ENaC proteins to one each of β-, and γ-ENaC (2a:1b:1g), and others favoring a higher-order assembly with a stoichiometry of 3a : 3b : 3g.[267] Regardless, the single channel characteristics of heterologously expressed ENaC are essentially identical to the amiloride-sensitive channel detectable at the apical membrane of CCD cells. [263] [264]



FIGURE 5-20  Maximal expression of the amiloride-sensitive epithelial Na+ channel (ENaC) at the plasma membrane requires the co-expression of all three subunits (a-, β-, and γ-ENaC). A, Amiloride-sensitive current in Xenopus oocytes expressing the individual subunits and various combinations thereof; channel activity is considerably enhanced in cells expressing all three subunits (“αβγ”). B, Surface expression is markedly enhanced in Xenopus oocytes that co-express all three sub-units. The individual cDNAs were engineered with an external epitope tag; expression of the channel proteins at the cell surface is measured by binding of a monoclonal antibody to the tag.  A (From Canessa CM, Schild L, Buell G, et al: Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467, 1994.); B (From Firsov D, Schild L, Gautschi I, et al: Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach. Proc Natl Acad Sci U S A 93:15370–15375, 1996.)




ENaC plays a critical role in renal Na+-Cl- reabsorption and maintenance of the extracellular fluid volume (see also Regulation of Na+-Cl- transport in the CNT and CCD). In particular, recessive loss-of-function mutations in the three subunits of ENaC are a cause of pseudohypoaldosteronism type I.[268] Patients with this syndrome typically present with severe neonatal salt wasting, hypotension, acidosis, and hyperkalemia; this dramatic phenotype underscores the critical roles of ENaC activity in renal Na+-Cl- reabsorption, K+ secretion, and H+ secretion. Gain-of-function mutations in the β- and γ-ENaC subunits are in turn a cause of Liddle syndrome, an autosomal-dominant hypertensive syndrome accompanied by suppressed aldosterone and variable hypokalemia.[269] With one exception,[270] ENaC mutations associated with Liddle syndrome disrupt interactions between a PPxY motif in the C-terminus of channel subunits with the Nedd4-2 ubiquitin-ligase (see also Regulation of Na+-Cl- transport in the CNT and CCD).

The ENaC protein is detectable at the apical membrane of CNT cells and principal cells within the CCD, OMCD, and IMCD. [228] [232] Notably, however, several lines of evidence support the hypothesis that the CNT makes the dominant contribution to amiloride-sensitive Na+ reabsorption by the distal nephron. First, amiloride-sensitive Na+ currents in the CNT are twofold to fourfold higher than in the CCD; the maximal capacity of the CNT for Na+reabsorption is estimated to be ≈10 times higher than that of the CCD.[226] Second, targeted deletion of α-ENaC in the collecting duct abolishes amiloride-sensitive currents in CCD principal cells, but does not affect Na+ or K+homeostasis; the residual ENaC expression in the late DCT and CNT of these knockout mice easily compensates for the loss of the channel in CCD cells.[271] Third, Na+/K+-ATPase activity in the CCD is considerably less than that of the DCT[14] (see also Fig. 5-4 ); this speaks to a greater capacity for transepithelial Na+-Cl- absorption by the DCT and CNT. Fourth, the apical recruitment of ENaC subunits in response to dietary Na+ restriction begins in the CNT, with progressive recruitment of subunits in the downstream CCD at lower levels of dietary Na+272; under conditions of high Na+-Cl- and low K+ intake, the bulk of aldosterone-stimulated Na+ transport likely occurs prior to the entry of tubular fluid into the CCD.[273]

Cl- Transport

There are two major pathways for Cl- absorption in the CNT and CCD; paracellular transport across the tight junction, and transcellular transport across type B intercalated cells ( Fig. 5-21 ). [227] [274] The CNT and CCD are “tight” epithelia, with comparatively low paracellular permeability that is not selective for Cl- over Na+; however, voltage-driven paracellular Cl- transport in the CCD may play a considerable role in transepithelial Na+-Cl- absorption.[275] The CNT, DCT, and collecting duct co-express claudin-3, -4, and -8 [26] [276]; claudin-8 in particular may function as a paracellular cation barrier that prevents backleak of Na+, K+, and H+ in this segment of the nephron.[276]Regulated changes in paracellular permeability may also contribute to Cl- absorption by the CNT and CCD. In particular, wild-type WNK4 appears to increase paracellular Cl- permeability in transfected MDCK II cell lines; a WNK4 PHA-II mutant has a much larger effect, with no effect seen in cells expressing kinase-dead WNK4 constructs.[261] Yamauchi and co-workers have also reported that PHA-II-associated WNK4 increases paracellular permeability, due perhaps to an associated hyper-phosphorylation of claudin proteins.[277]



FIGURE 5-21  Transepithelial Cl- transport by principal and intercalated cells. The lumen-negative PD generated by principal cells drives paracellular Cl- absorption. Alternatively, transepithelial transport occurs in type B intercalated cells, via apical Cl--HCO3- exchange (SLC26A4/pendrin) and basolateral Cl- exit via CLC-K2.



Transcellular Cl- absorption across intercalated cells is thought to play a quantitatively greater role in the CNT and CCD than that of paracellular transport.[274] In the simplest scheme, this process requires the concerted function of both type A and type B intercalated cells, achieving net electrogenic Cl- absorption without affecting HCO3- or H+ excretion[274] (see also Fig. 5-21 ). Chloride thus enters type B intercalated cells via apical Cl--HCO3- exchange, followed by exit from the cell via basolateral Cl- channels. Recycling of Cl- at the basolateral membrane of adjacent type A intercalated cells results in HCO3- absorption and extrusion of H+ at the apical membrane. The net effect of apical Cl--HCO3- exchange in type B intercalated cells, leading to apical secretion of HCO3-, and recycling of Cl- at the basolateral membrane type A intercalated cells, leading to apical secretion of H+, is electrogenic Cl-absorption across type B intercalated cells (see Fig. 5-21 ).

At the basolateral membrane, intercalated cells have a very robust Cl- conductance with transport characteristics similar to those of CLC-K2/Barttin.[195] CLC-K2 protein is also detected at the basolateral membrane of type A intercalated cells, although expression in type B cells was not clarified.[193] At the apical membrane, the SLC26A4 exchanger (also known as pendrin), has been conclusively identified as the elusive Cl--HCO3- exchanger of type B and non-A, non-B intercalated cells[227]; this exchanger functions as the apical entry site during transepithelial Cl- transport by the distal nephron. Human SLC26A4 is mutated in Pendred syndrome, which encompasses sensorineural hearing loss and goiter; these patients do not have an appreciable renal phenotype.[227] However, Slc26a4-deficient knockout mice are sensitive to restriction of dietary Na+-Cl-, developing hypotension during severe restriction.[278] Slc26a4 knockout mice are also resistant to mineralocorticoid-induced hypertension.[279] Finally, dietary Cl- restriction with provision of Na+-HCO3- results in Cl- wasting in Slc26a4 knockout mice and increased apical expression of Slc26a4 protein in the type B intercalated cells of normal littermate controls.[280] Therefore, Slc26a4 plays a critical role in distal nephron Cl- absorption, underlining the particular importance of transcellular Cl- transport in this process.

Regulation of Na+-Cl- Transport in the Connecting Tubule and Cortical Collecting Duct


The DCT, CNT, and collecting ducts collectively constitute the aldosterone-sensitive distal nephron, expressing both the mineralocorticoid receptor and the 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) enzyme that protects against illicit activation by glucocorticoids.[224] Aldosterone plays perhaps the dominant positive role in the regulation of distal nephron Na+-Cl- transport, with a plethora of mechanisms and transcriptional targets.[281] For example, aldosterone increases expression of the Na+/K+-ATPase α-1 and β-1 subunits in the CCD,[282] in addition to inducing Slc26a4, the apical Cl--HCO3- exchanger of intercalated cells.[279] Adosterone may also affect paracellular permeability of the distal nephron, via posttranscriptional modification of claudins and other components of the tight junction.[283] However, particularly impressive progress has been made in the understanding of the downstream effects of aldosterone on synthesis, trafficking, and membrane-associated activity of ENaC subunits.

Aldosterone increases abundance of α-ENaC, via a glucocorticoid-response element in promoter of the SCNN1A gene that encodes this subunit.[284] This transcriptional activation results in an increased abundance of α-ENaC protein in response to either exogenous aldosterone or dietary Na+-Cl- restriction [285] [286] ( Fig. 5-22 ); the response to Na+-Cl- restriction is blunted by spironolactone, indicating involvement of the mineralocorticoid receptor.[250] At baseline, α-ENaC transcripts in the kidney are less abundant than those encoding β- and γ-ENaC[287] (see Fig. 5-22 ). All three subunits are required for efficient processing of heteromeric channels in the endoplasmic reticulum and trafficking to the plasma membrane (see Fig. 5-20 ), such that the induction of α-ENaC is thought to relieve a major “bottleneck” in the processing and trafficking of active ENaC complexes.[287]



FIGURE 5-22  Immunofluorescence images of connecting tubule (CNT) profiles in kidneys from adrenalectomized rats (ADX) and from ADX rats 2 and 4 hours after aldosterone injection. Antibodies against the α-, β-, and γ-subunits of ENaC reveal absent expression of the former in ADX rats, with progressive induction by aldosterone. All three subunits traffic to the apical membrane in response to aldosterone. This coincides with rapid aldosterone induction of the SGK kinase in the same cells; SGK is known to increase the expression of ENaC at the plasma membrane (see text for details). Bar ≈ 15 mm.  (From Loffing J, Zecevic M, Feraille E, et al. Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: Possible role of SGK. Am J Physiol Renal Physiol 280:F675–682, 2001.)




Aldosterone also plays an indirect role in the regulated trafficking of ENaC subunits to the plasma membrane, via the regulation of accessory proteins that interact with pre-existing ENaC subunits. Aldosterone rapidly induces expression of a serine-threonine kinase denoted SGK-1 (serum and glucocorticoid-induced kinase-1) [288] [289]; co-expression of SGK-1 with ENaC subunits in Xenopus oocytes results in a dramatic activation of the channel, due to increased expression at the plasma membrane.[286] Notably, an analogous redistribution of ENaC subunits occurs in the CNT and early CCD, from a largely cytoplasmic location during dietary Na+-Cl- excess to a purely apical distribution after aldosterone or Na+-Cl- restriction (see Fig. 5-22 ). [250] [272] [286] Furthermore, there is a temporal correlation between the appearance of induced SGK-1 protein in the CNT and the redistribution of ENaC protein to the plasma membrane.[286]

SGK-1 modulates membrane expression of ENaC by interfering with regulated endocytosis of its channel subunits. Specifically, the kinase interferes with interactions between ENaC subunits and the ubiquitin-ligase Nedd4-2.[287]PPxY domains in the C-termini of all three ENaC subunits bind to WW domains of Nedd4-2[290]; these PPxY domains are deleted, truncated, or mutated in patients with Liddle syndrome,[269] leading to a gain-of-function in channel activity.[266] Co-expression of Nedd4-2 with wild-type ENaC channel results in a marked inhibition of channel activity due to retrieval from the cell membrane, whereas channels bearing Liddle syndrome mutations are resistant; Nedd4-2 is thought to ubiquitinate ENaC subunits, resulting in the removal of channel subunits from the cell membrane and degradation in lysosomes and the proteosome.[287] A PPxY domain in SGK-1 also binds to Nedd4-2, which is a phosphorylation substrate for the kinase; phosphorylation of Nedd4-2 by SGK-1 abrogates its inhibitory effect on ENaC subunits. [291] [292] Aldosterone also stimulates Nedd4-2 phosphorylation in vivo.[293]Nedd4-2 phosphorylation in turn results in ubiquitin-mediated degradation of SGK-1,[294] suggest that there is considerable feedback regulation in this system. Furthermore, the hormone reduces Nedd4-2 protein expression in cultured CCD cells,[295] suggesting additional levels of in vivo regulation.

The induction of SGK-1 by aldosterone thus appears to stimulate the redistribution of ENaC subunits from the cytoplasm to the apical membrane of CNT and CCD cells. This phenomenon involves SGK-1-dependent phosphorylation of the Nedd4-2 ubiquitin ligase, which is co-expressed with ENaC and SGK-1 in the distal nephron.[295] Of note, there is considerable axial heterogeneity in the recruitment and redistribution of ENaC to the plasma membrane, which begins in the CNT and only extends into the CCD and OMCD in Na+-Cl- restricted or aldosterone-treated animals. [224] [286] The underlying causes of this progressive axial recruitment are not as yet clear.[224]However, Nedd4-2 expression is inversely related to the apical distribution of ENaC, with low expression in the CNT and increased expression levels in the CCD[295]; in all likelihood, the relative balance between SGK-1, ENaC, and Nedd4-2 figures prominently in the recruitment of the channel subunits to the apical membrane.

Finally, aldosterone indirectly activates ENaC channels through the induction of “channel activating proteases”, which increase open channel probability by cleavage of the extracellular domain of α- and γ-ENaC. Western blotting of renal tissue from rats subjected to Na+-Cl- restriction or treatment with aldosterone reveals α- and γ-ENaC subunits of lower molecular mass than those detected in control animals, indicating that aldosterone induces proteolytic cleavage. [285] [296] Proteases that have been implicated in the processing of ENaC include furin, elastase, and three novel, membrane-associated proteases denoted CAP1-3 (channel activating proteases-1/2/3). [297] [298] CAP1 was initially identified from Xenopus A6 cells as an ENaC activating protease[299]; the mammalian ortholog is an aldosterone-induced protein in principal cells.[300] Urinary excretion of CAP1, also known as prostasin, is increased in hyperaldosteronism, with a reduction after adrenalectomy.[300] CAP1 is tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI) linkage,[299] whereas CAP2 and CAP3 are transmembrane proteases.[298] All three of these proteases activate ENaC by increasing the open probability of the channel, without increasing expression at the cell surface.[298] Proteolytic cleavage of ENaC appears to activate the channel by removing the “self-inhibitory” effect of external Na+297; in the case of furin-mediated proteolysis of aENaC, this appears to involve the removal of an inhibitory domain from within the extracellular loop.[301] Unprocessed channels at the plasma membrane are thought to function as a “reserve pool”, capable of rapid activation by membrane-associated luminal proteases.[297]

One would expect synergistic activation by co-expressed CAP1-3 and SGK-1, given that this kinase increases channel expression at the cell surface[286]; this is indeed the case.[298] Notably, the C-terminal mutations that cause Liddle syndrome, which abrogate interaction with Nedd4-2, appear to have a greater relative effect on channel activity than on surface expression[266]; this observation led to a longstanding controversy as to whether the gain-of-function in these mutant channels is partially due to an increase in channel activity at the membrane.[302] This issue has been dramatically resolved by the observation that Liddle syndrome mutations result in an increased proportion of cleaved channels at the plasma membrane; dominant-negative inhibition of Nedd4-2 leads to a similar increased in processing at the cell membrane, whereas over-expression of wild-type Nedd4-2 has the opposite effect.[302]Therefore, aldosterone-dependent induction of SGK-1 also affects protease-dependent cleavage and activation of ENaC at the cell membrane, in addition to reducing Nedd4-2-dependent degradation of the channel.

Vasopressin and Other Factors

Although not classically considered an anti-natriuretic hormone, vasopressin has well-characterized stimulatory effects on Na+-Cl- transport by the CCD. [82] [303] In perfused rat CCD segments, vasopressin and aldosterone can have synergistic effects on Na+ transport, with a combined effect that exceeds that of the individual hormones.[303] Prostaglandins inhibit this effect of vasopressin, particularly in the rabbit CCD; this inhibition occurs at least in part through reductions in vasopressin-generated cAMP. [82] [303] There are however considerable species-dependent differences in the interactions between vasopressin and negative modulators of Na+-Cl- transport in the CCD, which include prostaglandins, bradykinin, endothelin, and α2-adrenergic tone. [82] [303] Regardless, cyclic-AMP causes a rapid increase in the Na+ conductance of apical membranes in the CCD; this effect appears to be due to increases in surface expression of ENaC subunits at the plasma membrane,[304] in addition to effects on open channel probability (reviewed in Ref 305). Notably, cyclic-AMP inhibits retrieval of ENaC subunits from the plasma membrane, via PKA-dependent phosphorylation of the phosphoacceptor sites in Nedd4-2 that are targeted by SGK-1[306]; therefore, both aldosterone and vasopressin converge on Nedd4-2 in the regulation of ENaC activity in the distal nephron. Analogous to the effect on trafficking of aquaporin-2 in principal cells, cAMP also seems to stimulate exocytosis of ENaC subunits to the plasma membrane.[305] Finally, similar to the long-term effects of vasopressin on aquaporin-2 expression and NKCC2 expression,[207] chronic treatment with dDAVP results in an increase in abundance of the β- and γ-ENaC subunits.[307]

Systemic generation of circulating angiotensin II (ANGII) induces aldosterone release by the adrenal gland, with downstream activation of ENaC. However, ANGII also activates amiloride-sensitive Na+ transport directly in perfused CCDs; blockade by losartan or candesartan suggests that this activation is mediated by AT1 receptors.[308] Of particular significance, the effect of luminal ANGII (10-9) was greater than that of bath ANGII, suggesting that intra-tubular ANGII may regulate ENaC in the distal nephron. Similar stimulation of ENaC is seen when tubules are perfused with ANGI; this effect is blocked by ACE-inhibition with captopril, indicating that intraluminal conversion of ANGI to ANGII can occur in the CCD.[309] Notably, CNT cells express considerable amounts of immunoreactive renin, versus the vanishingly low expression of renin mRNA in the proximal tubule.[310] Angiotensinogen secreted into the tubule by proximal tubule cells[310] may thus be converted to ANGII in the CNT via locally generated renin and angiotensin converting enzyme, and/or related proteases.

As in other segments of the nephron, Na+-Cl- transport by the CNT and CCD is modulated by metabolites of arachidonic acid generated by cytochrome P450 mono-oxygenases. In particular, arachidonic acid inhibits ENaC channel activity in the rat CCD, via generation of the epoxygenase product 11,12-EET (epoxyeicosatreinoic acid) by the CYP2C23 enzyme expressed in principal cells.[311] Targeted deletion of the murine Cyp4a10 enzyme, another P450 mono-oxygenase, results in salt-sensitive hypertension; urinary excretion of 11,12-EET is reduced in these knockout mice, with a blunted effect of arachidonic acid on ENaC channel activity in the CCD.[312] These mice also became normotensive after treatment with amiloride, indicative of in vivo activation of ENaC. It appears that deletion of Cyp4a10 reduces activity of the murine ortholog of rat CYPC23 (Cyp2c44 in mouse), and/or related expoxygenases, via reduced generation of a ligand for PPARa (peroxisome proliferator-activated receptor-α) that induces expoxygenase activity.[312] The mechanism(s) whereby 11,12-EET inhibits ENaC are unknown as of yet. However, renal 11,12-EET production is known to be salt-sensitive, suggesting that generation of this mediator may serve to reduce ENaC activity during high dietary Na+-Cl- intake.[311]

Finally, activation of PPARg by thiazolidinediones results in amiloride-sensitive hypertension, suggesting in vivo activation of ENaC. [313] [314] Thiazolidinediones (TZDs—rosiglitazone, pioglitazone, and troglitazone) are insulin-sensitizing drugs utilized for the management of type II diabetes. Treatment with these agents is frequently associated with fluid retention, suggesting an effect on renal Na+-Cl- transport. Given robust expression of PPARg in the collecting duct, activation of ENaC was an attractive hypothesis for this TZD-associated edema syndrome. [313] [314] This appears to be the case, in that selective deletion of the murine PPARg gene in principal cells abrogates the increase in amiloride-sensitive transport seen in response to TZDs. [313] [314] TZDs appear to induce transcription of the Sccn1g gene encoding gENaC,[313] in addition to inducing SGK-1.[315] However, the mechanism(s) of this intriguing stimulation of ENaC activity are not completely clear, nor is the physiological renal ligand for PPARg known. Regardless, the beneficial effect of spironolactone in type II diabetics with TZD-associated volume expansion is consistent with in vivo activation of ENaC in the aldosterone-responsive distal nephron.[316] In addition, the risk of peripheral edema is increased considerably in patients treated with both TZDs and insulin therapy.[316] Notably, insulin appears to activate ENaC via SGK1-dependent mechanisms, [317] [318] such that this clinical observation may be a consequence of the syngergistic activation of ENaC by insulin and TZDs.


Maintenance of K+ balance is important for a multitude of physiological processes. Changes in intracellular K+ impact on cell volume regulation, regulation of intracellular pH, enzymatic function, protein synthesis,[319] DNA synthesis,[320] and apoptosis.[321] Changes in the ratio of intracellular to extracellular K+ affect the resting membrane potential, leading to depolarization in hyperkalemia and hyperpolarization in hypokalemia. In consequence, disorders of extracellular K+ have a dominant effect on excitable tissues, chiefly heart and muscle. In addition, a growing body of evidence implicates hypokalemia or reduced dietary K+ (or both) in the pathobiology of hypertension, heart failure, and stroke[322]; these and other clinical consequences of K+ disorders are reviewed in Chapter 15 .

Potassium is predominantly an intracellular cation, with only 2% of total body K+ residing in the extracellular fluid. Extracellular K+ is maintained within a very narrow range by three primary mechanisms. First, the distribution of K+ between the intracellular and extracellular space is determined by the activity of a number of transport pathways, namely Na+/K+-ATPase, the Na+-K+-2Cl- cotransporter NKCC1, the four K+-Cl- cotransporters, and a plethora of K+ channels. In particular, skeletal muscle contains as much as 75% of body potassium (see Fig. 15-1 ), and exerts considerable influence on extracellular K+. Short-term and long-term regulation of muscle Na+/K+-ATPase plays a dominant role in determining the distribution of K+ between the intracellular and extracellular space; the various hormones and physiological conditions that affect the uptake of K+ by skeletal muscle are reviewed in Chapter 15 (see Table 15-1 ). Second, the colon has the ability to absorb and secrete K+, with considerable mechanistic[323] and regulatory[324] similarities to renal K+ secretion. K+ secretion in the distal colon is increased after dietary loading[324] and in end-stage renal disease.[325] However, the colon has a relatively limited capacity for K+ excretion, such that changes in renal K+ excretion play the dominant role in responding to changes in K+ intake. In particular, regulated K+ secretion by the CNT and CCD play a critical role in the response to hyperkalemia and K+ loading; increases in the reabsorption of K+ by the CCD and OMCD function in the response to hypokalemia or K+deprivation.

This section will review the mechanisms and regulation of transepithelial K+ transport along the nephron. As in other sections of this chapter, the emphasis will be on particularly recent developments in the molecular physiology of renal K+ transport. Of note, transport pathways for K+ play important roles in renal Na+-Cl- transport, particularly within the TAL; furthermore, Na+ absorption via ENaC in the aldosterone-sensitive distal nephron generates a lumen-negative potential difference that drives distal K+ excretion. These pathways are primarily discussed in the section on renal Na+-Cl- transport; related issues relevant to K+ homeostasis per se will be specifically addressed in this section.

Proximal Tubule

The proximal tubule reabsorbs some 50% to 70% of filtered K+ ( Fig. 5-23 ). Proximal tubules generate minimal transepithelial K+ gradients, and fractional reabsorption of K+ is similar to that of Na+.[229] K+ absorption follows that of fluid, Na+, and other solutes, [326] [327] such that this nephron segment does not play a direct role in regulated renal excretion. Notably, however, changes in Na+-Cl- reabsorption by the proximal tubule have considerable effects on distal tubular flow and distal tubular Na+ delivery, with attendant effects on the excretory capacity for K+ (see K+ secretion by the DCT, CNT, and CCD).



FIGURE 5-23  K+ transport along the nephron. Approximately 90% of filtered K+ is reabsorbed by the proximal tubule and the loop of Henle. K+ is secreted along the initial and cortical collecting tubule; net reabsorption occurs in response to K+ depletion, primarily within the medullary collecting duct. PCT, proximal tubule; TAL, thick ascending limb; CCT, cortical collecting tubule; DCT, distal convoluted tubule; S, secretion; R, reabsorption; ALDO, aldosterone; ADH, antidiuretic hormone; MCD, medullary collecting duct; ICT, initial connecting tubule.



The mechanisms involved in transepithelial K+ transport by the proximal tubule are not completely clear, although active transport does not appear to play a major role. [327] [328] Luminal barium has modest effects on transepithelial K+ transport by the proximal tubule, suggesting a component of transcellular transport via barium-sensitive K+ channels.[329] However, the bulk of K+ transport is thought to occur via the paracellular pathway, [329] [330] driven by the lumen-positive potential difference in the mid-to-late proximal tubule (see Fig. 5-3 ). The total K+ permeability of the proximal tubule is thus rather high, apparently due to characteristics of the paracellular pathway. [329] [330]The combination of luminal K+ concentrations that are ≈10% higher than that of plasma, a lumen-positive PD of ≈2 mV (see Fig. 5-3 ), and high paracellular permeability leads to considerable paracellular absorption in the proximal tubule. This absorption is thought to primarily proceed via convective transport—“solvent drag” due to frictional interactions between water and K+—rather than diffusional transport.[331] Notably, however, the primary pathway for water movement in the proximal tubule is quite conclusively transcellular, via Aquaporin-1 and Aquaporin-7 water channels in the apical and basolateral membrane. [18] [28] [29] Therefore, the apparent convective transport of K+would have to constitute “pseudo-solvent drag”, with hypothetical, uncharacterized interactions between water traversing the transcellular route and diffusion of K+ along the paracellular pathway.[331]

The Loop of Henle and Medullary K+ Recycling

Transport by the loop of Henle plays a critical role in medullary K+ recycling ( Fig. 5-24 ). Several lines of evidence indicate that a considerable fraction of K+ secreted by the CCD is reabsorbed by the medullary collecting ducts and then secreted into the late proximal tubule or descending thin limbs of long-looped nephrons (or both).[332] In potassium-loaded rats there is thus a doubling of luminal K+ in terminal thin descending limbs, with a sharp drop after inhibition of CCD K+ secretion by amiloride.[333] Enhancement of CCD K+ secretion by treatment with dDAVP also results in an increase in luminal K+ in descending thin limbs.[334] This recycling pathway (secretion in CCD, absorption in OMCD and IMCD, secretion in descending thin limb) is associated with a marked increase in medullary interstitial K+. Passive transepithelial K+ absorption by the thin ascending limb and active absorption by the thick ascending limb (TAL)[171] also contribute to this increase in interstitial K+ (see Fig. 5-24 ). Specifically, the absorption of K+ by ascending thin limb, TAL, and OMCD exceeds the secretion by descending thin limbs, thus trapping K+ in the interestitium.



FIGURE 5-24  Schematic representation of medullary K+ recycling. Medullary interstitial K+ increases considerably after dietary K+ loading, due to the combined effects of secretion in the CCD, absorption in OMCD, TAL, and IMCD, and secretion in descending thin limb (see text for details).  (Redrawn from Stokes JB: Consequences of potassium recycling in the renal medulla. Effects of ion transport by the medullary thick ascending limb of Henle's loop. J Clin Invest 70:219–229, 1982.)




The physiological significance of medullary K+ recycling is not completely clear. However, an increase in interstitial K+ from 5 μM to 25 μM dramatically inhibits Cl- transport by perfused thick ascending limbs.[171] By inhibiting Na+-Cl- absorption by the TAL, increases in interstitial K+ would increase Na+ delivery to the CNT and CCD, thus enhancing the lumen-negative PD in these tubules and increasing K+ secretion.[171] Alternatively, the marked increase in medullary interstitial K+ after dietary K+ loading serves to limit the difference between luminal and peritubular K+ in the collecting duct, thus minimizing passive K+ loss from the collecting duct.

K+ is secreted into descending thin limbs by passive diffusion, driven by the high medullary interstitial K+ concentration. Descending thin limbs thus have a very high K+ permeability, without evidence for active transepithelial K+transport.[335] Transepithelial K+ transport by ascending thin limbs has not to our knowledge been measured; however, as is the case for Na+-Cl- transport (see Na+-Cl- transport in the thin ascending limb), the absorption of K+ by thin ascending limbs is presumably passive. Active transepithelial K+ transport across the TAL includes both a transcellular component, via apical Na+-K+-2Cl- cotransport mediated by NKCC2, and a paracellular pathway (seeFig. 5-15 ). Luminal K+ channels play a critical role in generating the lumen-positive PD in the TAL, as summarized in the section on Na+-Cl- transport (see Na+-Cl- transport in the TAL; apical K+ channels).

K+ Secretion by the Distal Convoluted Tubule, Connecting Tubule, and Cortical Collecting Duct

Approximately 90% of filtered K+ is reabsorbed by the proximal tubule and loop of Henle (see Fig. 5-23 ); the “fine tuning” of renal K+ excretion occurs in the remaining distal nephron. The bulk of regulated secretion occurs in principal cells within the CNT and CCD, whereas K+ reabsorption primarily occurs in the OMCD (see later). K+ secretion is initially detectable in the early DCT,[336] where NCC-positive cells express ROMK, the apical K+secretory channel.[178] Classically, the CCD is considered the primary site for distal K+ secretion, partially due to the greater ease with which this segment is perfused and studied. However, as is the case for Na+-Cl- absorption (see Na+-Cl- transport in the CNT and CCD; apical Na+ transport), the bulk of distal K+ secretion appears to occur prior to the CCD,[229] within the CNT.[337]

In principal cells, apical Na+ entry via ENaC generates a lumen-negative potential difference, which drives passive K+ exit through apical K+ channels. Distal K+ secretion is therefore dependent on delivery of adequate luminal Na+ to the CNT and CCD, [338] [339] essentially ceasing when luminal Na+ drops below 8 mmol/L.[340] Dietary Na+ intake also influences K+ excretion, such that excretion is enhanced by excess Na+ intake and reduced by Na+restriction (see Fig. 15-4 ). [338] [339] Secreted K+ enters principal cells via the basolateral Na+/K+-ATPase, which also generates the gradient that drives apical Na+ entry via ENaC (see Fig. 5-23 ).

Two major subtypes of apical K+ channels function in secretion by the CNT and CCD, +/- DCT; a small-conductance (SK) 30 pS channel [337] [341] and a large-conductance, Ca2+-activated 150 pS (“maxi-K”) channel. [179] [226]The density and high open probability of the SK channel indicates that this pathway alone is sufficient to mediate the bulk of K+ secretion in the CCD under baseline conditions,[342] hence its designation as the “secretory” K+channel. Notably, SK channel density is considerably higher in the CNT than in the CCD,[337] consistent with the greater capacity for Na+ absorption and K+ secretion in the CNT. The characteristics of the SK channel are similar to those of the ROMK K+ channel,[343] and ROMK protein has been localized at the apical membrane of principal cells.[178] SK channel activity is absent from apical membranes of the CCD in homozygous knockout mice with a targeted deletion of the Kcnj1 gene that encodes ROMK, definitive proof that ROMK is the SK channel.[179] The observation that these knockout mice are normokalemic with an increased excretion of K+ illustrates the considerable redundancy in distal K+ secretory pathways[179]; distal K+ secretion in these mice is mediated by apical maxi-K channels[344] (see later). Of interest, loss-of-function mutations in human KCNJ1 are associated with Bartter syndrome; ROMK expression is critical for the 30 pS and 70 pS channels that generate the lumen-positive PD in the TAL [179] [180] (see Fig. 5-15 ). These patients typically have slightly higher serum K+ than the other genetic forms of Bartter syndrome,[172] and affected patients with severe neonatal hyperkalemia have also been described[345]; this neonatal hyperkalemia is presumably the result of a transient developmental deficit in apical maxi-K channel activity.

The apical Ca2+-activated maxi-K channel plays a critical role in flow-dependent K+ secretion by the CNT and CCD.[341] Maxi-K channels have a heteromeric structure, with α-subunits that form the ion channel pore and modulatory β-subunits that affect the biophysical, regulatory, and pharmacological characteristics of the channel complex.[341] Maxi-K α-subunit transcripts are expressed in multiple nephron segments, and channel protein is detectable at the apical membrane of principal and intercalated cells in the CCD and CNT.[341] Increased distal flow has a well-established stimulatory effect on K+ secretion, due in part to both enhanced delivery and absorption of Na+ and to increased removal of secreted K+, [338] [339] but also due to the activation of apical K+ conductance. The pharmacology of flow-dependent K+ secretion in the CCD is consistent with dominant involvement of maxi-K channels,[346] and flow-dependent K+ secretion is reduced in mice with targeted deletion of the α1 and b1 subunits.[341] The physiological rationale for the presence of two apical secretory K+ channels—ROMK/SK and maxiK channels—is not completely clear. However, the high density and higher open probability of SK/ROMK channels is perhaps better suited for a role in basal K+ secretion, with additional recruitment of the higher capacity, flow-activated maxi-K channels when additional K+ secretion is required.[341]

Other K+ channels reportedly expressed at the luminal membranes of the CNT and CCD include voltage-sensitive channels such as Kv1.3, [347] [348] double-pore K+ channels such as TWIK-1,[349] and KCNQ1.[350] KCNQ1 mediates K+ secretion in the inner ear and is expressed at the apical membrane of principal cells in the CCD,[350] whereas TWIK-1 is expressed at the apical membrane of intercalated cells.[349] The role of these channels in renal K+ secretion or absorption is not as yet known.

K+ channels present at the basolateral membrane of principal cells appear to set the resting potential of the basolateral membrane, and function in K+ secretion and Na+ absorption at the apical membrane, the latter via K+ recycling at the basolateral membrane to maintain activity of the Na+/K+-ATPase. A variety of different K+ channels have been described in the electrophysiological characterization of the basolateral membrane of principal cells, which has a number of technical barriers to overcome (reviewed in Ref 351). However, a singe predominant activity can be identified in principal cells from the rat CCD, using whole-cell recording techniques under conditions in which ROMK is inhibited (low intracellular pH or presence of the ROMK inhibitor tertiapin-Q).[351] This basolateral current is TEA-insensitive, barium-sensitive, and acid-sensitive (pKa ≈ 6.5), with a conductance of ≈17 pS and weak inward-rectification. These properties do not correspond exactly to specific characterized K+ channels, or combinations thereof. However, candidate inward-rectifying K+ channel subunits that have been localized at the basolateral membrane of the CCD include Kir4.1, Kir5.1, Kir7.1, and Kir 2.3.[351] Notably, basolateral K+ channel activity increases on a high-K+ diet, suggesting a role in transepithelial K+ secretion.[351]

In addition to apical K+ channels, considerable evidence implicates apical K+-Cl- cotransport (or functionally equivalent pathways[352]) in distal K+ secretion. [59] [338] [353] Thus in rat distal tubules, a reduction in luminal Cl-markedly increases K+ secretion[354]; the replacement of luminal Cl- with SO4- or gluconate has an equivalent stimulatory effect on K+ secretion. This anion-dependent component of K+ secretion is not influenced by luminal Ba2+,[354] suggesting that it does not involve apical K+ channel activity. Perfused surface distal tubules are a mixture of distal convoluted tubule (DCT), connecting segment, and initial collecting duct (see Fig. 5-18 ); however, Cl--coupled K+ secretion is detectable in both the DCT and in early CNT.[355] In addition, similar pathways are detectable in rabbit CCD, where a decrease in luminal Cl- from 112 mmol/L to 5 mmol/L increases K+ secretion by 48%.[356] A reduction in basolateral Cl- also decreases K+ secretion without an effect on transepithelial voltage or Na+ transport, and the direction of K+ flux can be reversed by a lumen-to-bath Cl- gradient, resulting in K+ absorption.[356] In perfused CCDs from rats treated with mineralocorticoid, vasopressin increases K+ secretion[357]; because this increase in K+ secretion is resistant to luminal Ba2+ (2 mmol/L), vasopressin may stimulate Cl--dependent K+secretion.[356] Recent pharmacological studies of perfused tubules are consistent with K+-Cl- cotransport mediated by the KCCs [59] [353]; however, of the three renal KCCs, only KCC1 is apically expressed along the nephron (D.B.M., unpublished observations). Other functional possibilities for Cl--dependent K+ secretion include parallel operation of apical H+-K+-exchange and Cl--HCO3- exchange in type B intercalated cells.[352]

K+ Reabsorption by the Collecting Duct

In addition to K+ secretion, the distal nephron is capable of considerable reabsorption, primarily during restriction of dietary K+. [229] [230] [231] This reabsorption is accomplished in large part by intercalated cells in the outer medullary collecting duct (OMCD), via the activity of apical H+/K+-ATPase pumps. Under K+-replete conditions, apical H+/K+-ATPase activity recycles K+ with an apical K+ channel, without effect on transepithelial K+absorption. Under K+-restricted conditions, K+ absorbed via apical H+/K+-ATPase appears to exit intercalated cells via a basolateral K+ channel, thus achieving the transepithelial transport of K+.[358]

H+-K+-ATPase holoenzymes are members of the P-type family of ion transport ATPases, which also includes subunits of the basolateral Na+-K+-ATPase.[359] HKa-1 and HKa-2 are also referred to as the “gastric” and “colonic” subunits, respectively; humans also have an HKa-4 subunit. [359] [360] A specific HKb subunit interacts with the HKa subunits to ensure delivery to the cell surface and complete expression of H+-K+-ATPase activity[361]; HKa-2 and HKa-4 subunits are also capable of interaction with Na+-K+-ATPase b subunits. [362] [363] The pharmacology of H+-K+-ATPase holoenzymes differs considerably, such that the gastric HKa-1 is classically sensitive to the H+-K+-ATPase inhibitors SCH-28080 and omeprazole and resistant to ouabain; the colonic HKa-2 subunit is usually sensitive to ouabain and resistant to SCH-28080.[361] Within the kidney, the HKa-1 subunit is expressed at the apical membrane of at least a subset of type A intercalated cells in the distal nephron.[360] HKa-2 distribution in the distal nephron is more diffuse,[364] with robust expression at the apical membrane of type A and B intercalated cells and connecting segment cells and lesser expression in principal cells. [365] [366] The human HKa-4 subunit is reportedly expressed in intercalated cells.[360]

HKa-1 and HKa-2 are both constitutively expressed in the distal nephron. However, tubule perfusion of K+-replete animals suggests a functional dominance of omeprazole/SCH-28080-sensitive, oubain-resistant H+-K+-ATPase activity, consistent with holoenzymes containing HKa-1.[367] K+ deprivation increases the overall activity of H+-K+-ATPase in the collecting duct, with the emergence of a oubain-sensitive H+-K+-ATPase activity [368] [369]; this is consistent with a relative dominance of HKa-2 during K+-restricted conditions. K+-restriction also induces a dramatic up-regulation of HKa-2 transcript and protein in the outer and inner medulla during K+ depletion [370] [371]; HKa-1 expression is unaffected. [370] [371] Mice with a targeted deletion of HKa-2 exhibit lower plasma and muscle K+ than wild-type littermates when maintained on a K+-deficient diet. However, this appears to be due to marked loss of K+ in the colon rather than kidney because renal K+ excretion is appropriately reduced in the K+-depleted knockout mice.[372] Presumably the lack of an obvious renal phenotype in either HKa-1[373] or HKa-2[372]knockout mice reflects the marked redundancy in the expression of HKa subunits in the distal nephron. Indeed, collecting ducts from the HKa-1 knockout mice have significant residual ouabain-resistant and SCH-28080-sensitive H+-K+-ATPase activities, consistent with the expression of other HKa subunits that confer characteristics similar to the “gastric” H+-K+-ATPase.[374] However, more recent data from HKa-1 and HKa-2 knockout mice suggest that compensatory mechanisms in these mice are not accounted for by ATPase-type mechanisms.[375]

The importance of K+ reabsorption mediated by the collecting duct is dramatically illustrated by the phenotype of transgenic mice with generalized over-expression of a gain-of-function mutation in H+-K+-ATPase, effectively bypassing the redundancy and complexity of this reabsorptive pathway. This transgene expresses a mutant form of the HKb sub-unit, in which a tyrosine-to-alanine mutation within the C-terminal tail abrogates regulated endocytosis from the plasma membrane; these mice have higher plasma K+ than their wild-type littermates, with approximately half the fractional excretion of K+.[376]

Regulation of Distal K+ Transport

Aldosterone and K+ Loading

Aldosterone has a potent kaliuretic effect,[377] with important inter-relationships between circulating K+ and aldosterone. Aldosterone release by the adrenal is thus induced by hyperkalemia or a high K+ diet (or both), suggesting an important “feedback” effect of aldosterone on K+ homeostasis.[378] Aldosterone also has clinically relevant effects on K+ homeostasis, with a clear relationship at all levels of serum K+ between circulating levels of the hormone and the ability to excrete K+ (see Chapter 15 and Fig. 15-4 ).

Aldosterone has no effect on the density of apical SK channels in the CCD[379]; it does however induce a marked increase in the density of apical Na+ channels in the CNT and CCD.[379] This hormone activates ENaC via inter-related effects on the synthesis, trafficking, and membrane-associated activity of the subunits encoding the channel (see Regulation of Na+-Cl- transport in the CNT and CCD). Aldosterone is thus induced by high K+ diet (see Table 5-1 ), and strongly stimulates apical ENaC activity, which provides the lumen-negative PD that stimulates K+ secretion by principal cells.

TABLE 5-1   -- Effect of High K+ Diet, Aldosterone, and/or Na+-Cl- Restriction on SK Channel Density in the Rat Cortical Collecting Duct


K Channel Density/μm2

Plasma aldo (ng/dL)

Plasma K (mM)





High K diet, 6 h




High K diet, 48 h




Low Na diet, 7 days




Aldo infusion, 48 h




Aldo + High K diet




Modified from Palmer LG, Frindt G: Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake. Am J Physiol 277:F805–812, 1999.




The important relationships between K+ and aldosterone notwithstanding, it is increasingly clear that much of the adaptation to high K+ intake is aldosterone-independent. For example, a high K+ diet in adrenalectomized animals increases apical Na+ reabsorption and K+ secretion in the CCD.[380] At the tubular level, when basolateral K+ is increased there is a significant activation of the Na+/K+-ATPase, accompanied by a secondary activation of apical Na+ and K+ channels.[381] Increased dietary K+ also markedly increases the density of SK channels in the CCD, along with a modest increase in Na+ channel (ENaC) density[379]; this is associated with changes in the subcellular distribution of the ROMK protein, with an increase in apical expression.[382] Notably, this increase in ENaC and SK density in the CCD occurs within hours of assuming a high K+ diet, with a minimal associated increase in circulating aldosterone[383] ( Fig. 5-25 and Table 5-1 ). In contrast, a week of low Na+-Cl- intake, with almost a thousand-fold increase in aldosterone, has no effect on SK channel density; nor for that matter does 2 days of aldosterone infusion, despite the development of hypokalemia[383] ( Table 5-1 ). Of note, unlike the marked increase seen in the CCD, [379] [383] the density of SK channels in the CNT is not increased by high dietary K+337; this suggests that SK channels in the CNT are already maximally active, consistent with a progressive, axial recruitment of transport capacity for Na+ and K+ along the distal nephron (see Na+-Cl- transport in the CNT and CCD; apical Na+ transport).



FIGURE 5-25  High K+ diet rapidly activates SK channels in the CCD, mediated by the ROMK (Kir 1.1) K+ channel. Histograms of N (channels/patch) are shown for rats on control diet (A), on a high-K diet for 6 hours (B), and on a high-K diet for 48 hours (C). Each determination of N represents a single cell-attached patch. High K+ diet results in a progressive recruitment of SK channels at the apical membrane.  (From Palmer LG, Frindt G: Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake. Am J Physiol 277:F805–812, 1999.)




Maxi-K channels in the CNT and CCD play an important role in the flow-activated component of distal K+ excretion[341]; these channels are also activated by dietary K+ loading. Flow-stimulated K+ secretion by the CCD of both mice[344] and rats[384] is thus enhanced on a high-K+ diet, with an absence of flow-dependent K+ secretion in rats on a low-K+ diet.[384] This is accompanied by the appropriate changes in transcript levels for α- and β2–4-subunits of the maxi-K channel proteins in micro-dissected CCDs (β1 subunits are restricted to the CNT[341]). Trafficking of maxi-K subunits is also affected by dietary K+, with largely intracellular distribution of α-subunits in K+-restricted rats and prominent apical expression in K+-loaded rats.[384]

The changes in trafficking and/or activity of the ROMK channel that are induced by dietary K+ appear in large part to involve tyrosine phosphorylation/dephosphorylation of the ROMK protein (see later). However, a recent series of reports have linked changes in expression of WNK1 kinase subunits in the response to high K+ diet. WNK1 and WNK4 were initially identified as causative genes for pseudohypopaldosteronism type II (PHA-II), also known as Gordon syndrome or “hereditary hypertension with hyperkalemia” (see also Regulation of Na+-Cl- transport in the DCT). ROMK expression at the membrane of Xenopus oocytes is dramatically reduced by co-expression of WNK4; PHA-II-associated mutations dramatically increase this effect, suggesting a direction inhibition of SK channels in PHA-II.[260] The study of WNK1 is further complicated by the transcriptional complexity of its gene, which has at least three separate promoters and a number of alternative splice forms. In particular, the predominant intra-renal WNK1 isoform is generated by a distal nephron transcriptional site that bypasses the N-terminal exons that encode the kinase domain, yielding a kinase-deficient “short” isoform[385] (“WNK1-S”). Full-length WNK1 (WNK1-L) inhibits ROMK activity by inducing endocytosis of the channel protein [259] [386] [387]; kinase activity or the N-terminal kinase domain of WNK1 (or both) appear to be required for this effect, [259] [387] although Cope and colleagues have reported that a kinase-dead mutant of WNK1 is unimpaired.[386] The shorter WNK1-S isoform, which lacks the kinase domain, appears to inhibit the effect of WNK1-L. [259] [387] The ratio of WNK1-S to WNK1-L transcripts is reduced by K+ restriction (greater endocytosis of ROMK) [259] [388] and increased by K+ loading (reduced endocytosis of ROMK), [387] [388] suggesting that this ratio between WNK1-S and WNK1-L functions as a “switch” to regulate distal K+ secretion. Notably, in contrast to the prior data in Xenopus oocytes,[260] changes in the apical distribution of ROMK protein in CNT or CCD segments were reportedly not detected in BAC-transgenic mice overexpressing wild-type WNK4 or a PHA-II mutant (see Regulation of Na+-Cl- transport in the DCT)[251]; this does not, however, rule out a more direct role for WNK1 in trafficking of ROMK and/or the maxi-K channel.

K+ Deprivation

A reduction in dietary K+ leads within 24 hours to a dramatic drop in urinary K+ excretion. [388] [389] This drop in excretion is due to both an induction of reabsorption by intercalated cells in the OMCD [230] [231] and to a reduction in SK channel activity in principal cells.[390] The mechanisms involved in K+ reabsorption by intercalated cells are discussed earlier; notably, H+/K+-ATPase activity in the collecting duct does not appear to be regulated by aldosterone.[391]

Considerable progress has recently been made in defining the signaling pathways that regulate the activity of the SK channel (ROMK) in response to changes in dietary K+. Dietary K+ intake modulates trafficking of the ROMK channel protein to the plasma membrane of principal cells, with a marked increase in the relative proportion of intracellular channel protein in K+-depleted animals [382] [392] and clearly defined expression at the plasma membrane of CCD cells from animals on a high K+ diet.[382] The membrane insertion and activity of ROMK is modulated by tyrosine phosphorylation of the channel protein, such that phosphorylation of tyrosine residue[337] stimulates endocytosis and dephosphorylation induces exocytosis [393] [394]; this tyrosine phosphorylation appears to play a dominant role in the regulation of ROMK by dietary K+.[395] Whereas the levels of protein tyrosine phosphatase-1D do not vary with K+ intake, intra-renal activity of the cytoplasmic tyrosine kinases c-src and c-yes are inversely related to dietary K+ intake, with a decrease under high K+ conditions and a marked increase after several days of K+restriction. [390] [396] Localization studies indicate co-expression of c-src with ROMK in thick ascending limb and principal cells of the CCD.[382] Moreover, inhibition of protein tyrosine phosphatase activity, leading to a dominance of tyrosine phosphorylation, dramatically increases the proportion of intracellular ROMK in the CCD of animals on a high-K+ diet.[382]

The neurohumoral factors that induce the K+-dependent trafficking and expression of apical ROMK [382] [392] and maxi-K channels[384] are not as yet known. However, a landmark study recently implicated the intra-renal generation of superoxide anions in the activation of cytoplasmic tyrosine kinases and downstream phosphorylation of the ROMK channel protein by K+ depletion.[397] Potential candidates for the upstream kaliuretic factor include angiotensin II and growth factors such as IGF-1.[397] Regardless, reports of a marked post-prandial kaliuresis in sheep, independent of changes in plasma K+ or aldosterone, have led to the suggestion that an enteric or hepatoportal K+ “sensor” controls kaliuresis via a sympathetic reflex.[398] Changes in dietary K+ absorption may thus have a direct “anticipatory” effect on K+ homeostasis, in the absence of changes in plasma K+. Such a “feedfoward” control has the theoretical advantage of greater stability because it operates prior to changes in plasma K+.[399] Notably, changes in ROMK phosphorylation status and insulin-sensitive muscle uptake can be seen in K+-deficient animals in the absence of a change in plasma K+,[400] suggesting that upstream activation of the major mechanisms that serve to reduce K+ excretion (reduced K+ secretion in the CNT/CCD, decreased peripheral uptake, and increased K+reabsorption in the OMCD) does not require changes in plasma K+.


Vasopressin has a well-characterized stimulatory effect on K+ secretion by the distal nephron. [334] [401] Teleologically, this vasopressin-dependent activation serves to preserve K+ secretion during dehydration and extracellular volume depletion, when circulating levels of vasopressin are high and tubular delivery of Na+ and fluid is reduced. The stimulation of basolateral V2 receptors results in an activation of ENaC, which increases the driving force for K+ secretion by principal cells; the relevant mechanisms are discussed earlier in this chapter (see Regulation of Na+-Cl- transport in the CNT and cortical collecting duct; vasopressin). In addition, vasopressin activates SK channels directly in the CCD,[402] as does cAMP.[342] The ROMK protein is directly phosphorylated by protein kinase A on three serine residues (S25, S200, and S294 in the ROMK2 isoform), with phosphorylation of all three sites required for full activity in Xenopus oocytes (see Regulation of Na+-Cl- transport in TAL; activating influences). Finally, the stimulation of luminal V1 receptors also stimulate K+ secretion in the CCD, apparently via activation of maxi-K channels.[403]


Calcium Homeostasis

Total body Calcium

Calcium, which exists in the body as a divalent cation (Ca2+), plays an important structural role as a key component of the bony skeleton, and acts as an extracellular and intracellular signal. A total of 1 kg to 2 kg of Ca2+ is present in the body of an average adult, of which approximately 99% is in bone and teeth, and most of the remaining 1% is in soft tissues and the extracellular space. The normal total plasma Ca2+ concentration is 8.8 to 10.3 mg/dL (2.2 to 2.6 μM). (The atomic mass of calcium is 40.1. Thus, 1 mmol Ca2+ ≡ 2 mEq ≡ 40 mg; and 1 μM Ca2+ concentration ≡ 2 mEq/L ≡ 4 mg/dL.) Normally, approximately 40% of plasma Ca2+ is bound to plasma proteins, predominantly albumin. The remaining 60%, which is filterable through artificial and biological membranes (ultrafilterable Ca2+ or UFCa), consists of Ca2+ in complex with various anions, 10%, and free Ca2+ ions (ionized Ca2+ or iCa), 50%. The ionized Ca2+ is the fraction of plasma Ca2+ that is physiologically important and its concentration (1.05 μM to 1.23 μM) is tightly regulated by the hormones, parathyroid hormone (PTH), 1, 25-dihydroxyvitamin D (1,25(OH)2D, the active metabolite of vitamin D), calcitonin, and iCa itself, acting on three major organs, the kidneys, intestinal tract, and bone. The relative distribution of Ca2+ in plasma may be altered by changes in plasma protein concentration and pH. For example, the proportion of total plasma Ca2+ that is ionized is increased in hypoalbuminemia and acidemia. Under such circumstances, the total plasma Ca2+ concentration may not accurately reflect the status of physiologically relevant ionized Ca2+ in the extracellular fluid.

Within cells, Ca2+ is sequestered in the endoplasmic reticulum and mitochondria, or bound to cytoplasmic proteins and ligands, so that the basal intracellular iCa concentration is maintained at a very low level (0.1 μM to 1 μM). The steep gradient between the extracellular and intracellular iCa is maintained by active extrusion of Ca2+ across the plasma membrane mediated by a Ca2+-ATPase pump present in all cells, and also by a sodium-calcium exchanger in certain tissues.

Intake and Output

The daily dietary intake of Ca2+, of which the majority is obtained from milk and other dairy products, is 600 mg to 800 mg for adults in the United States. Many adults also take Ca2+ supplements, so that the average total intake of Ca2+ is approximately 1000 mg ( Fig. 5-26 ). Approximately 20% to 25% of dietary Ca2+ is absorbed by the intestine. In addition, the intestine also reabsorbs approximately 200 mg of Ca2+ from luminal secretions in the distal small intestine and colon. The efficiency of Ca2+ absorption is increased when dietary Ca2+ is reduced, as well as during periods of rapid growth in children, during pregnancy, and during lactation. Once absorbed, Ca2+ in the extracellular fluid may exchange with the pool in bone, with 300 mg of Ca2+ typically entering and leaving the skeleton daily as it is continuously remodeled. Ultimately, the kidneys are responsible for the excretion of about 200 mg of Ca2+ per day. More importantly, regulation of renal Ca2+ excretion is one of the principal ways in which the body regulates extracellular Ca2+ balance.



FIGURE 5-26  Typical daily Ca2+ intake and output for a normal adult in neutral Ca2+ balance (see text for details).



Overview of Calcium Regulation

The plasma concentration of iCa is meticulously maintained within a narrow range. This is accomplished by PTH, 1,25(OH)2D, calcitonin, and extracellular Ca2+ itself, acting on three major target organs, the kidneys, intestinal tract and bone ( Fig. 5-27 ). A fall in plasma iCa acutely stimulates secretion of PTH from the parathyroid gland, increases PTH gene expression, and chronically leads to parathyroid gland hyperplasia. These effects are thought to be mediated by the calcium-sensing receptor (CaSR),[404] a G-protein-coupled receptor located on the cell membrane of parathyroid glands. Extracellular Ca2+ acts as an agonist of the CaSR, thereby activating phospholipase C, generating inositol 1,4,5-triphosphate, and releasing intracellular Ca2+ stores, as well as inhibiting adenylate cyclase and reducing intracellular cyclic AMP levels.[404] Reduction in extracellular iCa leads to the opposite effects and thereby stimulates release of PTH. PTH acts to increase iCa and return it to normal by mobilizing Ca2+ from bone stores, and by stimulating renal Ca2+ reabsorption in the distal tubule (see later). In addition, PTH stimulates 25-hydroxyvitamin D-1α-hydroxylase, the enzyme in the proximal renal tubule that converts 25-hydroxyvitamin D to its active metabolite, 1,25(OH)2D, which in turn promotes intestinal absorption of Ca2+. An abnormal rise in iCa has the opposite effects on PTH and vitamin D metabolism. Hypercalcemia also activates the secretion from thyroid C-cells of calcitonin, a hormone that inhibits osteoclast-mediated bone resorption. However, calcitonin is not thought to play an important role in normal Ca2+ homeostasis in humans. In addition to its effects on the parathyroid gland, extracellular Ca2+ itself has hormone-like actions on other organs, which are mediated by the CaSR. For example, hypercalcemia is thought to directly inhibit renal tubule Ca2+ reabsorption, likely via engagement of the CaSR on the basolateral membrane of the thick ascending limb of Henle (see later).



FIGURE 5-27  Summary of overall Ca2+ homeostasis. The primary homeostatic mechanisms activated in response to a fall in extracellular fluid (ECF) ionized Ca2+ concentration are shown.



Renal Handling of Calcium

Only the ionized and complexed forms of plasma Ca2+ are ultrafiltered at the glomerulus. The Ca2+ concentration of fluid in Bowman space, determined by micropuncture in rodents, has generally been found to be 60% to 70% of the total plasma Ca2+ concentration,[405] consistent with the values for UFCa determined with artificial membranes. Renal clearance studies have demonstrated that 98% to 99% of the filtered load of Ca2+ is reabsorbed by the renal tubules, so that only about 200 mg/d is ultimately excreted. The contribution of individual nephron segments is summarized in Table 5-2 .

TABLE 5-2   -- Segmental Handling of Ca2+ Along the Renal Tubule

Nephron Segment

Fractional Reabsorption (%)

Cellular Transport Mechanism

Proximal tubule


Passive, paracellular

Thin descending and ascending limbs





Passive, paracellular
Active component stimulated by PTH



Active, transcellular

Collecting duct




TAL, thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule.




Calcium Handling by individual Nephron Segments

Proximal Convoluted Tubule (PCT) Segments, S1 and S2

Most of the filtered load of Ca2+ (50% to 60%) is reabsorbed in the PCT. The ratio of the concentration of Ca2+ in tubule fluid to its concentration in the glomerular ultrafiltrate (TF/UFCa), as determined by micropuncture, is 1.0 in the early convolutions of the superficial PCT, rising to 1.1 to 1.2 in the later convolutions.[405] The finding that the proximal tubule fluid Ca2+ concentration is close to UFCa suggests that Ca2+ is absorbed in parallel with Na+ and water. The modest increase in the later convolutions could be attributed either to a lag in the reabsorption of Ca2+ relative to water, creating a favorable concentration gradient for diffusive reabsorption downstream, or to a rising concentration of nonabsorbable complexed Ca2+.

The passive permeability of the PCT to Ca2+ is high, as demonstrated by a large backflux component in in vivo microperfusion experiments.[406] In vitro microperfusion studies of PCT S2 segments confirmed the high Ca2+permeability, and also showed that there was no net flux of Ca2+ in the absence of a transtubular electrochemical gradient.[407] These findings suggest that Ca2+ reabsorption in the PCT is primarily passive in nature. The observation that PCT Ca2+ reabsorption parallels closely that of Na+ and water not only under normal basal conditions, but also following maneuvers that change urinary Ca2+ excretion[405] such as administration of saline, diuretics, PTH, and acid-base disturbances, is also consistent with a passive mechanism. Passive reabsorption could potentially occur either by solvent drag or by diffusion. The reflection coefficient for Ca2+ in this segment is 0.9,[407] arguing against a major component of convective transport. Furthermore, recent evidence indicates that the majority of water reabsorption in the proximal tubule is transcellular and mediated by aquaporin-1 water channels. Given that transcellular transport would require uphill transport of Ca2+ against a steep, unfavorable electrochemical gradient at the basolateral membrane, and that aquaporin-1 channels are impermeable to cations, solvent drag is an unlikely mechanism for Ca2+ reabsorption in this segment.

By contrast, diffusion is thought to play the major role in Ca2+ reabsorption, particularly in the S2 and later segments of the proximal tubule where the TF/UFCa rises above 1.0 and the transepithelial electrical potential difference (PD) becomes lumen-positive, so that a favorable electrochemical gradient exists to drive vectorial transport ( Fig. 5-28 ). The unfavorable electrochemical gradient across the basolateral membrane precludes simple transcellular diffusion, so passive Ca2+ reabsorption by diffusion must occur via the paracellular route. The rate-limiting barrier in paracellular transport is the tight junction, and recent evidence suggests that a family of integral membrane tight junction proteins, the claudins, regulate paracellular permeability, possibly by constituting the pore of a paracellular channel. One of the members of this family, claudin-2, forms paracellular cation pores[408] and is strongly expressed in the tight junctions of the proximal nephron.[25] Claudin-2 is therefore a strong candidate for the paracellular Ca2+ channel.



FIGURE 5-28  Models depicting the putative cellular mechanism of Ca2+ reabsorption in different nephron segments. In the proximal tubule S2 segment (PT), transcellular Na+ and water generate a concentration gradient that drives passive paracellular Ca2+ reabsorption, likely through the tight junction channel protein, claudin-2 (CLDN2). In the thick ascending limb (TAL), transcellular Na+ reabsorption through the apical electroneutral Na-K-2Cl cotransporter (NKCC2), coupled to apical K+ recycling through the ATP-sensitive K+ channel, ROMK, generates a lumen-positive voltage that drives passive paracellular Ca2+ reabsorption through claudin-16/paracellin (CLDN16). In the distal convoluted tubule and connecting tubule (DCT/CNT), Ca2+ is reabsorbed transcellularly. Ca2+ enters apically through an epithelial Ca2+ channel (TRPV5), diffuses across the cytosol bound to calbindin-D28k (CBP-D28k), and exits basolaterally through a plasma membrane Ca2+ pump (PMCA), or Na-Ca exchanger (NCX1). Ca2+ backflux is blocked by a paracellular Ca2+ barrier constituted in part by claudin-8 (CLDN8). Other abbreviations: [iCa2+], ionized Ca2+ concentration; Ψ, electrical potential; NKA, Na-K-ATPase; CLC-Kb, basolateral Cl- channel.



The question of if, and how Ca2+ is reabsorbed in the earliest (S1) segment of the PCT remains open, as there are no direct studies of this segment. The TF/UFCa initially approximates 1.0 and the transepithelial electrical potential difference (PD) is lumen-negative due to the reabsorption of Na+ with uncharged organic solutes, so there is no driving force for Ca2+ diffusion. In certain situations, Ca2+ is apparently reabsorbed against an uphill electrochemical gradient in the PCT, suggesting that it may be actively transported in the PCT. However, the observation that TF/UFCa rises progressively to 1.1 to 1.2 along the length of this segment, and does so seemingly in parallel with an initially proportionate rise in the TF/UF of inulin, a nonreabsorbable filtration marker, suggests that more likely there is simply no Ca2+ reabsorption in the S1 segment.

In summary, the bulk of Ca2+ reabsorption in the PCT, probably mainly mediated by the S2 segment, is passive and diffusive, driven by a modest concentration gradient and lumen-positive PD.

Proximal Straight Tubule Segments, S2 and S3

Whereas the TF/UFCa is similar to TF/UFNa at the end of the PCT, tubular fluid obtained by micropuncture from the hairpin bend of the loop of Henle has a TF/UFCa that is 35% less than TF/UFNa, suggesting that there is disproportionate reabsorption of Ca2+, dissociated from that of Na+, in either the PST or the thin descending limb (TDL).[405] As the TDL has been found to be impermeable to Ca2+,[409] this implicates the PST as a site of Na+-independent Ca2+ reabsorption.

The S2 segment of the PST exhibits passive Ca2+ reabsorption driven by a lumen-positive PD and favorable concentration gradient,[410] identical to the S2 segment of the PCT. The S3 segment of the PST, when perfused in vitro with symmetrical solutions, reabsorbs Ca2+ despite the absence of a concentration gradient, and a lumen-negative PD,[411] indicating the active transport of Ca2+. Furthermore, this reabsorption is not affected by ouabain, confirming its dissociation from Na+ movement, and excluding any involvement of a Na-Ca exchanger. The molecular mechanisms for active Ca2+ reabsorption in this segment are currently unknown.

Thin Descending And Ascending Limbs of the Loop of Henle

The permeability of the thin limbs to Ca2+ is very low and no significant net transport of Ca2+ is believed to take place in these segments.[405]

Thick Ascending Limb of the Loop of Henle

Approximately 15% of the filtered load of Ca2+ is reabsorbed in the TAL. In this segment, active reabsorption of NaCl and luminal membrane recycling of K+ generate a lumen-positive PD (see Fig. 5-28 ). In vitro tubule perfusion studies have demonstrated that this segment has a significant permeability to Ca2+ and that the direction of net Ca2+ transport is voltage-dependent, consistent with passive, and presumably paracellular, diffusion.[405] Indeed, Bourdeau and Burg found no net flux of Ca2+ when the cortical TAL was perfused with symmetrical salt solutions and the transepithelial PD was abolished by administration of the loop diuretic, furosemide, which inhibits the apical Na-K-2Cl cotransporter and thereby establishes a chemical voltage clamp.[412] This suggests that transport of Ca2+ in this segment is entirely passive. Furthermore, they found that the Ussing flux ratio varied linearly with voltage, in a manner consistent with a model of single-file ionic diffusion. Mg2+ transport in the TAL is also thought to be primarily mediated by passive paracellular diffusion (see later). Such a model nicely explains why both hypercalciuria and hypermagnesuria occur in situations in which the ability to generate the TAL transepithelial PD is compromised, such as pharmacological inhibition of the Na-K-2Cl cotransporter with loop diuretics, and inherited loss-of-function mutations in either the Na-K-2Cl cotransporter, the ROMK K+ channel, or the ClC-Kb Cl- channel, as in Bartter syndrome.

Another disorder with both renal Ca2+ and Mg2+ wasting, familial hypomagnesemic hypercalcuria, has recently been found to be caused by mutations in claudin-16 (also known as paracellin), a member of the claudin family of integral membrane tight junction proteins that is expressed in the tight junctions of the TAL.[185] This suggests that claudin-16 plays an important role in the paracellular transport of divalent cations. Furthermore, careful analysis of the claudin-16 amino-acid sequence revealed a cluster of negatively charged residues in one of the two putative extracellular loops of the protein, which is not found in other homologous members of the claudin family. The extracellular domains of the protein would be predicted to protrude into the lateral intercellular space, potentially in directly contact with ions traversing the paracellular pathway. It is therefore tempting to speculate that claudin-16 may in fact constitute part of a paracellular Ca2+ and Mg2+ channel, with the negatively charged residues forming the mouth of the pore, as found in other transmembrane Ca2+ channels.

Controversy exists over whether there is an additional component of Ca2+ reabsorption in the TAL that is active. Suki and co-workers found that although furosemide decreased the tubular efflux of Ca2+ in the medullary TAL concomitant with abolition of the transepithelial PD, it actually increased Ca2+ efflux in the cortical TAL.[413] Imai varied the transepithelial PD of cortical TAL by perfusion with solutions of varying Na+ concentrations and showed that net reabsorption of Ca2+ occurred even when the transepithelial PD was zero.[414] Friedman demonstrated significant net Ca2+ reabsorption in the cortical TAL but not in medullary TAL under furosemide voltage-clamp conditions.[415] Furthermore, the residual, presumably active, component of net Ca2+ flux in the cortical TAL under these conditions was increased almost threefold by PTH.

Distal Convoluted Tubule and Connecting Tubule

Approximately 10% to 15% of filtered Ca2+ is reabsorbed in the distal tubule, which includes the DCT, CNT, and collecting tubule. The PD in the distal tubule is lumen-negative throughout, and the Ca2+ concentration below that of plasma, so any Ca2+ reabsorption must be active. By micropuncture, only the early and late convolutions of the superficial DCT are accessible. It is well established that there is Ca2+ reabsorption in the superficial DCT.[405]Costanzo and Windhager demonstrated that this reabsorption is active, and that backflux of Ca2+ is negligible, precluding a component of passive transport.[416] However, the fractional delivery of Ca2+ to the final urine is less than that to the late distal tubule, indicating that there must be further reabsorption of Ca2+ after the DCT. Indeed both Shareghi and Stoner,[417] and Imai[418] have perfused isolated segments of CNT and demonstrated active Ca2+reabsorption in these segments (as well as in the DCT). Importantly, Ca2+ reabsorption in both the DCT and CNT are stimulated by PTH and by cyclic AMP. Thus, these are the major segments for the anti-calciuric action of PTH, working through adenylate cyclase. The molecular mechanisms of distal tubule Ca2+ transport are discussed in the subsequent section and summarized in Figure 5-28 . It is important to note that although the absolute amount of Ca2+ reabsorbed in these segments is quite modest, the fraction excretion of Ca2+ into the final urine is only about 1% to 2%. Thus, even very small changes in DCT/CNT fractional reabsorption can have a large effect on the final amount excreted. This underscores the importance of the DCT and CNT for the hormonal regulation of renal Ca2+ excretion.

Collecting Duct

The collecting duct likely plays a minor role in renal Ca2+ reabsorption. In isolated perfused cortical collecting ducts (CCD), a small net reabsorption of Ca2+ has been observed by some, but not by others.[405] If there is indeed Ca2+ reabsorption in this segment, it would occur against a negative luminal voltage and uphill transepithelial Ca2+ concentration gradient, and would therefore have to be active and transcellular. Consistent with this, some of the components that could potentially mediate transcellular Ca2+ transport (see later) are also expressed in the CCD, including TRPV6[419] and calbindin-D28k in mice,[225] and NCX1, calbindin-D28k and the plasma membrane Ca-ATPase in humans.[420] Ca2+ reabsorption may also occur in the medullary collecting ducts.

Molecular Mechanisms of Transcellular Ca2+ Reabsorption

Transcellular Ca2+ reabsorption occurs primarily in the DCT and CNT, with perhaps minor components in the cortical TAL (when activated by PTH) and CCD. This process can be divided into three key steps: apical entry, cytosolic diffusion, and basolateral efflux (see Fig. 5-28 ). The intracellular free Ca2+ level in most cells is several orders of magnitude below that of extracellular fluid, and the membrane potential inside-negative. Thus, the basolateral efflux step must necessarily be active, whereas the apical entry step would rationally be expected to be passive.

Apical Ca2+ entry

The apical entry step is likely to be the rate-limiting step in vectorial Ca2+ transport. In rabbit CNT/CCD cells, apical Ca2+ entry and basolateral Ca2+ exit are closely coupled. [421] [422] Stimulation of adenylyl cyclase with forskolin increases intracellular Ca2+ levels and transcellular Ca2+ transport, suggesting that this occurs through stimulation of apical Ca2+ entry.[421] Conversely, inhibition of Ca2+ influx by apical acidification causes a decrease in intracellular Ca2+ and subsequent downregulation of transcellular Ca2+ transport.[422]

The apical entry step is mediated by a Ca2+ channel. Numerous groups have reported voltage-dependent Ca2+ channels with varying properties in the distal tubule by patch clamp study.[405] Bacskai and Friedman observed Ca2+entry into DCT/cortical TAL cells that was regulated by dihydropyridines, which are ligands of L-type voltage-dependent Ca2+ channels, and upregulated by PTH.[423] Expression of various L-type voltage-dependent Ca2+ channels has been found in distal renal tubule segments or cultured distal convoluted tubule cells. [424] [425] [426] However, the role of voltage-dependent Ca2+ channels in vectorial transepithelial Ca2+ transport remains unproven.

It is now clear that the principal apical epithelial Ca2+ channel in the distal tubule is TRPV5 (also known as ECaC1) a member of the TRP superfamily of cation channels. TRPV5 was identified by an expression cloning strategy[427] and found to be expressed on the apical membrane of CNT principal cells in the rabbit,[428] and in both the late distal convoluted tubule (DCT2) and the CNT in the mouse[225] and rat (see Fig. 5-18 ).[429] Toward the more distal segments, its location becomes intracellular, suggesting that its surface abundance may be regulated by vesicle trafficking.[225] Its functional properties are also consistent with a role in apical Ca2+ entry. When expressed in Xenopus oocytes or in human embryonic kidney cells, TRPV5 is highly selective for Ca2+ (PCa/PNa 100:1) and exhibits inwardly rectifying currents that would be expected to be constitutively activated at physiological membrane potentials.[430] Furthermore, entry of Ca2+ into the cell leads to inactivation of the channel.[431] This negative feedback mechanism may account in part for the tight coupling between apical Ca2+ entry and basolateral efflux that has been observed.[421] TRPV5 currents are also inhibited by reduction in extracellular pH.[432] This may account for the hypercalciuria observed in association with acidosis. There are no specific ligands of TRPV5, but it is blocked by ruthenium red, econazole, and inorganic polycations such as Gd3+ and Cd2+.[433]

TRPV6 (also referred to as CaT1 or ECaC2)[434] is a homolog of TRPV5 with similar functional properties. [435] [436] TRPV6 is the predominant apical Ca2+ entry channel in the duodenum, where it likely mediates transepithelial Ca2+ absorption. TRPV6 is also expressed in the kidney, specifically in DCT2, CNT, cortical and medullary collecting ducts (see Fig. 5-18 ).[419] When expressed in HEK293 cells, TRPV6 can assemble with TRPV5 into functional heterotetrameric Ca2+ channels.[437] However, the role of TRPV6 in the kidney in vivo remains undefined.

The critical role of TRPV5 in distal tubule Ca2+ reabsorption was demonstrated by the finding that TRPV5 knockout mice are hypercalciuric.[438] In these mice, renal TRPV6 expression is preserved. Fractional delivery of Ca2+ to the earliest segments of the distal convolution was found to be normal, but as more distal puncture sites were sampled, fractional delivery of Ca2+ appeared to rise, indicating not only the absence of reabsorption, but also significant secretion of Ca2+ into the tubule lumen. The latter is presumably due to passive backleak of Ca2+ via the paracellular pathway. These findings indicate that TRPV6 cannot compensate for absence of TRPV5 in the kidney.

TRPV5, particularly its C-terminus, participates in interactions with numerous proteins that may be important in its regulation, including S100A10, 80K-H, BSPRY, and calmodulin. S100A10 is a member of the S100 protein superfamily that forms a heterotetrameric complex with annexin-2, and serves to stabilize plasma membrane domains and provide a link to the actin cytoskeleton. This interaction is required for TRPV5 trafficking to the plasma membrane.[439] 80K-H is a protein with two EF hands that binds Ca2+ and appears to function as an intracellular Ca2+ sensor for TRPV5. Inactivation of the EF-hand pair reduces the TRPV5-mediated Ca2+ current and increases the TRPV5 sensitivity to intracellular Ca2+, accelerating feedback inhibition of the channel.[440] BSPRY (B-box and SPRY domain-containing protein) is a protein of unknown function that appears to inhibit Ca2+ influx in MDCK cells expressing TRPV5.[441] Both S100A10 and 80K-H mRNA are up-regulated by vitamin D, whereas BSPRY is down-regulated, suggesting these as possible modes of vitamin D regulation of TRPV5. Both TRPV5 and TRPV6 also bind calmodulin.[442] In TRPV6, calmodulin acts as a Ca2+ sensor and is responsible for slow inactivation of the channel by high levels of intracellular Ca2+.[443] The role of calmodulin in TRPV5 function has not been studied.

Cytosolic Diffusion of Ca2+

The baseline cytosolic free Ca2+ concentration in all cells is maintained at a very low level (submicromolar range). Against this low background, acute spikes in intracellular Ca2+ level can thereby be detected for signalling purposes. This poses a unique problem for Ca2+-transporting epithelia such as the distal renal tubule and the duodenum: at such low concentrations, the theoretical rate of diffusive flux of Ca2+ across the cytosol is insufficient to support the observed rates of transcellular Ca2+ transport.[444]

Both the distal tubule and duodenal epithelium contain the cytosolic calcium-binding protein, calbindin. Two isoforms exist, calbindin-D28k, which is predominantly expressed in the mammalian kidney (except in birds where it is also in the intestine), and calbindin-D9k, which is primarily in the intestine (but also in the kidney in the mouse).[405] The importance of calbindin-D28k for renal Ca2+ reabsorption is supported by three lines of evidence. First, it colocalizes to the exact same nephron segments as other Ca2+-transporting proteins (see Fig. 5-18 ). Calbindin-D28k is found in the DCT, particularly in the late DCT, and in principal cells of the CNT and CCD, [225] [420] coinciding with the localization of TRPV5 and NCX1 (see later). Second, its level of expression is induced by 1,25(OH)2D,[445] thus correlating with the increase in Ca2+ reabsorption stimulated by this hormone.[446] Finally, calbindin-D28k knockout mice[447] have a twofold to threefold increase in urinary Ca2+-creatinine ratio,[448] suggestive of a defect in renal tubule Ca2+ reabsorption.

It has been postulated that the role of calbindin is to buffer cytosolic Ca2+, so that a high total intracellular Ca2+ concentration may exist despite a very low free Ca2+ concentration. Bronner and Stein calculated that the predicted maximum transcellular flux across the distal tubule in the absence of calbindin is less than 2% of the actual transcellular flux observed experimentally. In the presence of calbindin, even though Ca2+ bound to it diffuses more slowly than unbound Ca2+, there is more total Ca2+ to diffuse, so that the predicted maximum transcellular flux is similar to the observed value.[444] Feher has performed in vitro studies using the intestinal isoform, calbindin-D9k, which confirm the predicted effect of Ca2+-binding protein to increase Ca2+ flux.[449] Calbindin may augment transcellular Ca2+ flux by an additional mechanism: by buffering intracellular Ca2+, it would prevent feedback inhibition of TRPV5 by high intracellular free Ca2+ concentrations and thereby increase apical Ca2+ entry. Finally it has been proposed, by analogy to calmodulin that calbindins may also act as Ca2+ sensors that directly bind to, and regulate, membrane transport proteins. Although there is no direct evidence for this hypothesis at this time, there are reports indicating that calbindin-D28k is not only in the cytosol but can be associated with the membrane fraction as well.[450] [451]

Basolateral Ca2+ Exit

Two classes of transport protein could potentially mediate active extrusion of Ca2+ at the basolateral membrane, plasma membrane Ca-ATPases (PMCA), and Na-Ca exchangers (NCX). Na-Ca exchange appears to be the major Ca extrusion mechanism in primary cultured CNT/CCD cells, with PMCA playing a minor role. [445] [452]

The Na-Ca exchanger is a secondary active transporter that utilizes the inwardly directed transmembrane Na+ gradient generated by the Na-K-ATPase to drive Ca2+ countertransport. Na+ gradient-dependent Ca2+ transport activity can be detected in renal cortical basolateral membranes,[453] and in liposomes reconstituted with partially purified protein.[454] Furthermore, it has been observed in isolated perfused tubules of the rabbit DCT and CNT,[455] and in membrane vesicles derived from the rat distal tubule but not from the proximal tubule,[456] consistent with a role in distal tubule transcellular Ca2+ reabsorption. The renal NCX shares similar properties to the NCX of excitable tissues; it is reversible, electrogenic with a stoichiometry of 3 to 4 Na+ ions per 1 Ca2+ ion, and the Km for Ca2+ is in the micromolar range.[454]

Three NCX genes have been identified. NCX1 is the gene expressed in the kidney. [457] [458] Alternative splicing within a large intracellular loop further increases the complexity of NCX1. Six small exons are used in different combinations in different tissues, giving rise to heart (NCX1.1), brain (NCX1.4), and kidney (NCX1.3) splice variants.[459] This has functional importance because the large intracellular loop is the site responsible for two regulatory mechanisms, Ca2+ activation, and Na+-dependent inactivation.[460] Intracellular Ca2+-activation is likely to be a mechanism to match exchanger activity with Ca2+ transport requirements. Specifically in the distal tubule, increased apical Ca2+ entry would increase intracellular Ca2+ levels, and thereby stimulate exchange activity to facilitate basolateral Ca2+ efflux. The physiological role of Na+-dependent inactivation is unclear.

NCX1 is expressed most strongly in the DCT2 with lesser expression in the CNT and late DCT1 in the mouse (see Fig. 5-4 ),[225] and most strongly in the CNT with lesser expression in the late DCT and CCD in humans.[420]

The other potential route for basolateral efflux of Ca2+ is via the PMCA. PMCA are primary active pumps of the P-type ATPase family. They are ubiquitous in eukaryotic cells where they serve to maintain low levels of intracellular free Ca2+. Biochemical evidence of Ca2+-dependent ATPase activity can be detected along the entire length of the nephron,[461] and ATP-dependent Ca2+ transport can be detected in membrane vesicles derived from both proximal and distal tubules.[456] If PMCA play an additional, more specialized role in transcellular Ca2+ reabsorption, one might expect that they would either be more strongly expressed, or a unique isoform would be expressed at the basolateral membrane of the DCT and CNT. Indeed early studies using a monoclonal antibody, raised against purified erythrocyte PMCA, stained exclusively the basolateral membrane of the DCT.[462] Four genes encode for the PMCA (named PMCA 1-4), and multiple alternatively spliced mRNA variants exist (denoted by a letter, a-d, after the gene number).[463]

So far, the distal tubule basolateral PMCA has not been identified. PMCA1 and PMCA4 are widely expressed throughout the body and are thought to play a housekeeping role. Consistent with this, PMCA1b and 4b are found ubiquitously within the kidney. [464] [465] By reverse-transcription polymerase chain reaction (RT-PCR) amplification of mRNA from individually microdissected nephron segments, PMCA2b was found to be most highly expressed in the DCT and cTAL. [464] [465] It is therefore the strongest candidate for the basolateral Ca2+ pump. However, a study using isoform-specific antibodies found that PMCA2 was confined to the brain.[466] Furthermore, in the distal tubule cell lines, mDCT[467] and MDCK,[467] the only PMCA transcripts and protein that have been detected are PMCA1b and 4b.

Paracellular Cation Barrier

In contrast to the proximal tubule and TAL, the distal nephron segments have a very low passive permeability to Ca2+ backflux,[416] and likewise to other cations including H+, K+, and Na+. This indicates that the paracellular pathway must be relatively impermeable to Ca2+, and indeed this would be expected to be a prerequisite for active transcellular reabsorption to proceed efficiently in these segments. Claudin-8, a tight junction membrane protein expressed in the distal renal tubule, can markedly impede paracellular permeability to cations, including Ca2+, implicating it as a key component of the paracellular Ca2+ barrier.[468]

Regulation of Renal Calcium Handling

Many factors influence renal Ca2+ handling. These are summarized in Table 5-3 , and only the most important are discussed here.

TABLE 5-3   -- Summary of Factors Affecting Ca2+ Reabsorption


Nephron Location





Volume expansion







↓ (PTH)










↑ (PTH)



↓ (PTH)

Acid-base status














 Vitamin D











 Loop diuretics









PTH, indirect effect mediated by parathyroid hormone.




Glomerular Filtration Rate

Increasing GFR with protein feeding, dexamethasone, or dopamine is associated with minimal change in renal Ca2+ excretion, presumably due to a compensatory increase in tubule reabsorption.[405] This demonstrates that glomerulotubular balance for Ca2+ is well maintained. A modest reduction in GFR, as in early renal failure, is associated with a decrease in the fractional excretion of Ca2+, perhaps in part due to secondary hyperparathyroidism. In advanced renal failure, however, although absolute Ca2+ excretion is low, fractional excretion increases, due to a reduction in Ca2+ reabsorption in the proximal tubule and TAL segments of the remaining intact nephrons.[469] This explains in part why serum Ca2+ does not normally rise during advanced renal failure.

Sodium and Extracellular Fluid Volume

Upon volume expansion with saline, the fractional excretion of Ca2+ increases in parallel with that of Na+. This is hardly surprising because Ca2+ reabsorption is dependent on Na+ reabsorption in the PCT and the TAL. However, Agus and co-workers observed that fractional delivery of Ca2+ to the late DCT was not increased with saline loading.[470] This seems to suggest that any effects in the proximal nephron are irrelevant and that the increased Ca2+excretion is actually due to decreased reabsorption in a segment downstream of the DCT, presumably either the CNT or CCD.


Hypercalcemia reduces the GFR and can cause a prerenal syndrome, as well as acute renal failure. In mild hypercalcemia the ultrafiltration coefficient, Kf, is reduced,[471] whereas more severe hypercalcemia causes renal arteriolar vasoconstriction and reduced renal plasma flow.[472]

Hypercalcemia also reduces renal tubule Ca2+ reabsorp-tion, an adaptive response that presumably serves to rid the body of the excess Ca2+.[405] In the PCT, hypercalcemia reduces reabsorption of Ca2+, presumably by increasing the peritubular Ca2+ concentration and thereby reducing the concentration gradient driving paracellular transport. Hypercalcemia also inhibits Ca2+ reabsorption in the TAL,[473] likely by activating the CaSR, which is located on the basolateral membrane.[220] One postulated mechanism is through activation of phospholipase A2, generating arachidonic acid metabolites, such as 20-hydroxyeicosatetraenoic acid (20-HETE), which inhibit the apical Na-K-2Cl cotransporter[474] and the apical K+ channel.[475] In this scheme, the generation of the normal lumen-positive voltage, which is the driving force for paracellular Ca2+ reabsorption, is impaired, and hence Ca2+ reabsorption inhibited. However, in a recent study in isolated, perfused cortical thick ascending limbs, activation of the CaSR with the agonists, Gd3+ or NPS R-467, did not seem to affect either active Na+ reabsorption or the transepithelial voltage generated by it, but rather inhibited PTH-stimulated transcellular Ca2+ reabsorption by an unexplained mechanism.[476]

Finally, hypercalcemia induced by Ca2+ infusion also inhibits Ca2+ reabsorption in the DCT/CNT.[477] This is probably not a direct effect of Ca2+, but is likely secondary to the suppression of PTH. In the in vitro perfused DCT that is not subjected to PTH, increased luminal Ca2+ delivery actually leads to increased Ca2+ reabsorption.[473]

Acidosis and Alkalosis

Acute and chronic metabolic acidosis cause hypercalciuria, primarily due to a decrease in distal tubule Ca2+ reabsorption.[478] Conversely, induction of metabolic alkalosis enhances tubule Ca2+ reabsorption and leads to hypocalciuria.[405] Furthermore, in the isolated perfused DCT, Ca2+ reabsorption is increased by a high perfusate pH and decreased by a low perfusate pH, suggesting that in metabolic acid-base disturbances it is the luminal pH of the distal tubule that is sensed.[479] The conductance of TRPV5, the apical Ca2+ entry channel in the distal tubule, is increased by an acute decrease in apical extracellular pH and decreased by a high pH.[432] Furthermore, chronic metabolic acidosis decreases renal TRPV5 and calbindin-D28k mRNA and protein abundance in wild-type mice, whereas chronic metabolic alkalosis has the opposite effect.[480] Thus, pH-regulation of TRPV5 expression and activity could potentially explain the effects of chronic and acute acidosis and alkalosis.


Parathyroid Hormone and Parathyroid Hormone-Related Peptide

Parathyroid hormone is secreted by the parathyroid gland and is one of the most important regulators of body Ca2+ homeostasis. Parathyroid hormone-related peptide (PTHrP) is a paracrine factor with PTH-like effects on Ca2+handling. Two types of PTH receptors have been identified, PTH1R, which binds both PTH and PTHrP, and PTH2R, which binds only to PTH. In the kidney, PTH1R mRNA is expressed in glomerular podocytes, PCT, PST, cortical TAL, and DCT.[481] By immunohistochemistry, it is expressed more strongly at the baso-lateral membrane of proximal tubules than at the apical membrane.[482] PTH2R is expressed in vascular endothelium and smooth muscle, and in lung and pancreas.[483] In the kidney, it is expressed only at the vascular pole of the glomerulus. For this reason, PTH1R is considered to be the receptor primarily responsible for the effects of PTH and PTHrP on renal tubule Ca2+ (and phosphate) transport.

Parathyroid hormone and PTHrP stimulation of PTH1R can activate at least two second messenger signaling systems: the classical adenylyl cyclase/protein kinase A (PKA) pathway and the phospholipase C/protein kinase C pathway (PKC). Chabardés and colleagues have shown that PTH-stimulated adenylyl cyclase is present in the PCT, PST, cortical TAL, CNT, and early CCD in rabbits.[405] There is some species variation, so that in mice it is also present in the DCT, but is absent from the CCD, whereas in humans it is primarily in the early DCT rather than the CNT, and is found in medullary as well as cortical TAL. The precise roles of the different signalling pathways in mediating the physiological effects of PTH and PTHrP are not well defined at present.

Parathyroid hormone has multiple different effects on the kidney ( Table 5-4 ). It directly decreases Kf,[471] thereby reducing GFR and the filtered load of Ca2+. PTH also stimulates tubular Ca2+ reabsorption, so that its overall effect is to reduce renal Ca2+ excretion.

TABLE 5-4   -- Parathyroid Effects on Renal Ca2+ Handling



Putative Mechanism


↓ Filtered Ca2+load

↓ Kf

Proximal tubule

↓ Ca2+ reabsorption

↓ Apical Na-H exchanger

Cortical TAL

↑Ca2+ reabsorption

↑Paracellular permeability


↑Ca2+ reabsorption

↑Apical Ca2+ channels



↑Luminal NaHCO3delivery


DCT, distal convoluted tubule; CNT, connecting tubule.




In the proximal tubule, the primary action of PTH is to inhibit NaHCO3 reabsorption via PKA-dependent phosphorylation and endocytosis of the Na-H exchanger, NHE3.[105] The reduced proximal tubule Na+ and water rebsorption is accompanied by a proportionate decrease in passive Ca2+ reabsorption in this segment.[484] However, this is counteracted by increased Ca2+ reabsorption in more distal segments.

In the TAL, PTH increases Ca2+ reabsorption. In mice, this effect has been shown to be confined to the cortical segment,[415] consistent with the known tubular distribution of PTH1R[481] and adenylyl cyclase. The mechanism of this effect is controversial. Several studies using isolated perfused tubules suggest that PTH affects the passive permeability of the cortical TAL. Bourdeau and Burg,[485] and Imai,[418] both found that PTH increased Ca2+reabsorption without increasing the transepithelial voltage. Wittner showed that the effect of PTH disappeared when the transepithelial voltage was abolished by administration of furosemide and perfusion of both tubule surfaces with symmetrical solutions (in effect, the imposition of a chemical voltage clamp).[486] Because the transepithelial voltage provides the driving force for passive paracellular Ca2+ reabsorption in this segment,[412] these studies suggest that the paracellular permeability of the cortical TAL can be regulated by PTH, perhaps via an effect on claudin-16. By contrast, Friedman found that PTH stimulated net Ca2+ reabsorption in cortical TAL tubules even when they were chemically voltage-clamped, suggesting that there may be a PTH-dependent component of active, transcellular Ca2+ transport in this segment.[415]

The primary site for PTH action is thought to be the DCT/CNT.[487] PTH increases Ca2+ reabsorption in these segments without affecting transepithelial voltage,[418] and its effects can be mimicked by cyclic AMP analogs,[487]suggesting that they are mediated by PKA. In the rabbit, Shimizu and co-workers[488] and Imai[418] observed that PTH-responsiveness was confined to the CNT, which would be consistent with the known distribution of adenylyl cyclase in this species, whereas Shareghi and Stoner found that both DCT and CNT were responsive to PTH.[417]

Because Ca2+ reabsorption in these segments is active and transcellular, PTH presumably regulates one or more of the steps involved in transcellular transport. In microdissected rabbit CNT,[489] and in isolated mouse cortical TAL/DCT cells,[423] PTH induces a sustained increase in intracellular Ca2+ within minutes that is due to extracellular Ca2+ entry, suggesting that PTH may regulate TRPV5. However, the mechanism of these acute effects is unknown. Chronic reduction in PTH in mice or rats, by parathyroidectomy or administration of a calcimimetic, reduces expression of TRPV5, calbindin-D28k and NCX1 over a period of 1 week, whereas PTH supplementation restores their expression. Furthermore, inhibition of Ca2+ entry by the TRPV5-specific inhibitor, ruthenium red, blocks the PTH-stimulated expression of calbindin-D28k and NCX1.[490] This suggests that PTH exerts its effects primarily on gene expression of TRPV5 and that the increased intracellular Ca2+ secondarily up-regulates expression of other components of transcellular Ca2+ reabsorption in a coordinated manner.

Vitamin D

Vitamin D metabolites play a minor role in regulating renal Ca2+ handling. The overall effect of vitamin D on renal Ca2+ clearance in parathyroidectomized animals is small and variable. However, there does appear to be good evidence for an effect of vitamin D metabolites to selectively increase Ca2+ reabsorption in the distal tubule. In primary cultures of rabbit CNT/CCD cells, 1,25(OH)2D significantly increases transepithelial Ca2+ reabsorption.[446]This is in part due to increased gene expression of Ca2+ transport proteins. In vitamin D-deficient knockout mouse models, there was downregulation of TRPV5, NCX1, and calbindin D28 mRNA that could be normalized by 1,25(OH)2-D supplementation.[491]

In addition, vitamin D appears to regulate TRPV5 activity through interaction with other proteins. The TRPV5-binding proteins, S100A10 and 80K-H are positive regulators of its activity, whereas BSPRY is a negative regulator of TRPV5 activity. Consistent with this, vitamin D up-regulates S100A10 and 80K-H, and down-regulates BSPRY. [439] [440] [441] Klotho is a newly described type I membrane protein of the β-glycosidase family that is expressed principally in distal tubule cells of the kidney. It hydrolyzes extracellular N-linked oligosaccharides on TRPV5 and stabilizes it at the plasma membrane, facilitating Ca2+ reabsorption.[492] Interestingly, Klotho is upregulated by 1,25(OH)2-D, which may be a novel mechanism by which vitamin D regulates Ca2+ metabolism.


Supraphysiologic doses of calcitonin are hypercalciuric. By contrast, at physiologic doses, calcitonin has been observed to decrease renal Ca2+ excretion.[405] Part of this effect is simply due to concomitant hypocalcemia, but there is also a direct effect of calcitonin to increase distal tubule Ca2+ reabsorption. Calcitonin receptors were detected in the TAL and DCT by ligand binding,[493] and in the TAL and CCD by RT-PCR,[494] whereas calcitonin-sensitive adenylyl cyclase has been demonstrated in the cortical and medullary TAL of rabbit, rat, and human, and in the DCT of mouse, rat, and human.[495] By isolated perfused tubule studies, calcitonin and cyclic AMP analogs enhance Ca2+ reabsorption in the rabbit medullary TAL[496] and DCT.[488]

Tissue Kallikrein

Kallikrein is a serine protease that cleaves kinninogens to activate kinins. Tissue kallikrein (TK) is expressed in the late DCT and CNT and is up-regulated on a low Ca2+ diet. TK knockout mice are hypercalciuric.[497] However, abolition of signalling by the two kinin receptors, B1 (by a receptor blocker) and B2 (by gene targeting), did not affect urine Ca2+ excretion. Thus, TK likely stimulates renal Ca2+ reabsorption by a kinin-independent mechanism.


Loop Diuretics

Loop diuretics inhibit the TAL apical Na-K-2Cl cotransporter, NKCC2. They cause profound natriuresis and calciuresis. The effect of loop diuretics on Ca2+ excretion is clearly a consequence of inhibition of NKCC2, because hypercalciuria is also seen in patients with type I Bartter syndrome, who have inactivating mutations in NKCC2.[498] Entry of Na+, K+, and Cl- through NKCC2 in a 1:1:2 ratio, in concert with apical K+ recycling through the K+channel, ROMK, generates the lumen-positive electrical potential that drives paracellular Ca2+ reabsorption in this segment (see Fig. 5-28 ). The effect of loop diuretics is due to dissipation of this electrical potential.[412]

Thiazide Diuretics

Acute administration of thiazides causes marked natriu-resis due to inhibition of the thiazide-sensitive NaCl cotransporter, NCC, in the DCT. At the same time, urinary Ca2+ excretion changes minimally.[499] This is likely because thiazides acutely stimulate Ca2+ reabsorption in the DCT, leading to a dissociation between Na+ and Ca2+ handling in this segment.[416]

Chronic administration of thiazides causes frank hypocalciuria. This effect is dependent on inhibition of NCC because hypocalciuria is also seen in patients with Gitelman syndrome. Several lines of evidence suggest that the likely mechanism is extracellular fluid volume contraction leading to increased proximal tubule reabsorption of Na+, and hence Ca2+. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic thiazide treatment, whereas Ca2+ reabsorption in the DCT was not affected.[500] Second, thiazides induce hypocalciuria in TRPV5 knockout mice, which have no active distal Ca2+ reabsorption.[500]Third, increased reabsorption of Ca2+ in the proximal tubule is also observed in NCC knockout mice.[241] Finally, the hypocalciuria in humans can be reversed by salt replacement.[501]


Magnesium Homeostasis

Total Body Magnesium

Magnesium, which exists in the body as a divalent cation (Mg2+), is the second most abundant intracellular cation.[405] It is a component of the bony skeleton, an essential cofactor for many metabolic enzymes, and a key regulator of ion channels and transporters in excitable tissues. The normal body content of Mg2+ is approximately 24 g (2000 mEq), of which 50% to 60% resides in mineralized bone, and 40 to 50% is in the intracellular compartment. (The atomic mass of Mg is 24.3. Thus, 1 mmol Mg2+ ≡ 2 mEq ≡ 24.3 mg; and 1 μM Mg2+ concentration ≡ 2 mEq/L ≡ 2.43 mg/dL.) Only about 1% of total body Mg2+ is present in extracellular fluid. The total concentration of Mg2+ in serum is normally 1.8 to 2.3 mg/dL (1.5 to 1.9 mEq/L). Approximately 70% to 80% is ultrafilterable, of which 90% is in an ionized form, and the rest is complexed to citrate, bicarbonate, and phosphate. Twenty percent to 30% of serum Mg2+ is bound to proteins, chiefly albumin. The ionized fraction of total Mg2+ is considered to be the physiologically important moiety. Unlike Ca2+, the relative distribution of Mg2+ in plasma does not vary much with pH.

Within cells, greater than 90% of Mg2+ is bound to anions such as ATP, ADP, citrate, proteins, RNA, and DNA, or is sequestered in subcellular compartments, chiefly mitochondria and the endoplasmic reticulum. Measurements of intracellular ionized Mg2+ by nuclear magnetic resonance or ion-sensitive fluorescent dyes in a variety of mammalian tissues have generally been found to be in the range of 0.25 to 1 μM.[405] No direct measurements have been performed in renal tubule epithelial cells, but in MDCK cells[502] and in immortalized mouse DCT cells,[503] intracellular free Mg2+ is approximately 0.5 μM. Thus, the intracellular free Mg2+ concentration is not substantially different from that of the extracellular fluid. However, most cells have an inside-negative resting membrane potential, so that the intracellular free Mg2+ concentration is actually maintained below its electrochemical equilibrium. Although most plasma membranes are relatively impermeable to Mg2+, there must exist active transport mechanisms to extrude excess Mg2+ that leaks into the cell. In heart and erthyrocytes, there is some evidence that this role is undertaken by a Na-Mg exchanger.

Intake and Output

The average daily dietary intake of Mg2+ in North America is 300 mg ( Fig. 5-29 ), the main sources of which are green vegetables, soybeans, nuts, whole grain cereals, and seafood. The minimum daily dietary Mg2+ intake needed to maintain Mg2+ balance in the average person is controversial, with estimates ranging from 12 mg up to 100 mg. Mg2+ is absorbed in the small intestine, primarily the jejunum and ileum. Of the dietary Mg2+ intake, 30% to 50% is normally absorbed, but this can increase to 75% on a low Mg2+ diet, and decrease to 24% on a high Mg2+ diet. Lower intestinal secretions can contain up to 16 mg/dL of Mg2+. Under normal circumstances, their contribution to overall Mg2+ elimination is minimal (about 20 mg/day), but these losses can become quite significant in diarrheal states. Urinary excretion normally accounts for about 100 mg of Mg2+ output per day.



FIGURE 5-29  Typical daily Mg2+ intake and output for a normal adult in neutral Mg2+ balance (see text for details).



Overview of Magnesium Regulation

Extracellular Mg2+ levels are regulated by three effector organs: intestine, bone, and kidney. Although several hormones have effects on Mg2+ homeostasis, none are specific for Mg2+, and no single hormone appears to play a very important role. For example, in contrast to their effects on Ca2+, the active metabolites of vitamin D have a slight, but probably physiologically insignificant, stimulatory effect on intestinal Mg2+ absorption. PTH induces a modest decrease in urinary Mg2+ excretion.[504] Mg2+ is also an agonist at the CaSR, but its affinity is relatively weak. In sharks and teleost fish, the CaSR senses Mg2+ at concentrations found in seawater in order to osmoregulate in response to environmental salinity.[505] Whether CaSR sensing of extracellular Mg2+ in mammals has any physiological significance is unknown.

Renal Magnesium Handling

Seventy percent to 80% of serum Mg2+ is freely filtered at the glomerulus, of which most is reabsorbed along the length of the nephron. Only about 3% of filtered Mg2+ normally appears in the final urine.[405] During severe dietary Mg2+ deprivation, the kidney avidly retains Mg2+. Under these circumstances, urinary Mg2+ excretion may be reduced to less than 24 mg/day (and often less than 12 mg/day), and the fractional excretion of filtered Mg2+ (FeMg) to less than 1%.[506] Conversely, with Mg2+ loading a threshold effect is observed[507]: when the threshold serum Mg2+ concentration (1.8 mg/dL in humans[507]) is exceeded, increasing Mg2+ spills into the urine in an amount equivalent to the excess filtered load ( Fig. 5-30 ). This observation of an apparent transport maximum (Tm) in such clearance studies initally suggested the existence of a saturable reabsorptive pathway. However, careful in vivo tubule perfusion studies have shown that Mg2+ reabsorption is not, in fact, saturable with respect to luminal Mg2+ concentration in any segment of the nephron, but is inhibited in a concentration-dependent manner by increasing peritubular Mg2+ levels in the TAL, giving rise to an apparent Tm effect in studies of whole kidney clearance.[508] The contribution of individual nephron segments is summarized in Table 5-5 .



FIGURE 5-30  Relationship of urinary Mg2+ excretion to serum ultrafiltrable Mg2+ in normal human subjects before and during Mg2+ infusion. The apparent threshold for appearance of urinary Mg2+ is a serum ultrafiltrable Mg2+ concentration of 1.4 mg/dL.  (From Rude RK, Bethune JE, Singer FR: Renal tubular maximum for magnesium in normal, hyperparathyroid, and hypoparathyroid man. J Clin Endocrinol Metabol 51:1425–1431, 1980.)




TABLE 5-5   -- Segmental Handling of Mg2+ Along the Renal Tubule

Nephron Segment

Fractional Reabsorption (%)

Cellular Transport Mechanism

Proximal tubule



Thin descending and ascending limbs





Passive, paracellular



Active, transcellular

Collecting duct




TAL, thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule.



Magnesium Handling by Individual Nephron Segments

Proximal Convoluted Tubule

Only 5% to 15% of filtered Mg2+ is reabsorbed in the PCT. Free-flow micropuncture studies have shown that the TF/UFMg is greater than 1, rises progressively along the length of the PCT, but is always less than the TF/UF for inulin.[405] This indicates that Mg2+ is reabsorbed in the PCT, but at a lower rate than that of Na+ and water. The cellular mechanism of Mg2+ reabsorption is unknown. In vivo tubule microperfusion studies showed that PCT Mg2+reabsorption is not saturable, but increases linearly in response to increasing luminal Mg2+ conentrations even up to ten times that of plasma UFMg.[508] Conversely, increasing basolateral Mg2+ by induction of hypermagnesemia inhibited Mg2+ reabsorption. These findings are consistent with passive Mg2+ transport. Furthermore, Mg2+ reabsorption in the PCT is inhibited by volume expansion, in parallel with changes in Na+ and water reabsorption in this segment. The model that best explains Mg2+ reabsorption in this segment is one in which the rise in luminal Mg2+ concentration due to water reabsorption provides a concentration gradient that drives diffusive paracellular flux. However, two studies have shown very little backflux of peritubular Mg2+ into the lumen, suggesting that the passive permeability of this segment is in fact very low. [406] [508] Thus, the mechanism for PCT Mg2+ reabsorption remains controversial.

In immature animals, the PCT plays a more important role, reabsorbing 60% to 70% of the filtered load.[509] The mechanism for this is unknown.

Proximal Straight Tubule

The only direct data on PST function come from a single study of isolated perfused PST segments, showing that they behave similarly to the PCT.[510] In this study, PST segments reabsorbed Mg2+ concomitant with, but at a lower fractional rate than, the reabsorption of Na+ and water.

Thin Descending and Ascending Limbs of the Loop of Henle

Mg2+ transport has not been studied directly in either the thin descending or ascending limbs. It is unlikely that there is any significant net reabsorption in these segments.

Thick Ascending Limb of the Loop of Henle

The TAL is the major site for tubular Mg2+ reabsorption and accounts for 60% to 70% of reabsorption along the nephron.[405] The TF/PMg decreases from 1.5 at the end of the PCT to 0.5 to 0.6 at the beginning of the DCT, indicating that there is net Mg2+ reabsorption without water reabsorption and that it can proceed against an uphill concentration gradient. The site of Mg2+ reabsorption is species-specific: in the mouse, it occurs in the cortical TAL only, whereas in the rabbit it has been found in both cortical and medullary segments.[405] Quamme and Dirks have shown by in vivo microperfusion that Mg2+ reabsorption in this segment increases linearly with luminal Mg2+concentration and is unsaturable.[508] When plasma Mg2+ and hence peritubular Mg2+ concentration is increased while luminal Mg2+ is held constant, Mg2+ reabsorption is inhibited. Similar results were obtained in in vitro perfused TAL segments.[511] These observations probably explain the apparent Tm observed in clearance studies (see Fig. 5-30 ).

The mechanism of Mg2+ transport in the TAL is primarily passive and driven by the transepithelial voltage. [486] [511] Under normal circumstances, there is a lumen-positive transepithelial potential difference that is generated by apical Na+, K+, and Cl- entry via NKCC2, concomitant with apical K+ recycling via ROMK ( Fig. 5-31 ).[486] This transepithelial voltage would be expected to drive passive reabsorption of Mg2+ even against an uphill concentration gradient. Loop diuretics, such as furosemide and bumetanide, inhibit the Na-K-2Cl cotransporter, and prevent the establishment of a transepithelial potential difference, thereby abolishing Mg2+ reabsorption. [486] [512]Similarly, extracellular volume expansion decreases Na+ reabsorption, transepithelial voltage, and hence Mg2+ reabsorption in this segment.[513] The route of transport is likely to be a paracellular pathway that is shared with Ca2+. Claudin-16, the tight junction protein mutated in familial hypercalciuric hypomagenesemia, is postulated to be the paracellular Mg2+ and Ca2+ pore.[185]



FIGURE 5-31  Models depicting the putative cellular mechanism of Mg2+ reabsorption in different nephron segments.



Most hormones regulate Mg2+ transport in the TAL by altering the transepithelial voltage, or the paracellular permeability (see later). However, glucagon and ADH appear to enchance Mg2+ reabsorption in the mouse cortical TAL with little or no change in the transepithelial voltage, [514] [515] suggesting the possibility of an additional, transcellular component.

The CaSR is present on the basolateral membrane in this segment[220] and when stimulated, inhibits Na+ reabsorption,[474] reduces the lumen-positive voltage, and hence inhibits Mg2+ reabsorption. This is presumably the mechanism by which hypercalcemia induces magnesuria.[473] The CaSR could also provide a convenient feedback loop for regulating hypermagnesemia. However, the affinity of the CaSR for Mg2+ is quite low (EC50 of 10 μM when expressed in Xenopus oocytes), so it is unlikely to play such a regulatory role under normal circumstances.

Distal Convoluted Tubule and Connecting Tubule

The DCT/CNT reabsorbs 5% to 10% of filtered Mg2+.[405] TF/PMg increases along the distal tubule, though to a lesser extent than that of inulin, indicating that there is net reabsorption at a lower rate than that of Na+ and water. Because Mg2+ transport in this segment normally operates close to maximum capacity, whereas fractional excretion of Mg2+ can vary over a very wide range, the DCT/CNT is unlikely to play a very important regulatory role in Mg2+ homeostasis. There are no direct studies to indicate whether reabsorption occurs in the DCT or the CNT, nor is the cellular mechanism known.

Quamme and colleagues have addressed the mechanisms of Mg2+ transport in a series of studies in the MDCT cell line. They found that Mg2+ entry is stimulated by hyperpolarization, amiloride, and PTH, and inhibited by dihydropyridine Ca2+ channel blockers (reviewed in Ref 516). Although these studies have yielded potentially interesting insights into the biology of Mg2+ transport, these cells have never been demonstrated to mediate vectorial transepithelial Mg2+ transport, nor have the observations ever been corroborated in the DCT itself. Thus, it is unclear at this point whether data obtained from the MDCT cell line have any direct relevance to the situation in the in vivo distal tubule.

Collecting Duct

Micropuncture studies comparing the Mg2+ content of late superficial distal tubule fluid to the final urine suggested that 1% to 3% of the filtered load may be reabsorbed in the collecting duct.[405] However, this interpretation is flawed because it does not account for the possibility of heterogeneity in handling of Mg2+ between superficial and juxtamedullary nephrons. There have been three direct studies of collecting tubule Mg2+ transport. Brunette and colleagues micropunctured early and late collecting tubule sites,[517] Shareghi and Agus isolated and perfused CCD segments,[518] and Bengele and co-workers microcatheterized the IMCD in vivo.[519] No significant Mg2+reabsorption was observed in any of these studies, indicating that the collecting duct system does not contribute to renal tubule Mg2+ handling.

Molecular Mechanisms of Transcellular Mg2+ Reabsorption

Apical Mg2+ Entry

The apical entry step is primarily mediated by the epithelial Mg2+ channel, TRPM6 (see Fig. 5-31 ). TRPM6, which is also a member of the TRP family, was first identified as the culprit gene for familial hypomagnesemia with secondary hypocalcemia, an autosomal recessive disease characterized by intestinal malabsorption of Mg2+ associated with renal Mg2+ wasting. [520] [521] TRPM6 is expressed at the apical membrane of DCT1 and DCT2 (see Fig. 5-18 ),[522] and also at the brush border of intestinal absorptive epithelial cells in the colon and cecum. When expressed in HEK cells, TRPM6 forms cation channels that permeate both Ca2+ and Mg2+. However, the affinity of the channel is much higher for Mg2+ than for Ca2+ suggesting that under physiological conditions, it primarily conducts Mg2+.[522]

TRPM7, a close homolog of TRPM6, is ubiquitously expressed, and also encodes divalent cation channels.[523] Interestingly, TRPM6 and TRPM7 are distinct from all other ion channels in that they are composed of a channel linked to an atypical protein α-kinase domain whose function is so far undefined.[523] TRPM6 associates with TRPM7. In Xenopus oocytes and in HEK cells, this appears to be necessary for trafficking of TRPM6 to the cell surface.[524]Thus, TRPM6/TRPM7 heteromultimers may represent the functional unit of the Mg2+ channel in the DCT.

Basolateral Mg2+ Exit

The identity of the basolateral Mg2+ transport protein(s) remains unknown. One hypothesis is that this step is mediated by a Na-Mg exchanger, such as has been observed in a variety of other tissues, including squid axon, myocardium, and erythrocytes. However, the molecular identity of such a Na-Mg exchanger has yet to be elucidated.

Interestingly, mutations in FXYD2,[525] which encodes a Na-K-ATPase γ-subunit, have been found in autosomal dominant isolated familial hypomagnesemia, which is characterized by hypomagnesemia with renal Mg2+ wasting.[526] Mutation of the γ-subunit was shown to abolish its ability to facilitate trafficking of the α- and β-subunits of the Na-K-ATPase to the cell surface.[525] A plausible explanation is that active, transcellular Mg2+ reabsorption in the distal tubule requires a secondary active transport step (i.e., Na-Mg exchange) that is energetically dependent on the Na+ gradient across the basolateral membrane, and hence on Na-K-ATPase activity (see Fig. 5-31 ). Mutation of the γ-subunit impairs Na-K-ATPase surface trafficking leading to loss of the basolateral Na+ gradient and therefore defective Na-Mg exchange.

Regulation of Renal Magnesium Handling

Many factors influence renal Mg2+ handling. These are summarized in Table 5-6 , and only the most important are discussed here.

TABLE 5-6   -- Summary of Factors Affecting Mg2+ Reabsorption


Nephron Location





Volume expansion







No Δ


No Δ


No Δ

Phosphate depletion

No Δ

Acid-base status














No Δ



No Δ


No Δ








 Loop diuretics

No Δ

No Δ

 Calcineurin inhibitors




TAL, thick ascending limb; PTH, parathyroid hormone.

No Δ, no change.




Sodium and Extracellular Fluid Volume

Saline expansion increases the excretion of Mg2+, concomitant with that of Na+ and water. This occurs even when the renal artery perfusion pressure is reduced to decrease the GFR, and is associated with an increase in the fractional excretion, indicating that tubule Mg2+ reabsorption is inhibited. In the PCT, Wen and colleagues showed that fractional reabsorption of Mg2+ declined from 30% to 15%.[527] This is consistent with the observation that Mg2+ handling in this segment generally parallels that of Na+ and water. There was also a substantial reduction in TAL fractional reabsorption so that the bulk of the increased load of Mg2+ delivered to the loop of Henle appeared in the urine.

Hypermagnesemia and Hypomagnesemia

Hypermagnesemia increases renal Mg2+ excretion, not only due to an increased filtered load, but also due to inhibition of tubule reabsorption. In the proximal tubule, absolute reabsorption increases, due to increased delivery, but fractional reabsorption decreases, in parallel with a decrease in Na+ and water reabsorption of unknown cause.[527] In the TAL, Mg2+ reabsorption is specifically inhibited by the high peritubular Mg2+ concentration.[508] Several possible mechanisms could explain this. First, the concentration gradient for passive diffusion of Mg2+ would be reduced if basolateral Mg2+ concentrations were high. However, in hypermagnesemia luminal Mg2+ concentrations would also be expected to be high, yet this does not seem to be sufficient to overcome the inhibition. Second, basolateral Mg2+ may direct inhibit Mg2+ transport, perhaps by binding to the basolateral opening of a paracellular Mg2+channel such as claudin-16, in the same way that Mg2+ blocks other cation channels. Third, basolateral Mg2+ may, like Ca2+, stimulate the TAL CaSR,[220] thereby triggering the generation of arachidonic acid metabolites that inhibit NKCC2 and ROMK and abolishing the transepithelial voltage that drives Mg2+ reabsorption.

During profound Mg2+ depletion, fractional excretion of Mg2+ falls markedly, usually to below 1% in humans. [506] [528] Studies of Mg2+ depletion in rats demonstrated that proximal tubule fractional reabsorption did not change; because the delivered load was less, though, the absolute reabsorption of Mg2+ in this segment was reduced.[529] The major site of adaptive change is the TAL, where fractional reabsorption was increased by 12%, so that only 3% of the filtered load exited the TAL.[529] The mechanism by which the TAL can conserve Mg2+ under these circumstances is not well understood. A recent study found that dietary Mg2+ restriction caused upregulation of TRPM6 mRNA in the kidney.[530] This could also increase Mg2+ reabsorption in the DCT.

Hypercalcemia and Hypocalcemia

Hypercalcemia causes an increase in renal excretion of Mg2+, concomitant with, but exceeding, the increase in excretion of Ca2+. In the proximal tubule, hypercalcemia reduces Na+ and water reabsorption, which is presumably the mechanism that then reduces reabsorption of Ca2+ and Mg2+.[473] In the TAL, both Ca2+ and Mg2+ reabsorption are inhibited by hypercalcemia, most likely due to stimulation of the basolateral CaSR. The DCT increases reabsorption of Ca2+ and Mg2+ in response to the increased delivered load.[473] In hypocalcemia, the opposite effects may be observed. Mg2+ excretion is reduced, consistent with an increase in reabsorption primarily in the TAL.[508]

Acid-Base Status

In most studies, metabolic acidosis has been found to increase Mg2+ excretion. This has been attributed to a decrease in TAL Mg2+ reabsorption.[506] However, chronic metabolic acidosis also decreases TRPM6 mRNA expression, suggesting an effect in the DCT.[480]

Metabolic alkalosis causes a consistent decrease in Mg2+ excretion. Mg2+ reabsorption is increased in the juxtamedullary loop and persists despite the administration of furosemide, excluding an effect in the TAL. Micropuncture studies suggested that the site of increased Mg2+ reabsorption is between the late PCT and the early DCT. However, TRPM6 mRNA expression is also increased, suggesting that alkalosis may also stimulate DCT Mg2+reabsorption.[480]


No single hormone plays a primary role in regulating Mg2+ homeostasis.

Parathyroid Hormone

Parathyroid hormone enhances the reabsorption of Mg2+, as it does for Ca2+, in hypoparathyroid humans and in experimental animals.[405] This appears to be due to increased reabsorption in the TAL.[508] In the in vitro perfused cortical TAL, PTH stimulates Mg2+ and Ca2+ reabsorption in parallel, both by increasing NaCl reabsorption and therefore the generation of a transepithelial potential difference, and by increasing the passive permeability of the TAL to paracellular transport.[486]


Menopausal women are magnesuric and estrogen replacement therapy decreases urinary Mg2+ excretion.[405] In rats, TRPM6 mRNA was downregulated by ovariectomy and restored to normal levels by estrogen administration, suggesting that estrogen effects on the DCT are responsible.[530]


Thiazide Diuretics

The administration of thiazide diuretics causes a very small and variable increase in Mg2+ excretion in animals and humans.[405] Thiazides primarily act on the NaCl cotransporter, NCC, which is located in the DCT. However, micropuncture studies in the hamster found that Mg2+ reabsorption in this segment was unchanged by chlorothiazide.[531] These findings are at odds with the observation that patients with Gitelman syndrome, who have inactivating mutations in NCC, universally have renal Mg2+ wasting.[498]

Loop Diuretics

Loop diuretics increase Mg2+ excretion by abolishing the lumen-positive voltage in the TAL (see Fig. 5-31 ).[499] Interestingly, the increase in Mg2+ excretion with furosemide is greater than that of Na+ or Ca2+.[512] The reasons for this are unknown, but it is possible that furosemide inhibits other aspects of Mg2+ transport, or that, unlike with Na+ or Ca2+, the distal nephron is unable to compensate for the increased Mg2+ delivered from the TAL.

Calcineurin Inhibitors

Transplant patients on calcineurin inhibitors such as cyclosporine and tacrolimus are frequently hypomagnesemic due to renal Mg2+ wasting.[532] One potential mechanism may be by downregulation of TRPM6 mRNA.[419]


Phosphate Homeostasis

Total Body Phosphate

Phosphate has multiple functions in the body. Like Ca2+, it is a key component of the bony skeleton. Phosphate is important for metabolic processes, including the formation of high energy phosphate bonds such as those in ATP. It is also an important component of nucleic acids. Phosphorylation of cellular proteins is an important mechanism for regulation of cellular function. Finally, phosphate is an important blood and urinary pH buffer.

The total body content of phosphorus is 700 g in an average adult, of which 85% is in bone and teeth, 14% in soft tissues, and only 1% in extracellular fluid. The normal concentration of phosphorus in plasma is 3 mg/dL to 4.5 mg/dL (1 to 1.5 μM). Phosphorus is present in plasma primarily as HPO4-2 and H2PO4-1, which exist in a pH-dependent equilibrium (pKa 6.8). Thus, at pH 7.4, the ratio of HPO4-2 to H2PO4-1 is 4:1 and the average valence is 1.8. (The atomic mass of phosphorus is 31. Thus, 1 mmol plasma phosphate ≡ 1.8 mEq ≡ 31 mg; and 1 μM phosphate ≡ 1.8 mEq/L ≡ 3.1 mg/dL.)

Plasma phosphate exists in ionized, complexed, and protein-bound forms. If phosphate were totally filterable through artificial and glomerular membranes, its concentration in the ultrafiltrate would be 1.18 times that of plasma (corrected for plasma water and the Gibbs-Donnan factor). Measured ultrafilterable phosphate to plasma phosphate ratios have been found to range from 0.89 to 0.96, indicating that about 25% of plasma phosphate is bound to protein. Of ultrafilterable phosphate, approximately 60% is ionized and 40% is complexed to the major plasma cations, chiefly Ca2+, Mg2+, and Na+. The fraction of total phosphate that is ultrafilterable declines with hypercalcemia, probably due to the formation of calcium-phosphate-proteinate complexes.

Intracellular phosphate is primarily sequestered in intracellular organelles, or incorporated into organic compounds such as creatine phosphate, adenosine phosphates and, in erythrocytes, 2,3-diphosphoglycerate. The cytosolic free inorganic phosphate concentration is only about 1 μM. Nevertheless, this is above its electrochemical equilibrium value as predicted from the membrane potential, suggesting that there must be active transport of phosphate into cells. The regulation of intracellular phosphate levels is closely linked to cellular metabolic activity. Inhibition of phosphate uptake impairs cellular metabolic function, whereas increasing extracellular phosphate concentration stimulates mitochondrial respiration. Conversely, bathing cells in glucose reduces phosphate uptake and in conditions of limited phosphate availability reduces mitochondrial respiration, oxidative phosphorylation, and ATP content, a phenomenon called the Crabtree effect.

Intake and Output

The daily dietary intake of phosphate is 800 mg to 1500 mg ( Fig. 5-32 ). Phosphate is found in many foods, including dairy products, meat, and cereal grains, so that dietary deficiency is rare. Approximately 65% of ingested phosphate is absorbed, primarily by the duodenum and jejunum. This varies proportionately with dietary intake. Dietary polyvalent cations such as Ca2+, Mg2+, and Al3+ bind to intestinal luminal phosphate and decrease its absorption. Secreted diges-tive juices contain about 3 mg/kg/day of phosphate. Once absorbed, phosphate in the extracellular fluid may exchange with the pool in bone, with 200 mg of phosphate typically entering and leaving the skeleton daily as it is continuously remodeled. Ultimately, the kidneys are responsible for the excretion of a substantial excess of phosphate, about 900 mg per day. During periods of growth, a greater proportion of phosphate is retained for bone deposition, but this still constitutes a small percentage of dietary intake. Thus, renal phosphate excretion is the principal mechanism by which the body regulates extracellular phosphate balance.



FIGURE 5-32  Typical daily phosphate intake and output for a normal adult in neutral phosphate balance (see text for details).



Overview of Phosphate Regulation

The plasma concentration of phosphate is maintained by 1,25(OH)2D, PTH and phosphatonins ( Fig. 5-33 ). The phosphatonins refer to a group of humoral phosphaturic factors, of which the most well characterized is fibroblast growth factor 23 (FGF-23),[533] which were first isolated from tumors of patients with tumor-induced osteomalacia,[534] and are produced primarily in bone. A rise in plasma phosphate stimulates the release of PTH in three ways. First, phosphate directly stimulates PTH synthesis and release from the parathyroid gland, as well as parathyroid cell growth. [535] [536] Second, a rise in serum phosphate causes a fall in serum free Ca2+, which stimulates PTH release via activation of the CaSR. Third, hyperphosphatemia decreases circulating 1,25(OH)2D, alleviating its inhibition of PTH secretion.[535] Hyperphosphatemia also stimulates FGF-23 expression and release.[537] Both PTH and FGF-23 (and perhaps other phosphatonins) then inhibit renal tubular phosphate reabsorption and hence increase phosphate excretion. In addition, hyperphosphatemia also inhibits expression of the enzyme, 25-hydroxyvitamin D 1α-hydroxylase, in the proximal tubule, perhaps mediated by FGF-23.[537] This decreases 1,25(OH)2D, a hormone that normally stimulates intestinal phosphate absorption. Decreased intestinal phosphate absorption then contributes to the restoration of normal plasma phosphate levels. A fall in plasma phosphate would trigger the opposite effects.



FIGURE 5-33  Summary of overall phosphate homeostasis. The primary homeostatic mechanisms activated in response to a rise in extracellular fluid (ECF) phosphate concentration are shown.



Renal Handling of Phosphate

Only the ionized and complexed forms of plasma phosphate are ultrafiltered at the glomerulus, so that the phosphate concentration of fluid in Bowman space is approximately 90% of the total plasma phosphate concentration.[405]Renal clearance studies have demonstrated that 80% to 97% of the filtered load of phosphate is reabsorbed by the renal tubules, so that only 3% to 20% is ultimately excreted. The contribution of individual nephron segments is summarized in Table 5-7 .

TABLE 5-7   -- Segmental Handling of Phosphate Along the Renal Tubule

Nephron Segment

Fractional Reabsorption (%)

Cellular TransportMechanism

Proximal tubule


Active, transcellular

Thin descending and ascending limbs








Active, transcellular

Collecting duct




TAL, thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule.




The relationship between plasma phosphate and phosphate excretion is shown in Figure 5-34 . Initially as the plasma phosphate, and hence filtered load, increases, there is a commensurate increase in the reabsorption of phosphate, so that minimal phosphate appears in the urine. When the plasma phosphate exceeds a certain level, the renal threshold, phosphate begins to appear in the urine, increasing in proportion to the filtered load. This indicates that tubular reabsorption of phosphate is saturable. In humans, with a GFR above 40 ml/min, the maximum tubular reabsorption rate of phosphate, TmP, varies proportionately with GFR. Thus, TmP/GFR, which is the theoretical renal threshold, is kept constant and is a reliable index of tubule reabsorptive capacity.



FIGURE 5-34  Relationship between urinary excretion rate and plasma concentration of phosphate in a normal human subject during fasting (open circle) and phosphate infusion (solid circles). Also shown is the excretion-concentration relationship for inulin (squares). The slope of both lines is the same and equal to the GFR. The vertical distance between the two lines represents the maximum tubular reabsorption rate of phosphate (TmP). The x-intercept, extrapolated from the line connecting the solid circles, represents the theoretical renal phosphate threshold (TmP/GFR).  (From Bijvoet OL: Relation of plasma phosphate concentration to renal tubular reabsorption of phosphate. Clin Sci 37:23–36, 1969.)




With advanced renal insufficiency (GFR <40 ml/min), TmP is further decreased (in part due to secondary hyperparathyroidism) and the fractional excretion of phosphate further increased. However, the decrease in TmP is less than the decrease in GFR, so that TmP/GFR rises and hyperphosphatemia ensues.

Phosphate Handling by individual Nephron Segments

Proximal Tubule

About 80% of filtered phosphate is reabsorbed in the proximal tubule. There is marked axial heterogeneity in reabsorptive activity. Micropuncture studies show that 60% to 70% of filtered phosphate is reabsorbed within the PCT itself.[538] Of this, most is reabsorbed in the S1 segment. The net reabsorptive rate has been estimated to be 11 to 14 pmol/min/mm in the S1 segment, 3 pmol/min/mm in the S2 segment, and 2 pmol/min/mm in the S3 segment. [538] [539] In thyroparathyroidectomized animals, the later convolutions become capable of higher reabsorptive rates, suggesting that the S2 segment may be more sensitive to suppression by PTH.[540]

These findings have been confirmed in primary cultured cell lines and in isolated membrane vesicles. Primary cell cultures derived from the S1 segments had higher reabsorptive rates than those from S3 segments.[541] Furthermore, PTH and cyclic AMP analogs suppressed phosphate reabsorption in S3 cells but not in S1 cells. Two studies have compared brush border membrane vesicles (BBMV) derived from the superficial cortex (primarily PCT segments), and those derived from the juxtamedullary cortex and/or outer stripe of outer medulla (primarily PST segments). [542] [543] These showed that the Vmax for Na-phosphate cotransport was four times higher in the superficial cortex BBMV than those from the juxtamedullary cortex, whereas the Km for phosphate was slightly lower. Furthermore, the density of Na-Pi transporters, as estimated by binding studies with phosphonoformic acid, was also higher in the superficial cortex.[543] These findings indicate that the earliest convolutions of the PCT have the highest density of phosphate transporters and the highest capacity to reabsorb phosphate. The PST has a lower reabsorptive capacity but higher affinity for phosphate. These are appropriate characteristics for a downstream segment that sees less delivered phosphate load and lower luminal concentrations than the PCT.

There is also some evidence for internephron heterogeneity in reabsorptive activity. The fractional delivery of phosphate to the micropuncture-accessible DCT, which is derived from superficial nephrons, is consistently greater than that delivered to the bend of the loop of Henle of juxtamedullary nephrons.[544] This suggests that the proximal reabsorptive capacity of juxtamedullary nephrons is greater than that of superficial nephrons. In rats, this discrepancy is observed only when they are thyroparathyroidectomized, but not when PTH is repleted, suggesting that juxtamedullary nephrons are more sensitive to the inhibitory effect of PTH.[544] In rats loaded with excess phosphate, the superficial nephrons reabsorb more than the juxtamedullary nephrons, suggesting that superficial nephrons adapt more effectively to increased filtered phosphate load than do juxtamedullary nephrons.[545] Conversely, in a study of isolated perfused tubule segments, no difference was found between the reabsorptive rates of superficial and juxtamedullary nephrons.[539]

Loop of Henle

There is likely no reabsorption of phosphate in the loop of Henle, other than the PST. In parathyroid-intact animals, micropuncture studies show no phosphate reabsorption between the late PCT and early DCT. In thyroparathyroidecomized animals there is some phosphate reabsorption in this region, but it could all be attributable to the transport occurring in the PST.[405] Consistent with this, the phosphate permeability measured in isolated perfused thin descending and ascending limbs, and cortical thick ascending limbs is extremely low.[409]

Distal Convoluted Tubule and Connecting Tubule

Micropuncture studies have observed reabsorption of phosphate between the early DCT and the final urine.[405] In the interpretation of these early micropuncture studies, one potential source of artifact that must be considered is the heterogeneity in proximal transport between nephrons. As mentioned previously, the proximal tubules of juxtamedullary nephrons reabsorb more phosphate than their superficial counterparts. Because the micropuncture fluid from the superficial DCT reflects delivery only from superficial nephrons while the final urine reflects delivery from both superficial and juxtamedullary nephrons, the phosphate concentration in the urine may be less than that in the early DCT, even when there is no distal tubule reabsorption.

Several lines of evidence suggest there may be true distal tubule phosphate reabsorption, at least under conditions of phosphate deprivation, and that this is not simply an artifact of nephron heterogeneity. First, micropuncture studies that have sampled fluid from both the early DCT (derived from superficial nephrons) and the papillary loop of Henle (derived from juxtamedullary nephrons) find that the fractional delivery of phosphate to both sites is similar and 6% to 8% greater than in the final urine, but only when the animal is on a low phosphate diet.[546] Second, micropuncture studies that have sampled fluid directly from both early and late convolutions of the DCT show that approximately 5% of filtered phosphate is reabsorbed between these two sites.[547] Third, in stop-flow studies of the DCT, the TF/UFphosphate concentration ratio, corrected for water absorption, doubled after PTH administration, consistent with a reduction in the normal phosphate reabsorption rate in this segment.[548]

Several studies have failed to find phosphate transport in the distal tubules from animals on a normal diet.[405] Because distal tubule phosphate transport is enhanced by low phosphate diet, it is possible that reabsorption in this segment is difficult to detect in animals on a normal phosphate diet.

The cellular mechanism for DCT phosphate reabsorption is unknown, but on thermodynamic grounds luminal uptake must occur by active transport. Whether transport occurs in both the DCT and CNT or in the DCT alone is also unclear.

Collecting Duct

The collecting duct accounts for very little, if any, phosphate reabsorption. In isolated perfused CCD, a small amount of phosphate reabsorption was found by some investigators but not by others.[405] One study found phosphate reabsorption by microcatheterization of the IMCD but only in thyroparathyroidectomized animals. In another study, no phosphate transport was found in the papillary collecting duct.

Molecular Mechanisms of Transcellular Phosphate Reabsorption

Transcellular phosphate reabsorption occurs primarily in the proximal tubule, where its mechanism has been studied in detail ( Fig. 5-35 ). Intracellular phosphate levels are higher than the level expected for electrochemical equilibrium with luminal fluid, so apical entry must occur by active transport, while basolateral exit may occur by diffusion.



FIGURE 5-35  Model depicting the putative cellular mechanisms of phosphate transport in the proximal tubule. Pi, phosphate; A-, inorganic anion.



Apical Phosphate Entry

The apical entry of phosphate is the rate-limiting step in transcellular phosphate transport, and the target for virtually all physiological mechanisms that alter tubule phosphate reabsorption. Hoffmann and colleagues[549] were the first to show that kidney cortex BBMV exhibit Na+-dependent phosphate cotransport. Some of the key features of the apical Na-phosphate cotransporter have emerged from this and subsequent vesicle studies. The coupling ratio of Na+to phosphate is greater than unity. Both HPO4-2 and H2PO4-1 are transported, so transport is partly electrogenic (net positive charge movement with phosphate entry); however, transport of the divalent form is preferred. There is normally a steep inward concentration gradient for Na+ across the plasma membrane that is maintained by the basolateral Na-K-ATPase, and an inside-negative membrane potential. Thus, the high coupling ratio and electrogenicity are both thermodynamically important for the secondary active transport of phosphate against its steep electrochemical gradient at the apical membrane, particularly in the late proximal tubule, where luminal phosphate levels are low. The transporter is stimulated by increasing extravesicular pH, which not only increases the proportion of phosphate in the preferred, divalent form, but also increases the affinity of the transporter for Na+, either by a competitive or allosteric effect. Extravesicular Na+ enhances the transporter's affinity for phosphate. Thus, there may be allosteric sites for modulation both by Na+ and by H+. Additionally, an inwardly directed H+ gradient stimulates transport, probably by trapping intravesicular phosphate in the monovalent (non-preferred) form and thereby impeding its efflux. Finally, transport is competitively inhibited by arsenate and foscarnet (phosphonoformic acid).

Three subclasses of the Na-phosphate (Na-Pi) cotransporters have now been identified at the molecular level, and named type I, II, and III ( Table 5-8 ). The type I Na-Pi cotransporter[550] is expressed on the brush border of the proximal tubule, [551] [552] as well as in liver and brain. However, the characteristics of the type I Na-Pi transporter do not resemble renal brush border Na-phosphate cotransport [553] [554] making it unlikely that the type I transporter plays an important role in transcellular phosphate transport. The type III Na-Pi cotransporters are ubiquitously expressed, including at the basolateral membrane of the proximal tubule,[555] and are probably housekeeping proteins required for phosphate uptake for cellular metabolic needs.

TABLE 5-8   -- The Three Families of Na-Pi Cotransporters


Type I

Type II

Type III



Type IIa

Type IIb

Type IIc


Protein name





PiT-1 (Glvr-1)
PiT-2 (Ram-1)

Gene name






Tissue expression

Kidney cortex/PT, liver, brain

Kidney cortex/PT

Small intestine, lung

Kidney cortex/PT



Phosphate, Cl-, organic anions





Affinity for phosphate

-1 μM

0.1–0.2 μM

0.05 μM

0.07 μM

0.025 μM

Affinity for Na+

50–60 μM

50–70 μM

33 μM

48 μM

40–50 μM

Na+-phosphate coupling ratio






pH dependence


Stimulated at high pH

Inhibited at high pH

Stimulated at high pH

Inhibited at high pH

Regulation by PTH or dietary phosphate


PTH and diet




Modified from Murer H, Hernando N, Forster I, Biber J: Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80:1373–1409, 2000.

PT, proximal tubule.





The type II Na-Pi cotransporters include three homologous isoforms, type IIa,[556] and IIc,[557] which are expressed at the proximal tubule brush border, and type IIb, which in mammals is expressed in the small intestine and in pneumocytes. [558] [559] The type IIa Na-Pi cotransporter is probably the predominant apical phosphate entry transporter in the proximal tubule. When expressed in Xenopus oocytes and studied by radioisotope uptake or by electrophysiology under whole cell voltage clamp, its functional characteristics match well those of the BBMV transport system (see Table 5-8 ).[556] Like the BBMV Na+-phosphate transport system, it is electrogenic.[560] By simultaneous measurement of substrate flux and charge movement under voltage-clamp conditions, the stoichiometry was determined to be 3 Na+:1 phosphate.[560] Furthermore, the dependence on external pH was confirmed, and shown to be due both to a competitive interaction of H+ with the Na+-binding site,[561] and to an additional effect on reorientation of the empty transporter.[562]

The role of the type IIa cotransporter is also supported by gene knockout studies. Mice with targeted inactivation of the Npt2 gene exhibited phosphaturia, hypophosphatemia, and an appropriate elevation in the serum concentration of 1,25(OH)2D with attendant hypercalcemia, hypercalciuria, and decreased serum PTH levels.[563] Na+-phosphate cotransport in BBMV derived from the knockout mice was reduced by 85% compared to wild-type controls, indicating that the cause of the phosphaturia was loss of the major brush border Na+-phosphate transport system.[564] Furthermore, two patients have now been described that have heterozygous inactivating mutations in this the type IIa cotransporter. Both have idiopathic hypophosphatemia with renal Pi wasting, associated in one case with recurrent nephrolithiasis and in the other with osteoporosis.[565]

The type IIa cotransporter participates in multiple protein interactions via a PDZ-binding motif on its C-terminal tail.[566] This is a peptide motif that binds to the PDZ domains on PDZ-containing proteins, including PDZK1 (NaPi-Cap1) and Na-H exchanger-regulatory factor 1 (NHERF1),[567] both of which are also located on the brush border membrane of the proximal tubule.[568] Disruption of these interactions by competition with PDZ domain peptides prevent normal trafficking of the type IIa cotransporter to the apical membrane.[569] The importance of NHERF1 is demonstrated by the finding that in knockout mice that lack NHERF1, the proximal tubule type IIa Na-Pi cotransporter is aberrantly localized intracellularly, and the mice are overtly phosphaturic.[570] Thus NHERF1 is required for normal trafficking of the type IIa cotransporter to the cell surface. By contrast, PDZK1-knockout mice had very little phenotype. When chronically fed a high-phosphate diet, the type IIa cotransporter expression at the brush border was decreased, and urinary excretion of phosphate was slightly elevated.[571] Thus the physiological role of PDZK1 is unclear.

The type IIc Na-Pi cotransporter is likely to be important in renal phosphate reabsorption primarily in early life. It exhibits Na-dependent phosphate cotransport that is electroneutral, with a stoichiometry of 2 Na+:1 phosphate, and is stimulated by alkaline extracellular pH. [557] [572] The type IIc cotransporter is expressed primarily in the kidney at the apical membrane of the proximal tubule, and at significantly higher levels in weaning animals than adults. mRNA hybrid depletion experiments suggest that both the type IIa and type IIc proteins function as Na-phosphate cotransporters in weaning animals, but only type IIa in adults.[557] The 2:1 stoichiometry, as well as the lack of coupled movement of net charge, would be expected to reduce the thermodynamic ability of the transporter to concentrate phosphate within the proximal tubule epithelial cell. However, weaning animals have substantially lower renal intracellular phosphate concentrations.[573] Thus, the type IIc cotransporter may be expressed in weaning animals to take advantage of the increased phosphate gradient and thereby transport phosphate more efficiently.

The physiological importance of the type IIc Na-Pi cotransporter has now been confirmed by the demonstration that inactivating mutations in its gene, SLC34A3, cause the autosomal recessive renal phosphate wasting disorder, hereditary hypophosphatemic rickets with hypercalciuria. [574] [575]

Basolateral Phosphate Exit

Transport of phosphate at the basolateral membrane in the proximal tubule must be sufficiently flexible to mediate two different functions. First, to mediate transcellular phosphate reabsorption it must be able to efflux some or all of the phosphate that enters from the luminal membrane. Second, it must be able to mediate influx of phosphate for intracellular metabolic processes, if apical phosphate entry is insufficient to meet cellular requirements.

The precise mechanisms for basolateral phosphate exit are poorly understood. Several studies have found evidence for Na+-independent phosphate uptake in basolateral membrane vesicles that is driven by an intravesicular-positive membrane potential, trans-stimulated by intravesicular loading with phosphate or other inorganic anions, and insensitive to distilbenes.[405] Thus, the evidence supports the existence of an electrogenic phosphate-anion exchanger as the probable basolateral phosphate efflux system.

In addition, Na+-phosphate cotransport has been found in basolateral membranes from dog kidney, but not from the rat. It has been suggested that such conflicting results may be caused by variable contamination of basolateral vesicle preparations with brush border membranes. If basolateral Na+-phosphate cotransport does occur, it would likely serve as the “housekeeping” phosphate influx system. As mentioned earlier, the most likely molecular candidate for such a transporter is the type III Na-Pi cotransporter.

Regulation of Renal Phosphate Handling

Many factors influence renal phosphate handling. These are summarized in Table 5-9 , and only the most important are discussed here.

TABLE 5-9   -- Summary of Factors Affecting Phosphate Reabsorption


Proximal Tubule Reabsorption

Effect on Type IIa Na-Pi Cotransporter

Putative Mechanism Acute/Chronic

Volume expansion









Activation of CaSR









Endocytosis/↓ mRNA


Exocytosis/↑ mRNA

Metabolic acidosis





No Δ




Endocytosis/↓ mRNA

Metabolic alkalosis






?Inhibition of basolateral Pi transport


Exocytosis/↑ mRNA

Respiratory acidosis


?↓ mRNA

Respiratory alkalosis


↓ filtered load/↑ mRNA






Endocytosis/↓ mRNA

 Vitamin D





↑ mRNA


↓ mRNA



Endocytosis of Na-K-ATPase α1 subunit







↓ mRNA















PTH, parathyroid hormone.




Dietary Phosphate

Clearance studies have demonstrated that phosphate excretion is remarkably responsive to antecedent phosphate intake. Fractional excretion of phosphate increases with a high phosphate diet and decreases with a low phosphate diet, independent of any effect on plasma phosphate, Ca2+, or PTH concentration.[405] By micropuncture, the major site of adaptation is the proximal tubule, though the distal tubule also shows upregulation of phosphate reabsorption during phosphate deprivation. Superficial nephrons demonstrate a greater suppression of phosphate reabsorption on a high phosphate diet than do juxtamedullary nephrons. BBMV transport studies demonstrate that this adaptation is due to changes in the Vmax of the apical Na+-phosphate cotransporter.

The acute adaptive changes in response to dietary phosphate are due to trafficking of the type IIa and type IIc Na-phosphate cotransporters, whereas chronic adaptive changes are mediated by changes in transcription and translation. In animals chronically fed a high phosphate diet, the type IIa transporter mRNA and protein abundance are low. By immunofluorescence the transporter is mostly found in juxtamedullary nephrons; within the proximal tubule it is largely localized to intracellular vesicles.[576] Upon acutely switching to a low phosphate diet, brush border membrane transporter protein is increased,[577] but without any change in mRNA abundance.[576] The transporter is recruited to more superficial nephrons and redistributed within the proximal tubule to the brush border.[576] This redistribution is due to microtubule-dependent insertion of membrane vesicles into the apical membrane.[578] These changes are not inhibited by actinomycin D nor by cycloheximide, confirming that new RNA and protein synthesis are not required.[578] With chronic feeding of a low phosphate diet, the type IIa transporter mRNA also increases. Refeeding a high phosphate diet to animals chronically adapted to a low phosphate diet reverses all these changes. [579] [580] Acutely after switching to a high phosphate diet, the type IIa transporter is internalized by membrane retrieval from the apical surface and sequestration in the subapical vacuolar network. This process was found to be dependent on microtubules in one study,[581] but not in another.[578] The endocytosed transporter is subsequently trafficked to lysosomes for degradation.[582] Chronic high phosphate diet also down-regulates transporter mRNA and protein. The effects of dietary phosphate are not mediated by PTH because they could be observed even in parathyroidectomized animals.[580]

Similarly a high phosphate diet causes acute internalization of type IIc transporter but, unlike type IIa Na-Pi cotransporters that are trafficked to lysosomes, type IIc transporters are trafficked to an intracellular, subapical pool and are not degraded.[583] Similar to type IIa cotransporters, type IIc cotransporter protein abundance was decreased in mice fed a chronic high-phosphate diet.[584]

The upstream signals that mediate the acute and chronic effects of dietary phosphate are not well understood. Dietary phosphate affects the concentrations of phosphate in plasma and in tubular fluid, so it is possible that that proximal tubule cells sense phosphate directly. In this regard, it is interesting to note that type IIa cotransporter adaptation can also be observed in opposum kidney (OK) cells simply by culturing in low or high phosphate media.[585] The basis of transcriptional activation of the NPT2 gene in response to chronic low phosphate diet has been investigated by two groups. Kido and colleagues used DNA footprinting analysis to identify a putative phosphate response element in the NPT2 promoter that binds to mouse transcription factor mE3 (TFE3).[586] TFE3 was found to be upregulated by a low phosphate diet, and stimulated transcription from the NPT2 promoter. Custer and colleagues[587] used differential display-PCR to identify PDZ1 as an mRNA upregulated by low phosphate diet. When PDZ1 was coexpressed with NaPi-2 cRNA in Xenopus oocytes, it stimulated Na+-phosphate cotransport, suggesting that PDZ1 may have a post-transcriptional regulatory role.


Acute hypercalcemia, especially when severe, decreases phosphate excretion by several mechanisms. Hypercalcemia decreases the plasma concentration of ultrafilterable phosphate because of the formation Ca2+-phosphate-proteinate complexes. Hypercalcemia also decreases renal blood flow and GFR. As a consequence, filtered load of phosphate falls. In most studies, tubule reabsorption of phosphate is increased with acute hypercalcemia. This is in part due to the decreased filtered phosphate load, and partly due to a reduction in circulating PTH.

Whether acute hypercalcemia also directly affects tubule phosphate reabsorption is controversial.[405] Micropuncture studies generally agree that hypercalcemia increases distal tubule reabsorption, but there are conflicting results regarding its effect on the proximal tubule. In vitro microperfused proximal tubule S2 segments showed increased phosphate reabsorption in response to increased luminal Ca2+, whereas S3 segments did not. Of note, the CaSR is expressed at the brush border of the proximal tubule, predominantly in S1 and S2 segments,[220] raising the possibility that it might mediate the effects of luminal Ca2+ on phosphate transport.

In contrast to its acute effects, chronic hypercalcemia decreases tubule phosphate reabsorption whereas chronic hypocalcemia increases phosphate reabsorption, independent of the effects of PTH, vitamin D, or serum Ca2+. The mechanism is unknown.

Sodium and Extracellular Volume

Extracellular volume expansion increases (and volume contraction decreases) phosphate excretion by several mechanisms.[405] First, volume expansion increases GFR and the filtered load of phosphate. Second, volume expansion inhibits proximal tubule Na+ and water reabsorption, diluting the concentration of luminal phosphate available for reabsorption. Third, volume expansion decreases plasma Ca2+ and increases PTH, which inhibits proximal tubule phosphate reabsorption (see later). Finally, there is probably a direct effect of volume expansion to inhibit tubule phosphate reabsorption, which is independent of filtered load, plasma Ca2+, or PTH.

Acid-Base Status

Acute metabolic acidosis (3 hours) has minimal effect on phosphate excretion but blunts the phosphaturic effect of PTH.[405] Acute metabolic alkalosis causes an increase in phosphate excretion. Paradoxically, alkalinization of proximal tubule luminal fluid would be expected to stimulate the type IIa and IIc Na-Pi cotransporters and increase phosphate reabsorption. Conversely, alkalinization of peritubular fluid has been shown to inhibit phosphate reabsorption in perfused PCT segments. Thus, the effects of acute metabolic alkalosis are best explained by regulation of basolateral rather than apical phosphate transport in the proximal tubule.

In contrast to the effects of acute acid-base disturbances, chronic metabolic acidosis decreases and chronic metabolic alkalosis increases renal tubule phosphate reabsorption. In early chronic metabolic acidosis (6 hours), the type IIa Na-Pi cotransporter protein expression at the brush border is decreased, but the protein abundance in total cortical homogenate, and the mRNA abundance are unchanged, suggesting that there is retrieval of transporters from the apical membrane.[588] Late chronic metabolic acidosis (12 hours to 10 days) is associated with progressive decrease in type IIa Na-Pi cotransporter mRNA abundance as well.[588]

Respiratory acidosis and alkalosis are associated with an increase and decrease in phosphate excretion, respectively. Acute respiratory alkalosis causes a redistribution of phosphate into cells, resulting in hypophosphatemia, so the effects on renal excretion may be attibutable to alterations in filtered load. Indeed in rats fed a high phosphate diet, which would be expected to induce saturating phosphate concentrations, the effect of acute respiratory alkalosis on fractional excretion of phosphate was abolished. By contrast, chronic respiratory alkalosis also causes reduced phosphate excretion, but is accompanied by hyperphosphatemia. In OK cells, decreasing the HCO3-/CO2 concentration of the media without changing the pH caused an increase in type IIa Na-Pi cotransporter expression due to transcriptional activation.[589] This suggests that the effects of respiratory acid-base status may be mediated by a direct effect of CO2 on proximal tubule cells.


Parathyroid Hormone

Parathyroid hormone is the major hormonal regulator of renal phosphate handling. It inhibits tubule phosphate reabsorption, primarily in the PCT.[405] The juxtamedullary nephrons are more sensitive to PTH than superficial nephrons,[544] and the S2 segment of the PCT may be more sensitive than the S1 segment.[540] PTH also inhibits phosphate transport in the PST, DCT, and the CCD.

The effects of PTH on phosphate reabsorption in proximal tubule, as well as in OK cells, are primarily mediated by its effects on the type IIa Na-Pi cotransporter. Acutely, PTH causes endocytosis of type IIa cotransporters from the apical surface in a microtubule-dependent manner.[590] Endocytosis occurs at the intermicrovillar clefts. There, the transporter is internalized into clathrin- and adapter protein-2 (AP2)-coated vesicles, where it colocalizes with endocytosed fluid-phase markers such as horseradish peroxidase, and is transiently trafficked to the subapical region.[591] Unlike other membrane transport proteins, type IIa cotransporters are not routed to a recycling compartment from which they can be recruited back to the surface. Instead, they are trafficked directly to the lysosomes and irreversibly degraded. [591] [592] The recognition signal for PTH-mediated endocytosis is a dibasic peptide “KR” motif located in the last intracellular loop of the type IIa Na-Pi cotransporter.[593] This KR motif interacts with PEX19, a protein normally involved in the binding and trafficking of peroxisomal proteins that stimulates endocytosis of the type IIa Na-Pi cotransporter.[594]

Chronic administration of PTH additionally causes a small decrease in type IIa cotransporter mRNA.[595] Withdrawal of PTH, or parathyroidectomy, reverses these changes and up-regulates brush border type IIa cotransporters, a process that requires de novo protein synthesis.[596] In knockout mice that lack the type IIa Na-Pi cotransporter, PTH has no effect on serum phosphate, fractional excretion of phosphate, or Na+-dependent phosphate transport in BBMV.[597]

Parathyroid hormone can signal in proximal tubules via several different signalling pathways, including the classical adenylate cyclase/PKA pathway, phospholipase C/PKC, and the extracellular signal-regulated kinase (ERK). PTH can inhibit Na+-phosphate cotransport via activation of either the PKA or PKC pathway alone.[598] In OK cells, the reduction in type IIa cotransporter expression by PTH has been shown to be mediated by PKA.[599] The type IIa cotransporter has now been shown to physically associate with PKA through an A kinase anchoring protein (AKAP) that is required for the regulation of phosphate transport by PTH.[600]

It has recently been recognized that proximal tubules have PTH receptors not only on their basolateral membrane, but also on their brush border membrane.[601] By perfusing either luminal or peritubular compartments of proximal tubules with PTH analogs, Traebert and colleagues[598] showed that activation of the PTH receptors at either surface was sufficient to cause Na+-phosphate cotransporter internalization. The luminal receptors signalled preferentially via PKC, while the basolateral receptors required activation of both PKA and PKC. As PTH is a small polypeptide that is probably freely filtered at the glomerulus, a sufficiently high concentration is likely to normally be present in the lumen of the proximal tubule to be sensed by these brush border receptors. The importance of luminal signaling is indicated by studies in mice with targeted inactivation of CLC5, the Dent disease gene.[602] These mice, which have defective endocytosis of luminal PTH and therefore high concentrations of PTH in proximal tubule luminal fluid, exhibit abnormal internalization of the type IIa Na+-phosphate cotransporter, and hence phosphaturia.

In addition to its effects on the type IIa Na+-phosphate cotransporter, PTH inhibits phosphate reabsorption by several other mechanisms. PTH inhibits the basolateral Na-K-ATPase, thereby indirectly preventing secondary active Na+-gradient dependent phosphate transport at the apical membrane. The signal transduction pathway for this is complex, and involves an early phase that depends on PKC, phospholipase A2, and ERK, and a late phase dependent on PKA, phospholipase A2, and ERK. [603] [604] ERK activation alone also inhibits phosphate transport.[605] It does not affect Na+-phosphate cotransporter expression, but its downstream target has not been defined. PTH-stimulated cyclic AMP generation can also inhibit phosphate transport via another pathway. It has been postulated that the cyclic AMP generated can be exported to the tubule lumen, where it is metabolized by 5′-ectonucleotidase to adenosine, re-enters the cell, and inhibits phosphate transport.[606] PTH-activated PKC acts synergistically by inhibit-ing phosphodiesterase,[607] the enzyme that degrades intracellular cyclic AMP, and also by activating the 5′-ectonucleotidase.[608]

Vitamin D

The effects of vitamin D metabolites on renal phosphate handling are complex. Chronic administration of vitamin D causes phosphaturia, a reduction in renal cortical BBMV phosphate transport, and reduced renal cortical expression of type IIa Na-Pi cotransporter mRNA and protein.[609] Because chronic vitamin D administration increases intestinal phosphate absorption, it is possible that the adaptive mechanism is the same as that of high dietary phosphate intake. The phosphaturic response to chronic vitamin D requires the presence of PTH,[609] whereas the downregulation of BBMV transport and cotransporter expression do not. This suggests that the vitamin D-induced decrease in proximal tubule phosphate reabsorption can be compensated by increased reabsorption at downstream nephron sites that are PTH-responsive.

Acute administration of vitamin D metabolites reduces renal phosphate excretion. This effect requires the presence of PTH, is associated with a decrease in urinary cyclic AMP and renal tubular adenylate cyclase activity, and can be inhibited by cycloheximide, which blocks de novo protein synthesis.[610] 1,25(OH)2D stimulated Na+-phosphate cotransport in a subclone of OK cells,[611] and phosphate uptake in isolated renal cells from vitamin D-deficient chicks,[612] both of which could be inhibited by actinomycin D and cycloheximide. In vitamin D-deficient rats, Taketani and colleagues showed that type IIa Na-Pi cotransporter mRNA and protein is down-regulated in the juxtamedullary cortex, but somewhat up-regulated in the superficial cortex.[613] Administration of 1,25(OH)2D caused upregulation of its mRNA and protein expression in the juxtamedullary cortex within 12 hours. Thus, the evidence suggests that vitamin D acts acutely by antagonizing the actions of PTH on adenylate cyclase, and thereby induces de novo synthesis of type IIa Na-Pi cotransporter, perhaps in the PST.


Dopamine is a renal paracrine phosphaturic hormone. Renal dopamine is produced in the proximal tubule from its precursor, L-dopa; a small amount is also released from nerve endings. Dopamine production is stimulated by a high phosphate diet and suppressed by a low phosphate diet. Exogenous dopamine inhibits phosphate transport in isolated perfused proximal tubule S3 segments,[614] and Na+-phosphate cotransport in BBMV.[615] Inhibition of endogenous dopamine production with carbidopa decreased renal phosphate excretion[616] and increased Na+-phosphate cotransport in BBMV.[616] Carbidopa also decreased Na+-gradient-dependent phosphate uptake into OK cells.[617] A synthetic L-dopa analog that is selectively activated in the proximal tubules was shown to inhibit BBMV Na+-phosphate cotransport and to cause phosphaturia.[618]

The action of dopamine is primarily mediated by DA1 receptors.[619] It is now clear that the primary action of dopamine in the proximal tubule is to inhibit the basolateral Na-K-ATPase, a process dependent on both PKA and PKC.[604] Activation of PKC causes phosphorylation of, and thereby induces endocytosis of the α1-subunit of the Na-K-ATPase. This would indirectly inhibit phosphate reabsorption because the apical Na+-phosphate transport step depends on the extracellular-to-intracellular Na+ gradient generated by the Na-K-ATPase. Whether dopamine also directly affects the type IIa Na-Pi cotransporter is unclear.

Insulin, Glucose, and Glucagon

Insulin reduces renal phosphate excretion,[620] independently of its effects on glucose. It increases phosphate uptake into cells, causing hypophosphatemia, a reduced filtered load, and hence increased proximal tubule reabsorption. Insulin has also been shown to directly stimulate phosphate uptake in BBMV[621] and in OK cells.[622] The effects of insulin may in part be explained by inhibition of gluconeogenesis, which causes intracellular phosphate depletion. Glucagon, which stimulates gluconeogenesis and increases cytosolic phosphate, is phosphaturic,[623] probably due to inhibition of phosphate reabsorption in the PST.[624] Glucose infusion is also phosphaturic.[620] It acts in part as an osmotic diuretic. Glucose also inhibits luminal phosphate uptake, probably because Na+-glucose cotransport depolarizes the apical membrane and dissipates the Na+ gradient, thereby inhibiting electrogenic Na+-phosphate cotransport.[625]


The mammalian stanniocalcins, STC1 and STC2, are homologs of a fish anticalcemic hormone. STC1 is ubiquitously expressed. The intrarenal sites of STC1 expression are controversial. In humans, STC1 protein appears to be expressed in the “distal tubule” and collecting duct. [626] [627] In rodents, STC1 mRNA has been detected in collecting ducts only, whereas STC1 protein is found in almost all tubule segments. [628] [629] STC1 mRNA expression in cortical and outer medullary collecting ducts was reduced by a low phosphate diet and increased on a high phosphate diet.[629] Expression of STC1 mRNA in the kidney is also increased by 1,25(OH)2D, whereas that of STC2 is decreased.[630] STC1 has a very short plasma half-life and circulating levels are not normally detectable, so it probably acts as a local paracrine factor.[631]

STC1 is an antiphosphaturic hormone. Recombinant human STC1, when injected into rats, caused a decrease in fractional excretion of phosphate, [626] [632] and an increase in Na+-phosphate cotransport in BBMV.[632] However, STC1-knockout mice have normal serum Ca2+ and phosphate and respond normally to acute injections of vitamin D, raising doubt as to the physiological significance of STC1.[633] No physiologic role has been proposed for STC2.


Phosphatonins are humoral factors, secreted by tumors of patients with tumor-induced osteomalacia, which have phosphaturic activity. Several proteins have now been found to be overexpressed by such tumors: FGF-23,[533] matrix extracellular phosphoglycoprotein (MEPE),[534] secreted frizzled related protein-4 (FRP-4),[634] and fibroblast growth factor-7 (FGF-7).[635]

The evidence for a role in regulating renal phosphate excretion is strongest for FGF-23. Mutations in FGF-23 that prevent its cleavage (and presumably increase functional levels) cause autosomal dominant hypophosphatemic rickets/osteomalacia, [636] [637] [638] [639] [640] whereas missense mutations in FGF-23 that presumably abolish its function have been identified in familial tumoral calcinosis, which is characterized by decreased urinary excretion of phosphorus, hyperphosphatemia, and ectopic calcification. [641] [642] Inactivating mutations in a metalloproteinase, PHEX, which inhibits FGF-23 expression and release, [643] [644] cause X-linked hypophosphatemic rickets.[645]Finally, targeted ablation of FGF-23 in mice causes hyperphosphatemia.[646]

FGF-23 is expressed in osteoblasts. Its expression is upregulated by hyperphosphatemia [537] [647] and also perhaps by 1,25-vit D[648] either directly, or via downregulation of PHEX gene transcription.[649] FGF-23[533] inhibits Na+-dependent phosphate transport in kidneys[650] and in OK cells. [651] [652] In FGF-23 transgenic mice, reduction in mRNA expression of both the type IIa and IIc Na-Pi cotransporters is the cause of phosphaturia.[653] In contrast, sFRP-4 increases renal phosphate excretion by inducing internalization of the type IIa Na-Pi cotransporter from the brush border of the proximal tubule.[654]


Most diuretics are somewhat phosphaturic.[405] Mannitol modestly increases phosphate excretion by decreasing Na+ and water reabsorption in the proximal tubule, and hence diluting luminal phosphate. Acetazolamide, which inhibits carbonic anhydrase, is quite phosphaturic, probably by setting up an acidic disequilibrium pH in the proximal tubule lumen that inhibits the type IIa Na-Pi cotransporter. Thiazides and fursosemide in high doses have a small phosphaturic effect, attributable to inhibition of carbonic anhydrase.


In contrast to other tubule transport processes, phosphate reabsorption is highest in infants, and declines with age.[405] This is important to maintain positive phosphate balance in the immature animal during active growth and development. The increased phosphate reabsorption in newborns is observed in both proximal and distal tubules. Furthermore, newborn animals exhibit a greater increase in phosphate reabsorption in response to a dietary phosphate deprivation, and a lesser decrease in response to high phosphate diet, or PTH. BBMV from neonates show a greater Vmax for Na+-phosphate transport than those of adults.[655] Type IIa Na-Pi cotransporter protein is concomitantly increased. [656] [657] [658] Early in development, the type IIa Na-Pi cotransporter is expressed in the brush borders of all proximal tubules, whereas in adulthood it is primarily expressed at the brush borders of juxtamedullary nephrons.[657] Furthermore, the type IIc Na-Pi cotransporter is expressed in weaning animals but not in adults, suggesting that it may supply added capacity for phosphate reabsorption just during development.[557] The mechanisms for regulating these developmental changes are unknown, but growth hormone[659] and triiodothyronine appear to play a role.[660]


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