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

CHAPTER 9. Urine Concentration and Dilution

Mark A. Knepper   Jason D. Hoffert   Randall K. Packer   Robert A. Fenton



The Kidney Can Regulate Water Excretion without Large Changes in Solute Excretion, 308



A Parallel Organization of Structures in the Renal Medulla is Critical to Urinary Concentrating and Diluting Process, 308



Renal Tubules, 308



Vasculature, 312



Medullary Interstitium, 312



Renal Pelvis, 312



General Features of the Urine Concentration and Dilution Process, 313



Sites of Urine Concentration and Dilution, 313



Mechanism of Tubule Fluid Dilution, 313



Mechanism of Tubule Fluid Concentration, 314



Molecular Physiology of Urinary Concentrating and Diluting Processes, 321



Transport Proteins Involved in Urinary Concentration and Dilution, 321



Use of Knockout Mice to Study the Urinary Concentrating Mechanism and Vasopressin Action, 323



Ammonium Accumulates in the Renal Medulla, 325


The tonicity of body fluids is controlled predominantly through the regulation of renal water excretion. The kidney also carries out several other homeostatic functions, including regulation of extracellular fluid volume (through control of NaCl excretion), regulation of systemic acid-base balance (through control of net acid excretion), regulation of systemic K balance (through the control of K+ excretion), and maintenance of nitrogen balance (through excretion of urea). Water excretion and the excretion of individual solutes must be regulated independently to allow all of the homeostatic functions of the kidney to be performed simultaneously. Thus, when water intake changes in the absence of changes in solute intake or of changes in metabolic production of waste solutes, the kidney can excrete the appropriate amount of water without marked perturbations in solute excretion (i.e., without disturbing the other homeostatic functions of the kidney). This phenomenon, shown in Figure 9-1 , occurs as a result of operation of the renal concentrating and diluting mechanism, the focus of this chapter.



FIGURE 9-1  Steady-state renal response to varying rates of vasopressin infusion in conscious rats.[212] A water load (4% of body weight) was maintained throughout the experiments to suppress endogenous vasopressin secretion. Although the urine flow rate was markedly reduced at higher vasopressin infusion rates, the osmolar clearance changed little.  (Data from Atherton JC, Green R, Thomas S: Influence of lysine-vasopressin dosage on the time course of changes in renal tissue and urinary composition in the conscious rat. J Physiol 213:291-309, 1971.)




Figure 9-1 highlights several important features of the concentrating and diluting mechanism viewed from the perspective of whole-kidney function. The major effector in the regulation of renal water excretion is the antidiuretic hormone vasopressin. Vasopressin is a peptide hormone secreted into the peripheral plasma by the posterior pituitary gland (see Chapter 8 ). As shown in the upper panel of Figure 9-1 , the kidney is capable of wide variations in water excretion (i.e., urine flow) in response to changing levels of vasopressin in the peripheral plasma. Water excretion is typically more than 100-fold lower in extreme antidiuresis (high vasopressin level) than in extreme water diuresis (low vasopressin level). These large changes in water excretion are achieved without substantial changes in the steady-state rate of total solute excretion (measured as osmolar clearance). As shown in the bottom panel ofFigure 9-1 , this behavior is dependent on the ability of the kidney to concentrate and dilute the urine. When water excretion is rapid because of a low circulating vasopressin level, the urine is diluted to an osmolality less than that of plasma (290 mOsm/kg H2O). When water excretion is low because of a high circulating vasopressin level, the urine is concentrated to an osmolality much higher than that of plasma.

Under normal circumstances, the circulating vasopressin level is determined by osmoreceptors in the hypothalamus that trigger increases in vasopressin secretion (by the posterior pituitary gland) when the osmolality of the blood rises above a threshold value, about 292 mOsm/kg H2O. This mechanism can be subverted when other inputs to the hypothalamus (e.g., associated with arterial underfilling, severe fatigue, or physical stress) override this osmotic mechanism. Such non-osmotic stimuli, coupled with continued water intake, explain the hyponatremia that occurs in severe congestive heart failure and cirrhosis.[1] Similar circumstances (stress induced vasopressin secretion coupled with continued water intake) are believed to be responsible for the hyponatremia seen in marathon runners[2] and for the high incidence of hyponatremia seen in Coalition troops during the 2003 invasion of Iraq.[3]


The ability of the kidney to vary water excretion over a broad range, without altering steady-state solute excretion, would not be predicted from simple consideration of sequential transport processes along the nephron.[4] Urine concentration and dilution cannot be explained by simple models based on the sequential action of several nephron segments. Instead, it is necessary to consider the parallel interactions between nephron segments that result from its folded or looped structure (see Chapter 2 ). An understanding of these interactions depends on knowledge of the regional architecture of the renal medulla and medullary rays illustrated in Figure 9-2 . The nomenclature used for the various renal tubule segments is summarized in Table 9-1 . Except where indicated, we follow the terminology recommended by Kriz and Bankir[5] on behalf of the Renal Commission of the International Union of Physiological Sciences.



FIGURE 9-2  Mammalian renal structure.[81] Major regions of the kidney are shown on the left. Configurations of a long-looped and a short-looped nephron are depicted. The major portions of the nephron are proximal tubules (medium blue), thin limbs of loops of Henle (single line), thick ascending limbs of loops of Henle (green), distal convoluted tubules (lavender), and the collecting duct system (yellow).  (Modified from Knepper MA, Stephenson JL: Urinary concentrating and diluting processes. In Andreoli TE, Fanestil DD, Hoffman JF, Schultz SG (eds): Physiology of Membrane Disorders, 2nd ed. New York, Plenum, 1986, pp 713-726.)




TABLE 9-1   -- Nomenclature for Renal Tubule Segments[*]

Major Segment



Region of Kidney

Proximal convoluted tubule

Early proximal convolution


Cortical labyrinth Late proximal convolution PCT(S2)

Cortical labyrinth

Loop of Henle

Early proximal straight


Medullary rays


Late proximal straight


Outer medulla (outer stripe)


Descending thin limb of short loops


Outer medulla (inner stripe)


Descending thin limb of long loops, outer medulla


Outer medulla (inner stripe)


Descending thin limb of long loops, inner medulla


Inner medulla


Ascending thin limb


Inner medulla


Medullary thick ascending limb


Outer medulla


Cortical thick ascending limb


Medullary rays


Distal convoluted tubule


Cortical labyrinth


Connecting tubule


Cortical labyrinth

Collecting ducts

Initial collecting tubule


Cortical labyrinth


Cortical collecting duct


Medullary rays


Outer medullary collecting duct, outer stripe


Outer medulla


Outer medullary collecting duct, inner stripe


Outer medulla


Inner medullary collecting duct, initial part


Inner medulla (base)


Inner medullary collecting duct, terminal part


Inner medulla (papilla)



Terminology is based on that proposed by the Renal Commission of the International Union of Physiological Sciences[5] with two exceptions: (1) Terminology for descending thin limb subsegments based on that proposed by Imai and colleagues[6] is used because it is more literally descriptive of the locations and topography of the segments; (2) Expanded terminology for the inner medullary collecting duct is based on studies [53] [218] demonstrating two distinct inner medullary collecting duct subsegments.

Renal Tubules

Loops of Henle

Two populations of nephrons merge to form a common collecting duct system (see Fig. 9-2 ). One population (short-looped nephrons) has loops that bend in the outer medulla. The other population (long-looped nephrons) has loops that bend at various levels of the inner medulla. Figure 9-3 shows three examples of long loops of Henle in mouse as traced using computer reconstruction techniques from serial histological sections through the kidney, providing a realistic view of the course of individual tubules. In rats, more than 70% of long loops of Henle bend in the outer half of the inner medulla, and progressively fewer loops extend to deeper levels of the inner medulla. The loops of Henle receive the effluent from the proximal convoluted tubules. They carry tubule fluid into and out of the renal medulla, establishing countercurrent flow between the two limbs of the hairpin loop as emphasized in Figure 9-3 .



FIGURE 9-3  The courses of three long-loop nephrons from mouse as determined by computer reconstruction from histological images. Color codes: proximal tubule segments, blue-green; thin limbs segments, green; thick ascending limb segments, red; distal convoluted tubule, purple; connecting tubules, yellow.  (From Zhai XY, Thomsen JS, Birn H, et al: Three-dimensional reconstruction of the mouse nephron. J Am Soc Nephrol 17:77-88, 2006.)




Several discrete nephron segments compose the loop of Henle (see Figs. 9-2 and 9-3 [2] [3], and Table 9-1 ). In Figure 9-3 , proximal tubule segments are depicted as blue-green and thin limbs of Henle are depicted as green. The descending part of the loop consists of the S2 proximal straight tubules in the medullary rays, the S3 proximal straight tubule in the outer stripe of the outer medulla, and the thin descending limbs in the inner stripe of the outer medulla and the inner medulla. The descending thin limbs of short loops of Henle (SDL) differ structurally and functionally from the descending thin limbs of long loops of Henle (LDL).[6] The SDLs are not depicted in Figure 9-3but their arrangement in the renal outer medulla is illustrated in Figure 9-4 (labeled in green). As can be seen, the SDLs tend to be organized in a ring-like pattern surrounding the vascular bundles of the outer medulla ( Fig. 9-4 , inset). Long-looped descending limbs in the outer medulla (LDLOM) differ morphologically and functionally from long-looped descending limbs in the inner medulla (LDLIM). [7] [8] [9] [10] The transition from the LDLOM to the LDLIM is gradual; it often occurs a considerable distance into the inner medulla. Figure 9-5 shows a computerized reconstruction of the inner medullary portions of several long loops of Henle from rats featuring labeling with antibodies to aquaporin-1 (AQP1) and the ClC-K1 chloride channel.[11] AQP1, a marker of the LDLOM in the outer medulla, is present in LDLs for a variable distance into the inner medulla. ClC-K1 labeling, marking the thin ascending limb-type epithelium, is first seen at variable distances before the loop bends, consistent with many morphological studies that have demonstrated that the DL-to-AL transition occurs before the loop bend. A substantial portion of the inner medullary LDL (presumably the LDLIM) did not express either AQP1 or ClC-K1. Overall, the ascending part of the loop of Henle consists of the ascending thin limbs (which are present only in long loops), the medullary thick ascending limbs in the inner stripe of the outer medulla, and the cortical thick ascending limbs in the medullary rays. (Medullary and cortical thick ascending limbs are shown in red in Figure 9-3 .)



FIGURE 9-4  Triple immunolabeling of rat renal medulla showing localization of UT-A2 (green) marking late SDL segments, von Willebrand factor (blue) marking endothelial cells of vasa recta, and aquaporin-1 (red) marking LDLOM segments and early SDL segments. Inset shows a cross section of a vascular bundle demonstrating that UT-A2 positive SDL segments surround the vascular bundles in the deep part of the outer medulla. Labels in this diagram: IM, inner medulla; IS, inner stripe of outer medulla; OS, outer stripe of outer medulla: VBa, vascular bundles in outer part of inner stripe; VBb, vascular bundles in inner part of inner stripe.  (From Wade JB, Lee AJ, Liu J, et al: UTA-2: A 55 kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin. Am J Physiol 278:F52–F62, 2000.)






FIGURE 9-5  The inner medullary courses of several long-loop nephrons from rat as determined by computer reconstruction from immunolabeled histological sections. Colors: aquaporin-1, red; ClC-K1, green; no labeling, light blue.  (From Pannabecker TL, Abbott DE, Dantzler WH: Three-dimensional functional reconstruction of inner medullary thin limbs of Henle's loop. Am J Physiol Renal Physiol 286:F38–F45, 2004 and Zhai XY, Thomsen JS, Birn H, et al: Three-dimensional reconstruction of the mouse nephron. J Am Soc Nephrol 17:77-88, 2006.)




Distal Tubule Segments in the Cortical Labyrinth

After exiting the loop of Henle, tubule fluid enters the distal convoluted tubules in the cortical labyrinth (violet tubules in Fig. 9-3 ). Several distal tubules merge to form a connecting tubule arcade in most mammalian species. (Connecting tubules are depicted as yellow in Figure 9-3 .) The arcades ascend upward through the cortical labyrinth in association with the interlobular arteries and veins.[12] The connecting tubule cells of the arcades express both aquaporin-2 (the vasopressin-regulated water channel) and the vasopressin V2-subtype receptor,[13] suggesting that, like the collecting ducts, the arcades are sites of regulated water absorption (see later). The arcades deliver their tubule fluid to initial collecting tubules in the superficial cortex and finally to the cortical collecting ducts. In rats and rabbits, five or six nephrons combine to form a single cortical collecting duct.[14] In mice, six to seven nephrons merge to form a single collecting duct.[15]

Collecting Duct System

The collecting duct system spans all the regions of the kidney between the superficial cortex and the tip of the inner medulla (see Fig. 9-2 ). The collecting ducts are arrayed parallel to the loops of Henle in the medulla and medullary rays. Like the loop of Henle, the collecting duct system is composed of several morphologically and functionally discrete tubule segments (see Table 9-1 ). In general, the collecting ducts descend straight through the medullary rays and outer medulla without joining other collecting ducts. However, repeated joinings occur in the inner medulla, which results in a progressive reduction in the number of inner medullary collecting ducts (IMCDs) toward the renal papillary tip.[14] This reduction in the number of collecting ducts, combined with the progressive reduction in the number of loops of Henle reaching successive levels of the inner medulla, accounts for the tapered structure of the renal papilla.


The major blood vessels that carry blood into and out of the renal medulla are called the vasa recta. The descending vasa recta receive blood from efferent arterioles of juxtamedullary nephrons and supply blood to the capillary plexuses at each level of the medulla. The capillary plexus in the outer medulla is considerably more dense and much better perfused than the plexus in the inner medulla.[16] Blood from the capillary plexus of the inner medulla feeds into ascending vasa recta. (Ascending vasa recta are never formed directly from descending vasa recta in a loop-like structure.) Ascending vasa recta from the inner medulla traverse the inner stripe of the outer medulla in close physical association with the descending vasa recta in vascular bundles.[17] In many animal species, the vascular bundles are surrounded by the thin limbs of short loops of Henle (SDLs) as shown in Figure 9-4 . Here the SDL segments are labeled with an antibody to the UT-A2 urea transporter, suggesting a route for urea recycling from the vasa recta to the short loops of Henle. The capillary plexus of the outer medulla is drained by vasa recta that ascend through the outer stripe of the outer medulla separate from the descending vasa recta.[18]

The counterflow arrangement of vasa recta in the medulla promotes countercurrent exchange of solutes and water. This exchange is abetted by the presence of aquaporin-1 water channels [19] [20] and the UT-B urea transporters [21] [22] in the endothelial cells of the descending portion of the vasa recta. Countercurrent exchange provides a means of reducing the effective blood flow to the medulla while maintaining a high absolute perfusion rate.[23] The low effective blood flow that results from countercurrent exchange is thought to be important to the preservation of solute concentration gradients in the medullary tissue (see later).

In contrast to the medulla, the cortical labyrinth has a high effective blood flow. The rapid vascular perfusion to this region promotes the rapid return of solutes and water absorbed from the nephron to the general circulation. The rapid perfusion is thought to maintain the interstitial concentrations of most solutes at levels close to those in the peripheral plasma. The medullary rays of the cortex have a capillary plexus that is considerably sparser than that of the cortical laby-rinth. Consequently, the effective blood flow to the medullary rays has been postulated to be lower than that of the cortical labyrinth.[4]

Medullary Interstitium

The renal medullary interstitium is a complex space that contains fluid, microfibrils, extracellular matrix, and interstitial cells.[24] In the outer medulla and the outer portion of the inner medulla, the interstitium is relatively small in volume,[4] which may be important in limiting diffusion of solutes upward along the medullary axis. The interstitial space is much larger in the inner half of the inner medulla.[4] A gelatinous matrix found in this region contains large amounts of highly polymerized hyaluronic acid (HA), consisting of alternating D-glucuronate and N-acetyl-D-glucosamine moieties.[25] Figure 9-6 shows a rat kidney labeled with a dye (Alcian Blue) that binds selectively to HA showing its distribution in the kidney. The inner medullary HA interstitial matrix (stained blue in Fig. 9-6 ) has recently been proposed to play a direct role in generation of an inner medullary osmotic gradient through its ability to store and transduce energy from the smooth muscle contractions of the renal pelvis[25] (see later).



FIGURE 9-6  Alcian blue staining of normal rat kidney showing distribution of hyaluronic acid with high levels in inner medulla. Bar is 2 mm.  (From Knepper MA, Saidel GM, Hascall VC, Dwyer T: Concentration of solutes in the renal inner medulla: Interstitial hyaluronan as a mechano-osmotic transducer. Am J Physiol Renal Physiol 284:F433–F446, 2003.)




Renal Pelvis

Urine exits the collecting duct system and enters the renal pelvis at the tip of the renal papilla ( Fig. 9-7 ; compare with Fig. 9-6 ). The renal pelvis (or the calyx in multipapillate kidneys) is a complex intrarenal urinary space surrounding the renal papilla. The renal pelvis (calyx) has extensions called fornices and secondary pouches whose lumens contact portions of the renal outer medulla. Although a transitional epithelium lines most of the pelvic space, a simple cuboidal epithelium separates the pelvic space from the renal parenchyma.[26] It has been proposed that solute and water transport could occur across this epithelium, modifying the composition of the renal medullary interstitial fluid.[27]



FIGURE 9-7  Pattern of urine flow in papillary collecting ducts and renal pelvis. Urine exits the papillary collecting ducts (ducts of Bellini) at the tip of the renal papilla and is carried to the urinary bladder by the ureter.  (Compare with Figure 9-6 .) Under some circumstances, a fraction of the urine may reflux backward in the pelvic space and contact the outer surface of the renal papilla. Solute and water exchange across the papillary surface epithelium has been postulated (see text).


The renal pelvic (calyceal) wall (see Fig. 9-6 ) contains two smooth muscle layers.[28] Contractions of these smooth muscle layers are responsible for powerful peristaltic waves that appear to displace the renal papilla downward with a “milking” action.[29] The peristaltic waves have been reported to intermittently propel the urine along the collecting ducts. The contractions compress all structures in the renal inner medulla including the collecting ducts, the loops of Henle, and the interstitium.[30] The contractions have been proposed to furnish part of the energy for concentrating solutes in the inner medulla,[25] as discussed subsequently.


Sites of Urine Concentration and Dilution

The sites of tubule fluid concentration and dilution along the mammalian nephron have been investigated by micropuncture studies in rats and other rodents. These results are summarized in Figure 9-8 . The tubule fluid in the proximal convoluted tubule is always approximately isosmotic with plasma, regardless of whether the kidney is concentrating or diluting the urine. [31] [32] In contrast, the fluid in the early distal convoluted tubule is always hypotonic, regardless of the osmolality of the urine. The earliest site along the nephron where differences in tubule fluid osmolality between antidiuresis and water diuresis can be detected is the late distal tubule. At this site, the tubule fluid becomes isosmotic with plasma during antidiuresis, but remains hypotonic during water diuresis. Between the late distal tubule and the final urine, the tubule fluid osmolality rises to a level greater than that of plasma during antidiuresis but remains hypotonic during water diuresis. On the basis of the foregoing observations, it has been concluded that the chief site of dilution of tubule fluid is the loop of Henle and that the dilution process in the loop occurs regardless of whether the final urine is dilute or concentrated. During water diuresis, further dilution occurs in the collecting ducts.[33] The chief site of urine concentration is beyond the distal tubule (i.e., in the collect-ing duct system). The following sections consider in turn the mechanism of urinary dilution and of urinary concentration.



FIGURE 9-8  Typical osmolalities (in mOsm/kg H2O) found in various vascular (left) and renal tubule (right) sites in rat kidneys. AD, antidiuresis (i.e., high vasopressin); WD, water diuresis (i.e., low vasopressin). Fluid in the proximal tubule is always isosmotic with plasma (290 mOsm/kg H2O). Fluid emerging from the loop of Henle (entering the early distal tubule) is always hypotonic. Osmolality in the late distal tubule increases to plasma level only during antidiuresis. Final urine is hypertonic when the circulating vasopressin level is high, and hypotonic when the vasopressin level is low. A high osmolality is always maintained in the loop of Henle and vasa recta. During antidiuresis, osmolalities in all inner medullary structures are nearly equal. Osmolalities are somewhat attenuated in the loop and vasa recta during water diuresis (not shown).  (Based on micropuncture studies; see text.)


Mechanism of Tubule Fluid Dilution

Micropuncture measurements in rats have revealed that the hypotonicity of the fluid in the early distal tubule is due chiefly to a reduction in luminal NaCl concentration relative to the proximal tubule.[34] In principle, a low luminal NaCl concentration could result from active NaCl absorption from the loop of Henle or water secretion into the loop. However, micropuncture studies, using inulin as a volume marker, demonstrated net water absorption from the superficial loop of Henle during antidiuresis,[35] which rules out the possibility of water secretion as a mechanism of tubule fluid dilution. Thus, we can conclude that luminal dilution occurs because of NaCl reabsorption in the loop of Henle in excess of water absorption. The mechanism of dilution has been demonstrated in classic studies of isolated perfused rabbit thick ascending limbs of loops of Henle. [36] [37] NaCl is rapidly absorbed by active transport, which lowers the luminal NaCl concentration and osmolality to levels below those in the peritubular fluid. The osmotic water permeability is low, which prevents dissipation of the transepithelial osmolality gradient by water fluxes. Details of the active NaCl transport process at a cellular level are discussed later in the section “Molecular Physiology of Urinary Concentrating and Diluting Processes.

The hypotonicity of tubule fluid is maintained throughout the distal tubule and collecting duct system during water diuresis, abetted by the low osmotic water permeability of the collecting ducts when circulating levels of vasopressin are low (see Chapter 8 ). Although the dilute state is sustained in the collecting ducts, the solute composition of the tubule fluid is modified in the collecting duct system, chiefly by Na absorption and K secretion. Active NaCl reabsorption by the collecting ducts is responsible for the further dilution of the collecting duct fluid beyond that achieved in the thick ascending limbs.[33]

Mechanism of Tubule Fluid Concentration

When circulating vasopressin levels are high, extensive net water absorption occurs at sites between the late distal tubule and the final urine (i.e., in the collecting ducts).[35] Measurements along the IMCDs of antidiuretic hamsters demonstrated directly that water is absorbed in excess of solutes, with a resulting rise in osmolality along the collecting ducts toward the papillary tip.[38] Thus, the collecting duct fluid is concentrated chiefly by water absorption, rather than by solute addition.

The osmotic driving force for water absorption along the collecting ducts is present because of the existence of an axial osmolality gradient in the renal medullary tissue, with the highest degree of hypertonicity at the papillary tip. Such an osmolality gradient was initially demonstrated by Wirz and colleagues[39] in a classic study that used an ingenious microcryoscopic method to measure the osmolality in the lumens of individual renal tubules in tissue slices from quick-frozen rat kidneys. The measurements revealed that in antidiuretic rats, there was a continuous osmolality gradient throughout the medullary axis, including both the outer medulla and inner medulla, with the highest osmolality in the deepest part of the inner medulla, the papillary tip. Furthermore, in the medulla, the osmolality was about as high in the large tubules (presumably collecting ducts) as in the small tubules (presumably loops of Henle); this demonstrates that the high tissue osmolality was not simply a manifestation of a high osmolality in a single structure, namely, the collecting duct. Consistent with this view, Wirz demonstrated by micropuncture that the osmolality of vasa recta blood, sampled from near the papillary tip in antidiuretic hamsters, was virtually equal to that of the final urine.[31] Subsequently, Gottschalk and Mylle,[32] using micropuncture in antidiuretic hamsters, confirmed that the osmolality of the fluid in the loops of Henle, the vasa recta, and the collecting ducts was approximately the same (see Fig. 9-8 ), in support of the view that the collecting duct fluid is concentrated by osmotic equilibration with a hypertonic medullary interstitium. Furthermore, in vitro studies demonstrated that collecting ducts have a high water permeability in the presence of vasopressin [40] [41] as is required for osmotic equilibration. The mechanism by which the corticomedullary osmolality gradient is generated is considered later.

The overall axial osmolality gradient in the renal medulla is composed of gradients of several individual solutes. However, the principal solutes responsible for the osmolality gradient are NaCl and urea, as demonstrated initially in dog kidneys by Ullrich and Jarausch[42] by use of the tissue slice analysis technique. These data are summarized in Figure 9-9 . The increase in NaCl concentration along the cortico-medullary axis occurs predominantly in the outer medulla, with only a small increase in the inner medulla. In contrast, the increase in urea concentration along the cortico-medullary axis occurs predominantly in the inner medulla, with little or no increase in the outer medulla. Although some aspects of the process that generates the renal medullary solute gradient are in doubt, the major aspects are well understood, viz. the mechanism of generation of the NaCl gradient in the outer medulla and the mechanism of urea accumulation in the inner medulla. In the following, we will emphasize these well understood aspects first and then briefly address the frontiers of our knowledge.



FIGURE 9-9  Cortico-medullary gradients of urea, sodium, and chloride in kidneys of antidiuretic dogs. Summary of data from Ullrich and Jarausch.[42]  (From Giebisch G, Windhager EE: Urine concentration and dilution. In Boron WF, Boulpaep EL (eds): Medical Physiology. Philadelphia, Saunders, 2006, pp 828-844.)


Generation of An Axial NaCl Gradient in the Renal Outer Medulla: Countercurrent Multiplication

The concept of countercurrent multiplication originally evolved from a consideration of industrial processes that separate and concentrate economically useful products (e.g., countercurrent extraction and distillation). In these processes, a single stage (given the appropriate energy input) is capable of modest concentration of one component. However, the effect of a single stage (“single effect”) can be multiplied by successive applications of the effect. Werner Kuhn, a Swiss applied physical chemist, and his colleagues used this concept to provide an explanation for the corticomedullary osmolality gradient in the renal medulla. [43] [44] [45] They showed, using mathematical techniques, that a small concentration difference (single effect) between the ascending and descending limbs of a hairpin counterflow system could be multiplied by the countercurrent flow to obtain an axial gradient much larger than the transverse concentration difference between the limbs. They demonstrated the feasibility of such a concept by constructing physical counterflow models that developed axial solute concentration gradients. In the following paragraphs, we develop in greater detail the conceptual basis of the countercurrent multiplier model.

Figure 9-10 A shows a hypothetical single-stage process that provides a starting point for consideration of the countercurrent multiplication concept. We assume that a volume of fluid containing a dissolved solute can be added to such a single stage and, with an appropriate energy input, can be divided into two smaller volumes, one slightly more concentrated than the input, one slightly less concentrated. If the more concentrated output were reintroduced into the same single stage and similarly divided into two smaller volumes, the overall action would be to concentrate a fraction of the original starting fluid more than could have been achieved by a single stage applied only once. If the concentrated output of a single stage is “y” times more concentrated than the input, then the two steps would concentrate the output R = y2 times more than the original input. That is, the single effect y is multiplied to obtain the overall concentration ratio R. Similarly, if there are “n” successive applications of a single stage, the single effect will be multiplied n times, and the overall concentration ratio R will be yn. It is evident that by such a scheme, the action of a single stage with a modest single-stage concentration ratio can be multiplied to yield any arbitrary overall concentration ratio R.



FIGURE 9-10  Conceptual development of the countercurrent multiplier hypothesis based on the work of Kuhn and associates. [43] [44] [45] See text for explanation.



Instead of successive applications of a single stage, it is theoretically feasible to stack several such stages in a cascade so that all can operate simultaneously in a steady-state operation ( Fig. 9-10B ). In this configuration, the concentrated output from a given stage is passed downward to the next stage to be further concentrated. As with the sequential operation of a single stage, this scheme can multiply the single effect of an individual stage to yield any arbitrary overall concentration ratio, given enough stages. The disadvantage of such a scheme is that the volume of fluid reaching each successive stage will be progressively smaller, and the volume of the overall effluent at the bottom of the stack would approach zero as the number of stages n became large. This drawback is avoided, however, if the more dilute output from each stage is passed upward to the next stage, allowing recycling of the fluid ( Fig. 9-10C ). This change results in a countercurrent arrangement, with the upward-flowing fluid interacting with the downward-flowing fluid at each stage. This countercurrent multiplication scheme allows several concentrating stages to interact to produce a relatively large volume of concentrated fluid at the bottom of the stack.

Kuhn and colleagues recognized that it was a simple matter to extend the countercurrent multiplier scheme involving several discrete stages shown in Figure 9-10C to a continuous-flow scheme in which discrete stages are replaced by ascending and descending streams whose interaction is distributed uniformly throughout their lengths, as would have to exist in the loop of Henle ( Fig. 9-10D ). A small concentration difference between the counterflowing descending and ascending streams could result in a large axial concentration gradient. The development of the concept by Kuhn and colleagues [43] [44] [45] that such a continuous countercurrent scheme could explain urine concentration was a landmark event in renal physiology. The terminology derived from the stagewise process was retained in the description of renal countercurrent multiplication. Thus, the term “single effect” (Einzeleffekt, individual effect), which referred to the action of an individual stage, was retained to denote a small solute concentration difference between ascending and descending limbs, although in fact discrete individual stages do not exist.

Hydrostatic pressure was initially considered a possible energy source for creation of a single effect in the loop of Henle.[43] High pressure on the descending limb side of the loop could theoretically force water to exit the lumen, thus concentrating the descending limb luminal fluid relative to the ascending limb fluid. The realization that hydrostatic pressures in the descending limb lumen were not likely to be high enough to provide a substantial osmolality difference between the two limbs led Kuhn and Ramel[45] to describe a model in which active NaCl transport out of the ascending limb lowered its concentration with respect to that of the descending limb (see Fig. 9-10D ). Later studies in isolated perfused thick ascending limbs demonstrated that this renal tubule segment indeed has the capability of generating a transepithelial osmotic gradient as required. [36] [37]

A continuous countercurrent multiplier is capable of producing a small volume of concentrated output, which in theory could be withdrawn from the bend of the hairpin loop (see Fig. 9-10D ). However, Hargitay and Kuhn[44]recognized that a more realistic scheme would include a third tube (a “collecting duct”) that equilibrates osmotically with the loop fluid to produce a concentrated output ( Fig. 9-10E ). Such a scheme has the advantage that it can concentrate solutes in the collecting ducts other than those responsible for the axial osmolality gradient in the loop. The volume flow into the collecting duct must be considerably less than in the loop for a significant overall concentrating effect to be maintained.

Figure 9-10 depicts a countercurrent multiplier that works strictly by recycling NaCl between ascending and descending limbs of the loop of Henle, thus increasing the mean residence time of Na and Cl ions in the renal medulla. Subsequent work has revealed that early portions of the descending limbs of long-loop nephrons are highly permeable to water [7] [8] [10] due to the expression of high levels of aquaporin-1 in the apical and basolateral plasma membrane of descending limb cells. [19] [46] [47] [48] Thus, as shown in Figure 9-11 , countercurrent multiplication works not only by NaCl recycling into the descending limb, but also by water short-circuiting from the descending limb.[49] The water reabsorbed from the descending limb is rapidly returned to the general circulation by the vasa recta, thus reducing the mean residence time for water molecules in the renal medulla. Either an increase in residence time of solute particles or a decrease in residence time of water molecules serves to concentrate the renal medulla.



FIGURE 9-11  Countercurrent multiplication in the renal outer medulla. The thick ascending limb actively reabsorbs NaCl, but because of low water permeability, water is not reabsorbed, resulting in luminal dilution necessary for ‘single effect’. The descending limb is highly permeable to water due to high levels of expression of aquaporin-1. Hypertonic NaCl reabsorbed from ascending limb drives osmotic water reabsorption from descending limb, resulting in short-circuiting of water back to the general circulation (see text).  (From Knepper MA, Nielsen S, Chou C-L: Physiological roles of aquaporins in the kidney. Curr Top Membr 51:121-153, 2001.)




It is now generally accepted that the axial osmolality gradient in the outer medulla is generated by countercurrent multiplication driven by active NaCl transport in the thick ascending limbs. However, as discussed in greater detail subsequently, this basic mechanism does not exist in the inner medulla of the kidney. Indeed, the ascending limb of Henle's loop in the inner medulla has thin limb morphology and little or no capacity for active NaCl transport. [41] [50] [51] [52] The solute responsible for most of the inner medullary osmolality gradient is urea (see Fig. 9-9 ). Mechanisms responsible for urea accumulation in the inner medulla are discussed next.

Accumulation of Urea in Renal Inner Medulla: Facilitated Urea Transport, Diffusion Trapping, and Urea Recycling

Urea accumulation in the inner medulla is dependent on differential urea permeability along the collecting duct system ( Fig. 9-12 ). The pattern of urea permeability differences among tubule segments has been defined chiefly using the isolated, perfused tubule technique. Figure 9-12 shows a long loop of Henle, a short loop of Henle, and the collecting ducts with each segment distorted so that its width is proportional to the urea permeability coefficient in that segment. Among collecting duct segments, a high urea permeability has been found only in the terminal part of the IMCD.[53] The urea permeability of the terminal IMCD is regulated by vasopressin, increasing to extremely high values within minutes of vasopressin exposure. [41] [54] [55] This action of vasopressin is mediated by cyclic adenosine monophosphate (cyclic AMP).[56] As discussed subsequently, the high urea permeability of the terminal part of the IMCD is due to the presence of specialized phloretin-sensitive urea transporters in the apical and basolateral plasma membranes of the IMCD cells. The low urea permeability of the collecting duct system proximal to the terminal IMCD is due to a lack of urea transporter expression.



FIGURE 9-12  Urea permeabilities of mammalian renal tubule segments. The width of each segment in the diagram is distorted to be proportional to the urea permeability of that segment. Numbers in parentheses are measured values for the permeability coefficient (×10-5 cm/sec). Values are from isolated perfused tubules studies. [54] [68] [70] [71] [214] [215] [216] Abbreviations for renal tubule segments are the same as in Table 9-1 .



The mechanism of urea accumulation in the renal medulla is shown in Figure 9-13 .[57] The accumulation process is a result of passive urea absorption from the IMCDs. The tubule fluid entering the collecting duct system in the renal cortex has a relatively low urea concentration. During antidiuresis, water is osmotically absorbed from the urea-impermeable parts of the collecting duct system in the cortex and outer medulla. This causes a progressive increase in luminal urea concentration along the connecting tubules, cortical collecting ducts, and outer medullary collecting ducts. Then, when the tubule fluid reaches the terminal IMCD, which is highly permeable to urea, urea can exit rapidly from the lumen to the inner medullary interstitium. The urea is trapped in the inner medullary interstitium because the effective blood flow is low owing to countercurrent exchange by the vasa recta (see later). Because the urea permeability of the terminal IMCD is extermely high, particularly in the presence of vasopressin, urea nearly equilibrates across the IMCD epithelium under steady-state conditions. This allows urea in the intersititum to almost completely balance osmotically the high urea concentration in the collecting duct lumen, preventing the osmotic diuresis that would otherwise occur ( Fig. 9-14 ).



FIGURE 9-13  Diagram of mammalian collecting duct system showing principal sites of water absorption and urea absorption. Water is absorbed in early part of the collecting duct system, driven by an osmotic gradient. Because urea permeabilities of cortical collecting duct, outer medullary collecting duct and initial IMCD are very low, the water absorption concentrates urea in the lumen of these segments. When the tubule fluid reaches the terminal IMCD, which is highly permeable to urea, urea rapidly exits from the lumen. This urea is trapped in the inner medulla as a result of countercurrent exchange.





FIGURE 9-14  Solutes that account for osmolality of medullary interstitium and tubule fluid in the inner medullary collecting duct during antidiuresis in rats. Urea nearly equilibrates across the IMCD epithelium as a result of rapid facilitated urea transport. Although the osmolalities of the fluid in the two spaces are nearly equal, the non-urea solutes can differ considerably between the two compartments. Typical values in untreated rats are presented. Values can differ considerably in other species and in the same species with different diets. NUN, non-urea nitrogen.



Close association of descending and ascending vasa recta facilitates countercurrent exchange of urea between the two structures.[23] The concentration of urea in the ascending vasa recta exiting the inner medulla approaches the concentration in the descending vasa recta entering the inner medulla, which minimizes the washout of urea from the inner medulla. The permeability of the vasa recta to urea is extremely high (>40 × 10-5 cm/sec), [21] [41] which abets the countercurrent exchange process. Countercurrent exchange cannot completely eliminate loss of urea from the inner medullary interstitium because the volume flow rate of blood in the ascending vasa recta normally exceeds that in the descending vasa recta.[58] The water added to the vasa recta derives from the IMCDs and descending limbs, both of which reabsorb water during antidiuresis. Because the mass flow rate of urea is the product of the urea concentration and the volume flow rate, the higher volume flow rate in the ascending vasa recta will ensure that the inner medullary vasculature continually removes urea from the inner medulla. Quantitatively, the most important loss of urea from the inner medullary interstitium is thought to occur via the vasa recta.[59]

Recycling pathways limit the loss of urea from the inner medulla. Flow in the ascending vasa recta and ascending limb of the loop of Henle tends to carry urea out of the inner medulla. These losses are minimized by urea recycling pathways, which return to the inner medulla much of the urea that leaves through the vasa recta or ascending limbs. Three major urea recycling pathways are described in Figure 9-15 .[59]



FIGURE 9-15  Pathways of urea recycling in the mammalian kidney. Solid lines represent a short-looped nephron (left) and a long-looped nephron (right). Transfer of urea between nephron segments is indicated by dashed arrows labeled a, b, and c corresponding to recycling pathways described in the text. tAL, thin ascending limb; CD, collecting duct; DCT, distal convoluted tubule; DL, descending limb; PST, proximal straight tubule; TAL, thick ascending limb; vr, vasa recta.  (From Knepper MA, Roch-Ramel F: Pathways of urea transport in the mammalian kidney. Kidney Int 31:629-633, 1987.)




Recycling of Urea through the Ascending Limbs, Distal Tubules, and Collecting Ducts

Urea exiting the inner medulla in the ascending limbs of the long loops of Henle is carried through the thick ascending limbs, the distal convoluted tubules, and the early part of the collecting duct system by the flow of tubule fluid[35] ( Fig. 9-15A ). When it reaches the urea-permeable part of the collecting duct in the inner medulla, it passively exits into the inner medullary interstitium, completing the cycle.

Recycling of Urea through the Vasa Recta, Short Loops of Henle, and Collecting Ducts

Micropuncture studies of non diuretic rats have revealed that the delivery of urea to the superficial distal tubule exceeds the delivery out of the superficial proximal tubule. [35] [60] [61] This implies that, in addition to urea recycling via the long loops as described in Figure 9-15A , net urea addition occurs along the short loops of Henle. To explain this finding, it has been proposed [60] [62] that urea leaving the inner medulla in the vasa recta is transferred to the descending limbs of the short loops of Henle ( Fig. 9-15B ). The urea that enters the short loops can then be carried through the superficial distal tubules and back to the inner medulla by the collecting ducts where it is reabsorbed, completing the recycling pathway. Transfer of urea from the vasa recta to the short loops of Henle is facilitated by a close physical association between the vasa recta and the descending limbs of the short loops in the vascular bundles of the inner stripe of the outer medulla. [18] [63] The recent finding that the urea transporter UT-A2 is selectively expressed in the thin descending limb of short loops of Henle [64] [65] provides support for this hypothesis. However, recent research using mathematical modeling[66] and UT-A2 knockout mice[67] (see later) has raised doubts about the importance of this pathway.

Recycling of Urea between Ascending Limb and Descending Limb

Studies in isolated perfused thick ascending limbs have revealed that the urea permeability of thick ascending limbs from the inner stripe of the outer medulla is too low to permit a substantial amount of urea absorption. [68] [69]However, similar studies in segments from the outer stripe and medullary rays have demonstrated a higher urea permeability. [68] [70] It has been proposed that urea reabsorbed from these thick ascending limbs enters neighboring proximal straight tubules, completing a recycling pathway between the ascending limb and descending limbs of the loop[4] ( Fig. 9-15C ). The urea transfer between thick ascending limbs and proximal straight tubules is facilitated by the parallel relationship between these two structures in the outer stripe and in the medullary rays. The transfer may also depend on a relatively attenuated effective blood flow in these regions. Urea secretion into the proximal straight tubules can occur by active transport,[71] by passive diffusion,[70] or by a combination of both. Urea presumably enters the proximal straight tubules of both short and long loops of Henle. The urea that enters the short-looped nephrons will be carried back to the inner medulla by the flow of tubule fluid through the superficial distal tubules and collecting ducts, reentering the inner medullary interstitium by reabsorption from the terminal IMCD. The urea that enters proximal straight tubules of long loops returns to the inner medulla directly through the descending limbs.

Collecting Duct Water Absorption

The process of urine concentration consists of two relatively independent components: (1) countercurrent processes that generate a hypertonic medullary interstitium by concentrating NaCl and urea (discussed earlier); and (2) osmotic equilibration of the tubule fluid in the medullary collecting ducts with the hypertonic medullary interstitium to form a hypertonic final urine. In this section, we discuss the mechanism of the latter.

Water excretion is regulated by vasopressin (see Fig. 9-1 ) largely as a result of its effect on the water permeability of the collecting ducts. The cellular mechanism of this response is discussed in considerable detail elsewhere (see Chapter 8 ). When the water permeability is low in collecting ducts because of a low circulating level of vasopressin, relatively little water is absorbed in the collecting ducts. The dilute fluid exiting the loops of Henle remains dilute as it passes through the collecting duct system, yielding a large volume of hypotonic urine. When the water permeability of the collecting ducts is high because of a high circulating level of vasopressin, water is rapidly reabsorbed along the collecting duct system by osmosis, drawn by the osmolality gradient between the lumen and the peritubular interstitium. The osmolality of the final urine approaches that of the inner medullary interstitium, which results in formation of a small volume of hypertonic urine.

Micropuncture studies have demonstrated that the late distal tubule is the earliest site along the renal tubule where water absorption increases during antidiuresis.[31] The “distal tubule”, as defined by micropuncturists, is made up of three segments, the distal convoluted tubule, the connecting tubule, and the initial collecting tubule. Osmotic water permeability is difficult to measure in these segments owing to their short length. The evidence available indicates that the distal convoluted tubule has a low water permeability and does not express any of the known water channels. In contrast, the connecting tubule expresses both the type 2 vasopressin receptor (V2R) and the vasopressin-regulated water channel aquaporin-2 (AQP2) and is presumably the segment responsible for distal tubular osmotic equilibration in micropuncture studies.[13] In addition, AQP2 is expressed in the initial collecting tubule as well as the cortical collecting duct.[72] Thus, among the segments making up the portion of the distal tubule accessible by cortical micropuncture (distal convoluted tubule, connecting segment, and initial collecting tubule), only the connecting segment and the initial collecting tubule appear to exhibit vasopressin-regulated water transport.

The amount of water absorption in the connecting segment and initial collecting tubule required to raise tubule fluid to isotonicity is considerably greater than the additional amount required to concentrate the urine above the osmolality of plasma in the medullary portion of the collecting duct system.[4] Consequently, most of the water reabsorbed from the collecting duct system during antidiuresis enters the cortical labyrinth, where the effective blood flow is high enough to return the reabsorbed water to the general circulation without diluting the interstitium. If such a large amount of water were absorbed along the medullary collecting ducts, it would be expected to have a significant dilutional effect on the medullary interstitium and impair concentrating ability. [73] [74]

During water diuresis, a corticomedullary osmolality gradient persists, although it is attenuated. [75] [76] [77] In the absence of vasopressin, the water permeability of the collecting ducts is low but not zero. [54] [78] Consequently, some water is absorbed by the collecting ducts during water diuresis. Most of the water absorption occurs from the terminal part of IMCDs, where the transepithelial osmolality gradient is highest and the basal water permeability is also highest. In fact, more water is absorbed from the terminal collecting ducts during water diuresis than during antidiuresis owing to a much larger transepithelial osmolality gradient.[73] A high rate of water absorption from the IMCDs is thought to contribute to the reduction of the medullary interstitial osmolality during water diuresis by its dilutional effect. The fall in inner medullary tissue osmolality during water diuresis results largely from an increase in tissue water content [74] [79] associated with the higher rate of water absorption from the collecting ducts, although reductions in the quantities of urea and NaCl in the medullary tissue have also been documented.

Determinants of Concentrating Ability

Figure 9-16 summarizes the major determinants of urinary concentrating ability based on the classical theoretical analysis of Stephenson.[80] We present this here because it provides a basis for understanding the effects of vasopressin and specific gene knockouts on the concentrating process discussed later (in section titled “Molecular Physiology of Urinary Concentrating and Diluting Processes”). Obviously, active transport of NaCl from the thick ascending limb (factor 2) and collecting duct water permeability (factor 4) are two determinants that are self evident from the foregoing description of the urinary concentrating mechanism. In addition, the “distal delivery” of NaCl and water to the loop of Henle (factor 1) is an important determinant because it places an upper bound on the amount of NaCl actively absorbed by the thick ascending limb to drive the countercurrent multiplier mechanism. Finally, the fluid delivery to the medullary collecting duct (factor 3) has an underappreciated effect on the concentrating process. Too much delivery saturates the water absorption process along the medullary collecting ducts and is associated with interstitial dilution owing to rapid osmotic water transport. Too little fluid delivery to the medullary collecting ducts, even in the absence of vasopressin, results in sustained osmotic equilibration across the collecting duct epithelium owing to the non-zero osmotic water permeability of the IMCD. [54] [73] [78]



FIGURE 9-16  Major determinants of urinary concentrating ability. See text for details.



An Unanswered Question: Concentration of NaCl in the Renal Inner Medulla

As described in Figure 9-9 , tissue slice studies have demonstrated that the corticomedullary osmolality gradient is made up largely of a NaCl gradient in the outer medulla and a urea gradient in the inner medulla. Accordingly, in the foregoing we have emphasized the process that concentrates NaCl in the outer medulla (classic countercurrent multiplication) and the process responsible for urea accumulation in the inner medulla (passive urea absorption from the inner medullary collecting duct plus diffusion trapping). Not addressed was the origin of the small NaCl gradient in the renal inner medulla (see Fig. 9-9 ), as well as the energy source for concentration of non-urea solutes in the inner medullary interstitium. These remain unanswered questions.

Presumably a countercurrent multiplication process is responsible for the inner medullary NaCl and osmolality gradient, but what is the single effect? As discussed earlier, the single-effect mechanism in the outer medulla is active NaCl transport out of the water-impermeable thick ascending limb, diluting the luminal fluid relative to the interstitium. However, repeated studies of thin ascending limbs [41] [50] [51] [52] have failed to show evidence for such an active transport process in the inner medulla. General analysis of inner medullary concentrating processes indicates that, to satisfy mass balance requirements, either an ascending stream (thin ascending limbs or ascending vasa recta) must be diluted relative to the inner medullary interstitium, or a descending stream (descending thin limbs, descending vasa recta, or collecting ducts) must be concentrated locally relative to the inner medulla. [25] [81] [82] In previous versions of this chapter (in earlier editions) and in published review papers, [25] [82] [83] we have categorized possible single effect mechanisms for inner medullary concentrating processes. In the following, we summarize the three mechanisms that garnered the greatest interest in the current literature.

The Kokko-Rector Stephenson Model: The “Passive Mechanism”

Kokko and Rector[84] and Stephenson[85] simultaneously proposed a model by which the osmolality in the ascending thin limb could be lowered below that of the interstitium entirely by passive transport processes in the inner medulla. This formulation is generally referred to as the “passive model” or the “passive countercurrent multiplier mechanism”. In this model, rapid efflux of urea from the inner medullary collecting duct causes osmotic withdrawal of water from the thin descending limb, concentrating NaCl in the lumen. The highly concentrated NaCl is then proposed to exit passively from the thin ascending limb, thus diluting the luminal fluid, providing a single effect for countercurrent multiplication. This model requires that the thin descending limb be highly permeable to water but not NaCl or urea, whereas the thin ascending limb would have to be permeable to NaCl but not water or urea. Previously objections to the model have been made largely on the basis of the high urea permeabilities that have been measured in the thin descending limb[86] and thin ascending limb.[25] Recent studies in mice [87] [88] in which facilitated urea transport from the inner medullary collecting duct was eliminated via a gene knockout strategy provided strong evidence that seemingly rules out the passive model (see later). Specifically, when the UT-A1 and UT-A3 urea transporter genes were deleted, urea accumulation in the inner medulla was largely eliminated, but inner medullary NaCl accumulation was not affected in contradiction to the passive model (see “UT-A1/3 knockout” later).

A more thorough discussion of the Kokko-Rector-Stephenson passive countercurrent multiplier model may be found in the corresponding chapter on urinary concentration and dilution in previous editions of this book.

Lactate-Driven Concentrating Mechanism

Because of a failure of models accounting only for urea, NaCl, and water to explain solute gradients in the inner medullary interstitium, consideration has been given to possible other solutes that could play a role.[89] In such a model, an unspecified solute may be assumed to be added continuously to the inner medullary interstitium. Such a solute would have to be generated de novo via a chemical reaction that generates more osmotically active particles than it consumes. In a subsequent mathematical modeling study, Thomas and Wexler[90] confirmed that addition of such a solute to the inner medullary interstitium could theoretically explain the concentration gradient along the inner medullary axis by driving water absorption from the thin descending limb. This action would concentrate NaCl in the descending limb, establishing a gradient for NaCl efflux from the ascending limb and dilution of the ascending limb lumen relative to the interstitium. This model is like the Kokko-Rector-Stephenson passive model discussed earlier except that the external solute substitutes for urea.

Thomas [91] [92] has proposed that the “external solute” is lactate, generated by anaerobic glycolysis (the predominant means of ATP generation in the inner medulla) in the proportion of two lactate ions per glucose molecule consumed:


The success of the proposed model in generating a medullary osmotic gradient depends on the fate of the H generated. If the H ions titrate bicarbonate, they will remove two osmotically active particles (HCO3 ions) causing net disappearance of osmotically active particles:


Because CO2 readily permeates lipid bilayers, it is unlikely to be osmotically effective. Alternatively, if the H ions titrate buffers other than bicarbonate, such as phosphate or NH3, net generation of osmotically active particles can occur. Thus far, this hypothesis has not been pursued experimentally. Recent mathematical modeling studies re-evaluating factors involved in inner medullary lactate accumulation suggest that countercurrent exchange of glucose from descending (DVR) to ascending vasa recta (AVR) in the outer medulla (OM) and upper inner medulla (IM) may severely limit lactate generation in the deepest part of the inner medulla,[93] raising doubts about the feasibility of the lactate generation model.

Hyaluronan as a Mechano-Osmotic Transducer

As proposed by Schmidt-Nielsen,[94] the contractions of the smooth muscle of the pelvic wall may provide energy for the inner medullary concentrating mechanism by compressing the spongelike interstitial hyaluronan matrix. Following this proposal, Knepper and collaborators[25] have described a periodic (non-steady state) concentrating model of the inner medulla (summarized later in this section) based on the assumption that the inner medullary interstitium consists of a semisolid viscoelastic hyaluronan gel rather than being a freely flowing aqueous medium.

Hyaluronan (or “hyaluronic acid”) is a glycosaminoglycan (GAG). GAGs consist of unbranched polysaccharide chains composed of repeating disaccharide units. Aside from hyaluronan, the family of mammalian GAGs include dermatan sulfate, chondroitin sulfates, keratan sulfate, heparan sulfate, and heparin. Hyaluronan differs from the other GAGs because it is not covalently linked to proteins to form proteoglycans and is not sulfated.[95] In contrast to the other GAGs, which are produced in the Golgi apparatus, hyaluronan is synthesized at the plasma membrane by an integral membrane protein, hyaluronan synthase (HAS). [96] [97] Three mammalian HAS genes are recognized, namely, HAS1, HAS2, and HAS3. All three HAS proteins produce hyaluronan on the cytoplasmic side of the plasma membrane and transport it across the plasma membrane to the extracellular space. Therefore, hyaluronan secretion is not dependent on vesicular trafficking. Because of the importance of GAGs in the structure of connective tissues such as cartilage, tendon, bone, synovial fluid, intervertebral disks, and skin, the physico chemical properties of these substances have been thoroughly investigated.[98]

As shown in Figure 9-6 , hyaluronan is extremely abundant in the renal inner medullary interstitium. [99] [100] Other GAGs are present in the inner medulla, but in much lower amounts. The hyaluronan in the inner medulla is produced by a specialized interstitial cell (the type 1 interstitial cell) that forms characteristic “bridges” between the thin limbs of Henle and vasa recta.[101] Thus, the inner medullary interstitium can be visualized as being composed of a compressible, viscoelastic hyaluronan matrix.

The compression of hyaluronan in the medullary interstitium by the peristaltic contractions of the pelvic wall can hypothetically serve to generate a single effect for inner medullary concentration without measurable changes in hydrostatic pressure.[25] During inner medullary compression due to the contraction of the pelvic wall, the compression of the hyaluronan matrix stores some of the mechanical energy generated from the smooth muscle contraction. This compression does not require a generalized increase in hydrostatic pressure, but simply involves a direct mechanical compression of the hyaluronan matrix as one would compress a steel spring. After passage of a peristaltic wave, the compressed hyaluronan springs back from its compressed state, exerting an elastic force that can theoretically drive water efflux from the descending limb of the loop of Henle and other water-permeable structures. This water efflux would concentrate solutes in the tubule lumens. In the descending limb, the total solute concentration would thereby rise above that of the surrounding interstitium, satisfying the requirement for an inner medullary single effect, which could be multiplied by the counterflow between ascending and descending limbs. Thus, hyaluronan compression and relaxation would facilitate an energy conversion starting with ATP hydrolysis in the smooth muscle cells of the pelvic wall, leading to an increase in electrochemical potential due to concentration of solutes in the tubule lumen.

Hyaluronan has other properties that would enhance the concentrating process.[102] It is a large (1000 kD to 10000 kD) polyanion. Its charge is due to the COO (carboxylate) groups of the glucuronic acid subunits. It is strongly hydrophilic and adopts a highly expanded, stiffened random-coil conformation that occupies a huge volume relative to its mass. The extended state of hyaluronan owes partly to repulsive electrostatic forces exerted by neighboring carboxylate groups, which maximize the distance between neighboring negative charges, and partly by the constraints of the glycosidic bonds that prefer somewhat extended conformations. This creates a swelling pressure (turgor) that allows the hyaluronan matrix to generate the elastic-like force (resilience) that resists compression. When hyaluronan is compressed, as occurs in a meniscus in the knee joint in response to load bearing, the repulsive force of neighboring carboxylate groups is overcome in part by condensation of cations (chiefly Na) forming a localized crystalloid structure. Thus, compression of a hyaluronan gel results in a decrease of the local sodium ion activity in the gel.[25] In aqueous solutions in equilibrium with the gel, the NaCl concentration will be decreased secondary to the compression-induced reduction in Na activity within the gel. Therefore, the free fluid that is expressed from the hyaluronan matrix during the contraction phase would have a lower total solute concentration than that of the gel as a whole. The slightly hypotonic fluid expressed from the interstitial matrix is likely to escape the inner medulla via the ascending vasa recta, the only structure that remains open during the compressive phase of the contraction cycle.[103] As a consequence, an ascending stream (the ascending vasa recta) would have a lower total solute concentration than the interstitium as a whole, creating a single effect for medullary concentration during the compression phase of the pelvic contraction cycle.

The HAS2 gene has been knocked out in mice.[104] However, the mice die during fetal development due to cardiac developmental abnormalities, preventing the evaluation of the inner medullary concentrating process in these mice. Thus, either a targeted deletion in the inner medullary interstitial cells or an inducible knockout of HAS2 would be needed for the studies to address a role for hyaluronan in the inner medullary concentrating mechanism.


Transport Proteins Involved in Urinary Concentration and Dilution

Figure 9-17 summarizes the renal tubule sites of expression of water channels (aquaporins), urea transporters, and ion transporters important to the urinary concentrating process. In the following, we summarize the roles of these transport proteins in urinary concentrating and diluting mechanisms. We emphasize these proteins as molecular targets for vasopressin action.



FIGURE 9-17  Grid showing sites of expression of water channels, urea transporters, and ion transporters important to the urinary concentrating process. See text for details.




Abundant expression of aquaporin-1 in the LDL-OM, LDL-IM, and the early part of the SDL accounts for the high water permeability in these segments. [19] [46] [47] [48] In contrast, the ascending limb segments (ATL, MTAL, and CTAL) do not express any known water channel, accounting for the low osmotic water permeability measured in these segments. [36] [37] [51] Aquaporin-2 is a major target for vasopressin action in the CNT and throughout the collecting duct system.[105] Aquaporin-2 is regulated in two ways by vasopressin: (1) short-term regulation of aquaporin-2 trafficking to and from the apical plasma membrane[106]; and (2) long-term regulation of aquaporin-2 abundance,[107] chiefly through transcriptional mechanisms.[108] Aquaporin-2 is chiefly expressed apically throughout the collecting duct system. The basolateral component of water transport across CNT cells and collecting duct principal cells is mediated by aquaporin-3[109] and aquaporin-4.[110] Aquaporin-3 is the dominant basolateral water channel in the CNT and early parts of the collecting duct system, whereas aquaporin-4 predominates in the outer medullary and inner medullary collecting ducts.[110] The abundance of aquaporin-3, but not that of aquaporin-4 is regulated by the long-term effect of vasopressin. [109] [110] [111] The regulation of aquaporin-3 occurs via changes in aquaporin-3 mRNA levels.[108]

Urea Transporters

The three urea transporters shown in Figure 9-17 are derived from the same gene, UT-A ( Fig. 9-18 ). The transcription of UT-A1 and UT-A3 is driven by the same promotor and are expressed in the terminal part of the IMCD. [64] [87] [112] [113] [114] [115] [116] In contrast, transcription of UT-A2 is driven by a downstream promotor present in an intron[117] and is expressed in the late portion of the SDL (see Fig. 9-4 ). [64] [65] The presence of UT-A2 is presumably responsible for the high urea permeability of the LDL-IM and the SDL (see Fig. 9-12 ) but the high urea permeability of the ATL is not attributable to any known urea transporter. We speculate that the high urea permeabil-ity of the ATL is due to paracellular urea movement. The extremely high urea permeability of the terminal IMCD corresponds to the localization of UT-A1 and UT-A3 (see also “Knockout Mice”, later).



FIGURE 9-18  Urea transporters derived from UT-A gene. UT-A1 and UT-A3 are driven by the same promotor and are identical through amino acid 459. Use of an alternative exon inserts a stop codon that terminates UT-A3 after amino acid 460 (an aspartic acid). UT-A2 is identical to the terminal 397 amino acids of UT-A1 and is driven by an alternative promotor in intron 13 of the mouse gene.[117] Numbers indicate amino acid sequence number.



All three of the UT-A urea transporters expressed in the kidney are regulated by vasopressin. UT-A2 protein abundance has been shown to be increased in the LDL-IM and SDL in response to long-term treatment with vasopressin.[65] This may be an indirect effect of vasopressin because V2 receptors have not been demonstrated in the LDL or SDL. Isolated perfused terminal IMCD segments exhibit a rapid increase in urea permeability in response to vasopressin. [54] [118] This may be in part due to direct phosphorylation of UT-A1[119] although the phosphorylation site has not yet been identified. Exposure of Xenopus oocytes injected with UT-A1 or UT-A3 cRNA to PKA agonists (cAMP/forskolin/IBMX) caused a significant increase in passive urea transport, whereas these agents had no effect in UT-A2 injected oocytes.[117] These results suggest that both UT-A1 and UT-A3 are targets for the short-term action of vasopressin working though cyclic AMP. New studies have demonstrated that UT-A2 activity can also be acutely regulated by both vasopressin and its second messengers in cultured MDCK cells.[120]

Long-term vasopressin stimulation or water deprivation has resulted in a decrease in UT-A1 protein abundance in the IMCD. [121] [122] Changes in osmolality could be responsible.

Na Transporters and Channels


The Na-H exchanger, NHE3, is the major absorptive pathway for Na in the proximal tubule. Immunocytochemical studies have demonstrated that it is also expressed in the thin descending limb of Henle (LDL-OM) and the thick ascending limb (MTAL and CTAL)[123] ( Fig. 9-19 ). NHE3 activity in the MTAL is increased by hypotonicity through activation of a PI 3-K-dependent pathway, which is inhibited by vasopressin working through cAMP.[124] Thus, vasopressin has the net effect to inhibit NHE3 activity in the thick ascending limb of Henle.



FIGURE 9-19  Ion transporters in thick ascending limb cell. Ion transporters and channels that account for net NaCl transport across the thick ascending limb epithelium. Transporters regulated by vasopressin indicated in green. Transporters not known to be regulated by vasopressin in red. Tight junctional pathway for cations is via a junctional protein called paracellin or claudin-16.[217]



Na-K-2Cl Cotransporters

Both of the known Na-K-2Cl cotransporters are expressed in the kidney. The ubiquitous form, NKCC1, is expressed in the basolateral plasma membrane of the inner medullary collecting duct,[125] where it is thought to play a role in NaCl secretion.[55] It is also present in the basolateral plasma membrane of outer medullary alpha-intercalated cells.[126]

The “renal” Na-K-Cl cotransporter isoform, NKCC2, is expressed in the apical plasma membrane of the cells of the MTAL (see Fig. 9-19 ) and the CTAL [127] [128] [129] as well as the macula densa.[129] NKCC2 is regulated on a long-term basis by vasopressin, which increases the abundance of NKCC2 protein in the thick ascending limb.[130] This effect is associated with an increase in maximal urinary concentrating capacity.[131] Vasopressin also acutely increases NaCl absorption in the MTAL, [132] [133] in part by regulating trafficking of NKCC2 to the apical plasma membrane [134] [135] in association with phosphorylation of the N-terminal tail of NKCC2.[134]

NCC and ENaC

The thiazide-sensitive Na-Cl cotransporter NCC and the amiloride-sensitive sodium channel ENaC are important targets for the action of aldosterone in the regulation of sodium excretion. [136] [137] NCC is expressed in distal convoluted tubule cells, [136] [138] [139] [140] whereas ENaC is expressed predominantly in the connecting tubule, initial collecting tubule, and cortical collecting duct. [141] [142] Both NCC[143] and the beta- and gamma- subunits of ENaC [143] [144] are increased in abundance by the long-term action of vasopressin. Furthermore, vasopressin acutely increases Na absorption in the rat cortical collecting duct [145] [146] by increasing apical Na entry via the amiloride-sensitive Na channel ENaC.[147] The increase in apical ENaC activity has been proposed to be due to vasopressin-induced trafficking of ENaC-containing vesicles from intracellular stores to the apical plasma membrane.[148]

Increasing NaCl absorption via the action of vasopressin on NCC in the distal convoluted tubule and ENaC activity in the connecting tubule and cortical collecting duct can have an important positive effect on urinary concentrating ability by reducing fluid delivery to the medullary collecting ducts (see Fig. 9-16 ).

Chloride Channels

Two closely related chloride channel (ClC) paralogs, ClC-K1 and ClC-K2 are expressed in renal tubule segments. Strong expression of ClC-K1 is found in both the apical and basolateral plasma membrane of the thin ascending limb of Henle.[149] Although generally viewed as being predominantly or exclusively expressed in the thin ascending limb, there is evidence from RT-PCR studies in microdissected tubules that ClC-K1 is expressed in the thick ascending limb and distal convoluted tubule as well.[150] In contrast, there is general agreement that the chloride channel ClC-K2 is broadly expressed basolaterally along the nephron from the thick ascending limb (see Fig. 9-19 ) through the collecting ducts. [150] [151] [152] Isolated perfused tubule studies have demonstrated that vasopressin increases chloride conductance in the thin ascending limb of hamster,[153] presumably by affecting unit conductance or localization of ClC-K1 chloride channels.

ROMK Potassium Channel

The ROMK potassium channel, an ATP-sensitive inwardly rectifier potassium channel, has been localized to the thick ascending limb, distal convoluted tubule, connecting tubule, and collecting duct system by in situ hybridization[154] and immunocytochemistry. [155] [156] ROMK is expressed predominantly or entirely in the apical plasma membrane in these segments. [155] [156] [157]

ROMK is critical in the active NaCl transport process in the thick ascending limb of Henle (see Fig. 9-19 ) and consequently plays an important role in urinary concentration and dilution. Vasopressin has a long-term effect to increase the abundance of ROMK protein in thick ascending limb cells,[158] thus contributing to the long-term effect of vasopressin to increase NaCl transport in this segment.[159]

In the connecting tubule and collecting duct, ROMK is responsible for the potassium secretion process that regulates urinary potassium excretion and systemic potassium balance. The later process is strongly regulated by vasopressin.[145] This secretory process may be an indirect consequence of vasopressin's action to increase Na entry via ENaC, which results in apical plasma membrane depolarization and an increase in the electrochemical driving force for K movement through ROMK.[160] An alternative view is that the open probability of the ROMK channel may be regulated by vasopressin in a process that is mediated by CFTR, a cAMP responsive protein. [161] [162]

K-Cl cotransporter, KCC4

K-Cl cotransport was first detected in the basolateral plasma membranes of isolated, perfused thick ascending limbs by Greger and Schlatter.[163] Molecular cloning[164] followed by immunohistochemical studies[165] demonstrated that KCC4 is likely to be the basolateral K-Cl cotransporter in the thick ascending limb (see Fig. 9-19 ). This cotransporter also appears to be expressed in the distal convoluted tubule and the connecting tubule.[165]

Use of Knockout Mice to Study the Urinary Concentrating Mechanism and Vasopressin Action

Expression of a number of the transporters depicted in Figures 9-17, 9-18, and 9-19 [17] [18] [19] as well as the vasopressin V2 receptor have been deleted in mice using targeted gene deletion approaches. The phenotypes of these mice have been informative with regard to the role of these gene products in the urinary concentrating mechanism. This section summarizes the key studies.

Aquaporin-1 Knockout (AQP1 KO) Mice

Verkman and colleagues developed a mouse model in which aquaporin-1 expression was deleted in all tissues.[166] In comparison with wild-type littermates, AQP1 knockout mice had reduced urinary osmolality that was not increased in response to water deprivation. Indeed, the urinary concentrating defect was so severe that after 36 hours of water deprivation, the average body weight decreased by 35% and serum osmolality increased to greater than 500 mOsm/kg H2O. Although proximal tubule fluid absorption is markedly impaired in these mice, distal delivery of NaCl and water was not impaired, owing to a TGF-mediated reduction in glomerular filtration rate.[167] However, the function of the thin descending limb (LDL-OM and LDL-IM), normally a site of aquaporin-1 expression, was markedly impaired. The osmotic water permeability of isolated perfused LDL segments from AQP1 KO mice was markedly reduced compared to control animals.[168] As discussed earlier (see Fig. 9-11 ), rapid water absorption from the thin descending limbs has been found to be a key component of the countercurrent multiplication process, and presumably impairment of LDL water absorption is largely responsible for the concentrating defect in AQP1 KO mice. In addition, descending vasa recta, a second renal medullary site of aquaporin-1 expression[19] also displayed a marked reduction in osmotic water permeability in AQP1 KO mice compared to controls.[20] Hence, countercurrent exchange processes involving the descending vasa recta are likely to be impaired in AQP1 KO mice. Thus, the concentrating defect in AQP1 KO mice is likely to be due to impairment of both countercurrent multiplication and countercurrent exchange in the renal medulla.

Aquaporin-2 Knockout (AQP2 KO) Mice

Despite the strong evidence implicating an essential role of AQP2 in the urinary concentrating mechanism, a suitable mouse model to examine its function has only recently been developed. In 2001, a mouse knock-in model of AQP2 dependent nephrogenic diabetes insipidus (NDI) was generated by inserting a T126M mutation into the mouse AQP2 gene.[169] This mutation results in a mouse equivalent of humans with a form of autosomal NDI. Although the mutant mice appeared normal at birth, they failed to thrive and generally died within 1 week. Analysis of the urine and serum revealed serum hyperosmolality and low urine osmolality, typical characteristics of a defective urinary concentrating mechanism. Forward genetic screening of ethylnitrosourea-mutagenized mice isolated another mouse model of NDI with a F204V mutation in AQP2. These mice survived beyond the neonatal period and had a much milder form of NDI.[170]

Recently, two other mouse models have been developed that allow the role of AQP2 in the adult mouse to be examined. One model, developed by Nielsen and colleagues, makes use of the Cre-loxP system of gene disruption to create a collecting duct specific deletion of AQP2.[171] Another model with complete AQP2 protein deletion in the CD was accomplished by tamoxifen-inducible Cre-recombinase expression in homozygous mice in which loxP sites were introduced in introns of the mouse AQP2 gene.[172] The major phenotype in both of these mouse lines is severe polyuria, with average basal urine volumes approximately equivalent to bodyweight. However, despite the polyuria, with free access to water, plasma concentrations of electrolytes, urea, and creatinine are not different in knockout mice compared to controls, and neither was the estimated GFR. Thus, despite having normal renal function (presumably normal active Na+ transport along the nephron), there is a major defect in the urinary concentrating mechanism in these mice. This defect confirms that AQP2 is responsible for the majority of transcellular water reabsorption in the collecting duct system.

Aquaporin-3 Knockout (AQP3 KO) Mice

AQP3 knockout mice have been generated by targeted gene deletion and found to have a greater than threefold reduced osmotic water permeability of the basolateral membrane of the cortical collecting duct compared to wild-type control mice.[173] AQP3 null mice are markedly polyuric (10-fold greater daily urine volume than controls), with an average urine osmolality of less than 300 mOsm/kg H2O. However, unlike AQP1 or AQP2 null mice, AQP3 KO mice are able to raise their urine osmolalities to a modest degree after either water deprivation or the administration of the vasopressin analog dDAVP. It is likely that when AQP3 is deleted, the reduced osmotic water permeability of the basolateral membrane results in a decrease in transepithelial water transport in the connecting tubule, initial collecting tubule and cortical collecting duct, where AQP3 is normally the predominant basolateral water channel. The relatively severe polyuria in this model is consistent with the view from micropuncture data that the majority of post-macula densa fluid reabsorption in the normal kidney is from the cortical portion of the collecting duct system.[4]

Aquaporin-4 Knockout (AQP4 KO) Mice

AQP4 null mice have been generated by standard gene deletion methods.[174] Isolated perfused tubule studies demonstrated a fourfold decrease in IMCD osmotic water permeability, indicating that AQP4 is responsible for most of the water movement across the basolateral membrane in this segment.[175] Despite this reduced water permeability in the IMCD, in hydrated mice, there was no difference in urine osmolality compared to controls and no difference in serum electrolyte concentrations. However, there was a small (15%–20%) but significant reduction in maximal urine osmolality in AQP4 null mice after 36 hours of water deprivation, and this reduced urine osmolality could not be further increased by vasopressin administration, indicating a mild urinary concentrating defect. Why does deletion of AQP4 manifest a modest decrease in urinary concentrating ability while another basolateral water channel AQP3 manifests a profound concentrating defect? The answer to this is based on the normal distribution of water transport along the collecting duct (discussed earlier).[4] The amount of water reabsorbed osmotically in the cortical portion of the collecting duct system (where AQP3 is predominant) is much greater than that absorbed in the medullary collecting ducts (where AQP4 is the predominant basolateral water channel).

UT-A1/3 Urea Transporter Knockout Mice

In 2004, a mouse model was reported in which the two collecting duct urea transporters, UT-A1 and UT-A3, were deleted by standard gene targeting techniques (UT-A1/3-/- mice).[87] Isolated perfused tubule studies demonstrated a complete absence of phloretin-sensitive and vasopressin-regulated urea transport in IMCD segments from UT-A1/3-/- mice. UT-A1/3-/- mice on either a normal protein (20% protein by weight) or high-protein (40%) diet had a significantly greater fluid intake and urine flow, resulting in a decreased urine osmolality, than wild-type animals. However, UT-A1/3-/- mice on a low-protein diet did not show a substantial degree of polyuria. In this latter condition, hepatic urea production is low and urea delivery to the IMCD is predicted to be low, thus rendering collecting duct urea transport largely immaterial to water balance. Studies investigating the maximal urinary concentrating capacity of UT-A1/3-/- mice showed that after an 18-hour water restriction, mice on a 20% or 40% protein intake are unable to reduce their urine flow to levels below those observed under basal conditions, resulting in volume depletion and loss of body weight. In contrast, UT-A1/3-/- mice on a 4% protein diet were able to maintain fluid balance. Thus, the concentrating defect in UT-A1/3-/- mice is caused by a urea-dependent osmotic diuresis; greater urea delivery to the IMCD results in greater levels of water excretion. These results are compatible with a model for the role of urea in the urinary concentrating mechanism proposed in the 1950s by Berliner and colleagues.[23] They hypothesized that luminal urea in the IMCD is normally osmotically ineffective because of the high concentrations of urea in the inner medullary interstitium, which balance osmotically the luminal urea and thus prevent the osmotic diuresis that would otherwise occur.

UT-A1/3-/- mice have been exploited to study the mechanism responsible for Na and Cl accumulation in the inner medulla. For many years, a model independently proposed by Stephenson and by Kokko and Rector in 1972 [84] [85]has been the chief paradigm for concentration of Na and Cl in the inner medulla (see earlier). In this mechanism, known colloquially as the ‘Passive Mechanism’, the generation of a passive electrochemical gradient that drives Na and Cl exit from the thin ascending limb is indirectly dependent on rapid absorption of urea from the IMCD (see earlier for full description). This model would predict that UT-A1/3-/- mice, which lack facilitated urea transport in the IMCD would fail to accumulate Na and Cl to a normal degree. However, two independent studies in UT-A1/3-/- mice failed to demonstrate the predicted decline in inner medullary Na and Cl concentration, despite a profound decrease in urea accumulation in the renal inner medulla. [87] [88] Thus, the passive\pard\plain concentrating model in the form originally proposed by Stephenson and by Kokko and Rector, where NaCl reabsorption from Henle's loop depends on a high IMCD urea permeability, is not the mechanism by which NaCl is concentrated in the inner medulla. Overall, the results with UT-A1/3-/- mice demonstrate that the primary role of IMCD urea transporters in the urinary concentrating mechanism is in their ability to prevent a urea-induced osmotic diuresis when urea excretion rates are high. [87] [88] [176]

UT-A2 and UT-B Urea Transporter Knockout Mice

UT-A2 knockout mice have recently been developed and some aspects of their renal phenotype have been described.[67] On a normal level of protein intake (20% protein), the UT-A2 null mice do not have significant differences in daily urine output compared to control mice and even after a 36-hour period of water deprivation, differences in urine output and urine osmolality are not observed. Furthermore, UT-A2 knockout mice do not have an impairment of urea or chloride accumulation in the inner medulla. Only on a low-protein diet (4% protein), did the UT-A2 knockout mice have a somewhat reduced maximal urinary concentrating capacity compared to wild-type controls, associated with a reduction in urea accumulation in the inner medulla. These results are surprising, considering the role that UT-A2 has been proposed to play in urea recycling in the renal medulla,[65] a process postulated to play a key role in maintenance of a high inner medullary urea concentration. Therefore, they call into question either the importance of UT-A2 in urea recycling or the importance of urea recycling in the concentrating mechanism.

In contrast, mice in which UT-B, the erythrocyte and vasa recta urea transporter, was deleted demonstrated a moderate decrease in maximal urinary osmolality (averaging 2403 mOsmol/kg H2O in UT-B null mice and 3438 mOsmol/kg H2O in wild-type mice). These findings suggest that urea transport by UT-B in the erythrocyte or vasa recta is important for the urinary concentrating process. [177] [178] In addition, a selective defect in urea accumulation is observed in the renal medullae of the UT-B knockout mice, compatible with the idea that UT-B is important for countercurrent exchange of urea in the renal medulla.

NHE3 and NKCC2 Knockout Mice

Both of the major apical Na transporters mediating Na entry in the thick ascending limb (see Fig. 9-19 ) have been knocked out in mice, namely NHE3[179] and NKCC2.[180] From the perspective of the urinary concentrating mechanism, the renal phenotypes are much different and a comparison is informative.

The NHE3 knockout mice are viable and the chief elements of the renal phenotype are associated with the fact that NHE3 is the major Na entry pathway in proximal tubule cells. These animals manifest a marked reduction in proximal tubule fluid absorption and a compensatory decrease in glomerular filtration rate owing to an intact tubulo-glomerular feedback mechanism.[181] On ad libitum water intake, they manifest a moderate increase in water intake associated with lower urinary osmolalities,[182] although urinary osmolality in the NHE3 knockout mice still averaged 1737 mOsmol/kg H2O and maximal urinary osmolality was not evaluated. The NHE3 knockout mice exhibited a marked decrease in renal NKCC2 expression [182] [183] despite elevated circulating levels of vasopressin. Thus, NHE3 mice retain the ability to concentrate the urine although they may exhibit a concentrating defect associated with a reduction in NKCC2 expression.

In contrast, NKCC2 knockout mice were not viable because of perinatal renal fluid wasting and dehydration resulting in death prior to weaning.[180] Although these mice could be induced to survive by treatment with indomethacin and fluid administration, extreme polyuria, hydronephrosis, and growth retardation could not be abrogated.

Why does deletion of NKCC2 result in such a severe phenotype, when deletion of NHE3, a transporter responsible for reabsorption of far more Na, results in a viable mouse capable of maintaining extracellular fluid volume? The answer appears to be in the special role that NKCC2 plays in the macula densa in the mediation of tubulo-glomerular feedback. Tubulo-glomerular feedback allows NHE3 knockout mice to maintain a relatively normal distal delivery through a decrease in glomerular filtration rate, whereas NKCC2 mice cannot compensate in this manner because the transporter is necessary for the feedback to occur.

NKCC1 Knockout Mice

NKCC1, expressed in the basolateral plasma membrane of the IMCD cells and intercalated cells, has also been knocked out in mice.[184] NKCC1 null mice had a reduced capacity to excrete free water relative to wild-type mice, and also had a blunted increase in urinary osmolality following vasopressin administration, suggesting abnormalities in vasopressin signaling in the collecting duct.[185]

NCC and ENaC Knockout Mice

Both of the major apical Na transporters mediating Na entry beyond the macula densa have been knocked out in mice, namely NCC[186] and ENaC. [187] [188] [189] The renal phenotypes are much different and a comparison is informative.

The NCC knockout mice appear to have a mildly altered phenotype with only a small decrease in blood pressure. On a normal diet they are not polyuric, but with restriction of potassium intake they develop hypokalemia and consequent polyuria, associated with an apparent central defect in the regulation of vasopressin secretion.[190] Only after prolonged hypokalemia do these animals develop evidence of NDI with suppressed aquaporin-2 expression in the kidney.[190]

In contrast to the NCC knockouts, knockout of any of the ENaC subunits results in a severe phenotype with neonatal death. In the alpha ENaC knockouts, early death appears to be due to failure to adequately clear fluid from the pulmonary alveoli after birth,[187] whereas the beta and gamma ENaC knockout mice appear to die of hyperkalemia and sodium chloride wasting. [188] [189] When alpha ENaC expression was deleted selectively from the renal collecting ducts, leaving intact ENaC expression in the renal connecting tubule and non-renal tissues, the mice were viable and exhibited only a very mild phenotype with little or no difficulty in maintaining homeostasis in the face of salt or water restriction.[191] Urinary osmolality after a 23-hour period of water restriction was not different from wild-type mice. Thus, Na absorption from the renal collecting duct via ENaC does not appear to be necessary for urinary concentration.

Thus, NCC deleted only from the distal convoluted tubule or ENaC deleted only from the collecting duct results in a very mild phenotype, presumably because one can compensate for the other with regard to sodium balance. It remains unclear whether the severity of the phenotype seen when any ENaC subunit is deleted globally is chiefly because of the importance of ENaC in non-renal tissues or is related to the role of ENaC in the connecting tubule, which is conserved in the collecting duct-only alpha ENaC deletion mice.

ClC-K1 Knockout Mice

In 1999, Matsumura and colleagues generated CLC-K1 null mice (Clcnk1-/-) and have made use of this model to examine the role of CLC-K1 in the urinary concentrating mechanism.[192] Microperfusion studies determined that there was drastically reduced transepithelial chloride transport in the tAL of knockout mice. Physiological studies revealed that Clcnk1-/- mice had significantly greater urine volume and lower urine osmolality compared to controls. Even after a 24-hour period of water deprivation knockout mice were unable to concentrate their urine. This observed polyuria was insensitive to dDAVP administration. The studies demonstrated that the polyuria observed in CLC-K1 null mice is due to water diuresis and not osmotic diuresis such as would be expected with NaCl wasting. Solute analysis of the inner medulla of Clcnk1-/- mice determined that the concentrations of urea, Na, and Cl were approximately half those of controls, resulting in a significantly reduced osmolality of the papilla. These studies demonstrate that the ClC-K1 chloride channel, expressed chiefly in the thin ascending limb, is necessary for maintenance of a maximal osmolality in the inner medullary tissue. The findings in the Clcnk1-/- mice, therefore emphasize the importance of rapid chloride exit (and presumably Na exit) from the thin ascending limb to the inner medullary concentrating process. As discussed earlier, all inner medullary models not including active NaCl transport from the thin ascending limb must include rapid NaCl (and urea) exit from the thin ascending limb as a component of the model.

ROMK Knockout Mice

Expression of the ROMK potassium channel (Kir1.1) has been deleted in mice by Lorenz and colleagues.[193] These mice manifest early death associated with hydronephrosis and severe dehydration, consistent with the known role of ROMK in active NaCl absorption in the thick ascending limb (see Fig. 9-19 ). About 5% of these mice survived the perinatal period, but surviving adults still manifested polydipsia, polyuria, impaired urinary concentrating ability, hypernatremia, and reduced blood pressure. From these animals, a line of mice has been derived that has a greater survival rate and no hydronephrosis in adults, albeit possessing higher water excretion rates.[194] Interestingly, these mice do not exhibit hyperkalemia, indicating that the connecting tubule or collecting duct principal cells (or both) must be capable of secreting K via some other pathway, presumably flow-dependent, Ca2+-activated K channels referred to as “Maxi-K” channels.[195]

Vasopressin V2 Receptor Knockout Mice

In 2000, Yun and colleagues created a mouse model of X-linked NDI (XNDI), by introducing a nonsense mutation (Glu242stop) into the mouse V2 receptor gene.[196] This mutation is known to cause XNDI in humans. This particular mutation was chosen as it has been shown that the encoded mutant receptor is retained intracellularly and completely lacks functional activity, thus mimicking the functional properties of many other disease-causing V2R mutants.

Male V2R mutant mice (V2R-/y) died within 7 days after birth. Urine osmolalities, collected from the bladders of 3-day-old pups, were significantly lower than controls. Serum electrolyte analysis revealed that V2R-/y pups have increased Na+ and Cl- levels, indicative of a severe state of hypernatremia. In control mice, an intraperitoneal injection of the V2R agonist dDAVP resulted in a significant increase in urine osmolality, whereas there was no effect in V2R-/y mice. Analysis of adult female V2R+/- mice revealed that the mice have polyuria, polydipsia, and a reduced urinary concentrating ability; consistent with NDI. Furthermore, they have an approximate 50% decrease in total AVP binding capacity, resulting in an approximately 50% decrease in dDAVP-induced intracellular cAMP levels. Taken together, the results obtained from this loss-of-function mutation in the V2R are consistent with the general view that the antidiuretic effects of AVP result from an initial interaction between AVP and the V2R, resulting in increased intracellular cAMP and eventually promoting water reabsorption in the kidney collecting duct via aquaporins. The implication is that there is no other significant compensatory event that can generate cAMP and increase water permeability in the renal collecting duct.


Ammonium can be concentrated in urine to concentrations of several hundred millimolar.[197] Production of a final urine with a high NH4+ concentration depends on two processes: (1) trapping of NH4+ in the renal medulla, which raises the medullary interstitial NH4+ concentration to well above that present in the cortex; and (2) diffusion trapping in the collecting ducts (i.e., parallel H+ and NH3 transport), which raises the luminal concentration of NH4+above that present in the medullary interstitium.

A substantial corticomedullary NH4+ gradient develops in antidiuretic rats and is regulated according to the acid-base state of rats, such that the gradient is markedly increased by systemic acid loading.[198] Similar medullary NH4+gradients have been found in dogs ( Fig. 9-20 ).[199] Micropuncture studies have shown that NH4+ concentrations in the inner medullary vasa recta of rats (and presumably in the medullary interstitium) greatly exceed values in the cortex or peripheral plasma.[200]



FIGURE 9-20  Corticomedullary NH4 gradient in canine renal medulla. NH4 concentrations were measured in tissue water from slices from cortex and medulla of untreated dogs.  (Plotted from data of Robinson RR, Owen EE: Intrarenal distribution of ammonia during diuresis and antidiuresis. Am J Physiol 208:1129-1134, 1965.)




NH4+ is produced in the mammalian proximal tubule as part of the overall renal process that regulates systemic acid-base balance. The increased NH4+ production in the proximal tubule in response to acid loading in rats is associated with increased activity of several ammoniagenic enzymes. Acid loading in rats increases glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase mRNA levels, [201] [202] contributing to increased NH4+ production and secretion in the proximal tubule.[203]

The trapping of NH4+ in the medullary interstitium is thought to occur by a countercurrent multiplication process analogous to the countercurrent multiplication of Na+ ( Fig. 9-21 ). Studies in isolated perfused tubules have demonstrated NH4+ absorption from the thick ascending limb of the loop of Henle against an NH4+ concentration gradient, [204] [205] which identifies a single effect for the countercurrent NH4+ multiplier. The NH4+ absorption from the thick ascending limb occurs by direct NH4+ transport. Most of the NH4+ absorption is active, resulting from coupled Na+-NH4+-Cl- transport in the apical membranes of the thick ascending limb cells. NH4+ absorbed from the thick ascending limb is presumably secreted into the proximal straight tubule and thin descending limb, which creates a recycling pathway around the loop of Henle (see Fig. 9-21 ). Studies by Flessner and colleagues[206]have demonstrated that the long-looped thin descending limb in the outer medulla is permeable to both NH4+ and NH3, providing a pathway for passive secretion of NH4+ actively absorbed from the thick ascending limb. For a high concentration of NH4+ to be delivered to the interstitium of the inner medulla, some of the NH4+ recycled around the loop must be reabsorbed by either the thin ascending limb or the inner medullary part of the descending limb. Studies of isolated perfused thin ascending limbs[207] from the inner medullae of chinchillas and rats suggest that direct passive NH4+ efflux from this segment is the most likely pathway of NH4+ delivery to the inner medullary interstitium.



FIGURE 9-21  Countercurrent multiplier for NH4 in renal medulla. Active absorption of NH4 from thick ascending limb of loop of Henle provides a single effect for countercurrent multiplication (see text).  (Modified from Good DW, Knepper MA: Ammonia transport in the mammalian kidney. Am J Physiol 248:F459–F471, 1985.)




The ammonium that accumulates in the medullary interstitium is then transported from the medullary interstitium into collecting duct cells and is subsequently secreted into the collecting duct lumen, where NH4+ can be concentrated to levels much higher than in the interstitium.[200] A number of specific mechanisms for transport of NH4+ across the collecting duct epithelium have been proposed. Wall and co-workers[208] demonstrated active NH4+ transport across basolateral membranes by substitution for K+ on the Na+,K+-ATPase. Recent studies by Weiner and associates [209] [210] suggest that both apical and basolateral transport of NH4+ into medullary collecting duct cells of rats is mediated in part by the putative NH4+ transporter RhB-glycoprotein. However, in mice, elimination of RhBG expression by genetic inactivation did not disrupt urinary NH4+ excretion nor did it inhibit NH4+uptake across the basolateral membrane of cortical collecting duct cells in microperfused tubules,[211] raising doubts about the role of RhBG in renal ammonia transport. In order for NH4+ to accumulate in urine to concentrations as high as several hundred millimolar,[197] the most likely mechanism appears to be parallel H+ and lipid phase NH3 diffusion (diffusion trapping) across the plasma membranes of collecting duct cells.


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