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

CHAPTER 11. Arachidonic Acid Metabolites and the Kidney

Raymond C. Harris Jr   Matthew D. Breyer

  

 

Cellular Origin of Eicosanoids, 363

  

 

The Cyclooxygenase (COX) Pathway, 364

  

 

Renal COX-1 and COX-2 Expression, 366

  

 

Renal Complications of NSAIDs, 369

  

 

Cardiovascular Effects of COX-2 Inhibitors, 371

  

 

Prostanoid Synthases, 372

  

 

Cellular Origin of Eicosanoids Prostanoid Receptors, 374

  

 

Involvement of Cyclooxygenase Metabolites in Renal Pathophysiology, 382

  

 

The Lipoxygenase Pathway, 385

  

 

The Cytochrome P450 Pathway, 387

CELLULAR ORIGIN OF EICOSANOIDS

Eicosanoids comprise a family of biologically active, oxygenated arachidonic acid (AA) metabolites. Arachidonic acid is a polyunsaturated fatty acid possessing 20 carbon atoms and 4 double bonds (C20:4) and is formed from linoleic acid (C18:2) by addition of two carbons to the chain and further desaturation. In mammals, linoleic acid is derived strictly from dietary sources. Essential fatty acid (EFA) deficiency occurs when dietary fatty acid precursors, including linoleic acid, are omitted, thereby depleting the hormone-responsive pool of AA. EFA deficiency thereby reduces the intracellular availability of AA in response to hormonal stimulation and abrogates many biological actions of hormone-induced eicosanoid release.[1]

Of an approximate 10 gm of linoleic acid ingested per day, only about 1 mg/day is eliminated as end products of AA metabolism. Following its formation, AA is esterified into cell membrane phospholipids, principally at the 2 position of the phosphatidylinositol fraction (i.e., sn-2 esterified AA), the major hormone-sensitive pool of AA that is susceptible to release by phospholipases.

Multiple stimuli lead to release of membrane-bound AA, via activation of cellular phospholipases, principally phospholipase A2's (PLA2).[2] This cleavage step is rate limiting in the production of biologically relevant arachidonate metabolites. In the case of PLA2 activation, membrane receptors activate guanine nucleotide-binding (G) proteins, leading to release of AA directly from membrane phospholipids. Activation of phospholipase C or PLD, on the other hand, releases AA via the sequential action of the phospholipase-mediated production of diacylglycerol (DAG) with subsequent release of AA from DAG by DAG lipase.[3] When considering eicosanoid formation, the physiological significance of AA release by these other phospholipases remains uncertain because at least in the setting of inflammation, phospholipase A2 action appears essential for the generation of biologically active AA metabolites.[4]

More than 15 proteins with PLA2 activity are known to exist, including secreted (sPLA2) and cytoplasmic PLA2 (cPLA2) isoforms. [5] [6] A mitogen activated cytoplasmic PLA2 has been found to mediate AA release in a calcium/calmodulin-dependent manner. Other hormones and growth factors, including epidermal growth factor (EGF) and platelet derived growth factors, activate PLA2 directly through tyrosine residue kinase activity, allowing the recruitment of co-activators to the enzyme without an absolute requirement for the intermediate action of Ca++/calmodulin or other cellular kinases.

Following de-esterification, AA is rapidly re-esterified into membrane lipids or avidly bound by intracellular proteins in which case it becomes unavailable to further metabolism. Should it escape re-esterification and protein binding, free AA becomes available as a substrate for one of three major enzymatic transformations, the common result of which is the incorporation of oxygen atoms at various sites of the fatty acid backbone, with accompanying changes in its molecular structure (such as ring formation). [7] [8] This results in the formation of biologically active molecules, referred to as “eicosanoids”. The specific nature of the products generated is a function of the initial stimuli for AA release, as well as the metabolic enzyme available, which is determined in part by the cell type involved. [8] [9]

These products, in turn, either mediate or modulate the biologic actions of the agonist in question. AA release may also result from non-specific stimuli, such as cellular trauma including ischemia and hypoxia,[10] oxygen free radicals,[11] or osmotic stress.[12] The identity of the specific AA metabolite generated in a particular cell system depends on both the proximate stimulus and the availability of the down stream AA metabolizing enzymes present in that cell.

Three major enzymatic pathways of AA metabolism are present in the kid-ney: cyclooxygenases, lipoxygenases, and cytochrome P450s ( Fig. 11-1 ). The cyclooxygenase pathway mediates the formation of prostaglandins (PGs) and thromboxanes, the lipoxygenase pathway mediates the formation of mono-, di-, and trihydroxyeicosatetraenoic acids (HETEs) leukotrienes (LTs), and lipoxins (LXs) and the cytochrome P-450-dependent oxygenation of AA mediates the formation of epoxyeicosatrienoic acids (EETs), their corresponding diols, HETEs, and monooxygenated AA derivatives. Fish oil diets, rich in w-3 polyunsaturated fatty acids[13] interfere with metabolism via all three pathways by competing with AA oxygenation, resulting in the formation of biologically inactive end products.[14] Interference with the production of pro-inflammatory lipids has been hypothesized to underlie the beneficial effects of fish-oil in IgA nephropathy and other cardiovascular diseases.[15] The following sections deal with the current understanding of the chemistry, biosynthesis, renal metabolism, mechanisms of release, receptor biology, signal transduction pathways, biologic activities, and functional significance of each of the metabolites generated by the three major routes of AA metabolism in the kidney.

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FIGURE 11-1  Pathways of enzymatically mediated arachidonic acid metabolism. Arachidonic acid can be converted into biologically active compounds by cyclooxygenase- (COX), lipoxygenase- (LO), or cytochrome P450- (CYP450) mediated metabolism.

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THE CYCLOOXYGENASE (COX) PATHWAY

Molecular Biology

The cyclooxygenase enzyme system is the major pathway for AA metabolism in the kidney ( Fig. 11-2 ). Cyclooxygenase (prostaglandin synthase G2/H2) is the enzyme responsible for the initial conversion of arachidonic acid to prostaglandin G2 and subsequently to prostaglandin H2. Cyclooxygenase was first purified from ram seminal vesicles and cloned in 1988. The protein was found to be widely expressed and the level of activity not dynamically regulated. Other studies supported the presence of a cyclooxygenase that was dynamically regulated and responsible for increased prostanoid production in inflammation. This second, inducible cyclooxygenase isoform was identified shortly after the cloning of the initial enzyme and designated cyclooxygenase-2 (COX-2), whereas the initially isolated isoform is now designated COX-1. [7] [16] [17] COX-1 and COX-2 are encoded by distinct genes located on different chromosomes. The human COX-1 gene (PTGS1=prostaglandin synthase 1) is distributed over 40 kB on 11 exons on chromosome 9, whereas COX-2 is localized on chromosome 1 and spans approximately 9 kB. The genes are also subject to dramatically different regulatory signals.

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FIGURE 11-2  Cyclooxygenase metabolism of arachidonic acid. Both COX-1 and COX-2 convert AA to PGH2, which is then acted upon by specific synthases to produce prostanoids that act at G-protein coupled receptors that either increase or decrease cAMP or increase intracellular calcium.

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Regulation of Cyclooxygenase Gene Expression

At the cellular level, COX-2 expression is highly regulated by several processes that alter its transcription rate, message export from the nucleus, message stability, and efficiency of message translation. [18] [19] These processes tightly control the expression of COX-2 in response to many of the same cellular stresses that activate arachidonate release (e.g., cell volume changes, shear stress, hypoxia), [10] [20] as well as a variety of cytokines and growth factors, including tumor necrosis factor (TNF) interleukin 1b, epidermal growth factor, and platelet derived growth factor (PDGF). Activation of COX-2 gene transcription is mediated via the coordinated activation of several transcription factors that bind to and activate consensus sequences in the 5′ flanking region of the COX-2 gene for NF-kB, and NF-IL6/C-EBP, and a cyclic AMP response element (CRE).[21] Induction of COX-2 mRNA transcription by endotoxin (lipopolysaccharide) may also involve CRE sites[22] and NF-kB sites.[23]

Regulation of Cyclooxygenase Expression by Anti-inflammatory Steroids

A molecular basis linking the anti-inflammatory effects of cyclooxygenase inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs) and anti-inflammatory glucocorticoids has long been sought. A novel mechanism for the suppression of arachidonate metabolism by corticosteroids involving translational inhibition of COX formation had been suggested prior to the molecular recognition of COX-2. With the cloning of COX-2 it became well established that glucocorticoids suppress COX-2 expression and prostaglandin synthesis, an effect now viewed as central to the anti-inflammatory effects of glucocorticoids. Post-transcriptional control of COX-2 expression represents another robust mechanism by which adrenal steroids regulate COX-2 expression.[24] Accumulating evidence suggests COX-2 is modulated at multiple steps in addition to transcription rate, including stabilization of the mRNA and enhanced translation. [18] [25] Glucocorticoids, including dexamethasone, down regulate COX-2 mRNA in part by destabilizing the mRNA.[25] The 3′untranslated region of COX-2 mRNA contains 22 copies of an AUUUA motif, which are important in destabilizing COX-2 message in response to dexamethasone, whereas other 3′ sequences appear important for COX-2 mRNA stabilization in response to interleukin-1β.[25] Effects of the 3′UTR as well as other factors regulating efficiency of COX-2 translation have also been suggested.[18] The factors determining the expression of COX1 are more obscure.

Enzymatic Chemistry

Despite these differences, both prostaglandin (PG) synthases catalyze a similar reaction, resulting in cyclization of C-8 to C-12 of the AA backbone forming cyclic endoperoxide, accompanied by the concomitant insertion of two oxygen atoms at C-15 to form PGG2 (a 15-hydroperoxide). In the presence of a reduced glutathione-dependent peroxidase, PGG2 is converted to the 15-hydroxy derivative, PGH2. The endoperoxides (PGG2 and PGH2) have very short half lives of about 5 minutes and are biologically active in inducing aortic contraction and platelet aggregation.[26] However under some circumstances, the formation of these endoperoxides may be strictly limited, via the self-deactivating properties of the enzyme.

Expression of recombinant enzymes and determination of the crystal structure of COX-2 have provided further insight into the observed physiologic and pharmacologic similarities to, and differences from, COX-1. It is now clear that cyclooxygenase inhibiting NSAIDs work by sterically blocking access of AA to the heme containing, active enzymatic site.[27] Particularly well conserved are sequences surrounding the aspirin-sensitive serine residues, at which acetylation by aspirin irreversibly inhibits activity.[28] More recent evidence has developed showing that COX-1 and COX-2 are capable of forming heterodimers and sterically modulating each other's function.[29] The substrate binding pocket of COX-2 is larger and therefore accepting of bulkier inhibitors and substrates. This difference has allowed the development and marketing of both relatively and highly selective COX-2 inhibitors for clinical use as analgesics,[30] antipyretics,[31] and anti-inflammatory agents.[30] In addition to its central role in inflammation, aberrantly up-regulated COX-2 expression has been implicated in the pathogenesis of a number of epithelial cell carcinomas[32] and in Alzheimer disease and other degenerative neurologic conditions.[33]

RENAL COX-1 AND COX-2 EXPRESSION

COX-2 Expression in the Kidney

There is now definitive evidence for significant COX-2 expression in the mammalian kidney ( Fig. 11-3 ). COX-2 mRNA and immunoreactive COX-2 are present at low but detectable levels in normal adult mammalian kidney, where in situ hybridization and immunolocalization demonstrated localized expression of COX-2 mRNA and immunoreactive protein in the cells of the macula densa and a few cells in the cortical thick ascending limb cells immediately adjacent to the macula densa.[34] COX-2 expression is also abundant in the lipid-laden medullary interstitial cells in the tip of the papilla. [34] [35] [36] Some investigators have reported that COX-2 may be expressed in inner medullary collecting duct cells or intercalated cells in the renal cortex.[37] Nevertheless COX-1 expression is constitutive and clearly the most abundant isoform in the collecting duct, so potential existence and physiological significance of COX-2 co-expression in this segment remains uncertain.

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FIGURE 11-3  Localization (indicated in shaded areas) of immunoreactive COX-1, COX-2, and microsomal prostaglandin E synthase along the rat nephron.  (Reproduced with permission from S. Bachmann.)

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COX-2 Expression in the Renal Cortex

It is now well documented that COX-2 is expressed in macula densa/cTALH and in kidney of mouse, rat, rabbit, and dog. Furthermore, despite initial controversy regarding COX-2 localization in primate and human kidney, more recent studies confirm a similar distribution of COX-2 in macula densa (as well as medullary interstitial cells) especially in kidneys of the elderly, [38] [39] patients with dia-betes mellitus, congestive heart failure,[40] and Bartter-like Syndrome.[41]

The presence of COX-2 in the unique group of cells comprising the macula densa, points to a potential role for COX-2 derived prostanoids in regulating glomerular function. Studies of the prostanoid-dependent control of glomerular filtration rate by the macula densa suggest effects via both dilator and constrictor effects of prostanoids contributing to tubuloglomerular feedback (TGF). [42] [43] Some studies suggest that COX-2 derived prostanoids are predominantly vasodilators. [44] [45] By inhibiting production of dilator prostanoids contributing to the patency of adjacent afferent arteriole, COX-2 inhibition may contribute to the decline in GFR observed in patients taking NSAIDs or selective COX-2 inhibitors[46] (see later discussion). The identity of the specific prostanoids elaborated by the COX-2 expressing macula densa cells remains uncertain.

The volume-depleted state is typified by low NaCl delivery to the macula densa, and COX-2 expression in the macula densa is also increased in states associated with volume depletion ( Fig. 11-4 ).[34] Of note, COX-2 expression in cultured macula densa cells and cTAL cells is also increased in vitro by reducing extracellular Cl- concentration. Studies in which cortical thick limbs and associated glomeruli were removed and perfused from rabbits pretreated with a low salt diet to upregulate macula densa COX-2 demonstrated COX-2-dependent release of PGE2 from the macula densae in response to decreased chloride perfusate.[47] Furthermore the induction of COX2 by low Cl- can be blocked by a specific p38 MAP kinase inhibitor. [48] [49] Finally, in vivo, renal cortical immunoreactive pp38 expression (the active form of p38) predominantly localized to the macula densa and cTALH and increases in response to a low salt diet.[48] These findings point to a molecular pathway whereby enhanced COX-2 expression occurring in circumstances associated with intracellular volume depletion could result from decreased luminal chloride delivery. Recent studies have also indicated that the carbonic anhydrase inhibitor, acetazolamide, and dopamine may both indirectly regulate macula densa COX-2 expression by inhibiting proximal reabsorption and thereby increasing luminal macula densa chloride delivery.[50]

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FIGURE 11-4  COX-2 expression is regulated in renal cortex in rats. Left, Under basal conditions, sparse immunoreactive COX-2 is localized to the macula densa and surrounding cortical thick ascending limb. Right, Following chronic administration of a sodium deficient diet, macula densa/cTAL COX-2 expression increases markedly.

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In the mammalian kidney, the macula densa is involved in regulating renin release by sensing alterations in luminal chloride via changes in the rate of Na+/K+/2Cl- cotransport ( Fig. 11-5 ).[51] Measurements in vivo, in isolated perfused kidney and in isolated perfused juxtaglomerular prepara-tion all indicated that that administration of non-specific cyclooxygenase inhibitors prevented the increases in renin release mediated by macula densa sensing of decreases in luminal NaCl.[51] Induction of a high renin state by imposition of a salt deficient diet, ACE inhibition, diuretic administration, or experimental renovascular hypertension all significantly increase macula densa/cTALH COX-2 mRNA, and immunoreactive protein. COX-2 selective inhibitors blocked elevations in plasma renin activity, renal renin activity, and renal cortical renin mRNA in response to loop diuretics, ACE inhibitors, or a low salt diet,[52] [53] [54] [55] and in an isolated perfused juxtaglomerular preparation, increased renin release in response to lowering the perfusate NaCl concentration was blocked by COX-2 inhibition.[56] In COX-2 knockout mice, increases in renin in response to low salt or ACE inhibitors were significantly blunted [57] [58] but were unaffected in COX-1 knockout mice. [59] [60] COX-2 inhibitors have also been shown to decrease renin production in models of renovascular hypertension[61] and recent studies in mice with targeted deletion of the prostacyclin receptor suggest a predominant role for prostacyclin in mediating renin production and release in these models.[62]

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FIGURE 11-5  Proposed intrarenal roles for vasodilatory prostaglandins to regulate renal function and blood pressure control. Prostaglandins released from the macula densa and/or the afferent arteriole can both vasodilate the afferent arteriole and modulate renin release from juxtaglomerular cells.

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COX-2 Expression in the Renal Medulla

The renal medulla is a major site of prostaglandin synthesis and abundant COX-1 and COX-2 expression ( Fig. 11-6 ).[63] COX-1 and COX-2 exhibit differential compartmentalization within the medulla, with COX-1 predominating in the medullary collecting ducts and COX-2 predominating in medullary interstitial cells. COX-2 may also be expressed in endothelial cells of the vasa recta supplying the inner medulla.

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FIGURE 11-6  Differential immunolocalization of COX-1 (left) and COX-2 (right) in the renal medulla of rodents. COX-1 is predominantly localized to the collecting duct and is also found in a subset of medullary interstitial cells, whereas COX-2 is predominantly localized to a subset of interstitial cells.

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The factors determining this differential tissue expression of COX-2 remain uncertain but likely include distinct upstream promoter elements and gene organization. In the collecting duct or human ureter and bladder epithelium, which are also derived from ureteric bud, COX-2 expression is only detected in the setting of malignant transformation.[64] Because of the potential chemopreventive and therapeutic effects of NSAIDs in epithelial cancers[65] the factors contributing to the aberrant expression of COX-2 in malignant epithelia is an area of intense investigation.[32] Aberrant methylation of COX-2 DNA has been associated with silencing of COX-2 expression in some colon cancers,[66] but whether differential methylation contributes to the cellular compartmentalization of COX-2 in the normal kidney is unknown.

In those cells normally expressing COX-2, dynamic regulation of its expression appears to be an important adaptive response to physiological stresses, including water deprivation and exposure to endotoxin. [37] [63] [67] Following dehydration renal medullary COX-2 mRNA and protein expression are significantly induced, [37] [63] primarily in medullary interstitial cells.[63] In contrast, COX-1 expression is unaffected by water deprivation. Although hormonal factors could also contribute to COX-2 induction, shifting cultured renal medullary interstitial cells to hypertonic media (using either NaCl or mannitol) is sufficient to induce COX-2 expression directly. Because prostaglandins play an important role in maintaining renal function during volume depletion or water deprivation, induction of COX-2 by hypertonicity provides an important adaptive response.

As is the case for the macula densa, medullary interstitial cell COX-2 expression is transcriptionally regulated in response to renal extracellular salt and tonicity. Water deprivation activates COX-2 expression in medullary interstitial cells by activating the NF-kB pathway.[63] Other studies suggest roles for MAP kinase/JNK in COX-2 induction following hypertonicity.[68]

COX-1 Expression in the Kidney

Whereas well-defined factors regulating COX-2 and determining the role of COX-2 expression in the kidney are coming to light, the role of renal COX-1 remains more obscure. COX-1 is constitutively expressed in platelets[69] in renal microvasculature, and glomerular parietal epithelial cells ( Fig. 11-7 ). In addition COX-1 is abundantly expressed in the collecting duct, but there is little COX-1 expressed in the proximal tubule or thick ascending limb.[44]COX-1 expression levels do not appear to be dynamically regulated, and consistent with this observation, the COX-1 promoter does not possess a TATA box. The factors accounting for the tissue specific expression of COX-1 are uncertain but may involve histone acetylation and the presence of two tandem Sp1 sites in the upstream region of the gene.[70]

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FIGURE 11-7  Renal cortical COX-1 expression. Immunoreactive COX-1 is predominantly localized to the afferent arteriole, glomerular mesangial cells and parietal glomerular epithelial cells, and the cortical collecting duct.  (From Hirata M, Hayashi Y, Ushikubi F, et al: Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature 349:617-620, 1991.)

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RENAL COMPLICATIONS OF NSAIDs

Na+ Retention, Edema, and Hypertension

Use of non-selective NSAIDs may be complicated by the development of significant Na+ retention, edema, congestive heart failure, and hypertension.[71] These complications are also apparent in patients using COX-2 selective NSAIDs. Studies with celecoxib and rofecoxib demonstrate that like non-selective NSAIDs, these COX-2 selective NSAIDs reduce urinary Na+ excretion, and are associated with modest Na+ retention in otherwise healthy subjects.[72] [73] COX-2 inhibition likely promotes salt retention via multiple mechanisms ( Fig. 11-8 ). Reduced glomerular filtration rate may limit the filtered Na+ load and salt excretion. [74] [75] In addition, PGE2 directly inhibits Na+absorption in the thick ascending limb and collecting duct.[76] The relative abundance of COX-2 in medullary interstitial cells places this enzyme adjacent to both these nephron segments, allowing for COX-2 derived PGE2 to modulate salt absorption. COX-2 inhibitors decrease renal PGE2 production [72] [77] and thereby may enhance renal sodium retention. Finally, reduction in renal medullary blood flow by inhibition of vasodilator prostanoids may significantly reduce renal salt excretion and promote the development of edema and hypertension. COX-2 selective NSAIDs have been demonstrated to exacerbate salt dependent hypertension in rats.[78] Similarly, patients with pre-existing treated hypertension commonly experience hypertensive exacerbations with COX-2 selective NSAIDs.[73] Taken together these data suggest that COX-2 selective NSAIDs have similar effects as non-selective NSAIDs with respect to salt excretion.

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FIGURE 11-8  Integrated role of PGE2 on regulation of salt and water excretion. PGE2 can both increase medullary blood flow and directly inhibit NaCl reabsorption in mTAL and water reabsorption in collecting duct.

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Hyperkalemia

Non-selective NSAIDs cause hyperkalemia due to suppression of the renin/aldosterone axis. Both decreased GFR and inhibition of renal renin release may compromise renal K+ excretion. Two recent studies in patients on a salt-restricted diet demonstrated that a COX-2 selective inhibitor (either rofecoxib or celecoxib) decreased urinary potassium excretion. [74] [75] In sub-populations of patients at risk, development of overt hyperkalemia with COX-2 selective inhibitors seems likely.

Papillary Necrosis

Both acute and sub-acute forms of papillary necrosis have been observed with NSAID use. [79] [80] [81] Acute NSAID-associated renal papillary injury is more likely to occur in the setting of dehydration, suggesting a critical dependence of renal function upon COX metabolism in this setting.[63] Long-term use of COX-inhibiting NSAIDs has been associated with papillary necrosis and progressive renal structural and functional deterioration much like the syndrome of analgesic nephropathy observed with acetaminophen, aspirin, and caffeine combinations.[80] Experimental studies suggest that renal medullary interstitial cells are an early target of injury in analgesic nephropathy.[82] COX-2 has been shown to be an important survival factor for cells exposed to a hypertonic medium. [36] [63] [83] The coincident localized expression of COX-2 in these interstitial cells [36] [63] raises the possibility that, like non-selective NSAIDs, long-term use of COX-2 selective NSAIDs may contribute to development of papillary necrosis and analge-sic nephropathy.[84] Because the development of analgesic nephropathy requires the regular ingestion of NSAIDs or analgesics over years, this possibility remains to be verified.

Acute Renal Insufficiency

Acute renal failure is a well-described complication of NSAID use.[71] This is generally considered to be a result of altered intra-renal microcirculation and glomerular filtration secondary to the inability to produce beneficial endogenous prostanoids when the kidney is dependent on them for normal function. Recent reports suggest that like the traditional, non-selective NSAIDs, COX-2 selective NSAIDs will also reduce glomerular filtration in susceptible patients.[71] Although rare overall, NSAID-associated renal insufficiency occurs in a significant proportion of patients with underlying volume depletion, renal insufficiency, congestive heart failure, diabetes, and old age.[71] These risk factors are additive and rarely are present in patients included in study cohorts used for safety assessment of these drugs. It is therefore relevant that both celecoxib and rofecoxib caused a slight but significant fall in glomerular filtration rate in salt depleted but otherwise healthy subjects. [74] [75] More than 200 cases of acute renal insufficiency due to COX-2 selective NSAIDs have now been reported. [46] [85] Pre-clinical studies support the concept that inhibition of COX-2 derived prostanoids generated in the macula densa contributes to a fall in GFR by reducing the diameter of the afferent arteriole. In vivo video microscopy studies document reduced afferent arteriolar diameter following administration of a COX-2 inhibitor.[45] Taken together these animal data not only support the concept that COX-2 plays an important role regulating glomerular filtration rate but also the clinical observations that COX-2 selective inhibitors can cause renal insufficiency similar to that reported with non-selective NSAIDs.

Interstitial Nephritis

The gradual development of renal insufficiency characterized by a subacute inflammatory interstitial infiltrate may occur after several months of continuous NSAID ingestion. Less commonly, the interstitial nephritis and renal failure may be fulminant. The infiltrate is typically accompanied by eosinophils; however, the clinical picture is typically much less dramatic than the allergic interstitial nephritis associated with β-lactam antibiotics, lacking fever or rash.[86] This syndrome has also been reported with the COX-2 selective drug, celecoxib. [87] [88] Dysregulation of the immune system is thought to play an important role in the syndrome, which typically abates rapidly following discontinuation of the NSAID or COX-2 inhibitor.

Nephrotic Syndrome

Like interstitial nephritis, nephrotic syndrome typically occurs in patients chronically ingesting any one of a myriad of NSAIDs over course of months. [86] [89] The renal pathology is usually consistent with minimal change disease with foot process fusion of glomerular podocytes observed on EM, but membranous nephropathy has also been reported.[90] Typically, the nephrotic syndrome occurs together with the interstitial nephritis.[86] Nephrotic syndrome without interstitial nephritis may occur, as well as immune-complex glomerulopathy, in a small subset of patients receiving NSAIDs. It remains uncertain whether this syndrome results from mechanism-based cyclooxygenase inhibition by these drugs, an idiosyncratic immune drug reaction, or a combination of both.

Renal Dysgenesis

Reports of renal dysgenesis and oligohydramnios in offspring of women administered non-selective NSAIDs during the third trimester of pregnancy[91] have implicated prostaglandins in the process of normal renal development. A similar syndrome of renal dysgenesis has been reported in mice with targeted disruption of the COX-2 gene, as well as mice treated with the specific COX-2 inhibitor SC58236.[92] Since neither COX-1-/- mice or mice treated with the COX-1 selective inhibitor SC58560 exhibited altered renal development, a specific role for COX-2 in nephrogenesis is suggested. [93] [94] [95] A report of renal dysgenesis in the infant of a woman exposed to the COX-2 selective inhibitor nimesulide suggests COX-2 also plays a role in renal development in humans.[91]

The intra-renal expression of COX-2 in the developing kidney peaks in mouse at post-natal day 4 and in the rat in the second post-natal week. [92] [96] It has not yet been determined if a similar pattern of COX-2 is seen in humans. Although the most intense staining is observed in a small subset of cells in the nascent macula densa and cortical thick ascending limb, expression in the papilla is also observed. [92] [96] Considering the similar glomerular developmental defects observed in rodents treated with the COX-2 inhibitor and in mice with targeted disruption of the COX-2 gene, it seems likely that prostanoids or other products resulting from COX-2 activity in cortical thick limb (and macula densa) act in a paracrine manner to influence glomerular development. The identity of the COX-2 derived prostanoids that promote glomerulogenesis, remains uncertain. In vitro studies show that exogenous PGE1promotes renal metanephric development,[97] and is a critical growth factor for renal epithelia cells. Nevertheless, none of the prostaglandin receptor knockout mice recapitulate the phenotype of the COX-2 knockout mouse.[98]

CARDIOVASCULAR EFFECTS OF COX-2 INHIBITORS

Effects of COX-2 Inhibition on Vascular Tone

In addition to their propensity to reduce renal salt excretion and decrease medullary blood flow, NSAIDs and selective COX-2 inhibitors have been shown to exert direct effects on systemic resistance vessels. The acute pressor effect of angiotensin infusion in human subjects was significantly increased by pretreatment with the non-selective NSAID, indomethacin, at all angiotensin II doses studied. More recently, administration of selective COX-2 inhibitors or COX-2 gene knockout has been shown to accentuate the pressor effects of angiotensin II in mice.[44] These studies also demonstrated that Ang II-mediated blood pressure increases were markedly reduced by administration of a selective COX-1 inhibitor or in COX-1 gene knockout mice.[44] These findings support the conclusion that COX-1 derived prostaglandins participate in, and are integral to the pressor activity of angiotensin II, whereas COX-2 derived prostaglandins are vasodilators that oppose and mitigate the pressor activity of angiotensin II. Other animal studies more directly show that that both NSAIDs and COX-2 inhibitors blunt arteriolar dilation and decrease flow through resistance vessels.[99]

Increased Cardiovascular Thrombotic Events

COX-2 is known to be induced in vascular endothelial cells in response to shear stress, and selective COX-2 inhibition reduces circulating prostacyclin levels in normal human subjects.[100] Therefore, increasing evidence indicates that COX-2 selective antagonism may carry increased thrombogenic risks due to selective inhibition of the endothelial-derived anti-thrombogenic prostacyclin without any inhibition of the prothrombotic platelet-derived thromboxane generated by COX-1.[101] Although animal studies have provided conflicting results about the role of COX-2 inhibition on development of atherosclerosis, [102] [103] [104] [105] [106] there are recent indications that COX-2 inhibition may destabilize atherosclerotic plaques,[107] as suggested by studies indicating increased COX-2 expression and colocalization with microsomal PGE synthase-1 and metalloproteinases-2 and -9 in carotid plaques from individuals with symptomatic disease before endarterectomy.[108] Because of the concerns about increased cardiovascular risk, two selective COX-2 inhibitors, rofecoxib and valdecoxib, have been withdrawn from the market, and remaining coxibs and other NSAIDs have been relabeled to highlight the increased risk for cardiovascular events.

PROSTANOID SYNTHASES

Once PGH2 is formed in the cell, it can undergo a number of possible transformations, yielding biologically active prostaglandins and thromboxane A2. As seen in Figure 11-9 , in the presence of isomerase and reductase enzymes, PGH2 is converted to PGE2 and PGF, respectively. Thromboxane synthase converts PGH2 into a bicyclic oxetane-oxane ring metabolite, thromboxane A2 (TxA2), a prominent reaction product in the platelet and an established synthetic pathway in the glomerulus. Prostacyclin synthase, a 50 kD protein located in plasma and nuclear membranes and found mostly in vascular endothelial cells, catalyzes the biosynthesis of prostacyclin (PGI2). PGD2, the major prostaglandin product in mast cells, is also derived directly from PGH2, but its role in the kidney is uncertain. The enzymatic machinery and their localization in the kidney are discussed in detail later.

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FIGURE 11-9  Prostaglandin synthases.

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Sources and Nephronal Distribution of Cyclooxygenase Products

COX activity is present in arterial and arteriolar endothelial cells, including glomerular afferent and efferent arterioles. The predominant metabolite from these vascular endothelial cells is PGI2.[109] Whole glomeruli generate PGE2, PGI2, PGF, and TxA2. The predominant products in rat and rabbit glomeruli are PGE2, followed by PGI2 and PGF and finally TxA2.

Analysis of individual cultured glomerular cell sub-populations has also provided insight into the localization of prostanoid synthesis. Cultured mesangial cells are capable of generating PGE2, and in some cases PGF and PGI2have also been detected.[110] Other studies suggest mesangial cells may produce the endoperoxide PGH2 as a major cyclooxygenase product.[111] Glomerular epithelial cells also appear to participate in prostaglandin synthesis, but the profile of COX products generated in these cells remains controversial. Immunocytochemical studies of rabbit kidney demonstrate intense staining for COX-1 predominantly in the parietal epithelial cells. Glomerular capillary endothelial cell PG generation profiles remain undefined but may well include prostacyclin.

The predominant synthetic site of prostaglandin synthesis along the nephron is the collecting duct (CD), particularly its medullary portion (MCT).[112] In the presence of exogenous arachidonic acid, PGE2 is the predominant PG formed in collecting duct, the variations among the other products being insignificant. PGE2 is also the major COX metabolite generated in medullary interstitial cells.[113] The role that specific prostanoid synthases may play in the generation of these products is outlined later.

Thromboxane Synthase

Thromboxane A2 (TxA2) is produced from PGH2 by thromboxane synthase (TxAS), a microsomal protein of 533 amino acids with a predicted molecular weight of ∼60 kDa. The amino acid of sequence of the enzyme exhibits homology to the cytochrome P450s and is now classified as CYP5A1.[114] The human gene is localized on Chromosome 7q and spans 180 kB. TxAS mRNA is highly expressed in hematopoietic cells, including platelets, macrophages, and leukocytes. TxAS mRNA is expressed in thymus, kidney, lung, spleen, prostate, and placenta. Immunolocalization of TxA synthase demonstrates high expression in the dendritic cells of the interstitium, with lower expression in glomerular podocytes of human kidney.[115] TxA2 synthase expression is regulated by dietary salt intake.[116] Furthermore experimental use of ridogrel, a specific thromboxane synthase inhibitor, reduced blood pressure in spontaneously hypertensive rats.[117] The clinical use of TxA2 synthase inhibitors is complicated by the fact that its endoperoxide precursors (PGG2/PGH2) are also capable of activating its downstream target, the TP receptor.[26]

Prostacyclin Synthase

The biological effects of prostacyclin are numerous and include nociception, anti-thrombosis, and vasodilator actions, which have been targeted therapeutically to manage pulmonary hypertension.

Prostacyclin (PGI2) is derived by the enzymatic conversion of PGH2 via prostacyclin synthase (PGIS). The cloned cDNA contains a 1500 base pair open reading frame that encodes a 500 amino acid protein of approximately 56 kDa. The human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q. Northern blot analysis shows prostacyclin synthase mRNA is widely expressed in human tissues and is particularly abundant in ovary, heart, skeletal muscle, lung, and prostate. PGI synthase expression exhibits segmental expression in the kidney especially in kidney inner medulla tubules and interstitial cells.

Recently, PGI2 synthase-null mice were generated.[118] PGI2 levels in the plasma, kidneys, and lungs, were reduced, documenting the role of this enzyme as an in vivo source of PGI2. Blood pressure and blood urea nitrogen and creatinine in the PGIS knockout mice were significantly increased and renal pathological findings included surface irregularity, fibrosis, cysts, arterial sclerosis, and hypertrophy of vessel walls. Thickening of the thoracic aortic media and adventitia were observed in aged PGI null mice.[118] Interestingly this is a phenotype different from that reported for the IP receptor knockout mouse.[119] These differences points to the presence of additional IP independent PGI2 activated signaling pathways. Regardless, these findings demonstrate the importance of PGI2 to maintenance of blood vessels and to the kidney.

Prostaglandin Synthase

Prostaglandin D2 is derived from PGH2 via the action of specific enzymes designated PGD synthases. Two major enzymes are capable of transforming PGH2 to PGD2 including a lipocalin type PGD synthase and a hematopoietic type PGDS. [120] [121] Mice lacking the lipocalin D synthase gene exhibit altered sleep and pain sensation.[122] PGD2 is the major prostanoid released from mast cells following challenge with IgE. The kidney also appears capable of synthesizing PGD2. RNA for the lipocalin type PGD synthase has been reported to be widely expressed along the rat nephron, whereas the hematopoietic type PGD synthase is restricted to the collecting duct.[123] Urinary excretion of lipocalin D synthase has recently been proposed as a biomarker predictive of renal injury[124] and lipocalin D synthase knockout mice appear to be more prone to diabetic nephropathy.[125] However the physiologic roles of these enzymes in the kidney remain less certain. Once synthesized, PGD2 is available to interact with the either the DP or CRTH2 receptors (see later discussion) or undergo further metabolism to a PGF2 like compound.

Prostaglandin F Synthesis

Prostaglandin F is a major urinary cyclooxygenase product. Its synthesis may derive either directly from PGH2 via a PGF synthase[126] or indirectly by metabolism of PGE2 via a 9-keto-reductase.[126] Another more obscure pathway for PGF formation is by the action of a PGD2 ketoreductase, yield-ing a stereoisomer of PGF2, 9a, 11β-PGF2 (11epi-PGF).[126] This reaction, and conversion of PGD2 into an apparently biologically active metabolite (9a,11β-PGF) has been documented in vivo.[127] Interestingly this isomer can also ligate and activate the FP receptor.[128] The physiologically relevant enzymes responsible for renal PGF formation remain incompletely characterized.

Prostaglandin 9 Ketoreductase

Physiologically relevant transformations of COX products occur in the kidney via a NADPH-dependent 9-ketoreductase, which converts PGE2 into PGF. This enzymatic activity is typically cytosolic[126] and may be detected in homogenates from renal cortex, medulla, or papilla. The activity appears to be particularly robust in suspensions from the thick ascending limb of Henle (TALH). Renal PGE2 9 keto-reductase also exhibits 20a hydroxysteroid reductase activity that could affect steroid metabolism.[126] This enzyme appears to be a member of the aldo-keto reductase family 1C.[129]

Interestingly, some studies suggest activity of a 9-keto-reductase may be modulated by salt intake and AT2 receptor activation, and may play an important role in hypertension.[130] Mice deficient in the AT2 receptor exhibit salt sensitive hypertension, increased PGE2 production, and reduced production of PGF,[131] consistent with reduced 9-ketoreductase activity. Other studies suggest dietary potassium intake may also enhance the activity of conversion from PGE2 to PGF.[132] The intra-renal sites of expression of this enzymatic activity remain to be characterized.

Prostaglandin E Synthases

PGE2 is the other major product of cyclooxygenase-initiated arachidonic acid metabolism in the kidney and is synthesized at high rates along the nephron, particularly in the collecting duct. Two membrane associated PGE2 synthases have been identified: a 33 kDa and a 16 kDa membrane associated enzyme. [133] [134] The initial report describing the cloning of a glutathione dependent microsomal enzyme (the 16 kDa form) that specifically converts PGH2 to PGE2[134] showed mRNA for this enzyme is highly expressed in reproductive tissues as well as in kidney. Genetic disruption confirms that mPGES1-/- mice exhibit a marked reduction in inflammatory responses compared with mPGES1+/+135 and indicate that mPGES1 is also critical for the induction of inflammatory fever.[136]

Intra-renal expression of mPGES1 has been demonstrated and mapped to collecting duct with lower expression in the medullary interstitial cells and macula densa (see Fig. 11-3 ). [112] [137] Thus in the kidney this isoform co-localizes with both cyclooxygenase 1 and -2. In contrast, in inflammatory cells, this PGE synthase is co-induced with COX-2 and appears to be functionally coupled to it.[138] Notably, the kidneys of mPGES1-/- mice are normal and do not exhibit the renal dysgenesis observed in COX2-/- mice. [94] [139] Nor do these mice exhibit perinatal death from patent ductus arteriosus observed with the prostaglandin EP4 receptor knockout mouse.[140]

More recently another membrane associated PGE synthase with a relative mass of ∼33 kDa was purified from heart. The recombinant enzyme was activated by several SH-reducing reagents, including dithiothreitol, glutathione (GSH), and beta-mercaptoethanol. Moreover, the mRNA distribution was high in the heart and brain, and was also expressed in the kidney, but the mRNA was not expressed in the seminal vesicles. The intra-renal distribution of this enzyme is, at present, uncharacterized.[133]

Other cytosolic proteins exhibit lower prostaglandin E synthase activity, including a 23 kDa GST requiring cytoplasmic PGES[141] that is expressed in the kidney and lower genitourinary tract.[142] Some evidence suggests this isozyme may constitutively couple to COX-1 in inflammatory cells. In addition several cytosolic glutathione-S-transferases have the capability to convert PGH2 to PGE2; however their physiologic role in this process remains uncertain.[143]

CELLULAR ORIGIN OF EICOSANOIDS PROSTANOID RECEPTORS

TP Receptors

The TP receptor was originally purified by chromatography using a high affinity ligand to capture the receptor (Figs. 11-10 and 11-11 [10] [11]).[144] This was the first eicosanoid receptor cloned and is a G-protein coupled transmembrane receptor capable of activating a calcium coupled signaling mechanism ( Fig. 11-12 ). The cloning of other prostanoid receptors was achieved by finding cDNAs homologous to this TP receptor cDNA. Two alternatively spliced variants of the human thromboxane receptor have been described[145] that differ in their carboxyl-terminal tail distal to Arg[328]. Similar patterns of alternative splicing have been described for both the EP3receptor and the FP receptor.[146] Heterologous cAMP mediated signaling of the thromboxane receptor may occur via its heterodimerization with the prostacyclin (IP) receptor.[147]

000661

000519

FIGURE 11-10  Tissue distribution of prostanoid receptor mRNA.  (Adapted from Bek M, Nusing R, Kowark P, et al: Characterization of prostanoid receptors in podocytes. J Am Soc Nephrol 10:2084-2093, 1999.)

000519

 

 

000658

000519

FIGURE 11-11  Intrarenal localization of prostanoid receptors.

000519

 

000662

000519

FIGURE 11-12  Prostaglandin receptors are 7-transmembrane G-protein coupled receptors.

000519

 

Either the endoperoxide, PGH2 or its metabolite, TxA2 can activate the TP receptor.[26] Competition radioligand binding studies have demonstrated a rank order of potency on human platelet TP receptor of the ligands I-BOP, S145>SQ29548>STA2>U-46619. [148] [149] Whereas I-BOP, STA2, and U-46619 are agonists, SQ29548 and S145, are potent TP receptor antagonists.[150] Studies have suggested that the TP receptor may mediate some of the biological effects of the non-enzymatically derived isoprostanes,[151] including modulation of tubuloglomerular feedback.[152] This latter finding may have significance in pathophysiological conditions associated with increased oxidative stress.[153] Signal transduction studies show the TP receptor activates phosphatidylinositol hydrolysis (PIP2) dependent Ca++ influx. [144] [154] Northern analysis of mouse tissues revealed that the highest level of TP mRNA expression is in the thymus followed by spleen, lung, and kidney, with lower levels of expression in heart, uterus, and brain.[155]

Thromboxane is a potent modulator of platelet shape change and aggregation as well as smooth muscle contraction and proliferation. Moreover, a point mutation (Arg[60] to Leu) in the first cytoplasmic loop of the TXA2 receptor was identified in a dominantly inherited bleeding disorder in humans, characterized by defective platelet response to TXA2.[156] Targeted gene disruption of the murine TP receptor also resulted in prolonged bleeding times and reduction in collagen stimulated platelet aggregation ( Table 11-1 ). Conversely, overexpression of the TP receptor in vascular tissue increases the severity of vascular pathology following injury. Increased thromboxane synthesis has been linked to cardiovascular diseases, including acute myocardial ischemia, heart failure, and inflammatory renal diseases.


TABLE 11-1   -- Published Phenotypes of Prostanoid Receptor Knockout Mice

 

Renal Expression

Knockout Phenotype

References

DP

Minimal?

No

185

Reduced allergic asthma

IP

++ Afferent arteriole

±

119

Reduced inflammation, pain; increased thrombosis

TP

+ Glomerulus, tubules?

No

158

Prolonged bleeding time, platelet defect

FP

+++ Distal tubules

No

193

Failure of parturition

EP1

++++ MCD

No

K. Watanabe et al, 1997

Decreased pain, sensation

EP2

++ Interstitial, stromal

Impaired ovulation, salt sensitive hypertension (?)

Hizaki et al, 1999;215, 217, 253 [215] [217] [253]

EP3

++++ TAL, MCD

±

140, 236 [140] [236]

Impaired febrile response, Mild diluting defect

EP4

+++ Glomerulus, + distal tubules

±

140, 236 [140] [236]

Perinatal death from persistent patent ductus arteriosus

 

 

 

In the kidney, TP receptor mRNA has been reported in glomeruli and vasculature. Radioligand autoradiography using 125I-BOP suggests a similar distribution of binding sites in mouse renal cortex, but additional renal medullary binding sites were observed.[157] These medullary TxA2 binding sites are absent following disruption of the TP receptor gene, suggesting they also represent authentic TP receptors.[158] Glomerular TP receptors may participate in potent vasoconstrictor effects of TxA2 analogs on the glomerular microcirculation associated with reduced glomerular filtration rate. Mesangial TP receptors coupled to phosphatidylinositol hydrolysis, protein kinase C activation, and glomerular mesangial cell contraction may contribute to these effects.[159]

An important role for TP receptors in regulating renal hemodynamics and systemic blood pressure has also been suggested. Administration of a TP receptor antagonist reduces blood pressure in spontaneously hypertensive rats (SHRs)[117] and in angiotensin-dependent hypertension.[160] The TP receptor also appears to modulate renal blood flow in AngII dependent hypertension[161] and in endotoxemia-induced renal failure.[162] Modulation of renal TP receptor mRNA expression and function by dietary salt intake has also been reported.[163] These studies also suggested an important role for luminal TP receptors in the distal tubule to enhance glomerular vasoconstriction indirectly via effects on the macula densa and tubuloglomerular feedback (TGF).[164] However, recent studies reveal no significant difference in tubuloglomerular feedback between wild type and TP receptor knockout mice.[43]

Despite the renal effects of thromboxane mimetics, the major phenotype of TP receptor disruption in mice and humans appears to be reduced platelet aggregation and prolonged bleeding time.[158] Thromboxane may also modulate the glomerular fibrinolytic system by increasing the production of an inhibitor of plasminogen activator (PAI-1) in mesangial cells.[165] Although a specific renal phenotype in the TP receptor knockout mouse has not yet been reported, important pathogenic roles for TxA2 and glomerular TP receptors in mediating renal dysfunction in glomerulonephritis, diabetes mellitus, and sepsis seem likely.

Prostacyclin Receptors

The cDNA for the IP receptor encodes a transmembrane protein of approximately 41 kDa. The IP receptor is selectively activated by the analog cicaprost.[166] Iloprost and carbaprostacyclin potently activate the IP receptor but also activate the EP1 receptor. Most evidence suggests the PGI2 receptor signals via stimulation of cAMP generation; however at 1000 fold higher concentrations the cloned mouse PGI2 receptor also signaled via PIP2.[167] It remains unclear whether PIP2 hydrolysis plays any significant role in the physiologic action of PGI2.

IP receptor mRNA is highly expressed in mouse thymus, heart, and spleen[167] and in human kidney, liver, and lung.[168] In situ hybridization shows IP receptor mRNA predominantly in neurons of the dorsal root ganglia and vascular tissue including aorta, pulmonary artery, and renal interlobular and glomerular afferent arterioles.[169] The expression of IP receptor mRNA in the dorsal root ganglia is consistent with a role for prostacyclin in pain sensation. Mice with IP receptor gene disruption exhibit a predisposition to arterial thrombosis, diminished pain perception, and inflammatory responses.[119]

PGI2 has been demonstrated to play an important vasodilator role in the kidney[170] including in the glomerular microvasculature[171] as well as regulating renin release. [172] [173] The capacity of PGI2 and PGE2 to stimulate cAMP generation in the glomerular microvasculature is distinct and additive,[174] demonstrating the effects of these two prostanoids are mediated via separate receptors. IP receptor knockout mice also exhibit salt sensitive hypertension.[175] Prostacyclin is a potent stimulus of renal renin release, and studies using IP-/- mice confirm an important role for the IP receptor in the development of renin dependent hypertension of renal artery stenosis.[62]

Renal epithelial effects of PGI2 in the thick ascending limb have also been suggested[176] and IP receptors have been reported in the collecting duct[177] but the potential expression and role of prostacyclin in these segments are less well established. Of interest, in situ hybridization also demonstrated significant expression of prostacyclin synthase in medullary collecting ducts,[178] consistent with a role for this metabolite in this region of the kidney. In summary, whereas IP receptors appear to play an important role regulating renin release and a vasodilator in the kidney, their role in regulating renal epithelial function remains to be firmly established.

DP Receptors

The DP receptor has been cloned and like the IP and EP2/4 receptors, the DP receptor predominantly signals by increasing cAMP generation. The human DP receptor binds PGD2 with a high affinity binding of 300 pM, and a lower affinity site of 13.4 nM.[179] DP selective PGD2 analogs, include the agonist BW 245C.[180] DP receptor mRNA is highly expressed in leptomeninges, retina, and ileum but was not detected in the kidney.[181] Northern blot analysis of the human DP receptor demonstrated mRNA expression in the small intestine and retina,[182] whereas in the mouse the DP receptor mRNA was detected in the ileum and lung.[179] PGD2 has also been shown to affect the sleep-wake cycle,[183] pain sensation,[122] and body temperature.[184] Peripherally, PGD2 has been shown to mediate vasodilation as well as possibly inhibiting platelet aggregation. Consistent with this latter finding, the DP receptor knockout displayed reduced inflammation in the ovalbumin model of allergic asthma.[185] Although the kidney appears capable of synthesizing PGD2, its role in the kidney remains poorly defined. Intra-renal infusion of PGD2resulted in dose-dependent increase in renal artery flow, urine output, creatinine clearance, and sodium and potassium excretion.[186]

Recently, another G-protein coupled receptor capable of binding and being activated by PGD2 was cloned from eosinophils and T-cells (TH2 subset) and designated the CRTH2 receptor.[187] This receptor, also referred to by some as the DP2 receptor, bears no significant sequence homology to the family of prostanoid receptors discussed earlier, and couples to increased cell calcium rather than increased cAMP. The use of DP selective agonists should help clarify whether renal effects of PGD2 are mediated by authentic DP receptors or the CRTH2 receptor. The recognition of this molecularly un-related receptor allows for the possibility of existence of a distinct and new family of prostanoid activated membrane receptors.

FP Receptors

The cDNA encoding the PGF receptor (FP receptor) was cloned from a human kidney cDNA library and encodes a protein of 359 amino acid residues. The bovine and murine FP receptors, cloned from corpora lutea similarly encode proteins of 362 and 366 amino acid residues, respectively. Transfection of HEK293 cells with the human FP receptor cDNA, conferred preferential 3H-PGF binding with a KD of 4.3±1.0 nM. [150] [188] Selective activation of the FP receptor may be achieved using fluprostenol or latanoprost.[150] 3H-PGF binding was displaced by a panel of ligands with a rank order potency of: PGF=fluprostenol>PGD2>PGE2>U46619>iloprost.[166] When expressed in oocytes, PGF or fluprostenol induced a Ca++ dependent Cl- current. Increased cell calcium has also been observed in fibroblasts expressing an endogenous FP receptor.[189] Recent studies suggest FP receptors may also activate protein kinase C dependent and Rho mediated/PKC independent signaling pathways.[190] An alternatively spliced isoform with a shorter carboxy-terminal tail, has been identified that appears to signal via a similar manner as the originally described FP receptor.[191] More recent studies suggest these two isoforms may exhibit differential desensitization and may also activate a glycogen synthase kinase/b-catenin coupled signaling pathway.[192]

Tissue distribution of FP receptor mRNA shows highest expression in ovarian corpus luteum followed by kidney, with lower expression in lung, stomach, and heart.[193] Expression of the FP receptor in corpora lutea is critical for normal birth, and homozygous disruption of the murine FP receptor gene results in failure of parturition in females apparently due to failure of the normal pre-term decline in progesterone levels.[194] PGF is a potent constrictor of smooth muscle in the uterus, bronchi, and blood vessels; however an endothelial FP receptor may also play a dilator role.[195] The FP receptor is also highly expressed in skin, where it may play an important role in carcinogenesis.[196] A clinically important role for the FP receptor in the eye has been demonstrated to increase uveoscleral outflow and reduce ocular pressure. The FP selective agonist latanoprost has been used clinically as an effective treatment for glaucoma.[197]

The role of FP receptors in regulating renal function is only partially defined. FP receptor expression has been mapped to the cortical collecting duct in mouse and rabbit kidney.[198] FP receptor activation in the collecting duct inhibits vasopressin-stimulated water absorption via a pertussis toxin sensitive (presumably Gi) dependent mechanism. Although PGF increases cell Ca++ in cortical collecting duct, the FP selective agonists latanoprost and fluprostenol did not increase calcium.[199] Because PGF can also bind to EP1 and EP3 receptors [166] [200] [201] these data suggest that the calcium increase activated by PGF in the collecting duct may be mediated via an EP receptor. PGF also increases Ca++ in cultured glomerular mesangial cells and podocytes, [202] [203] suggesting an FP receptor may modulate glomerular contraction. In contrast to these findings, demonstration of glomerular FP receptors at the molecular level has not been forthcoming. Other vascular effects of PGF have been described, including selective modulation of renal production of PGF by sodium or potassium loading and AT2 receptor activation.[130]

Multiple EP Receptors

Four EP receptor subtypes have been identified.[204] Although these four receptors uniformly bind PGE2 with a higher affinity than other endogenous prostanoids, the amino-acid homology of each is more closely related to other prostanoid receptors that signal through similar mechanisms.[149] Thus the relaxant/cAMP coupled EP2 receptor is more closely related to other relaxant prostanoid receptors such as the IP and DP receptors, whereas the constrictor/Ca++ coupled EP1 receptor is more closely related to the other Ca++ coupled prostanoid receptors such as the TP and FP receptors.[205] These receptors may also be selectively activated or antagonized by different analogs. EP receptor subtypes also exhibit differential expression along the nephron, suggesting distinct functional consequences of activating each EP receptor subtype in the kidney.[204]

EP1 Receptors

The human EP1 receptor cDNA encodes a 402 amino acid polypeptide that signals via IP3 generation and increased cell Ca++ with IP3 generation. Studies of EP1 receptors may utilize one of several relatively selective antagonists including SC51089, SC19220, or SC53122. EP1 receptor mRNA predominates in the kidney>>gastric muscularis mucosae>adrenal.[206] Renal EP1 mRNA expression determined by in situ hybridization is expressed primarily in the collecting duct, and increases from the cortex to the papillae.[206] Activation of the EP1 receptor increases intracellular calcium and inhibits Na+ and water reabsorption absorption in the collecting duct,[206] suggesting renal EP1receptor activation might contribute to the natriuretic and diuretic effects of PGE2.

Hemodynamic, microvascular effects of EP1 receptors have also been supported. The EP1 receptor was originally described as a smooth muscle constrictor.[207] A recent report suggests the EP1 receptor may also be present in cultured glomerular mesangial cells[208] where it could play a role as a vasoconstrictor and a stimulus for mesangial cell proliferation. Although a constrictor PGE2 effect has been reported in the afferent arteriole of rat,[209] it remains unclear whether this is mediated by an EP1 or EP3 receptor. There does not appear to be very high expression of the EP1 receptor mRNA in pre-glomerular vasculature or other arterial resistance vessels in either mice or rabbits.[210] Other reports suggest EP1 receptor knockout mice exhibit hypotension and hyperreninemia, supporting a role for this receptor in maintaining blood pressure.[211]

EP2 Receptors

Two cAMP stimulating EP receptors, designated EP2 and EP4 have been identified. The EP2 receptor can be pharmacologically distinguished from the EP4 receptor by its sensitivity to butaprost.[212] Prior to 1995 literature the cloned EP4 receptor was designated the EP2 receptor, but then a butaprost sensitive EP receptor was cloned,[213] and the original receptor reclassified as the EP4 receptor and the newer butaprost sensitive protein designated the EP2receptor.[214] A pharmacologically defined EP2 receptor has now also been cloned for the mouse, rat, rabbit, dog, and cow.[215] The human EP2 receptor cDNA encodes a 358 amino acid polypeptide, which signals through increased cAMP. The EP2 receptor may also be distinguished from the EP4 receptor, the other major relaxant EP receptor, by its relative insensitivity to the EP4 agonist PGE1-OH and insensitivity to the weak EP4 antagonist AH23848.[212]

The precise distribution of the EP2 receptor mRNA has been partially characterized. This reveals a major mRNA species of ∼3.1 kb, which is most abundant in the uterus, lung, and spleen, exhibiting only low levels of expression in the kidney.[215] EP2 mRNA is expressed at much lower levels than EP4 mRNA in most tissues.[216] There is scant evidence to suggest segmental distribution of the EP2 receptor along the nephron.[215] Interestingly it is expressed in cultured renal interstitial cells, supporting the possibility that the EP2 receptor is predominantly expressed in this portion of the nephron.[215] Studies in knockout mice demonstrate a critical role for the EP2 receptor role in ovulation and fertilization.[217] In addition these studies suggest a potential role for the EP2 receptor in salt sensitive hypertension.[217] This latter finding supports an important role for the EP2 receptor in protecting systemic blood pressure, perhaps via its vasodilator effect or effects on renal salt excretion.

EP3 Receptors

The EP3 receptor generally acts as a constrictor of smooth muscle.[218] Nuclease protection and northern analysis demonstrate relatively high levels of EP3 receptor expression in several tissues including kidney, uterus, adrenal, and stomach, with riboprobes hybridizing to major mRNA species at ∼2.4 and ∼7.0 kb.[219] This receptor is unique in that multiple (more than eight) alternatively spliced variants differing only in their C-terminal cytoplasmic tails, exist. [220] [221] [222] The EP3 splice variants bind PGE2, and the EP3 agonists MB28767 and sulprostone with similar affinity, and although they exhibit common inhibition of cAMP generation via a pertussis toxin sensitive Gi-coupled mechanism, the tails may recruit different signaling pathways, including Ca++ dependent signaling [149] [212] and the small G-protein, rho.[223] Differences in agonist independent activity have been observed for several of the splice variants, suggesting that they may play a role in constitutive regulation of cellular events.[224] The physiologic roles of these different C-terminal splice variants and sites of expression within the kidney remains uncertain.

In situ hybridization demonstrates EP3 receptor mRNA is abundant in the thick ascending limb and collecting duct.[225] This distribution has been confirmed by RT-PCR on microdissected rat and mouse collecting ducts and corresponds to the major binding sites for radioactive PGE2 in the kidney.[226] An important role for a Gi coupled prostaglandin E receptor in regulating water and salt transport along the nephron has been recognized for many years. PGE2 directly inhibits salt and water absorption in both microperfused thick ascending limbs (TAL) and collecting ducts (CD). PGE2 directly inhibits Cl- absorption in the mouse or rabbit medullary TAL from either the luminal or basolateral surfaces.[227] PGE2 also inhibits hormone stimulated cAMP generation in TAL. Good demonstrated that PGE2 modulates ion transport in the rat TAL by a pertussis toxin sensitive mechanism.[227] Interestingly these effects also appear to involve protein kinase C activation,[228] possibly reflecting activation of a novel EP3 receptor signaling pathway, possibly corresponding to alternative signaling pathways as described earlier.[223] Taken together, these data support a role for the EP3 receptor in regulating transport in both the collecting duct and TAL.

Blockade of endogenous PGE2 synthesis by NSAIDs enhances urinary concentration. It is likely PGE2 mediated antagonism of vasopressin-stimulated salt absorption in the TAL and water absorption in the collecting duct contributes to its diuretic effect. In the in vitro microperfused collecting duct, PGE2 inhibits both vasopressin-stimulated osmotic water absorption and vasopressin-stimulated cAMP generation.[199] Furthermore PGE2 inhibition of water absorption and cAMP generation are both blocked by pertussis toxin, suggesting effects mediated by the inhibitory G protein, Gi.[199] When administered in the absence of vasopressin, PGE2 actually stimulates water absorption in the collecting duct from either the luminal or the basolateral side.[229] These stimulatory effects of PGE2 on transport in the collecting duct appear to be related to activation of the EP4 receptor.[229] Despite the presence of this absorption enhancing EP receptor, in vivo studies suggest that, in the presence of vasopressin, the predominant effects of endogenous PGE2 on water transport are diuretic.

Based on the preceding functional considerations, one would expect EP3-/- mice to exhibit inappropriately enhanced urinary concentration. Surprisingly EP3-/- mice exhibited a comparable urinary concentration following dDAVP, similar 24 hour water intake, and similar maximal and minimal urinary osmolality.[230] The only clear difference was that in mice allowed free access to water, indomethacin increased urinary osmolality in normal mice but not in the knockout animals. These findings raise the possibility that some of the renal actions of PGE2 normally mediated by the EP3 receptor have been co-opted by other receptors (such as the EP1 or FP receptor) in the EP3 knockout mouse. This remains to be formally tested.

The significance of EP3 receptor activation to animal physiology has been significantly advanced by the availability of mice with targeted disruption of this gene. [230] [231] Mice with targeted deletion of the EP3 receptor exhibit an impaired febrile response, suggesting that EP3 receptor antagonists could be effective antipyretic agents.[231] Other studies suggest the EP3 receptor plays an important vasopressor role in the peripheral circulation of mice.[210]Studies in knockout mice also support a potential role for the EP3 receptor as an important systemic vasopressor. [210] [232]

The EP4 Receptor

Like the EP2 receptor, the EP4 receptor signals through increased cAMP.[233] The human EP4 receptor cDNA encodes a 488 amino acid polypeptide with a predicted molecular mass of ∼53 kDa.[234] Note, care must be taken in reviewing the literature prior to 1995, when this receptor was generally referred to as the EP2 receptor.[214] In addition to the human receptor, EP4 receptors for the mouse, rat, rabbit, and dog have been cloned. EP4 receptors can be pharmacologically distinguished from the EP1 and EP3 receptors by insensitivity to sulprostone and from EP2 receptors by its insensitivity to butaprost and relatively selective activation by PGE1-OH.[150] Recently an EP4 selective agonist (ONO-AE1-329) and antagonist have been generated[212]; however to date, their use has not been widely reported.

EP4 receptor mRNA is highly expressed relative to the EP2 receptor and widely distributed, with a major species of ∼3.8 kb detected by northern analysis in thymus, ileum, lung, spleen, adrenal, and kidney. [216] [235] Dominant vasodilator effects of EP4 receptor activation have been described in venous and arterial beds. [180] [218] A critical role for the EP4 receptor in regulating the peri-natal closure of the pulmonary ductus arteriosus has also been suggested by the recent studies of mice with targeted disruption of the EP4 receptor gene. [140] [236] On a 129 strain background, EP4-/- mice had nearly 100% peri-natal mortality due to persistent patent ductus arteriosus.[236]Interestingly, when bred on a mixed genetic background, only 80% of EP4-/- mice died whereas ∼21% underwent closure of the ductus and survived.[140] Preliminary studies in these survivors support an important role for the EP4receptor as a systemic vasodepressor[237]; however, their heterogeneous genetic background complicates the interpretation of these results because survival may select for modifier genes that not only allow ductus closure but also alter other hemodynamic responses.

Other roles for the EP4 receptor in controlling blood pressure have been suggested, including the ability to stimulate aldosterone release from zona glomerulosa cells.[238] In the kidney, EP4 receptor mRNA expression is primarily in the glomerulus, where its precise function is uncharacterized [235] [239] but might contribute to regulation of the renal micro-circulation as well as renin release.[240] Recent studies in mice with genetic deletion of selective prostanoid receptors indicated that EP4-/- mice, as well as IP-/- mice to a lesser extent, failed to increase renin production in response to loop diuretic administration, indicating that macula densa-derived PGE2 increased renin primarily through EP4 activation.[241] This corresponds to recent studies suggesting EP4 receptors are expressed in cultured podocytes and juxtaglomerular apparatus cells. [202] [240] Finally, the EP4 receptor in the renal pelvis may participate in regulation of salt excretion by altering afferent renal nerve output.[242]

Regulation of Renal Function by EP Receptors

PGE2 exerts myriad effects in the kidney, presumably mediated by EP receptors. PGE2 not only dilates the glomerular microcirculation and vasa rectae, supplying the renal medulla,[243] but also modulates salt and water transport in the distal tubule (see Fig. 11-5 ).[244] The maintenance of normal renal function during physiologic stress is particularly dependent on endogenous prostaglandin synthesis. In this setting, the vasoconstrictor effects of angiotensin II, catecholamines, and vasopressin are more effectively buffered by prostaglandins in the kidney than in other vascular beds, preserving normal renal blood flow, glomerular filtration rate (GFR), and salt excretion. Administration of cyclooxygenase inhibiting NSAIDs in the setting of volume depletion interferes with these dilator effects and may result in a catastrophic decline in GFR, resulting in overt renal failure.[245]

Other evidence points to vasoconstrictor and pro-hypertensive effects of endogenous PGE2. PGE2 stimulates renin release from the juxtaglomerular apparatus[246] leading to a subsequent increase in the vasoconstrictor, angiotensin II. In conscious dogs, chronic intra-renal PGE2 infusion increases renal renin secretion resulting in hypertension.[247] Treatment of salt depleted rats with indomethacin not only decreases plasma renin activity, but also reduces blood pressure, suggesting prostaglandins support blood pressure during salt depletion, via their capacity to increase renin.[248] Direct vasoconstrictor effects of PGE2 on vasculature have also been observed.[210] It is conceivable these latter effects might predominate in circumstances where the kidney is exposed to excessively high perfusion pressures. Thus depending on the setting, the primary effect of PGE2 may be either to increase or decrease vascular tone, effects that appear to be mediated by distinct EP receptors.

Renal Cortical Hemodynamics

The expression of the EP4 receptor in the glomerulus suggests it may play an important role regulating renal hemodynamics. Prostaglandins regulate the renal cortical microcirculation and as alluded to earlier, both glomerular constrictor and dilator effects of prostaglandins have been observed. [210] [249] In the setting of volume depletion, endogenous PGE2 helps maintain GFR by dilating the afferent arteriole.[249] Some data suggest roles for EP and IP receptors coupled to increased cAMP generation in mediating vasodilator effects in the pre-glomerular circulation. [42] [240] [250] PGE2 exerts a dilator effect on the afferent arteriole but not the efferent arteriole, consistent with the presence of an EP2 or EP4 receptor in the pre-glomerular microcirculation.

Renin Release

Other data suggest the EP4 receptor may also stimulate renin release. Soon after the introduction of NSAIDs it was recognized that endogenous prostaglandins play an important role in stimulating renin release.[42] Treatment of salt depleted rats with indomethacin not only decreases plasma renin activity, but also causes blood pressure to fall, suggesting prostaglandins support blood pressure during salt depletion, via their capacity to increase renin. Prostanoids also play a central role in the pathogenesis of renovascular hypertension, and administration of NSAIDs lowers blood pressure in both animals and humans with renal artery stenosis.[251] PGE2 induces renin release in isolated pre-glomerular juxtaglomerular apparatus cells.[246] Like the effect of β-adrenergic agents, this effect appears to be through a cAMP coupled response, supporting a role for an EP4 or EP2 receptor.[246] EP4 receptor mRNA has been detected in microdissected JGAs,[252] supporting the possibility that renal EP4 receptor activation contributes to enhanced renin release. Finally regulation of plasma renin activity and intra-renal renin mRNA does not appear to be different in wild-type and EP2 knockout mice,[253] arguing against a major role for the EP2 receptor in regulating renin release. Conversely, one report suggests EP3 receptor mRNA is localized to the macula densa, suggesting this cAMP inhibiting receptor may also contribute to the control of renin release.[239]

Renal Microcirculation

The EP2 receptor also appears to play an important role in regulating afferent arteriolar tone.[249] In the setting of systemic hypertension, the normal response of the kidney is to increase salt excretion, thereby mitigating the increase in blood pressure. This so-called pressure natriuresis plays a key role in the ability of the kidney to protect against hypertension.[254] Increased blood pressure is accompanied by increased renal perfusion pressure and enhanced urinary PGE2 excretion.[255] Inhibition of prostaglandin synthesis markedly blunts (although it does not eliminate) pressure natriuresis.[256] The mechanism by which PGE2 contributes to pressure natriuresis may involve changes in resistance of the renal medullary microcirculation.[257] PGE2 directly dilates descending vasa recta, and increased medullary blood flow may contribute to increased interstitial pressure observed as renal perfusion pressure increases, leading to enhanced salt excretion.[243] The identity of the dilator PGE2 receptor controlling the contractile properties of the descending vasa recta remains uncertain, but EP2 or EP4 receptors seem likely candidates.[180] Recent studies demonstrating salt sensitive hypertension in mice with targeted disruption of the EP2 receptor[217] suggests the EP2 receptor facilitates the ability of the kidney to increase sodium excretion, thereby protecting systemic blood pressure from a high salt diet. Given its defined role in vascular smooth muscle,[217] these effects of the EP2 receptor disruption seem more likely to relate to its effects on renal vascular tone. In particular, loss of a vasodilator effect in the renal medulla might modify pressure natriuresis and could contribute to hypertension in EP2 knockout mice. Nonetheless a role for either the EP2 or EP4 receptor in regulating renal medullary blood flow remains to be established. In conclusion, direct vasomotor effects of EP4 receptors as well as effects on renin release may play critical roles in regulating systemic blood pressure and renal hemodynamics.

Effects on Salt and Water Transport

COX-1 and COX-2 metabolites of arachidonate have important direct epithelial effects on salt and water transport in along the nephron.[258] Thus, functional effects can be observed that are thought to be independent of any hemodynamic changes produced by these compounds. Because biologically active arachidonic acid metabolites are rapidly metabolized, they act predominantly in an autocrine or paracrine fashion and, thus, their locus of action will be quite close to their point of generation. Thus, one can expect that direct epithelial effects of these compounds will result when they are produced by the tubule cells themselves or the neighboring interstitial cells and the tubules possess an appropriate receptor for the ligand.

Proximal Tubule

Neither the proximal convoluted tubule nor the proximal straight tubule appears to produce amounts of biologically active cyclooxygenase metabolites of arachidonic acid. As will be discussed in a subsequent section, the dominant arachidonate metabolites produced by proximal convoluted and straight tubules are metabolites of the cytochrome P-450 pathway.[259]

Early whole animal studies suggested that PGE2 might have an action in the proximal tubule because of its effects on urinary phosphate excretion. PGE2 blocked the phosphaturic action of calcitonin infusion in thyroparathyroidectomized rats. Nevertheless, studies utilizing in vitro perfused proximal tubules failed to show an effect of PGE2 on sodium chloride or phosphate transport in the proximal convoluted tubule. More recent studies suggest PGE2 may play a key role in the phosphaturic action of FGF23,[260] because phosphaturia in hyp mice with X-linked hyperphosphaturia is associated with markedly increased urine PGE2 excretion and phosphaturia was normalized by indomethacin.[261] Nevertheless, there are very little data on the actions of other cyclooxygenase metabolites in proximal tubules and scant molecular evidence for expression of classic G-protein coupled prostaglandin receptors in this segment of the nephron.

Loop of Henle

The nephron segments making up the loop of Henle also display limited metabolism of exogenous arachidonic acid through the cyclooxygenase pathway, although given the realization that COX-2 is expressed in this segment, it is of note that PGE2 was uniformly greater in the cortical segment than the medullary thick ascending limb. The thick ascending limb has been shown to exhibit PGE2 receptors in high density.[262] Studies have also demonstrated high expression levels of mRNA for the EP3 receptor in medullary TAL of both rabbit and rat[201] (see earlier section on the EP3 receptor). Subsequent to the demonstration that PGE2 inhibits sodium chloride absorption in the medullary thick ascending limb of the rabbit TAL perfused in vitro, it was shown that PGE2 blocks ADH but not cyclic AMP stimulated sodium chloride absorption in the medullary thick ascending limb of the mouse. It is likely that the mechanism involves activation of Gi and inhibition of adenyl cyclase by PGE2, possibly via the EP3 receptors expressed in this segment.

Collecting Duct System

In vitro perfusion studies of rabbit cortical collecting tubule demonstrated that PGE2 directly inhibits sodium transport in the collecting duct when applied to the basolateral surface of this nephron segment. It is now apparent that PGE2 utilizes multiple signal transduction pathways in the cortical collecting duct, including those that modulate intracellular cyclic AMP levels and Ca++. PGE2 can stimulate or suppress cyclic AMP accumulation. The latter may also involve stimulation of phosphodiesterase. Although modulation of cyclic AMP levels appears to play an important role in PGE2 effects on water transport in the cortical collecting duct (see following section), it is less clear that PGE2 affects sodium transport via modulation of cyclic AMP levels.[199] PGE2 has been shown to increase cell calcium possibly coupled with PKC activation in in vitro perfused cortical collecting ducts.[263] This effect may be mediated by the EP1 receptor subtype coupled to phosphatidylinositol hydrolysis.[206]

Water Transport

Vasopressin regulated water transport in the collecting duct is markedly influenced by cyclooxygenase products, especially prostaglandins. When cyclooxygenase inhibitors are administered to humans, rats, or dogs, the antidiuretic action of arginine vasopressin is markedly augmented. Because vasopressin also stimulates endogenous PGE2 production by the collecting duct, these results suggest that PGE2 participates in a negative feedback loop, whereby endogenous PGE2 production dampens the action of AVP.[264] In agreement with this model, the early classical studies of Grantham and Orloff directly demonstrated that PGE1 blunted the water permeability response of the cortical collecting duct to vasopressin. In these early studies, the action of PGE1 appeared to be at a pre-cyclic AMP step. Interestingly, when administered by itself PGE1 modestly augmented basal water permeability. These earlier studies have been confirmed with respect to PGE2. PGE2 also stimulates basal hydraulic conductivity and suppresses the hydraulic conductivity response to AVP in rabbit cortical collecting duct. [265] [266] Inhibition of both AVP stimulated cAMP generation and water permeability appears to be mediated by the EP1 and EP3 receptors, whereas the increase in basal water permeability may be mediated by the EP4 receptor.[229] These data are consistent with functional redundancy between the EP1 and EP3 with respect to their effects on vasopressin stimulated water absorption in the collecting duct.

Metabolism of Prostaglandins

15-keto Dehydrogenase

The half life of prostaglandins is 3 to 5 minutes and that of TxA2 is approximately 30 seconds. Elimination of PGE2, PGF and PGI2 proceeds through enzymatic and non-enzymatic pathways, whereas that of TxA2 is non-enzymatic. The end products of all of these degradative reactions generally possess minimal biologic activity, although this is not uniformly the case (see later discussion). The principal enzyme involved in the transformation of PGE2, PGI2, and PGF is 15-hydroxyprostaglandin dehydrogenase (PGDH), which converts the 15 alcohol group to a ketone.[267]

15-PGDH is an NAD+/NADP+-dependent enzyme that is 30 to 49 times more active in the kidney of the young rat (3 weeks of age) than in the adult. It is mainly localized in cortical and juxtamedullary zones,[268] with little activity detected in papillary slices. Its Km for PGE2 is 8.4 μM and 22.6 μM for PGF.[267] Disruption of this gene in mice results in persistent patent ductus arteriosus PDA, thought to be a result of failure of circulating PGE2 levels to fall in the immediate peripartum period.[269] Thus administration of cyclooxygenase inhibiting NSAIDs rescues the knockout mice by decreasing prostaglandins and allowing the animals to survive.

Subsequent catalysis of 15-hydroxy products by a delta-13 reductase leads to the formation of 13,14 dihydro compounds. PGI2 and TxA2 undergo rapid degradation to 6-keto-PGF and TxB2 respectively.[267] These stable metabolites are usually measured and their rates of formation taken as representative of those of the parent molecules.

ω/ω-1-Hydroxylation of Prostaglandins

Both PGA2 and PGE2 have been shown to undergo hydroxylation of the terminal or sub-termi nal carbons by a cytochrome P450 dependent mechanism.[270] This reaction may be mediated by a CYP4A family members or CYP4F enzyme. Both CYP4A[271] and CYP4F members have been mapped along the nephron.[272] Some of these derivatives have been shown to exhibit biological activity.

Cyclopentenone Prostaglandins

The cyclopentenone prostaglandins include PGA2, a PGE2 derivative, and PGJ2, a derivative of PGD2. Although it remains uncertain whether these compounds are actually produced in vivo, this possibility has received increasing attention because some cyclopentenone prostanoids been shown to be activating ligands for nuclear transcription factors, including PPARd and PPARg. [273] [274] [275] The realization that the antidiabetic thiazolidinedione drugs act through PPARg to exert their antihyperglycemic and insulin sensitizing effects[276] has generated intense interest in the possibility that the cyclopentenone prostaglandins might serve as the endogenous ligands for these receptors. An alternative biologic activity of these compounds has been recognized in their capacity to covalently modify thiol groups, forming adducts with cysteine of several intracellular proteins including thioredoxin 1, vimentin, actin, and tubulin.[277] Studies regarding biological activity of cyclopentenone prostanoids abound and the reader is referred to several excellent sources in the literature. [278] [279] [280] Although evidence supporting the presence of these compounds in vivo exists,[281] it remains uncertain whether they can form enzymatically or are an unstable spontaneous dehydration product of the E and D ring prostaglandins.[282]

Non-enzymatic Metabolism of Arachidonic Acid

It has long been recognized that oxidant injury can result in peroxidation of lipids. In 1990, Morrow and Roberts reported that a series of prostaglandin-like compounds can be produced by free radical catalyzed peroxidation of arachidonic acid that is independent of cyclooxygenase activity.[283] These compounds, which are termed “isoprostanes”, are increasingly utilized as a sensitive marker of oxidant injury in vitro and in vivo.[284] In addition, at least two of these compounds, 8-iso-PGF (15-F2-isoprostane) and 8-iso-PGE2 (15-E2-isoprostane) are potent vasoconstrictors when administered exogenously.[285] 8-iso-PGF has been shown to constrict the renal microvasculature and decrease GFR, an effect that is prevented by thromboxane receptor antagonism.[286] However, the role of endogenous isoprostanes as mediators of biologic responses remains unclear.

Prostaglandin Transport and Urinary Excretion

It is notable that most of the prostaglandin synthetic enzymes have been localized to the intracellular compartment, yet extracellular prostaglandins are potent autocoids and paracrine factors. Thus, prostanoids must be transported extra-cellularly to achieve efficient metabolism and termination of their signaling. Similarly, enzymes that metabolize PGE2 to inactive compounds are also intra-cellular, requiring uptake of the prostaglandin for its metabolic inactivation. The molecular basis of these extrusion and uptake processes are only now being defined.

As a fatty acid, prostaglandins may be classified as an organic anion at physiological pH. Early microperfusion studies documented that basolateral PGE2 could be taken up into proximal tubules cells and actively secreted into the lumen. Furthermore this process could be inhibited by a variety of inhibitors of organic anion transport including PAH, probenecid, and indomethacin. Studies of basolateral renal membrane vesicles also supported the notion that this transport process was via an electroneutral anion exchanger. These studies are of note because renal prostaglandins enter the urine in Henle's loop, and late proximal tubule secretion could provide an important entry mechanism.

Recently a molecule that mediates PGE2 uptake in exchange for lactate has been cloned and christened “PGT” for prostaglandin transporter.[287] PGT is a member of SLC21/SLCO: organic anion transporting family (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=5032094000672) and its cDNA encodes a transmembrane protein of 100 amino acids that exhibits broad tissue distribution heart, placenta, brain, lung, liver, skeletal muscle, pancreas, kidney, spleen, prostate, ovary, small intestine, and colon. [288] [289] [290] Immunocytochemical studies of PGT expression in rat kidneys suggest expression primarily in glomerular endothelial and mesangial cells, arteriolar endothelial and muscularis cells, principal cells of the collecting duct, medullary interstitial cells, medullary vasa rectae endothelia, and papillary surface epithelium.[291] PGT appears to mediate PGE2 uptake rather than release,[292] allowing target cells to metabolize this molecule and terminate signaling.[293]

Other members of the organic cation/anion/zwitterion transporter family SLC22 family have also been shown to transport prostaglandins[287] and have been suggested to mediate prostaglandin excretion into the urine. Specifically OAT1 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=24497474000672) and OAT3 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=24497498000672) are localized on the basolateral proximal tubule membrane, where they likely participate in urinary excretion of PGE2. [294] [295] Conversely members of the multidrug resistance protein (MRP) have been shown to transport prostaglandins in an ATP dependent fashion. [296] [297] MRP2 (also designated ABBC2) is expressed in kidney proximal tubule brush borders and may contribute to the transport (and urinary excretion) of glutathione conjugated prostaglandins. [298] [299] This transporter has more limited tissue expression, restricted to the kidney, liver, and small intestine and could contribute not only to renal PAH excretion but also to prostaglandin excretion as well.[300]

INVOLVEMENT OF CYCLOOXYGENASE METABOLITES IN RENAL PATHOPHYSIOLOGY

Experimental and Human Glomerular Injury

Glomerular Inflammatory Injury

Cyclooxygenase metabolites have been implicated in functional and structural alterations in glomerular and tubulointerstitial inflammatory diseases.[301] Essential fatty acid deficiency totally prevents the structural and functional consequences of administration of nephrotoxic serum (NTS) to rats, an experimental model of antiglomerular basement membrane glomerulonephritis.[302] Changes in arteriolar tone during the course of this inflammatory lesion are mediated principally by locally released COX and lipoxygenase (LO) metabolites of AA.[302]

TxA2 release appears to play an essential role in mediating the increased renovascular resistance observed during the early phase of this disease. Subsequently, increasing rates of PGE2 generation may account for progressive dilation of renal arterioles and increases in renal blood flow at later stages of the disease. Consistent with this hypothesis, TxA2 antagonism ameliorated the falls in RBF and GFR two hours post-NTS administration, but not at one day. During the later, heterologous, phase of NTS, COX metabolites mediate both the renal vasodilation as well as the reduction in Kf that characterize this phase.[302] The net functional result of COX inhibition during this phase of experimental glomerulonephritis, therefore, would depend on the relative importance of renal perfusion versus the preservation of Kf to the maintenance of GFR. Evidence also indicates that COX metabolites are mediators of pathologic lesions and the accompanying proteinuria in this model. COX-2 expression in the kidney increases in experimental anti-GBM glomerulonephritis [303] [304] and after systemic administration of lipopolysaccharide.[305]

A beneficial effect of fish oil diets (enriched in eicosapentaenoic acid), with an accompanying reduction in the generation of COX products, has been demonstrated on the course of genetic murine lupus (MRL-lpr mice). In subsequent studies, enhanced renal TxA2 and PGE2 generation was demonstrated in this model, as well as in NZB mice, another genetic model of lupus. In addition, studies in humans demonstrated an inverse relation between TxA2biosynthesis and glomerular filtration rate and improvement of renal function following short-term therapy with a thromboxane receptor antagonist in patients with lupus nephritis. More recently, studies have indicated that in humans, as well as NZB mice, COX-2 expression was up-regulated in patients with active lupus nephritis, with colocalization to infiltrating monocytes, suggesting that monocytes infiltrating the glomeruli contribute to the exaggerated local synthesis of TXA2. [306] [307] COX-2 inhibition selectively decreased thromboxane production, and chronic treatment of NZB mice with a COX-2 inhibitor and mycophenolate mofetil significantly prolonged survival.[307] Taken together, these data, as well as others from animal and human studies support a major role for the intrarenal generation of TxA2 in mediating renal vasoconstric-tion during inflammatory and lupus-associated glomerular injury.

The demonstration of a functionally significant role for COX metabolites in experimental and human inflammatory glomerular injury has raised the question of the cellular sources of these eicosanoids in the glomerulus. In addition to infiltrating inflammatory cells, resident glomerular macrophages, glomerular mesangial cells, and glomerular epithelial cells represent likely sources for eicosanoid generation. In the anti-Thy1.1 model of mesangioproliferative glomerulonephritis, COX-1 staining was transiently increased in diseased glomeruli at day 6, and was localized mainly to proliferating mesangial cells. COX-2 expression in the macula densa region also transiently increased at day 6. [308] [309] Glomerular COX-2 expression in this model has been controversial, with one group reporting increased podocyte COX-2 expression[304] and two other groups reporting minimal, if any glomerular COX-2 expression.[308] [309] However, it is of interest that selective COX-2 inhibitors have been reported to inhibit glomerular repair in the anti-Thy1.1 model.[309] In both anti-Thy1.1 and anti-GBM models of glomerulonephritis, the non-selective COX inhibitor, indomethacin, increased monocyte chemoattractant protein-1 (MCP-1), suggesting that prostaglandins may repress recruitment of monocytes/macrophages in experimental glomerulonephritis.[310]

A variety of cytokines have been reported to stimulate PGE2 synthesis and COX-2 expression in cultured mesangial cells. Furthermore, complement components, in particular C5b-9, which are known to be involved in the inflammatory models described earlier, have been implicated in the stimulation of PGE2 synthesis in glomerular epithelial cells. Cultured GEC express predominantly COX-1, but exposure to C5b-9 significantly increased COX-2 expression.

Glomerular Non-Inflammatory Injury

Studies have suggested that prostanoids may also mediate altered renal function and glomerular damage following subtotal renal ablation, and glomerular prostaglandin production may be altered in such conditions. Glomeruli from remnant kidneys, as well as animals fed a high protein diet, have increased prostanoid production. These studies suggested an increase in cyclooxygenase enzyme activity per se rather than, or in addition to, increased substrate availability because increases in prostanoid production were noted when excess exogenous arachidonic acid was added.

Following subtotal renal ablation, there are selective increases in renal cortical and glomerular COX-2 mRNA and immunoreactive protein expression, without significant alterations in COX-1 expression.[311] This increased COX-2 expression was most prominent in the macula densa and surrounding cTALH. In addition, COX-2 immunoreactivity was also present in podocytes of remnant glomeruli, and increased prostaglandin production in isolated glomeruli from remnant kidneys was inhibited by a COX-2 selective inhibitor but was not decreased by a COX-1 selective inhibitor.[311] Of interest, Weichert and colleagues have recently reported that in the fawn-hooded rat, which develops spontaneous glomerulosclerosis, there is increased cTALH/macula densa COX-2 and nNOS and juxtaglomerular cell renin expression preceding development of sclerotic lesions.[312]

When given 24 hours after subtotal renal ablation, a non-selective NSAID, indomethacin, normalized increases in renal blood flow and single nephron GFR; similar decreases in hyperfiltration were noted when indomethacin was given acutely to rats 14 days after subtotal nephrectomy, although in this latter study, the increased glomerular capillary pressure (PGC) was not altered because both afferent and efferent arteriolar resistances increased. Previous studies have also suggested that non-selective cyclooxygenase inhibitors may acutely decrease hyperfiltration in diabetes and inhibit proteinuria and/or structural injury; more recent studies have indicated selective COX-2 inhibitors will decrease the hyperfiltration seen in experimental diabetes or increased dietary protein. [313] [314] Of note, NSAIDs have also been reported to be effective in reducing proteinuria in patients with refractory nephrotic syndrome.

The prostanoids involved have not yet been completely characterized, although it is presumed that vasodilatory prostanoids are involved in mediation of the altered renal hemodynamics. Defective autoregulation of renal blood flow due to decreased myogenic tone of the afferent arteriole is seen after either subtotal ablation or excessive dietary protein and is corrected by inhibition of cyclooxygenase activity. In these hyperfiltering states, tubuloglomerular feedback (TGF) is reset at a higher distal tubular flow rate. Such a resetting dictates that afferent arteriolar vasodilatation will be maintained in the face of increased distal solute delivery. It has previously been shown that the alterations in TGF sensitivity after reduction in renal mass are prevented with the non-selective cyclooxygenase inhibitor, indomethacin. An important role has been suggested for neuronal nitric oxide synthase, which is localized to the macula densa, in the vasodilatory component of TGF. [315] [316] [317] Of interest, studies by Ishihara and co-workers have determined that this nNOS-mediated vasodilation is inhibited by the selective COX-2 inhibitor, NS398, suggesting that COX-2-mediated prostanoids may be essential for arteriolar vasodilation. [45] [318]

Administration of COX-2 selective inhibitors decreased proteinuria and inhibited development of glomerular sclerosis in rats with reduced functioning renal mass. [319] [320] In addition, COX-2 inhibition decreased mRNA expression of TGF-β1 and types III and IV collagen in the remnant kidney.[319] Similar protection was observed with administration of nitroflurbiprofen (NOF), a NO-releasing NSAID without gastrointestinal toxicity.[321] Prior studies have also demonstrated that thromboxane synthase inhibitors retarded progression of glomerulosclerosis, with decreased proteinuria and glomerulosclerosis in rats with remnant kidneys and in diabetic nephropathy, in association with increased renal prostacyclin production and lower systolic blood pressure. [322] [323] Studies in models of type I and type II diabetes have indicated that COX-2 selective inhibitors retarded progression of diabetic nephropathy. [324] [325] Schmitz and associates confirmed increases in thromboxane B2 excretion in the remnant kidney and correlated decreased arachidonic and linoleic acid levels with increased thromboxane production because the thromboxane synthase inhibitor U63557A restored fatty acid levels and retarded progressive glomerular destruction.[322]

Enhanced glomerular synthesis and/or urinary excretion of both PGE2 and TxA2 have been demonstrated in passive Heymann nephritis (PHN), and Adriamycin-induced glomerulopathies in rats. Both COX-1 and COX-2 expression are increased in glomeruli with PHN.[326] Both thromboxane synthase inhibitors and selective COX-2 inhibitors also decreased proteinuria in PHN.

In contrast to the putative deleterious effects of thromboxane, the prostacyclin analog, cicaprost, retarded renal damage in uninephrectomized dogs fed a high sodium and high protein diet, an effect that was not mediated by amelioration of systemic hypertension.[327]

Prostanoids have also been shown to alter extracellular matrix production by mesangial cells in culture. Thromboxane A2 stimulates matrix production by both TGF-b-dependent and -independent pathways.[328] PGE2 has been reported to decrease steady state mRNA levels of alpha 1(I) and alpha 1(III) procollagens, but not alpha 1(IV) procollagen and fibronectin mRNA, and to reduce secretion of all studied collagen types into the cell culture supernatants. Of interest, this effect did not appear to be mediated by cAMP.[329] PGE2 has also been reported to increase production of matrix metalloproteinase-2 and to mediate angiotensin II-induced increases in MMP-2.[330]Whether vasodilatory prostaglandins mediate decreased fibrillar collagen production and increased matrix degrading activity in glomeruli in vivo has not yet been studied; however, there is compelling evidence in nonrenal cells that prostanoids may either mediate or modulate matrix production.[331] Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis exhibit decreased ability to express COX-2 and to synthesize PGE2.[332]

Acute Renal Failure (ARF)

When cardiac output is compromised, as in extracellular fluid volume depletion or congestive heart failure, systemic blood pressure is preserved by the action of high circulating levels of systemic vasoconstrictors (norepinephrine, angiotensin II, AVP). Amelioration of their effects within the renal vasculature serves to blunt the development of otherwise concomitant marked depression of renal blood flow. Intrarenal generation of vasodilator products of AA, including PGE2 and PGI2, is a central part of this protective adaptation. Increased renal vascular resistance induced by exogenously administered angiotensin II or renal nerve stimulation (increased adrenergic tone) is exaggerated during concomitant inhibition of prostaglandin synthesis. Experiments in animals with volume depletion have demonstrated the existence of intrarenal AVP-prostaglandin interactions similar to those described earlier for angiotensin II. Studies in patients with congestive heart failure have confirmed that enhanced prostaglandin synthesis is crucial in protecting kidneys from various vasoconstrictor influences in this condition.

Acute renal failure accompanying the acute administration of endotoxin in rats is characterized by progressive reductions in RBF and GFR in the absence of hypotension. Renal histology in such animals is normal, but cortical generation of COX metabolites is markedly elevated. A number of reports have provided evidence for a role for TxA2-induced renal vasoconstriction in this model of renal dysfunction.[333] In addition, roles for PGs and TxA2 in modulating or mediating renal injury have been suggested in ischemia/reperfusion[334] and models of toxin-mediated acute tubular injury including those induced by uranyl nitrate,[335] amphotericin B,[336] aminoglycosides,[337]and glycerol.[338] In experimental acute renal failure, administration of vasodilator prostaglandins has been shown to ameliorate injury.[339]

Urinary Tract Obstruction

Following induction of chronic (more than 24 hours) ureteral obstruction, renal PG and TxA2 synthesis is markedly enhanced, particularly in response to stimuli such as endotoxin or bradykinin. Enhanced prostanoid synthesis likely arises from infiltrating mononuclear cells, proliferating fibroblast-like cells, interstitial macrophages, and interstitial medullary cells. Considerable evidence, derived from studies utilizing specific enzyme inhibitors, suggests a causal relationship between increased renal generation of this eicosanoid and the intense vasoconstriction that characterizes the hydronephrotic or post-obstructed kidney (reviewed in Ref. 301). In this sense, therefore, hydronephrotic injury can be regarded as a form of sub-acute inflammatory insult in which intrarenal eicosanoid generation from infiltrating leukocytes contributes to the pathophysiologic process. Finally, TxA2 has been implicated in the resetting of the tubuloglomerular feedback mechanism observed in hydronephrotic kidneys.[340] Recent studies have also suggested that selective COX-2 inhibitors may prevent renal damage in response to unilateral ureteral obstruction. [341] [342]

Allograft Rejection and Cyclosporine Nephrotoxicity

Allograft Rejection

Coffman and colleagues demonstrated that acute administration of a TxA2 synthesis inhibitor was associated with significant improvement in rat renal allograft function.[343] A number of other experimental and clinical studies have also demonstrated increased TxA2 synthesis during allograft rejection, [344] [345] leading some to suggest that increased urinary TxA2 excretion may be an early indicator in renal and cardiac allograft rejection.

Calcineurin Inhibitor Nephrotoxicity

Numerous investigators have demonstrated effects for cyclosporine A (CY-A) on renal prostaglandin/TxA2 synthesis, and provided evidence for a major role for renal and leukocyte TxA2 synthesis in mediating acute as well as chronic CY-A nephrotoxicity in rats.[346] Fish oil-rich diets, TxA2 antagonists, or administration of CY-A in fish oil as vehicle have all been shown to reduce renal TxA2 synthesis and afford protection against nephrotoxicity. Moreover, CY-A has been reported to decrease renal COX-2 expression.[347]

Hepatic Cirrhosis and Hepatorenal Syndrome

Patients with cirrhosis of the liver show an increased renal synthesis of vasodilating PGs, as indicated by the high urinary excretion of PGs and/or their metabolites. Urinary excretion of 2-3-dinor 6-keto-PGF, an index of systemic PGI2 synthesis, is increased in patients with cirrhosis and hyperdynamic circulation, thus raising the possibility that systemic synthesis of PGI2 may contribute to the arterial vasodilatation of these patients. Inhibition of cyclooxygenase activity in these patients may cause a profound reduction in renal blood flow and glomerular filtration rate, a reduction in sodium excretion, and an impairment of free water clearance.[348] The sodium-retaining properties of NSAIDs are particularly exaggerated in patients with cirrhosis of the liver, attesting to the dependence of renal salt excretion on vasodilatory PGs. In the kidneys of rats with cirrhosis, COX-2 expression increases while COX-1 expression is unchanged; however, in these animals, selective inhibition of COX-1 leads to impaired renal hemodynamics and natriuresis, whereas COX-2 inhibition has no effect. [349] [350]

Diminished renal PG synthesis has been implicated in the pathogenesis of the severe sodium retention seen in hepatorenal syndrome, as well as in the resistance to diuretic therapy. [351] [352] There is reduced renal synthesis of vasodilating PGE2 in the face of activation of endogenous vasoconstrictors and a maintained or increased renal production of thromboxane A2. [348] [353] Therefore, an imbalance between vasoconstricting systems and the renal vasodilator PGE2 has been proposed as a contributing factor to the renal failure observed in this condition. However, administration of exogenous prostanoids to patients with cirrhosis is not effective either in ameliorating renal function or in preventing the deleterious effect of NSAIDs.[348]

Diabetes Mellitus

In the streptozotocin-induced model of diabetes in rats, COX-2 expression is increased in the cTALH/macula densa region. [313] [357] COX-2 immunoreactivity has also been detected in the macula densa region in human diabetic nephropathy.[354] Studies suggest that vasodilator prostanoids, PGI2 and PGE2, play an important role in the hyperfiltration seen early in diabetes mellitus.[355] In streptozotocin-induced diabetes in rats, previous studies indicated that non-selective cyclooxygenase inhibitors acutely decrease hyperfiltration in diabetes and inhibit proteinuria and/or structural injury,[356] and more recent studies have also indicated that acute administration of a selective COX-2 inhibitor decreased hyperfiltration.[313] Chronic administration of a selective COX-2 inhibitor significantly decreased proteinuria and reduced extracellular matrix deposition, as indicated by decreases in immunoreactive fibronectin expression and in mesangial matrix expansion. In addition, COX-2 inhibition reduced expression of TGF-b, PAI-1, and VEGF in the kidneys of the diabetic hypertensive animals.[357] The vasoconstrictor thromboxane A2 may play a role in the development of albuminuria and basement membrane changes with diabetic nephropathy. In addition, administration of a selective PGE2 EP1 receptor antagonist prevented development of experimental diabetic nephropathy.[358] In contrast to the proposed detrimental effects of these “vasoconstrictor” prostanoids, administration of a prostacyclin analog decreased hyperfiltration and reduced macrophage infiltration in early diabetic nephropathy by increasing eNOS expression in afferent arterioles and glomerular capillaries.[359]

Pregnancy

Most, but not all, investigators do not report increases in vasodilator PG synthesis or suggest an essential role for prostanoids in the mediation of the increased GFR and RPF of normal pregnancy[360]; however, diminished synthesis of PGI2 has been demonstrated in humans and in animal models of pregnancy-induced hypertension.[361] In the latter, inhibition of TxA2 synthetase has been associated with resolution of the hypertension, suggesting a possible pathophysiologic role.[362] A moderate beneficial effect of reducing TxA2 gener-ation, while preserving PGI2 synthesis, by low dose (60-100 mg/day) aspirin therapy has been demonstrated in patients at high risk for pregnancy-induced hypertension and pre-eclampsia. [363] [364]

THE LIPOXYGENASE PATHWAY

The lipoxygenase enzymes metabolize arachidonic acid to form leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and lipoxins (LXs) ( Fig. 11-13 ). These lipoxygenase metabolites are primarily produced by leukocytes, mast cells, and macrophages in response to inflammation and injury. There are three lipoxygenase enzymes, 5-, 12-, and 15-lipoxygenase, so named for the carbon of arachidonic acid where they insert an oxygen. The lipoxygenases are products of separate genes and have distinct distributions and patterns of regulation. Glomeruli, mesangial cells, cortical tubules, and vessels also produce the 12-lipoxygenase (12-LOX) product, 12(S)-HETE and the 15-LOX product, 15-HETE. Recent studies have localized 15-LO mRNA primarily to the distal nephron, and 12-LO mRNA to the glomerulus. 5-LO mRNA and 5-Lipoxygenase Activating Protein (FLAP) mRNA were expressed in the glomerulus and the vasa recta.[365] In polymorphonuclear leukocytes (PMNs) macrophages and mast cells, 5-lipoxygenase (5-LO) mediates the formation of leukotrienes.[366] 5-LO, which is regulated by FLAP, catalyzes the conversion of arachidonic acid to 5-HpETE and then to leukotriene A4 (LTA4).[367] LTA4 is then further metabolized to either the peptidyl-leukotrienes (LTC4 and LTD4) by glutathione-S-transferase or to LTB4 by LTA4 hydrolase. Although glutathione-S-transferase expression is limited to inflammatory cells, LTA4 hydrolase is also expressed in glomerular mesangial cells and endothelial cells[368]; PCR analysis has actually demonstrated ubiquitous LTA4hydrolase mRNA expression throughout the rat nephron.[365] Leukotriene C4 synthase mRNA could not be found in any nephron segment.[365]

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FIGURE 11-13  Pathways of lipoxygenase metabolism of arachidonic acid.

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Recently two cysteinyl leukotriene receptors (CysLTR) have been cloned and have been identified as members of the G protein coupled superfamily of receptors. They have been localized to vascular smooth muscle and endothelium of the pulmonary vasculature. [369] [370] [371] In the kidney the cysteinyl leukotriene receptor type 1 is expressed in the glomerulus, whereas cysteinyl receptor type 2 mRNA has not been detected in any nephron segment to date.[365]

The peptidyl-leukotrienes are potent mediators of inflammation and vasoconstrictors of vascular, pulmonary, and gastrointestinal smooth muscle. In addition, they increase vascular permeability and promote mucus secretion.[372]Because of the central role that peptidyl-leukotrienes play in the inflammatory trigger of asthma exacerbation, effective receptor antagonists have been developed and are now an important component of management of asthma.[373]

In the kidney, LTD4 administration has been shown to decrease renal blood flow and GFR, and peptidyl leuko-trienes are thought to be mediators of decreased RBF and GFR associated with acute glomerular inflammation. Micropuncture studies revealed that the decreases in GFR are the result of both afferent and arteriolar vasoconstriction, with more pronounced efferent vasoconstriction and a decrease in Kf.

In addition both LTC4 and LTD4 increase proliferation of cultured mesangial cells. The LTB4 receptor is also a seven-transmembrane G protein coupled receptor. On PMNs, receptor activation promotes chemotaxis, aggregation and attachment to endothelium. In the kidney LTB4 mRNA is localized to the glomerulus.[365] A second, low affinity LTB4 receptor is also expressed,[374] which may mediate calcium influx into PMNs, thereby leading to activation. LTB4 receptor blockers lessen acute renal ischemic-reperfusion injury[375] and nephrotoxic nephritis in rats,[376] and PMN infiltration and structural and functional evidence of organ injury by ischemia/reperfusion are magnified in transgenic mice overexpressing the LTB4 receptor.[377] In addition to activation of cell surface receptors, LTB4 has also been shown to be a ligand for the nuclear receptor PPARa.[378]

15-lipoxygenase (15-LO) leads to the formation of 15-S-HETE. In addition, dual oxygenation in activated PMNs and macrophages by 5- and 15-LO leads to formation of the lipoxins. LX synthesis also can occur via transcellular metabolism of the leukocyte-generated intermediate, LTA4, by 12-LO in platelets or adjoining cells including glomerular endothelial cells. [379] [380]

15-S-HETE is a potent vasoconstrictor in the renal microcirculation[381]; however, 15-LO-derived metabolites antagonize proinflammatory actions of leukotrienes, both by inhibiting PMN chemotaxis, aggregation, and adherence and by counteracting the vasoconstrictive effects of the peptidyl-leukotrienes. [382] [383] Administration of 15-S-HETE reduced LTB4 production by glomeruli isolated from rats with acute nephrotoxic serum-induced glomerulonephritis, and it has been proposed that 15-LO may regulate 5-LO activity in chronic glomerular inflammation because it is known that in experimental glomerulonephritis, lipoxin A4 (LXA4) administration increased renal blood flow and GFR in large part by inducing afferent arteriolar vasodilation, an effect mediated in part by release of vasodilator prostaglandins. LXA4 also antagonized the effects of LTD4 to decrease GFR, although not renal blood flow, even though administration of LXA4 and LXB4 directly into the renal artery induced vasoconstriction. Glomerular micropuncture studies revealed that LXA4 led to moderate decreases in Kf.[382] Lipoxins signal through a specific G-protein coupled receptor G protein-coupled receptor denoted ALXR. This receptor is related at the nucleotide sequence level to both chemokine and chemotactic peptide receptors, such as N-formyl peptide receptor.[384] It is also noteworthy that in isolated perfused canine renal arteries and veins, LTC4 and LTD4 were found to be vasodilators, which were partially dependent upon an intact endothelium and was mediated by nitric oxide production.[385]

Recently, a potential interaction between cyclooxygenase- and lipoxygenase-mediated pathways has been reported. Whereas aspirin inhibits prostaglandin formation by both COX-1 and COX-2, aspirin-induced acetylation converts COX-2 to a selective generator of 15-R-HETE. This product can then be released, taken up in a transcellular route by PMNs and converted to 15-epi-lipoxins, which have similar biological actions as the lipoxins.[386]

Similar to 15-HETE, 12(S)-HETE also potently vasoconstricts glomerular and renal vasculature.[379] 12(S)-HETE increases protein kinase C and depolarizes cultured vascular smooth muscle cells. Afferent arteriolar vasoconstriction and increases in smooth muscle calcium in response to 12(S)-HETE, were partially inhibited by voltage-gated L-type calcium channel inhibitors.[387] 12(S)-HETE has also been proposed to be an angiogenic factor because in cultured endothelial cells, 12-LO inhibition reduces cell proliferation and 12-LO overexpression stimulates cell migration and endothelial tube formation.[388] 12/15 LO inhibitors and elective elimination of the leukocyte 12-LO enzyme also ameliorate the development of diabetic nephropathy in mice.[389] There is also interaction between 12/15-LO pathways and TGF-b-mediated pathways in the diabetic kidney.[390] 12(S)-HETE has also been proposed to be a mediator of renal vasoconstriction by angiotensin II, with inhibition of the 12-LO pathway attenuating angiotensin II-mediated afferent arteriolar vasoconstriction and decreased renal blood flow.[391] Lipoxygenase inhibition also blunted renal arcuate artery vasoconstriction by norepinephrine and KCl.[392] However, 12-LO products have also been implicated as inhibitors of renal renin release. [393] [394]

Although the major significance of LO products in the kidney derives from their release from infiltrating leukocytes or resident cells of macrophage/monocyte origin, there is evidence to suggest that intrinsic renal cells are capable of generating LTs and LXs either directly or through transcellular metabolism of intermediates.[395] Human and rat glomeruli can generate 12- and 15-HETE, though the cells of origin are unclear. LTB4 can be detected in supernatants of normal rat glomeruli, and its synthesis could be markedly diminished by maneuvers that depleted glomeruli of resident macrophages, such as irradiation or fatty acid deficiency. In addition, 5, 12, and 15-HETEs were detected from pig glomeruli, and their structural identity confirmed by mass spectrometry. 12-LO products are increased in mesangial cells exposed to hyperglycemia and in diabetic nephropathy.[396] Glomeruli subjected to immune injury release LTB4,[397] and LTB4 generation was suppressed by resident macrophage depletion. Synthesis of peptido-LTs by inflamed glomeruli has also been demonstrated,[398] but leukocytes could not be excluded as its primary source LXA4 is generated by immune-injured glomeruli.[399] Rat mesangial cells generate LXA4 when provided with LTA4 as substrate, thereby providing a potential intraglomerular source of LXs during inflammatory reactions. In non-glomerular tissue, 12-HETE production has been reported from rat cortical tubules and epithelial cells and 12- and 15-HETE from rabbit medulla.

Biological Activities of Lipoxygenase Products in the Kidney

In early experiments, systemic administration of LTC4 in the rat and administration of LTC4 and LTD4 in the isolated perfused kidney revealed potent renal vasoconstrictor actions of these eicosanoids. Subsequently, micropuncture measurements revealed that LTD4 exerts preferential constrictor effects on post-glomerular arteriolar resistance and depresses Kf and GFR. The latter is likely due to receptor-mediated contraction of glomerular mesangial cells, which has been demonstrated for LTC4 and LTD4 in vitro (see above). These actions of LTD4 in the kidney are consistent with its known smooth-muscle contractile properties. LTB4, a potent chemotactic and leukocyte-activating agent, is devoid of constrictor action in the normal rat kidney. Lipoxin A4 dilates afferent arterioles when infused into the renal artery, without affecting efferent arteriolar tone. This results in elevations in intraglomerular pressure and plasma flow rate, thereby augmenting GFR.

Involvement of Lipoxygenase Products in Renal Pathophysiology

Increased generation rates of LTC4 and LTD4 have been documented in glomeruli from rats with immune complex nephritis and mice with spontaneously developing lupus nephritis. [366] [399] Moreover, results from numerous physiologic studies utilizing specific LTD4 receptor antagonists have provided strong evidence for the release of these eicosanoids during glomerular inflammation. In four animal models of glomerular immune injury (anti-GBM nephritis, anti-Thy1.1 antibody-mediated mesangiolysis, passive Heymann nephritis, and murine lupus nephritis) acute antagonism of LTD4 by receptor binding competition or inhibition of LTD4 synthesis led to highly significant increases in GFR in nephritic animals.[400] The principal mechanism underlying the im-provement in GFR was reversal of the depressed values of the glomerular ultrafiltration coefficient (Kf), which is characteristically compromised in immune injured glomeruli. In other studies in PHN, Katoh and colleagues provided evidence that endogenous LTD4 not only mediates reductions in Kf and GFR, but that LTD4-evoked increases in intraglomerular pressure underlie, to a large extent, the accompanying proteinuria.[400] Cysteinyl-leukotrienes have been implicated in cyclosporine nephrotoxicity.[401] Of interest, 5-lipoxygenase deficiency accelerates renal allograft rejection.[402]

LTB4 synthesis, measured in the supernates of isolated glomeruli, is markedly enhanced early in the course of several forms of glomerular immune injury.[403] Cellular sources of LTB4 in injured glomeruli include PMNs and macrophages. All studies concur as to the transient nature of LTB4 release. LTB4 production decreases 24 hours after onset of the inflammation, which coincides with macrophage infiltration, a major source of 15-LO activity.[404]15-HPETE incubation decreased lipopolysaccharide-induced tumor necrosis factor (TNF) expression in a human monocytic cell line,[405] and HVJ-liposome-mediated glomerular transfection of 15-LO in rats decreased markers of injury (BUN, proteinuria) and accelerated functional (GFR, renal blood flow) recovery in experimental glomerulonephritis.[406] In addition, MK501, a FLAP antagonist, restored size selectivity and decreased glomerular permeability in acute GN.[407]

The suppression of LTB4 synthesis beyond the first 24 hours of injury is rather surprising, since both PMN and macrophages are capable of effecting the total synthesis of LTB4 (they contain the two necessary enzymes that convert arachidonic acid to LTB4, namely 5-LO and LTA4-hydrolase). It has therefore been suggested, based on in vitro evidence that the major route for LTB4 synthesis in inflamed glomeruli is through transcellular metabolism of leukocyte-generated LTA4 to LTB4 by LTA4-hydrolase present in glomerular mesangial, endothelial, and epithelial cells. Since the transformation of LTA4 to LTB4 is rate-limiting, regulation of LTB4 synthetic rate might relate to regulation of LTA4-hydrolase gene expression or catalytic activity in these parenchymal cells, rather than to the number of infiltrating leukocytes. In any case, leukocytes represent an indispensable source for LTA4, the initial 5-LO product and the precursor for LTB4, since endogenous glomerular cells do not express the 5-LO gene.[408] Thus, it was demonstrated that the polymorphonuclear (PMN) cell-specific activator, N-Formyl-Met-Leu-Phe, stimulated LTB4 production from isolated perfused kidneys harvested from NTS-treated rats to a significantly greater degree than from control animals treated with non-immune rabbit serum.[409] The renal production of LTB4 correlated directly with renal myeloperoxidase activity, suggesting interdependence of LTB4 generation and PMN infiltration.

The acute and long-term significance of LTB4 generation in conditioning the extent of glomerular structural and functional deterioration has been highlighted in studies in which LTB4 was exogenously administered or in which its endogenous synthesis was inhibited. Intrarenal administration of LTB4 to rats with mild NTS-induced injury was associated with an increase in PMN infiltration, reduction in renal plasma flow rate, and marked exacerbation of the fall in glomerular filtration rate, the latter correlating strongly with the number of infiltrating PMNs/glomerulus, while inhibition of 5-lipoxygenase led to preservation of GFR and abrogation of proteinuria.[409] Similarly, both 5-LO knockout mice and wild type mice treated with the 5-LO inhibitor, zileuton, had reduced renal injury in response to ischemia/reperfusion.[410] Thus, while devoid of vasoconstrictor actions in the normal kidney, increased intrarenal generation of LTB4 during early glomerular injury amplifies leukocyte-dependent reductions in glomerular perfusion and filtration rates and inflammatory injury, likely due to enhancement of PMN recruitment/activation.

THE CYTOCHROME P450 PATHWAY

Following their elucidation and characterization as endogenous metabolites of arachidonic acid, numerous studies have investigated the possibility that cytochrome P450 (CYP450) arachidonic acid metabolites subserve physiologic and/or pathophysiologic roles in the kidney ( Fig. 11-14 ). In whole animal physiology, these compounds have been implicated in the mediation of release of peptide hormones, regulation of vascular tone, and regulation of volume homeostasis. On the cellular level, CYP arachidonic acid metabolites have been proposed to regulate ion channels and transporters and to act as mitogens.

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FIGURE 11-14  Pathways of CYP450 metabolism of arachidonic acid.

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CYP450 monooxygenases are mixed-function oxidases that utilize molecular oxygen and NADPH as cofactors [411] [412] and will add an oxygen molecule to arachidonic acid in a regio- and stereo-specific geometry. CYP450 monooxy-genase pathways metabolize arachidonic acid to generate HETEs and epoxyeicosatrienoic acids (EETs), the latter of which can be hydrolyzed to dihydroxyeicosatrienoic acids (DHETs). [411] [412] [413] The kidney displays one of the highest CYP450 activities of any organ and produces CYP450 arachidonic acid metabolites in significant amounts. [387] [411] [414] HETEs are formed primarily via CYP450 hydroxylase enzymes and EETs and DHETs are formed primarily via CYP450 epoxygenase enzymes.[414] The CYP450 4A gene family is the major pathway for synthesis of hydroxylase metabolites, especially 20-HETE and 19-HETE, [413] [414] whereas the production of epoxygenase metabolites is primarily via the 2C gene family. [387] [411] A member of the 2J family that is an active epoxygenase is also expressed in the kidney.[415] CYP450 enzymes have been localized to both vasculature and tubules.[413] The 4A family of hydroxylases is expressed in preglomerular renal arterioles, glomeruli, proximal tubules, the TALH, and macula densa.[416]

The 2C and 2J families of epoxygenases are expressed at highest levels in proximal tubule and collecting duct. [415] [417] When isolated nephron segments expressing CYP450 protein have been incubated with arachidonic acid, production of CYP450 arachidonic acid metabolites can be detected. 20-HETE and EETs are both produced in the afferent arterioles,[418] glomerulus,[419] and proximal tubule.[420] 20-HETE is the predominant CYP450 AA metabolite produced by the TALH and in the pericytes surrounding vasa recta capillaries, [421] [422] whereas EETs are the predominant CYP450 AA metabolites produced by the collecting duct.[423]

Renal production of both epoxygenase and hydroxylase metabolites has been shown to be regulated by hormones and growth factors, including angiotensin II, endothelin, bradykinin, parathyroid hormone, and epidermal growth factor. [387] [412] [413] Alterations in dietary salt intake also modulate CYP450 expression and activity.[424] Alterations in the production of CYP450 metabolites have also been reported with uninephrectomy, diabetes mellitus, and hypertension. [412] [413]

Vasculature

20-HETE

In rat and dog renal arteries and afferent arterioles, 20-HETE is a potent vasoconstrictor,[418] whereas it is a vasodilator in rabbit renal arterioles. The vasoconstriction is associated with membrane depolarization and a sustained rise in intracellular calcium. 20-HETE is produced in the smooth muscle cells, and its afferent arteriolar vasoconstrictive effects are mediated by closure of KCa channels through a tyrosine kinase- and ERK-dependent mechanism ( Fig. 11-15 ).

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FIGURE 11-15  Proposed interactions of CYP450 arachidonic acid metabolites derived from vascular endothelial cells and smooth muscle cells to regulate vascular tone.

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An interaction between CYP450 arachidonic acid metabolites and nitric oxide has also been demonstrated. NO can inhibit the formation of 20-HETE in renal VSM cells; a significant portion of NO's vasodilator effects in the preglomerular vasculature appear to be mediated by the inhibition of tonic 20-HETE vasoconstriction, and inhibition of 20-HETE formation attenuates the pressor response and fall in renal blood flow seen with NO synthase inhibition. [425] [426]

Epoxides

Unlike CYP450 hydroxylase metabolites, epoxygenase metabolites of arachidonic acid increase renal blood flow and glomerular filtration rate. [387] [412] [413] 11,12-EET and 14,15-EET vasodilate the preglomerular arterioles independently of COX activity, whereas 5,6-EET and 8,9-EET cause COX-dependent vasodilation or vasoconstriction.[427] It is possible that these COX-dependent effects are mediated by COX conversion of 5,6-EET and 8,9-EET to prostaglandin-or thromboxane-like compounds.[428] EETs are produced primarily in the endothelial cells and exert their vasoactive effects on the adjacent smooth muscle cells. In this regard, it has been suggested that EETs, and specifically 11,12-EET, may serve as an endothelium-derived hyperpolarizing factor (EDHF) in the renal microcirculation. [387] [429] EET-induced vasodilation is mediated by activation of KCa channels, through cAMP-dependent stimulation of protein kinase C.

CYP450 metabolites may serve as either second messengers or modulators of the actions of hormonal and paracrine agents. Vasopressin increases renal production of CYP450 metabolites, and increases in intracellular calcium and proliferation in cultured renal mesangial cells are augmented by EET administration.[430] CYP450 metabolites also may serve to modulate the renal hemodynamic responses of endothelin-1, with 20-HETE as a possible mediator of the vasoconstrictive effects and EETs counteracting the vasoconstriction. [431] [432] Formation of 20-HETE does not affect the ability of ET-1 to increase free intracellular calcium transients in renal vascular smooth muscle intracellular but appears to enhance the sustained elevations that represent calcium influx through voltage-sensitive channels.

CYP450 metabolites have also been implicated in mediation of renal vascular responses to angiotensin II. In the presence of AT1 receptor blockers, angiotensin II produces an endothelial-dependent vasodilation in rabbit afferent arterioles that is dependent on CYP450 epoxygenase metabolites production by AT2 receptor activation.[433] With intact AT1 receptors, angiotensin II increases 20-HETE release from isolated preglomerular microvessels through an endothelium-independent mechanism.[434] Angiotensin II's vasoconstrictive effects are in part the result of 20-HETE-mediated inhibition of KCa, which enhances sustained increases in intracellular calcium concentration by calcium influx through voltage-sensitive channels. Inhibition of 20-HETE production reduces the vasoconstrictor response to ANG II by >50% in rat renal interlobular arteries in which the endothelium has been removed.[434]

Autoregulation

CYP450 metabolites of AA have been shown to be mediators of renal blood flow autoregulatory mechanisms. When prostaglandin production was blocked in canine arcuate arteries, arachidonic acid administration enhanced myogenic responsiveness, and renal blood flow autoregulation was blocked by CYP450 inhibitors. [387] [413] Similarly, in the rat juxtamedullary preparation, selective blockade of 20-HETE formation significantly decreased afferent arteriolar vasoconstrictor responses to elevations in perfusion pressure, and inhibition of epoxygenase activity enhanced vasoconstriction,[435] suggesting that 20-HETE is involved in afferent arteriolar autoregulatory adjustment, whereas release of vasodilatory epoxygenase metabolites in response to increases in renal perfusion pressure acts to attenuate the vasoconstriction. In vivo studies have also implicated 20-HETE as a mediator of the autoregulatory response to increased perfusion pressure.[436] Bradykinin-induced efferent arteriolar vasodilation has been shown to be mediated in part by direct release of EETs from this vascular segment. In addition, bradykinin-induced release of 20-HETE from the glomerulus can modulate the EET-mediated vasodilation.[437]

Tubuloglomerular Feedback

CYP450 metabolites may also be involved in the tubuloglomerular feedback response.[413] As noted, 20-HETE is produced by both the afferent arteriole and macula densa, and studies have suggested the possibility that 20-HETE may either serve as a vasoconstrictive mediator of tubuloglomerular feedback (TGF) released by the macula densa or a second messenger in the afferent arteriole in response to mediators released by the macula densa, such as adenosine or ATP.[438] 20-HETE may also be a mediator of regulation of intrarenal distribution of blood flow. [439] [440]

Tubules

20-HETE and EETs both inhibit tubular sodium reabsorption. [412] [413] Renal cortical interstitial infusion of the nonselective CYP450 inhibitor 17-ODYA increases papillary blood flow, renal interstitial hydrostatic pressure, and sodium excretion without affecting total renal blood flow or glomerular filtration rate. High dietary salt intake in rats increases expression of the renal epoxygenase 2C23 and production and urinary excretion of EETs, while decreasing 20-HETE production in renal cortex. [411] [424] 14,15-EET has also been shown to inhibit renin secretion[441]; furthermore, clotrimazole, which is a relatively selective epoxygenase inhibitor, induced hypertension in rats fed a high salt diet, suggesting a role in regulation of blood pressure.[424]

Proximal Tubule

The proximal tubule contains the highest concentration of CYP450 within the mammalian kidney and expresses minimal cyclooxygenase and lipoxygenase activity. The 4A CYP450 family of hydroxylases that produce 19- and 20-HETE is highly expressed in mammalian proximal tubule.[271] CYP450 enzymes of both the 2C and 2J family that catalyze the formation of EETs are also expressed in the proximal tubule.[411] Both EETs and 20-HETE have been shown to be produced in the proximal tubule and have been proposed to be modulators of sodium reabsorption in the proximal tubule.

Studies in isolated perfused proximal tubule indicate that 20-HETE inhibits sodium transport whereas 19-HETE stimulates sodium transport, suggesting that 19-HETE may serve as competitive antagonist of 20-HETE. [420] [442]Administration of EETs inhibits amiloride-sensitive sodium transport in primary cultures of proximal tubule cells[443] and in LLC-PK1 cells, a non-transformed, immortalized cell line from pig kidney with proximal tubule characteristics. [444] [445]

20-HETE has been proposed to be a mediator of hormonal inhibition of proximal tubule reabsorption by PTH, dopamine, angiotensin II, and EGF. Although the mechanisms of 20-HETE's inhibition have not yet been completely elucidated, there is evidence that it can inhibit Na+/K+-ATPase activity by phosphorylation of the Na+/K+-ATPase alpha subunit through a protein kinase C dependent pathway. [446] [447]

Epoxyeicosatrienoic acids (EETs) may also serve as second messengers in the proximal tubule for EGF[448] and angiotensin II.[449] In the proximal tubule, angiotensin II has been noted to exert a biphasic response on net sodium uptake via AT1 receptors, with low (10-10–10-11) concentrations stimulating and high (10-7) concentrations inhibiting net uptake.[449] Such high concentrations are not normally seen in plasma but may exist in the proximal tubule lumen as a result of the local production of angiotensin II by proximal tubule.[450] The mechanisms by which CYPP450 AA metabolites modulate proximal tubule reabsorption have not been completely elucidated, and may involve both luminal (NHE3) and basolateral (Na+/K+ATPase) transporters. [443] [446] CYP450 arachidonic acid metabolites may modulate the proximal tubule component of the pressure-natriuresis response.[451]

TALH

20-HETE also serves as a second messenger to regulation transport in the thick ascending limb. It is produced in this nephron segment[416] and can inhibit net Na-K-Cl cotransport, by direct inhibition of the transporter and by blocking the 70-pS apical K+ channel. [452] [453] In addition, 20-HETE has been implicated as a mediator of the inhibitory effects of angiotensin II[454] and bradykinin[455] on TALH transport.

Collecting Duct

In the collecting duct, EETs and/or their diol metabolites serve as inhibitors of the hydroosmotic effects of vasopressin, as well as inhibitors of sodium transport in this segment. [423] [456] The latter effects were specific for 5,6-EET and were blocked by cyclooxygenase inhibitors.[456] Patch clamp studies have indicated that the eNaC sodium channel activity in the cortical collecting duct is inhibited by 11,12-EET.[457]

Role in Mitogenesis

In rat mesangial cells, endogenous non-cyclooxygenase metabolites of arachidonic acid modulate the proliferative responses to phorbol esters, vasopressin, and EGF, and agonist-induced expression of the immediate early response genes c-fos and Egr-1 is inhibited by ketoconazole or nordihydroguaiaretic acid (NDGA), but not specific lipoxygenase inhibitors.[458] EET-mediated increases in rat mesangial cell proliferation was the first direct evidence that CYP450 arachidonic acid metabolites are cellular mitogens.[459] In cultured rabbit proximal tubule cells, CYP450 inhibitors blunted EGF-stimulated proliferation in proximal tubule cells.[448] In LLCPKcl4, EETs were found to be potent mitogens, cytoprotective agents, and second messengers for EGF signaling. 14,15-EET-mediated signaling and mitogenesis are dependent upon EGF receptor transactivation, which is mediated by metalloproteinase-dependent release of HB-EGF.[460] In addition to the EETs, 20-HETE has been shown to increase thymidine incorporation in primary cultures of rat proximal tubule and LLC-PK1 cells[461] and vascular smooth muscle cells.[462]

Role in Hypertension

There is increasing evidence that the renal production of CYP450 AA metabolites is altered in a variety of models of hypertension and that blockade of the formation of compounds can alter blood pressure in several of these models. CYP450 AA metabolites may have both pro- and antihypertensive properties. At the level of the renal tubule, both 20-HETE and EETs inhibit sodium transport. However, in the vasculature, 20-HETE promotes vasoconstriction and hypertension, whereas EETs are endothelial-derived vasodilators that have antihypertensive properties. Rats fed a high salt diet increase expression of the CYP450 epoxygenase 2C23[463] and develop hypertension if treated with a relatively selective epoxygenase inhibitor. Because EETs have antihypertensive properties, efforts are underway to develop selective inhibitors of soluble epoxide hydrolase (sEH), which converts active EETs to their inactive metabolites, DHETs, and thereby increase EET levels. Studies in rats indicated that one such sEH inhibitor, 1-cyclohexyl-3-dodecylurea, lowered blood pressure and reduced glomerular and tubulointerstitial injury in an angiotensin II-mediated model of hypertension in rats.[464]

In DOCA/salt hypertension, administration of a CYP450 inhibitor prevented the development of hypertension. [465] [466] Angiotensin II stimulates the formation of 20-HETE in the renal circulation,[467] and 20-HETE synthesis inhibition attenuates angiotensin II mediated renal vasoconstriction[434] and reduced angiotensin II-mediated hypertension.[466]

The CYP450 4A2 gene is regulated by salt and is overexpressed in spontaneously hypertensive rats (SHR),[468] and production of both 20HETE and diHETEs is increased and production of EETs is reduced. [271] [469] CYP450 inhibitors or antisense oligonucleotides directed against CYP4A1 and 4A2 lowered blood pressure in SHR. [470] [471] Conversely, recent studies in humans have indicated that a variant of the human CYP4A11 with reduced 20-HETE synthase activity is associated with hypertension.[472]

In Dahl salt sensitive rats (Dahl S), pressure-natriuresis in response to salt loading is shifted such that the kidney requires a higher perfusion pressure to excrete the same amount of sodium as normotensive salt resistant (Dahl R) rats,[411] [412] [413] which is due at least in part to increased TALH reabsorption. The production of 20-HETE and expression of CYP4A protein are reduced in the outer medulla and TALH of Dahl S rats relative to Dahl R, which is consistent with the observed effect of 20-HETE to inhibit TALH transport. In addition, Dahl S rats do not increase EET production in response to salt loading.

Studies have indicated that angiotensin II acts on AT2 receptors on renal vascular endothelial cells to release EETs that may then counteract AT1-induced renal vasoconstriction and may influence pressure natriuresis. [427] [473] [474]AT2 receptor knockout mice develop hypertension,[475] which is associated with blunted pressure natriuresis, reduced renal blood flow, and glomerular filtration rate and defects in kidney 20-HETE production.[475]

ACKNOWLEDGMENTS

The writing of this chapter was supported by grants from the Veterans Administration and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to RCH (DK39261 and DK62794) and MDB (DK37097 and DK39261).

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