Molecular Biology

The cyclo-oxygenase enzyme system is the major pathway for AA metabolism in the kidney ( Figure 14.2 ). Cyclo-oxygenase (prostaglandin [PG] synthase G ₂ /H ₂ ) is the enzyme responsible for the initial conversion of arachidonic acid to PGG ₂ and subsequently to PGH ₂ . COX was first purified from ram seminal vesicles and cloned in 1988. The protein is widely expressed, and the level of activity is not dynamically regulated. Other studies supported the presence of a COX that was dynamically regulated and responsible for increased prostanoid production in inflammation. This second inducible COX isoform was identified shortly after the cloning of the initial enzyme and designated COX-2, whereas the initially isolated isoform is now designated COX-1. COX-1 and COX-2 are encoded by distinct genes located on different chromosomes. The human COX-1 gene ( PTGS1, PG synthase 1) is on chromosome 9, whereas COX-2 is localized on chromosome 1. The genes are also subject to dramatically different regulatory signals.

Cyclo-oxygenase metabolism of arachidonic acid. Both COX-1 and COX-2 convert arachidonic acid to PGH ₂ , which is then acted on by specific synthases (TXAS, PGDS, PGES, PGFS, PGIS) to produce prostanoids that act at G protein–coupled receptors (TP. DP1, DP2, EP1-4. FP, IP) that increase or decrease cAMP or increase intracellular calcium levels. cAMP, Cyclic adenosine monophospate; COX, cyclo-oxygenase; NSAIDs, nonsteroidal antiinflammatory drugs; PGG ₂ , prostaglandin G ₂ ; PGH ₂ , prostaglandin H ₂ .

Regulation of Cyclo-Oxygenase 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. 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), as well as a variety of cytokines and growth factors, including tumor necrosis factor (TNF), interleukin-1β, EGF, 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 nuclear factor-kappaB (NF-κB), and NF–interleukin-6 (IL-6)/CCAAT-enhancer binding proteins (C-EBP), and a cyclic adenosine monophosphate (cAMP) response element (CRE). Induction of COX-2 messenger RNA (mRNA) transcription by endotoxin (lipopolysaccharide) may also involve CRE sites and NF-κB sites.

Regulation of Cyclo-Oxygenase Expression By Antiinflammatory Steroids

A molecular basis linking the antiinflammatory effects of COX-inhibiting nonsteroidal antiinflammatory drugs (NSAIDs) and antiinflammatory 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 antiinflammatory effects of glucocorticoids. Posttranscriptional control of COX-2 expression represents another robust mechanism whereby adrenal steroids regulate COX-2 expression. Accumulating evidence has suggested that COX-2 is modulated at multiple steps in addition to transcription rate, including stabilization of the mRNA and enhanced translation. Glucocorticoids, including dexamethasone, downregulate COX-2 mRNA in part by destabilizing the mRNA. The 3′ untranslated region of COX-2 mRNA contains 22 copies of an AUUUA motif, which are important in destabilizing the COX-2 message in response to dexamethasone, whereas other 3′ sequences appear important for COX-2 mRNA stabilization in response to IL-1β. Effects of the 3′ untranslated region (UTR), as well as other factors regulating the efficiency of COX-2 translation, have also been suggested. The factors determining the expression of COX-1 are more obscure.

Enzymatic Chemistry

Despite these differences, both PG synthases catalyze a similar reaction, resulting in cyclization of carbons 8 to 12 of the AA backbone, forming cyclic endoperoxide, accompanied by the concomitant insertion of two oxygen atoms at carbon 15 to form PGG ₂ (a 15-hydroperoxide). In the presence of a reduced glutathione-dependent peroxidase, PGG ₂ is converted to the 15-hydroxy derivative, PGH ₂ . The endoperoxides (PGG ₂ and PGH ₂ ) have very short half-lives, about 5 minutes, and are biologically active in inducing aortic contraction and platelet aggregation. However, under some circumstances, the formation of these endoperoxides may be strictly limited via the self-deactivating properties of the enzyme.

The 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 COX-inhibiting NSAIDs work by sterically blocking access of AA to the heme-containing, active enzymatic site. Particularly well-conserved are sequences surrounding the aspirin-sensitive serine residues, at which acetylation by aspirin irreversibly inhibits activity. More recent evidence showed that COX-1 and COX-2 are capable of forming heterodimers and sterically modulating each other’s function. 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 relatively highly selective COX-2 inhibitors for clinical use as analgesics, antipyretics, and antiinflammatory agents. In addition to its central role in inflammation, aberrantly upregulated COX-2 expression has been implicated in the pathogenesis of a number of epithelial cell carcinomas and in Alzheimer’s disease and other degenerative neurologic conditions.

COX-2 Expression in the Kidney

There is now definitive evidence for significant COX-2 expression in the mammalian kidney ( Figure 14.3 ). 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 have 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. COX-2 expression is also abundant in the lipid-laden medullary interstitial cells in the tip of the papilla. Some investigators have reported that COX-2 may be expressed in inner medullary collecting duct cells or intercalated cells in the renal cortex. Nevertheless, COX-1 expression is constitutive and clearly the most abundant isoform in the collecting duct, so the potential existence and physiologic significance of COX-2 coexpression in this segment remains uncertain.

Localization of immunoreactive cyclo-oxgenases 1 and 2 (COX-1, COX-2) and microsomal prostaglandin E synthase (PGES) along the rat nephron ( shaded areas ). IR, Immunoreactive.

(Courtesy S. Bachmann.)

COX-2 Expression in the Renal Cortex

It is now well documented that COX-2 is expressed in the macula densa and cortical thick ascending limb (CTAL). It is expressed in human kidney, especially in kidneys of older adults, and in patients with diabetes mellitus, congestive heart failure, and Bartter-like syndrome.

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 have suggested effects via vasodilator and vasoconstrictor effects of prostanoids contributing to tubuloglomerular feedback (TGF). Some studies have suggested COX-2–derived prostanoids are predominantly vasodilators. By inhibiting production of dilator prostanoids contributing to the patency of the adjacent afferent arteriole, COX-2 inhibition may contribute to the decline in glomerular filtration rate (GFR) observed in patients taking NSAIDs or selective COX-2 inhibitors (see later).

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 ( Figure 14.4 ). 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 PGE ₂ from the macula densa in response to decreased chloride perfusate. Furthermore the induction of COX-2 by a low Cl ⁻ level can be blocked by a specific p38 mitogen-activated protein (MAP) kinase inhibitor. Finally , in vivo, renal cortical immunoreactive pp38 expression (the active form of p38) is predominantly localized to the macula densa and CTAL and increases in response to a low-salt diet. 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 Cl ⁻ delivery. Some 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 Cl ⁻ delivery. In mice deficient in the Na ⁺ -H ⁺ exchanger subtype 2 (NHE2), the macula densa is shrunken, accompanied by increased COX-2 expression and juxtaglomerular renin expression, suggesting that NHE2 appears to be the major isoform associated with macula densa cell volume regulation.

COX-2 expression is regulated in renal cortex in rats. Under basal conditions, sparse immunoreactive COX-2 is localized to the macula densa and surrounds the cortical thick ascending limb (CTAL). Following chronic administration of a sodium-deficient diet, macula densa, CTAL, and COX-2 expression increase markedly.

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 ( Figure 14.5 ). In vivo measurements in isolated perfused kidney and isolated perfused juxtaglomerular preparation have all indicated that administration of nonspecific COX inhibitors prevents the increases in renin release mediated by macula densa sensing of decreases in luminal NaCl. Induction of a high renin state by imposition of a salt-deficient diet, angiotensin-converting enzyme (ACE) inhibition, diuretic administration, or experimental renovascular hypertension all significantly increase macula densa–CTAL 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 and, in an isolated perfused juxtaglomerular preparation, increased renin release in response to lowering the perfusate NaCl concentration was blocked by COX-2 inhibition. In COX-2 knockout mice, increases in renin in response to low salt or ACE inhibitors were significantly blunted but were unaffected in COX-1 knockout mice. COX-2-derived PGE ₂ , activating the type 4 (EP4) receptors on juxtaglomerular cells, has been shown to be important for the macula densa regulation of renin release. Macula densa COX-2–derived prostanoids appear to be predominantly involved in setting tonic levels of juxtaglomerular renin expression rather than necessarily mediating acute renin release. There is evidence that the effect of ACE inhibitors and angiotensin receptor blockers (ARBs) to increase macula densa COX-2 expression is mediated by feedback of angiotensin II (Ang II) on the macula densa, with Ang II type 1 (AT ₁ ) receptor activation inhibiting and AT ₂ receptor activation stimulating COX-2 expression. In addition, prorenin and/or renin may stimulate macula densa COX-2 expression through activation of the prorenin receptor.

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 vasodilate the afferent arteriole and modulate renin release from juxtaglomerular cells. ACE, Angiotensin-converting enzyme; COX-2, cyclo-oxygenase-2.

COX-2 inhibitors have also been shown to decrease renin production in models of renovascular hypertension, and studies in mice with targeted deletion of the prostacyclin receptor have suggested a predominant role for prostacyclin in mediating renin production and release in these models. In a model of sepsis, COX-2 expression increased in the macula densa and both cortical and medullary TAL. This increased COX-2 expression was mediated by Toll-like receptor 4 (TLR4) and in TLR4 ^−/− mice, juxtaglomerular apparatus renin expression was absent.

In addition to mediating juxtaglomerular renin expression, COX-2 metabolites may also modulate tubuloglomerular feedback (TGF). However, using different methodologies, investigators have reported that COX-2 metabolites predominantly modulate TGF by the production of vasodilatory prostanoids or mediate afferent arteriolar vasoconstriction by activating thromboxane receptors through the generation of thromboxane A ₂ (TXA ₂ ) and/or PGH ₂ . Further studies will be required to reconcile these divergent results.

There is evidence that macula densa COX-2 expression is sensitive not only to alterations in intravascular volume, but also to alterations in renal metabolism. Specifically, the G protein–coupled receptor, GPR91, has been shown to be a receptor for succinate, an intermediate of the citric acid cycle. GPR91 is expressed in the macula densa, and GPR91, and intrarenal production of succinate are increased in diabetes. Studies have suggested that succinate activation of GPR91 leads to increased macula densa COX-2 expression.

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 ( Figure 14.6 ). 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. In the collecting duct, COX-2 has also been localized to intercalated cells but is absent in principal cells. COX-2 may also be expressed in endothelial cells of the vasa recta supplying the inner medulla.

Differential immunolocalization of COX-1 and COX-2 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.

In medullary interstitial cells, dynamic regulation of COX-2 expression appears to be an important adaptive response to physiologic stresses, including water deprivation, increased dietary sodium, and exposure to endotoxins. 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 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 and a high-sodium diet both induce COX-2 expression in medullary interstitial cells by activating the NF-κB pathway. There is also evidence that nitric oxide may modulate medullary COX-2 expression through MAP kinase–dependent pathways. The mechanisms underlying the upregulation of medullary COX-2 expression in response to volume expansion are probably multifactorial. There is clear evidence that increased medullary tonicity increases medullary COX-2 expression. Different studies have indicated a role for NF-κB, EGF receptor (EGFR) transactivation, and mitochondrial-generated ROS. Whether these represent parallel pathways or are all interrelated is not yet clear; however, it should be noted that the described EGFR transactivation is mediated by cleavage of the EGFR ligand, transforning growth factor-α, by ADAM17 (known as TACE [tumor necrosis factor-α–converting enzyme]). This is known to be activated by src, which can be activated by reactive oxygen species (ROS). In addition to medullary COX-2, cortical COX-2 expression increases in salt-sensitive hypertension, especially in the glomerulus and is inhibited by the superoxide dismutase mimetic, tempol, or an ARB.

COX-1 Expression in the Kidney

Although well-defined factors regulating COX-2 and determining the role of COX-2 expression in the kidney are being delineated, the role of renal COX-1 remains more obscure. COX-1 is constitutively expressed in platelets in renal microvasculature and glomerular parietal epithelial cells ( Figure 14.7 ). In addition, COX-1 is abundantly expressed in the collecting duct, but there is little COX-1 expressed in the proximal tubule or TAL. Although 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, vasopressin does increase COX-1 expression in collecting duct epithelial cells and in interstitial cells in the inner medulla. 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.

Renal cortical COX-1 expression. Immunoreactive COX-1 is predominantly localized to the afferent arteriole (AE), glomerular mesangial cells (G), parietal glomerular epithelial cells (P), and cortical collecting duct (CT).

(From Eguchi N, Minami T, Shirafuji N, et al: Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase–deficient mice. Proc Natl Acad Sci U S A 96:726-773, 1999.)

Na ⁺ Retention, Edema, and Hypertension

Use of nonselective NSAIDs may be complicated by the development of significant Na ⁺ retention, edema, congestive heart failure and hypertension. These complications are also apparent in patients using COX-2–selective NSAIDs. Studies with celecoxib and rofecoxib have demonstrated that like nonselective NSAIDs, these COX-2–selective NSAIDs reduce urinary Na ⁺ excretion and are associated with modest Na ⁺ retention in otherwise healthy subjects. COX-2 inhibition likely promotes salt retention via multiple mechanisms ( Figure 14.8 ). A reduced GFR may limit the filtered Na ⁺ load and salt excretion. In addition, PGE ₂ directly inhibits Na ⁺ absorption in the thick ascending limb (TAL) and collecting duct. The relative abundance of COX-2 in medullary interstitial cells places this enzyme adjacent to both these nephron segments, allowing for COX-2–derived PGE ₂ to modulate salt absorption. COX-2 inhibitors decrease renal PGE ₂ production and thereby may enhance renal sodium retention. Finally, reduction in renal medullary blood flow by the 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. Similarly, patients with preexisting treated hypertension commonly experience hypertensive exacerbations with COX-2–selective NSAIDs. Taken together, these data suggest that COX-2–selective NSAIDs have effects similar to those of nonselective NSAIDs with respect to salt excretion.

Integrated role of PGE ₂ on regulation of salt and water excretion. PGE ₂ can increase medullary blood flow and directly inhibit NaCl reabsorption in the medullary thick ascending limb and water reabsorption in the collecting duct.

Hyperkalemia

Nonselective NSAIDs cause hyperkalemia due to suppression of the renin angiotensin aldosterone axis. Both a decreased GFR and inhibition of renal renin release may compromise renal K ⁺ excretion. Patients on a salt-restricted diet also have decreased urinary potassium excretion when treated with a COX-2–selective inhibitor, and COX-2–selective inhibitors may pose an equal or greater risk as nonselective NSAIDs for the development of hyperkalemia.

Papillary Necrosis

Acute and subacute forms of papillary necrosis have been observed with NSAID use. Acute NSAID-associated renal papillary injury is more likely to occur in the setting of volume depletion, suggesting a critical dependence of renal function on COX metabolism in this setting. Long-term use of NSAIDs has been associated with papillary necrosis and progressive renal structural and functional deterioration, much like the syndrome of analgesic nephropathy observed with an acetaminophen, aspirin, and caffeine combination. Experimental studies have suggested that renal medullary interstitial cells are an early target of injury in analgesic nephropathy. COX-2 has been shown to be an important survival factor for cells exposed to a hypertonic medium. The coincident localized expression of COX-2 in these interstitial cells raises the possibility that, like nonselective NSAIDs, long-term use of COX-2–selective NSAIDs may contribute to the development of papillary necrosis.

Acute Kidney Injury

Acute kidney injury (AKI) is a well-described complication of NSAID use. This is generally considered to be a result of altered intrarenal microcirculation and glomerular filtration secondary to the inability to produce beneficial endogenous prostanoids when the kidney is dependent on them for normal function. Like the traditional, nonselective NSAIDs, COX-2– selective NSAIDs will also reduce glomerular filtration in susceptible patients. Although generally rare, NSAID-associated renal insufficiency occurs in a significant proportion of patients with underlying volume depletion, renal insufficiency, congestive heart failure, diabetes, and advanced age. 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 celecoxib and rofecoxib caused a slight but significant fall in the GFR in salt-depleted but otherwise healthy subjects. Similar to nonselective NSAIDs, AKI can occur secondary to the use of COX-2–selective NSAIDs. Preclinical studies have supported 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 have documented reduced afferent arteriolar diameter following administration of a COX-2 inhibitor. These animal data not only support the concept that COX-2 plays an important role in regulating the GFR, but also the clinical observations that COX-2–selective inhibitors can cause renal insufficiency similar to that reported with nonselective 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 that of classic drug-induced allergic interstitial nephritis, lacking fever or rash. This syndrome has also been reported with the COX-2–selective drug celecoxib. 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 a course of months. The renal pathology is usually consistent with minimal change disease, with foot process fusion of glomerular podocytes observed on electron microscopy (EM), but membranous nephropathy has also been reported. Typically, the nephrotic syndrome occurs together with the interstitial nephritis. 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 COX inhibition by these drugs, an idiosyncratic immune drug reaction, or a combination of both.

Renal Dysgenesis

Reports of renal dysgenesis and oligohydramnios in the offspring of women given nonselective NSAIDs during the third trimester of pregnancy 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. Because neither COX-1 ^−/− mice nor mice treated with the COX-1– selective inhibitor SC58560 exhibited altered renal development, a specific role for COX-2 in nephrogenesis has been suggested. A report of renal dysgenesis in the infant of a woman exposed to the COX-2–selective inhibitor nimesulide suggested that COX-2 also plays a role in renal development in humans.

The intrarenal expression of COX-2 in the developing kidney peaks in the mouse at postnatal day 4 and in the rat in the second postnatal week. 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 CTAL, expression in the papilla has also been observed. 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 the CTAL (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 have shown that exogenous PGE ₂ promotes renal metanephric development and is a critical growth factor for renal epithelia cells. Nevertheless, none of the prostaglandin receptor knockout mice recapitulated the phenotype of the COX-2 knockout mice.

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 nonselective NSAID indomethacin at all Ang II doses studied. Administration of selective COX-2 inhibitors or COX-2 gene knockout has been shown to accentuate the pressor effects of angiotensin II in mice. 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. These findings support the conclusion that COX-1–derived prostaglandins participate in, and are integral to, the pressor activity of Ang II, whereas COX-2–derived prostaglandins are vasodilators that oppose and mitigate the pressor activity of Ang II. Other animal studies have more directly shown that NSAIDs and COX-2 inhibitors blunt arteriolar dilation and decrease flow through resistance vessels.

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. Therefore, increasing evidence has indicated that COX-2–selective antagonism may carry increased thrombogenic risks due to selective inhibition of the endothelial-derived, antithrombogenic prostacyclin without any inhibition of the prothrombotic, platelet-derived thromboxane generated by COX-1. Although animal studies have provided conflicting results about the role of COX-2 inhibition on development of atherosclerosis, there have been indications that COX-2 inhibition may destabilize atherosclerotic plaques. This has been 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. Because of the concerns about increased cardiovascular risk, two selective COX-2 inhibitors, rofecoxib and valdecoxib, were withdrawn from the market, and remaining coxibs and other NSAIDs have been relabeled to highlight the increased risk for cardiovascular events.

Sources and Nephronal Distribution of Cyclo-Oxygenase 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 PGI ₂ . Whole glomeruli generate PGE ₂ , PGI ₂ , PGF _2α and TXA ₂ . The predominant products in rat and rabbit glomeruli are PGE ₂ , followed by PGI ₂ and PGF _2α and, finally, TXA ₂ .

Analysis of individual cultured glomerular cell subpopulations has also provided insight into the localization of prostanoid synthesis. Cultured mesangial cells are capable of generating PGE ₂ , and, in some cases, PGF _2α and PGI ₂ have also been detected. Other studies have suggested that mesangial cells may produce the endoperoxide PGH ₂ as a major COX product. 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 have demonstrated intense staining for COX-1, predominantly in parietal epithelial cells. Glomerular capillary endothelial cell PG generation profiles remain undefined but may include prostacyclin.

The predominant synthetic site of prostaglandin synthesis along the nephron is the cortical collecting duct (CCD), particularly its medullary portion (MCT). In the presence of exogenous arachidonic acid, PGE ₂ is the predominant PG formed in the collecting duct, with variations among the other products being insignificant. PGE ₂ is also the major COX metabolite generated in medullary interstitial cells. The role that specific prostanoid synthases may play in the generation of these products is outlined below.

TP Receptors

The TP receptor was originally purified by chromatography using a high-affinity ligand to capture the receptor. This was the first eicosanoid receptor cloned and is a G protein–coupled transmembrane receptor capable of activating a calcium-coupled signaling mechanism ( Figure 14.12 ). 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 that differ in their C-terminal tail distal to Arg. Similar patterns of alternative splicing have been described for the EP3 and FP receptors. Heterologous cAMP-mediated signaling of the thromboxane receptor may occur via its heterodimerization with the prostacyclin (IP) receptor.

Prostaglandin receptors are seven-transmembrane, G protein–coupled receptors.

The endoperoxide PGH ₂ or its metabolite, TXA ₂ , can activate the TP receptor. Competition radioligand binding studies have demonstrated a rank order of potency on the human platelet TP receptor of the ligands I-BOP, S145 > SQ29548 > STA ₂ > U-46619. Whereas I-BOP, STA ₂ , and U-46619 are agonists, SQ29548 and S145 are high-affinity TP receptor antagonists. Studies have suggested that the TP receptor may mediate some of the biologic effects of the nonenzymatically derived isoprostanes, including modulation of tubuloglomerular feedback. This latter finding may have significance in pathophysiologic conditions associated with increased oxidative stress. Signal transduction studies have shown that the TP receptor activates phosphatidylinositol bisphosphate (PIP ₂ ) hydrolysis–dependent Ca ²⁺ influx . Northern blot analysis of mouse tissues has revealed that the highest level of TP mRNA expression is in the thymus, followed by the spleen, lung, and kidney, with lower levels of expression in the heart, uterus, and brain.

Thromboxane is a potent modulator of platelet shape change and aggregation, as well as smooth muscle contraction and proliferation. Moreover, a point mutation (Arg to Leu) in the first cytoplasmic loop of the TXA ₂ receptor was identified in a dominantly inherited bleeding disorder in humans, characterized by a defective platelet response to TXA ₂ . Targeted gene disruption of the murine TP receptor also resulted in prolonged bleeding times and reduction in collagen-stimulated platelet aggregation ( Figure 14.13 ). 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.

Published phenotypes of prostanoid receptor knockout mice. MCD, Medullary collecting duct; TAL, thick ascending limb; UUO, unilateral ureteral obstruction.

In the kidney, TP receptor mRNA has been reported in glomeruli and vasculature. Radioligand autoradiography using ¹²⁵ I-BOP has suggested a similar distribution of binding sites in the mouse renal cortex, but additional renal medullary binding sites were observed. These medullary TXA ₂ binding sites are absent following disruption of the TP receptor gene, suggesting that they also represent authentic TP receptors. Glomerular TP receptors may participate in potent vasoconstrictor effects of TXA ₂ analogs on the glomerular microcirculation associated with a reduced GFR. Mesangial TP receptors coupled to phosphatidylinositol hydrolysis, protein kinase C activation, and glomerular mesangial cell contraction may contribute to these effects.

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) and in angiotensin-dependent hypertension. The TP receptor also appears to modulate renal blood flow in Ang II–dependent hypertension and in endotoxemia-induced renal failure. Modulation of renal TP receptor mRNA expression and function by dietary salt intake has also been reported. 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 TGF. However, other studies revealed no significant difference in TGF between wild-type and TP receptor knockout mice.

A major phenotype of TP receptor disruption in mice and humans appears to be reduced platelet aggregation and prolonged bleeding time. Thromboxane may also modulate the glomerular fibrinolytic system by increasing the production of an inhibitor of plasminogen activator (PAI-1) in mesangial cells. Although a specific renal phenotype in the TP receptor knockout mouse has not yet been reported, important pathogenic roles for TXA ₂ and glomerular TP receptors in mediating renal dysfunction in glomerulonephritis, diabetes mellitus, and sepsis seem likely.

In an Ang II–dependent mouse model of hypertension, deletion of the TP receptor gene ameliorated hypertension and reduced cardiac hypertrophy, but had no effect on proteinuria. In contrast, although blockade of NO synthase in an l -NAME ( N ^G -nitro- l -arginine methyl ester) model of hypertension, in which deletion of the TP receptor also ameliorated hypertension and did not decrease GFR, it led to an increased worsening of histopathology and significant renal hypertrophy. This suggests that the TP receptor may play a renal protective role in some settings.

IP 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. Iloprost and carbaprostacyclin potently activate the IP receptor but also activate the EP1 receptor. Most evidence suggests that the PGI ₂ receptor signals via stimulation of cAMP generation; however, at 1000-fold higher concentrations, the cloned mouse PGI ₂ receptor also signaled via PIP ₂ . It remains unclear whether PIP ₂ hydrolysis plays any significant role in the physiologic action of PGI ₂ .

IP receptor mRNA is highly expressed in the mouse thymus, heart, and spleen and in human kidney, liver, and lung. In situ hybridization shows IP receptor mRNA predominantly in neurons of the dorsal root ganglia and vascular tissue, including the aorta, pulmonary artery, and renal interlobular and glomerular afferent arterioles. 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.

PGI ₂ has been demonstrated to play an important vasodilator role in the kidney, including in the glomerular microvasculature, as well as regulating renin release. The capacity of PGI ₂ and PGE ₂ to stimulate cAMP generation in the glomerular microvasculature is distinct and additive, demonstrating that the effects of these two prostanoids are mediated via separate receptors. IP receptor knockout mice also exhibit salt-sensitive hypertension. Prostacyclin is a potent stimulus of renal renin release, and studies using IP ^−/− mice have confirmed an important role for the IP receptor in the development of renin-dependent hypertension of renal artery stenosis.

Renal epithelial effects of PGI ₂ in the thick ascending limb have also been suggested, and IP receptors have been reported in the collecting duct, 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, consistent with a role for this metabolite in this region of the kidney. Thus, although IP receptors appear to play an important role regulating renin release and as a vasodilator in the kidney, their role in regulating renal epithelial function remains to be firmly established.

DP Receptors

The DP1 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 PGD ₂ with a high-affinity binding of 300 pM and a lower affinity site of 13.4 nM. DP-selective PGD ₂ analogs include the agonist BW 245C. DP receptor mRNA is highly expressed in leptomeninges, retina, and ileum but was not detected in the kidney. Northern blot analysis of the human DP receptor demonstrated mRNA expression in the small intestine and retina, whereas in the mouse the DP receptor mRNA was detected in the ileum and lung. PGD ₂ has also been shown to affect the sleep-wake cycle, pain sensation, and body temperature. Peripherally, PGD ₂ 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. Development of the antagonist laropiprant was undertaken to inhibit the niacin-induced vasodilation flushing response. Although the kidney appears capable of synthesizing PGD ₂ , its role in the kidney remains poorly defined. Intrarenal infusion of PGD ₂ resulted in a dose-dependent increase in renal artery flow, urine output, creatinine clearance, and sodium and potassium excretion.

A second G protein–coupled receptor capable of binding and being activated by PGD ₂ was cloned as an orphan chemoattractant receptor from eosinophils and T cells (type 2 helper T cell subset) and designated the CRTH2 receptor. This receptor, now referred to as the DP2 receptor, bears no significant sequence homology to the family of prostanoid receptors discussed earlier, and couples to G _i /G _o inhibition of intracellular cAMP. It binds agonists with an order of potency PGD ₂ ≫ PGF _2α , PGE ₂ > PGI ₂ , TXA ₂ . DP2 receptor action is blocked by the antagonist ramatroban, a drug used to treat allergic rhinitis and originally described as a TP receptor antagonist. Deletion of the DP2 receptor gene was protective in a mouse UUO model of fibrosis. The recognition of this molecularly unrelated receptor allows for the possibility of the existence of a distinct and new family of prostanoid-activated membrane receptors.

FP Receptors

The cDNA encoding the PGF _2α 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 ³ H-PGF _2α binding with a K _D of 4.3 ± 1.0 nM. Selective activation of the FP receptor may be achieved using fluprostenol or latanoprost. ³ H-PGF _2α binding was displaced by a panel of ligands with a rank order potency of PGF _2α = fluprostenol > PGD ₂ > PGE ₂ > U46619 > iloprost. When expressed in oocytes, PGF _2α or fluprostenol induced a Ca ²⁺ -dependent Cl ⁻ current. Increased cell calcium has also been observed in fibroblasts expressing an endogenous FP receptor. Other studies have suggested that FP receptors may also activate protein kinase C (PKC)–dependent and Rho -mediated/PKC–independent signaling pathways. An alternatively spliced isoform with a shorter C-terminal tail has been identified that appears to signal via a similar manner as the originally described FP receptor, and other studies have suggested that these two isoforms may exhibit differential desensitization and may also activate a glycogen synthase kinase/β-catenin–coupled signaling pathway.

Tissue distribution of FP receptor mRNA shows highest expression in ovarian corpus luteum followed by kidney, with lower expression in the lung, stomach, and heart. Expression of the FP receptor in corpora lutea is critical for normal birth, and homozygous disruption of the mouse FP receptor gene results in failure of parturition in females, apparently due to failure of the normal preterm decline in progesterone levels. PGF _2α 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. The FP receptor is also highly expressed in skin, where it may play an important role in carcinogenesis. 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.

The role of FP receptors in regulating renal function is only partially defined. FP receptor expression has been mapped to the CCD in mouse and rabbit kidney. FP receptor activation in the collecting duct inhibits vasopressin-stimulated water absorption via a pertussis toxin–sensitive (presumably G _i ) dependent mechanism. Although PGF _2α increases cell Ca ²⁺ in cortical collecting duct, the FP–selective agonists latanoprost and fluprostenol did not increase calcium. Because PGF _2α can also bind to EP1 and EP3 receptors, these data suggest that the calcium increase activated by PGF _2α in the collecting duct may be mediated via an EP receptor. PGF _2α also increases Ca ²⁺ in cultured glomerular mesangial cells and podocytes, suggesting that an FP receptor may modulate glomerular contraction. In contrast to these findings, glomerular FP receptors at the molecular level have not been demonstrated. Other vascular effects of PGF _2α have been described, including selective modulation of renal production of PGF _2α by sodium or potassium loading and AT ₂ receptor activation.

Some studies have uncovered a role for the FP receptor in regulating renin expression. Interestingly, FP agonists increased renin mRNA expression in the JGA in a dose-dependent manner but, unlike IP receptor agonists, did not increase intracellular cAMP. Deletion of the FP receptor resulted in decreased renin levels and decreased systemic blood pressure. These data suggest that FP receptor blockade may be a novel target for the treatment of hypertension.

Multiple EP Receptors

Four EP receptor subtypes have been identified. Although these four receptors uniformly bind PGE ₂ 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. 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 ²⁺ –coupled EP1 receptor is more closely related to the other Ca ²⁺ –coupled prostanoid receptors (e.g., TP and FP receptors). 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.

Proximal Tubule

Neither the proximal convoluted tubule nor the proximal straight tubule appears to produce amounts of biologically active COX 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 P450 pathway.

Early whole-animal studies suggested that PGE ₂ might have an action in the proximal tubule because of its effects on urinary phosphate excretion. PGE ₂ blocked the phosphaturic action of calcitonin infusion in thyroparathyroidectomized rats. Nevertheless, studies using in vitro perfused proximal tubules failed to show an effect of PGE ₂ on sodium chloride or phosphate transport in the proximal convoluted tubule. More recent studies have suggested that PGE ₂ may play a key role in the phosphaturic action of fibroblast growth factor-23 because phosphaturia in hyp mice with X-linked hyperphosphaturia is associated with markedly increased urine PGE ₂ excretion, and phosphaturia was normalized by indomethacin. Nevertheless, there are very little data on the actions of other COX metabolites in proximal tubules and scant molecular evidence for the 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 COX pathway although, given the realization that COX-2 is expressed in this segment, it is of note that PGE ₂ was uniformly greater in the cortical segment than the medullary thick ascending limb. The TAL has been shown to exhibit high- density PGE ₂ receptors. Studies have also demonstrated high expression levels of mRNA for the EP3 receptor in medullary TAL of both rabbit and rat (see earlier, “ EP3 Receptors ”). Subsequent to the demonstration that PGE ₂ inhibits sodium chloride absorption in the medullary TAL of the rabbit perfused in vitro, it was shown that PGE ₂ blocks vasopressin (AVP) but not cAMP-stimulated sodium chloride absorption in the medullary TAL of the mouse. It is likely that the mechanism involves activation of G _i and inhibition of adenyl cyclase by PGE ₂ , possibly via the EP3 receptors expressed in this segment.

Collecting Duct System

In vitro perfusion studies of rabbit cortical collecting tubule have demonstrated that PGE ₂ directly inhibits sodium transport in the collecting duct when applied to the basolateral surface of this nephron segment. It is now apparent that PGE ₂ uses multiple signal transduction pathways in the cortical collecting duct, including those that modulate intracellular cAMP levels and Ca ²⁺ . PGE ₂ can stimulate or suppress cAMP accumulation. The latter may also involve stimulation of phosphodiesterase. Although modulation of cAMP levels appears to play an important role in PGE ₂ effects on water transport in the CCD (see following section), it is less clear that PGE ₂ affects sodium transport via modulation of cAMP levels. PGE ₂ has been shown to increase cell calcium, possibly coupled with PKC activation, in in vitro perfused cortical collecting ducts. This effect may be mediated by the EP1 receptor subtype coupled to phosphatidylinositol hydrolysis.

Water Transport

AVP-regulated water transport in the collecting duct is markedly influenced by COX products, especially prostaglandins. When COX inhibitors are administered to humans, rat, or dog, the antidiuretic action of AVP is markedly augmented. Because vasopressin also stimulates endogenous PGE ₂ production by the collecting duct, these results suggest that PGE ₂ participates in a negative feedback loop, whereby endogenous PGE ₂ production dampens the action of AVP. In agreement with this model, the early classic studies of Grantham and Orloff directly demonstrated that PGE ₁ blunted the water permeability response of the CCD to vasopressin. In these early studies, the action of PGE ₁ appeared to be at a pre-cAMP step. Interestingly, when administered by itself, PGE ₁ modestly augmented basal water permeability. These earlier studies have been confirmed with respect to PGE ₂ . PGE ₂ also stimulates basal hydraulic conductivity and suppresses the hydraulic conductivity response to AVP in the rabbit cortical collecting duct. Inhibition of 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. These data are evidence of consistent functional redundancy between the EP1 and EP3 with respect to their effects on AVP-stimulated water absorption in the collecting duct.

15-Ketodehydrogenase

The half-life of prostaglandins is 3 to 5 minutes and that of TXA ₂ is approximately 30 seconds. Elimination of PGE ₂ , PGF _2α , and PGI ₂ proceeds through enzymatic and nonenzymatic pathways, whereas that of TXA ₂ is nonenzymatic. The end products of all these degradative reactions generally possess minimal biologic activity, although this is not uniformly true (see below). The principal enzyme involved in the transformation of PGE ₂ , PGI ₂ , and PGF _2α is 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which converts the 15 alcohol group to a ketone.

15-PGDH is an oxidized nicotinamide adenine dinucl­eotide–oxidized nicotinamide adenine dinucleotide phosphate (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. Its K _m for PGE ₂ is 8.4 µmol/L and 22.6 µmol/L for PGF _2α . It is mainly localized in cortical and juxtamedullary zones, with little activity detected in papillary slices. At baseline, it is found in the proximal tubule, TAL, and CD. However, it was present in the macula densa in COX-2 knockout mice and in the presence of high-salt diet and, in cultured macula densa cells, COX inhibition increased expression. Disruption of the 15-PGDH gene in mice results in persistent patent ductus arteriosus (PDA), thought to be a result of failure of circulating PGE ₂ levels to fall in the immediate peripartum period. Thus, administration of COX-inhibiting NSAIDs rescues the knockout mice by decreasing PGs and allowing the animals to survive.

Subsequent catalysis of 15-hydroxy products by a δ-13 reductase leads to the formation of 13,14-dihydro compounds. PGI ₂ and TXA ₂ undergo rapid degradation to 6-keto-PGF _1α and TXB ₂ , respectively. 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 PGA ₂ and PGE ₂ have been shown to undergo hydroxylation of their terminal or subterminal carbons by a cytochrome P450–dependent mechanism. This reaction may be mediated by CYP4A family members or a CYP4F enzyme. CYP4A and CYP4F members have been mapped along the nephron. Some of these derivatives have been shown to exhibit biologic activity.

Cyclopentenone Prostaglandins

The cyclopentenone prostaglandins include PGA ₂ , a PGE ₂ derivative, and PGJ ₂ , a derivative of PGD ₂ . Although it remains uncertain whether these compounds are actually produced in vivo, this possibility has received increasing attention because some cyclopentenone prostanoids have been shown to be activating ligands for nuclear transcription factors, including peroxisome proliferator–activated receptors δ and γ (PPARδ and PPARγ). The realization that the antidiabetic thiazolidinedione drugs act through PPARγ to exert their antihyperglycemic and insulin-sensitizing effects has generated intense interest in the possibility that the cyclopentenone PGs might serve as the endogenous ligands for these receptors. Interestingly, DP2, unlike DP1 or other members of the prostaglandin GPCR family, binds and is activated by PGD ₂ metabolites such as 15-deoxy-Δ12,14-PGJ ₂ , which acts at nanomolar concentrations. An alternative biologic activity of these compounds has been recognized in their capacity to modify thiol groups covalently, forming adducts with cysteine of several intracellular proteins, including thioredoxin 1, vimentin, actin, and tubulin. Studies regarding the biologic activity of cyclopentenone prostanoids abound and the reader is referred to several excellent sources in the literature. Although there is evidence supporting the presence of these compounds in vivo, it remains uncertain whether they can form enzymatically or are an unstable spontaneous dehydration product of the E and D ring prostaglandins.

Nonenzymatic Metabolism of Arachidonic Acid

It has long been recognized that oxidant injury can result in peroxidation of lipids. In 1990, Morrow and coworkers reported that a series of prostaglandin-like compounds could be produced by free radical catalyzed peroxidation of arachidonic acid that is independent of COX activity. These compounds, termed isoprostanes, have been increasingly used as sensitive markers of oxidant injury in vitro and in vivo. In addition, at least two of these compounds, 8-iso-PGF _2α (15-F ₂ -isoprostane) and 8-iso-PGE ₂ (15-E ₂ -isoprostane) are potent vasoconstrictors when administered exogenously. 8-Iso-PGF _2α has been shown to constrict the renal microvasculature and decrease GFR, an effect that is prevented by thromboxane receptor antagonism. 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 extracellularly to achieve efficient metabolism and termination of their signaling. Similarly, enzymes that metabolize PGE ₂ to inactive compounds are also intracellular, requiring uptake of the PG for its metabolic inactivation. The molecular basis of these extrusion and uptake processes are now being defined.

As a fatty acid, prostaglandins may be classified as an organic anion at physiologic pH. Early microperfusion studies documented that basolateral PGE ₂ could be taken up into proximal tubule cells and actively secreted into the lumen. Furthermore this process could be inhibited by a variety of inhibitors of organic anion transport, including p -aminohippurate (PAH), probenecid, and indomethacin. Studies of basolateral renal membrane vesicles also supported the notion that this transport process occurs 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.

A molecule that mediates PGE ₂ uptake in exchange for lactate has been cloned and termed prostaglandin transporter (PGT). PGT is a member of the SLC21/SLCO organic anion transporting family, 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). Immunocytochemical studies of PGT expression in rat kidneys have suggested expression primarily in glomerular endothelial and mesangial cells, arteriolar endothelial and muscularis cells, principal cells of the CD, medullary interstitial cells, medullary vasa recta endothelia, and papillary surface epithelium. PGT appears to mediate PGE ₂ uptake rather than release, allowing target cells to metabolize this molecule and terminate signaling. PGT expression is decreased with low salt and increased with high salt in the CD, which may allow regulation of PG excretion by taking up more prostaglandins excreted from luminal surface, the site of the PGT, thereby allowing more accumulation at the basolateral surface.

Other members of the organic cation, anion, and zwitterion transporter family SLC22 have also been shown to transport prostaglandins and have been suggested to mediate prostaglandin excretion into the urine. Specifically, OAT1 and OAT3 are localized on the basolateral proximal tubule membrane, where they likely participate in urinary excretion of PGE ₂ . Conversely members of the multidrug resistance protein (MRP) have been shown to transport PGs in an adenosine triphosphate–dependent fashion. 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. 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.

Experimental and Human Glomerular Injury

Acute Kidney Injury

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 (e.g., norepinephrine, Ang II, AVP). Amelioration of their effects in 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 PGE ₂ and PGI ₂ , is a central part of this protective adaptation. Increased renal vascular resistance induced by exogenously administered Ang 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 Ang II. Studies in patients with congestive heart failure have confirmed that enhanced prostaglandin synthesis is crucial in protecting the kidneys from various vasoconstrictor influences in this condition.

Renal dysfunction 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 TXA ₂ -induced renal vasoconstriction in this model of renal dysfunction. In addition, roles for PGs and TXA ₂ in modulating or mediating renal injury have been suggested in ischemia and reperfusion and models of toxin-mediated acute tubular injury, including those induced by uranyl nitrate, amphotericin B, aminoglycosides, and glycerol. In experimental acute renal failure, administration of vasodilator PGs has been shown to ameliorate injury. Similarly, administration of nonselective or COX-2–selective NSAIDs exacerbates experimental ischemia-reperfusion injury.

COX-2 expression decreases in the kidney in response to acute ischemic injury. There is some controversy about the role of COX products in ischemia-reperfusion injury. Furthermore, fibrosis resulting from prolonged ischemic injury has been shown to be ameliorated by nonspecific COX inhibition. In contrast, renal injury in response to ischemia-reperfusion is worsened by COX-2–selective inhibitors or in COX-2 ^−/− mice, and administration of vasodilator PGs has been shown to ameliorate injury, possibly through a PPARδ-dependent mechanism.

Urinary Tract Obstruction

Following the induction of chronic (>24 hours) ureteral obstruction, renal PG and TXA ₂ synthesis is markedly enhanced, particularly in response to stimuli such as endotoxins or bradykinins. Enhanced prostanoid synthesis, especially thromboxane, likely arises from infiltrating mononuclear cells, proliferating fibroblast-like cells, interstitial macrophages, and interstitial medullary cells. Selective COX-2 inhibitors may prevent renal damage in response to unilateral ureteral obstruction (UUO). However, PGE ₂ acting through the EP4 receptor can limit tubuloin­terstitial fibrosis resulting from UUO. Prostaglandins derived from medullary COX-2 are mediators of the early phase of diuresis seen after relief of ureteral obstruction, because COX-2 inhibition prevents the acute (24-hour) phase of postobstructive diuresis. However, more persistent, chronic, postobstructive diuresis is not prostaglandin- dependent but results from downregulation of NKCC2 (Na ⁺ -K ⁺ -2Cl ⁻ cotransporter type 2) and decreases aquaporin-2 (AQP2) phosphorylation and translocation to the CCD membrane.

Allograft Rejection and Cyclosporine Nephrotoxicity

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 _1α , an index of systemic PGI ₂ synthesis, is increased in patients with cirrhosis and hyperdynamic circulation, thus raising the possibility that systemic synthesis of PGI ₂ may contribute to the arterial vasodilatation of these patients. Inhibition of COX activity in these patients may cause a profound reduction in RBF and GFR, a reduction in sodium excretion, and an impairment of free water clearance. 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 but 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.

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. There is reduced renal synthesis of vasodilating PGE ₂ when there is activation of endogenous vasoconstrictors and a maintained or increased renal production of TXA ₂ . Therefore, an imbalance between vasoconstricting systems and the renal vasodilator PGE ₂ 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 for ameliorating renal function or preventing the deleterious effect of NSAIDs.

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 RBF of normal pregnancy ; however, diminished synthesis of PGI ₂ has been demonstrated in humans and in animal models of pregnancy-induced hypertension, which is associated with decreased expression of COX-2 and PGI ₂ synthase in the placental villi. In animal models, inhibition of TXA ₂ synthetase has been associated with resolution of the hypertension, suggesting a possible pathophysiologic role. A moderate beneficial effect of reducing TXA ₂ generation, while preserving PGI ₂ synthesis, by low-dose aspirin therapy (60 to 100 mg/day) has been demonstrated in patients at high risk for pregnancy-induced hypertension and preeclampsia.

Lithium Nephrotoxicity

Lithium chloride is a mainstay of treatment in the psychiatric treatment of bipolar illness. However, it is routinely complicated by polyuria and even frank nephrogenic diabetes insipidus. In vitro and in vivo studies have demonstrated lithium-induced renal medullary interstitial cell COX-2 protein expression via inhibition of glycogen synthase kinase-3β (GSK-3β). COX-2 inhibition prevented lithium-induced polyuria and also resulted in the upregulation of AQP2 and NKCC2.

Role of Reactive Oxygen Species as Mediators of COX-2 Actions

In addition to NADPH oxidase, nitric oxide synthase, and xanthine oxidase, COX-2 can also be a source of oxygen radicals. COX-2 enzymatic activity is commonly accompanied by associated oxidative mechanisms (co-oxidation) and free radical production. The catalytic activity of COX consists of a series of radical reactions that use molecular oxygen and generate intermediate ROS. Elevated levels of COX-2 protein are associated with increased ROS production and apoptosis in cultured renal cortical cells and human mesangial cells . It has been suggested that COX-2–mediated lipid peroxidation, rather than prostaglandins, can induce DNA damage via adduct formation. A COX-2 specific inhibitor, NS-398, was able to reduce the oxidative activity, with prevention of oxidant stress.

In addition to ROS generated by cyclo-oxygenase per se, prostanoids may also activate intracellular pathways that generate ROS. Locally generated ROS may damage cell membranes, leading to lipid peroxidation and release of arachidonic acid. Prostanoids released during inflammatory reactions cause rapid degenerative changes in some cultured cells, and their potential cytotoxic effect has been suggested to occur by accelerating intracellular oxidative stress. Thromboxane and PGE ₂ acting through the EP1 receptor have been reported to induce NADPH oxidase and ROS production. Of interest, PGE ₂ acting through the EP4 receptor inhibits macrophage oxidase activity. As noted, there is also evidence for cross-talk between COX-2 and ROS, such that ROS may induce COX-2 expression. Interestingly, during aging there is ROS-mediated NF-kB expression, which increases COX-2 expression in the kidney. Furthermore, this appears to induce a vicious cycle, since COX-2 then serves as a source of ROS. This interaction of COX-derived prostaglandin and ROS production has posited to play a role in development of hypertension. The amount of renal ROS resulting from COX activity increases with age, such that up to 25% of total kidney ROS production in aged rat kidneys is inhibited by NSAID administration.