INTRODUCTION

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

ARACHIDONIC ACID THAT IS ESTERIFIED at the sn-2 position of the glycerol backbone of membrane phospholipids can be released intracellulary, then enzymatically converted to a family of metabolites known as eicosanoids. Three major enzymatic pathways, cyclooxygenase (COX), lipoxygenase, and cytochrome P-450 (CYP450), are responsible for the generation of biologically active eicosanoids. The arachidonic acid metabolites formed are determined by factors including species, tissue, and hormonal background. In recent years, studies have provided convincing evidence that metabolites of the major enzymatic pathways contribute importantly to the regulation of renal hemodynamics (70, 99, 110). This review will focus on the emerging concepts related to the control of renal blood flow and glomerular filtration by eicosanoids.

	COX METABOLITES: VASODILATOR PROSTAGLANDINS

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

Prostaglandins PGE₂ and PGI₂ are synthesized by vascular and tubular structures throughout the kidney and regulate renal hemodynamics and tubular transport function (23, 110, 142). Overall, the COX metabolites PGE₂ and PGI₂ have been demonstrated to increase renal blood flow and glomerular filtration rate (70, 110). Iloprost, a PGI₂ analog, increases renal blood flow and decreases urinary sodium excretion when administered into the dog renal artery (165). The antinatriuretic effect of iloprost appears to be due to its effect on arterial pressure because the PGI₂ analog causes natriuresis when administered at a dose that does not decrease arterial pressure (70, 110, 165). Direct comparison of the two prostaglandins has demonstrated that the renal vasodilatory response to PGI₂ is approximately one-half that demonstrated to PGE₂ (165)_. Similarly, the PGE₂ analog viprostol is a more potent renal vasodilator compared with iloprost (31). Besides increasing renal blood flow, PGE₂ elicits increases in sodium excretion and renal interstitial hydrostatic pressure, suggesting a role for this prostanoid in natriuretic responses (110, 131, 165).

The regulation of water and electrolyte homeostasis by prostaglandins is partly mediated through their renal hemodynamic actions. A number of studies have demonstrated that the increase in sodium excretion in response to increased renal perfusion pressure is markedly attenuated by inhibition of the COX pathway (90, 110, 132). The natriuretic response to increasing renal interstitial hydrostatic pressure is blunted by indomethacin or meclofenamate (110, 131), but not during COX-2 inhibition (51), suggesting involvement of a COX-1-derived metabolite. PGE₂ may be the COX-derived metabolite that mediates pressure-induced natriuresis because PGE₂ is a renal vasodilator and inhibits epithelial sodium transport in the medullary thick ascending limb (mTAL) and cortical collecting duct (131, 132, 165). The actions of PGE₂ on renal medullary blood flow could also explain its contribution to the pressure-natriuretic response (131, 148). PGE₂ dilates endothelin-precontracted isolated outer medullary descending vasa recta vessels where PGE₂ is synthesized by neighboring collecting duct epithelial and interstitial cells (148). Additionally, intrarenal infusion of PGE₂, but not PGI₂, restores the pressure-natriuretic response during COX blockade (70, 110). Thus PGE₂ is necessary for the full expression of the pressure-natriuretic response and is important for the kidney's ability to maintain water and electrolyte balance.

PGE₂ is the major renal COX metabolite in the kidney, and the PGE₂ receptors (EP) are the most abundant prostanoid receptors in the kidney (22, 23, 70, 110). Four seven-transmembrane-spanning domain receptor subtypes have been identified, and the cDNA for each EP receptor has been determined (22, 23). Renal localization of the mRNA and immunohistochemical expression for the EP receptors have been studied. Collecting ducts of the cortex and papilla contain the EP₁ receptor, and tubules of the outer medulla and cortex express the EP₃ receptor (151). The EP₂ receptor protein is also detectable in the media of human renal arteries and arterioles (101). High levels of EP₃ receptor mRNA expression is localized to the mTAL, with lower levels detected in the cortical collecting duct (23, 101, 158). Glomeruli also have been demonstrated to have strong EP₃ protein expression in the human kidney (101). The EP₄ receptor is expressed in the glomerulus, media of renal arteries, and renin-secreting juxtaglomerular granular cells, with lower levels of expression detected in the outer medulla (22, 24, 101). This renal localization of the EP receptors is intimately associated with the specific physiological actions of PGE₂.

Characterization of the EP receptors and their intracellular signaling mechanisms have been extensively studied in nonrenal cells, but the contribution of these receptors to the control of renal blood flow remains unresolved. Renal vascular smooth muscle cells contain the receptor protein for the EP₁ and EP₃ receptors (22). Because EP₁ and EP₃ receptor activation stimulates cellular signaling mechanisms that would oppose vasodilation, these receptors may antagonize the PGE₂-mediated increase in renal blood flow. Activation of the EP₁ receptor stimulates phosphatidylinositol hydrolysis, resulting in receptor-operated calcium mobilization, and evidence suggests that PGE₂ acts through the EP₁ receptor in rabbit cortical collecting duct cells to inhibit sodium transport (58, 59). PGE₂ activation of the EP₁ receptor causes vascular smooth muscle contraction in a variety of tissues and may be responsible for the PGE₂-evoked increase in intracellular calcium observed in cultured mesangial cells (22, 36). Early studies that localized the EP₃ receptor to the mTAL cells and cortical collecting duct focused investigation on its contribution to tubular transport processes (22, 23). The EP₃ receptor signals via pertusssis toxin-sensitive G_i and inhibition of adenylate cyclase and may oppose the vasodilatory response to PGE₂ (22, 170). This possibility has been suggested by studies in EP₃ gene-disrupted mice that show a prolonged systemic vasodepressor response to PGE₂ administration that is more pronounced in male mice (10). EP₃ receptor activation may also have renal hemodynamic actions because misoprostol, an EP₂-, EP₃-, and EP₄-receptor agonist, causes a slight vasoconstriction and decreased glomerular filtration rate in humans (109). Additionally, mice that lack the EP₃ receptor have elevated renal blood flows compared with wild-type mice (9). This is consistent with the renal vasoconstriction to PGE₂ seen in the juxtamedullary vasculature of the rat (75) and supports the concept that the EP₁ and EP₃ receptors contribute to the control of renal blood flow.

EP₂ and EP₄ receptors will be considered together because they share similar signaling and physiological characteristics. Stimulation of these EP receptors activates G_s coupled to adenylate cyclase and elevates intracellular cAMP levels in rabbit and rat preglomerular vessels (22, 23, 33, 135). PGE₂ stimulates cAMP production, resulting in mesangial cell relaxation, and is thought to be the mechanism by which PGE₂ causes an increase in glomerular filtration rate (100). Disruption of the EP₂ receptor in mice does not alter renal blood flow or vascular resistance (9) but unmasks a systemic vasoconstriction in response to PGE₂ (85). PGE₂ also vasoconstricts the afferent arterioles of EP₂ receptor-deficient mice (66). These mice lacking an EP₂ receptor develop hypertension when fed a high-salt diet (85). Investigation of EP₂ receptor regulation is of extreme interest because the vascular and transport effects of PGE₂ are altered in renal pathophysiological states.

PGI₂ exerts its biological effects via stimulation of the PGI₂ receptor (IP) found throughout the renal cortex and medulla (22, 70, 110). The IP receptor is a seven-transmembrane-spanning receptor coupled to the generation of intracellular cAMP (22). PGI₂ dose dependently stimulates adenylate cyclase activity in freshly isolated preglomerular vessels (33, 135). Cicaprost and iloprost are PGI₂ analogs that selectively activate the IP receptor and vasodilate the glomerular microvasculature (31, 32, 110). IP receptor activation is not as effective as EP receptor activation in causing renal vasodilation (31, 32, 165). Even with this being the case, IP receptor activation in response to PGI₂ may be the primary mechanism that opposes vasoconstrictor agonists because renal vascular smooth muscle cell PGI₂ production is substantially greater than that of PGE₂ (126).

Studies investigating PGE₂, PGI₂, or analogs of these COX metabolites have demonstrated that vasodilatory prostaglandins oppose the effects of renal vasoconstrictors (17, 31, 32, 126). Renal hemodynamic interactions between prostaglandins and angiotensin II have been extensively studied (Fig. 1). Angiotensin II increases the production of prostaglandins by the kidney (110, 126), and these COX metabolites buffer the angiotensin II-mediated decrease in renal blood flow (31, 32, 79). Although prostaglandins buffer the whole kidney renal blood flow responses to angiotensin II, there may be differences between superficial and juxtamedullary afferent arteriolar populations. Microdissected superficial afferent arterioles demonstrate an enhanced responsiveness to angiotensin II during COX inhibition (81, 179). In contrast, the angiotensin II vasoconstrictor response of juxtamedullary afferent arterioles (67) and descending vasa recta is not altered by COX blockade (56). The cellular signaling mechanism by which prostaglandins modulate the cytosolic calcium response to angiotensin II has been investigated by using cultured renal arteriolar vascular smooth muscle cells. Purdy and Arendshorst (126) demonstrated that the renal vascular smooth muscle cell calcium response to angiotensin II was enhanced during indomethacin treatment. In this study, PGE₂ and PGI₂ attenuated the angiotensin II-mediated intracellular calcium response (126). These results suggested that a vascular smooth muscle cell-derived COX metabolite was involved in the angiotensin II-induced calcium response (Fig. 1). Although prostaglandins are considered to be primarily endothelial derived, renal preglomerular vascular smooth muscle cells produce PGE₂ and PGI₂ (87, 126). Renal vascular smooth muscle cell PGI₂ production is greater than that of PGE₂, and angiotensin II increases the production of these prostaglandins (126). Taken together, these studies suggest that endothelial- and vascular smooth muscle cell-derived vasodilatory prostaglandins activate adenylate cyclase and attenuate the renal vascular response to angiotensin II.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Cell diagram depicting the renal vascular smooth muscle and endothelial cell interactions between angiotensin II and arachidonic acid metabolites. HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; CYP450, cytochrome P-450; 12-LO, 12-lipoxygenase; AT1 and AT2, type 1 and type 2 angiotensin II receptor, respectively; B2, bradykinin type 2 receptor.

Similarly, vasodilator prostaglandins have been implicated in the modulation of the vasoconstrictor responses to norepinephrine and vasopressin. Norepinephrine stimulates the release of prostaglandins from the kidney (76, 110). The renal vasoconstrictor response to norepinephrine is enhanced by COX blockade (8, 15, 67), but addition of prostaglandin metabolites can either enhance or attenuate this response (110). Norepinephrine reactivity of isolated perfused rabbit afferent and efferent arterioles is enhanced by indomethacin (8, 67). This modulation of the afferent arteriolar vasoconstriction in response to norepinephrine appears to be mediated via the COX-2 enzyme because the decrease in vascular diameter is enhanced to the same extent by the nonselective COX inhibitor indomethacin or the COX-2 inhibitor NS-398 (67). Like norepinephrine, vasopressin stimulates renal and glomerular prostaglandin production (2, 140). In addition, systemic administration of COX inhibitors enhances and prolongs the renal vasoconstrictor response to vasopressin (15, 164). Hence, COX-derived prostaglandins contribute to the renal blood flow responses elicited by norepinephrine and vasopressin.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide that plays a crucial role in maintaining fluid and electrolyte homeostasis, and its response is modulated by COX metabolites (49, 118, 124). The involvement of the COX pathway in the renal vasoconstrictor response to ET-1 appears to be complex. Actions of ET-1 on the ET_A and ET_B receptors include phospholipase A₂ (PLA₂) stimulation and the resultant increase in COX-derived mediators (16, 118, 141). Oyekan et al. (118) demonstrated that the ET-1-evoked decreases in renal blood flow and glomerular filtration rate were enhanced by COX blockade when studied in vivo. The same group of investigators showed in the isolated perfused kidney that COX inhibition attenuated the renal vasoconstrictor response to ET-1 (117). In a recent study, the COX inhibitor indomethacin attenuated the afferent arteriolar decrease in diameter and renal microvascular smooth muscle cell calcium response to ET-1 (72). Because indomethacin had a greater effect in the juxtamedullary preparation with an intact endothelium than in freshly isolated vascular smooth muscle cells, these results suggest that endothelial-derived COX vasoconstrictor metabolites contribute importantly to the ET-1-mediated afferent arteriolar response (72). The opposing findings of interactions between COX and ET-1 may be due to the absence of bloodborne elements such as platelets or sympathetic innervation when studied in vitro. The relative influence of ET_A and ET_B receptors could also be different with the ET_A receptor-mediated activation of arachidonic acid pathways predominating under certain experimental conditions. These paradoxical findings demonstrating positive and negative contributions of COX-derived metabolites to ET-1 are not limited to the kidney (40, 92, 141) and most likely reflect in vitro vs. in vivo experimental conditions and the prevailing COX metabolites.

On the other hand, renal vasodilatory responses to bradykinin and acetylcholine have been demonstrated to involve the generation and actions of prostaglandins (70, 110) (Fig. 2). Bradykinin has been demonstrated to increase the renal production of prostaglandins, and one-third of the bradykinin-mediated vasodilation is prostaglandin dependent (98). Release of endothelial PGI₂ and nitric oxide occurs after acetylcholine administration (43), and combined COX and nitric oxide blockade prevents the acetylcholine-induced increase in renal blood flow (177). Interestingly, the actions of COX and nitric oxide overlap, and one system can fully compensate for the other.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Cell diagram depicting the endothelial-derived metabolites contributing to the bradykinin-evoked afferent arteriolar vasodilation. Renal microvascular smooth muscle cell signaling pathways activated by nitric oxide (NO), prostacyclin (PGI2), and 11,12-EET that result in vasodilation are shown. NOS, nitric oxide synthase; COX, cyclooxygenase; AC, adenylate cyclase; GC, guanylate cyclase; PKG and PKA, protein kinase G and A, respectively;

	COX METABOLITE: THROMBOXANE

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

Thomboxane A₂ (TxA₂) and PGH₂ are two vasoconstrictors produced in small quantities by the kidney under normal physiological conditions (120). A major renal site for TxA₂ synthesis is the glomerular mesangial and podocyte cells (70, 93, 142). COX-derived vasoconstrictors act on thromboxane/enderperoxide (TP) receptors and influence renal hemodynamics to a greater extent during pathophysiological states (70, 110, 149). Administration of TxA₂ analogs either in vivo or in vitro has been consistently shown to decrease renal blood flow and glomerular filtration rate, but the relative influence on pre- and postglomerular resistance could not be resolved in studies evaluating whole kidney hemodynamics (70, 110). Hayashi et al. (57) directly evaluated the actions of the TxA₂ mimetic U-44069 on afferent and efferent arteriolar diameter in the isolated perfused rat hydronephrotic kidney. These investigators found that U-44069 constricted the afferent arteriole to a greater degree than the efferent arteriole (57). TxA₂ and TxA₂-mimetic actions are the result of TP receptor activation because TP-receptor antagonists block all the renal hemodynamic effects of these agents (70, 110).

The primary TP receptor (TPa) has been cloned and is expressed in the kidney (1, 61). Human intrarenal arteries and glomeruli have also been demonstrated to possess TP receptors (25). TPa and the splice variant TPK are coupled to G_q that signals through phospholipase C (PLC), resulting in elevated intracellular inositol triphosphate (IP₃) levels and mobilization of calcium from intracellular stores (115, 128). This signaling pathway has been demonstrated in cultured rat glomerular mesangial cells that respond to the TxA₂ mimetic U-46619 by increasing cellular IP₃ and calcium levels (150). Like other vasoconstrictors, TP receptor-mediated decreases in afferent and efferent arteriolar diameter are the result of activating different intracellular signaling mechanisms. Afferent arteriolar vasoconstriction in response to U-44069 is blocked by L-type calcium channel inhibition (57). In contrast, the smaller decrease in efferent arteriolar diameter evoked by TP receptor activation is unaltered by the presence of the L-type calcium channel inhibitors diltiazem or nifedipine (57). Thus TxA₂-mediated afferent arteriolar vasoconstriction involves calcium influx via L-type channels, whereas the decrease in efferent arteriolar diameter is independent of L-type calcium channel activation.

TP receptor activation can also contribute to the angiotensin II-mediated renal vasoconstriction. Involvement of TxA₂ and TP receptor activation in the renal vascular actions of angiotensin II has been associated with conditions in which the renin-angiotensin system is elevated and circulating angiotensin II levels are high (70, 110). Along these lines, kidney TxA₂ synthesis increases during various renal disorders including nephritis, cyclosporin nephrotoxicity, and renal transplant rejection (70, 110). A recent study demonstrated that ureteral obstruction led to a greater intratubular pressure in TP receptor-deficient mice compared with wild-type mice, suggesting that TxA₂ is an important regulator of renal vascular tone in this pathological state (145). Therefore, TP-receptor antagonists have possible clinical utility in the treatment of renal hemodynamic dysfunction, particularly when the renin-angiotensin system is activated.

	COX METABOLITES: RENAL BLOOD FLOW AND FLUID-ELECTROLYTE HOMEOSTASIS

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

The extremely efficient autoregulation of renal blood flow and glomerular filtration rate is the result of the complicated interplay between both the tubuloglomerular feedback and myogenic responses of the renal microvasculature (70, 110). The contribution of COX metabolites to renal blood flow autoregulation has been studied by using inhibitors of COX in a number of species. In cases where renal blood flow autoregulation has been assessed, COX inhibition has failed to alter the autoregulatory responses to changes in perfusion pressure (70, 110). Nevertheless, COX metabolites may be involved in the modulation of tubuloglomerular feedback responses. Several micropuncture studies have demonstrated that COX inhibition blunts tubuloglomerular feedback responses (123, 144, 171). In addition, tubular prostaglandin infusion alters the sensitivity of the tubuloglomerular feedback response (70, 110). Systemic or tubular perfusion of TxA₂ mimetic U-46619 enhanced the sensitivity of the tubuloglomerular feedback response (172). The TxA₂ mimetic has also been demonstrated to enhance the tubuloglomerular feedback response in wild-type mice but does not alter this response in TP receptor-deficient mice (145). Interestingly, TP receptor gene disruption did not attenuate the tubuloglomerular feedback response under control conditions (145). Thus COX metabolites do not appear to mediate renal autoregulatory responses but do have modulatory influence on tubuloglomerular feedback responses. Interest in the involvement of the COX pathway and renal autoregulatory responses has been revitalized by the discovery of COX-2 in the macula densa and adjacent epithelial cells (55, 178).

Gene-disruption studies would appear to be ideally suited to investigations aimed at determining the contribution of COX-1 and COX-2 on renal blood flow autoregulatory responses. This has not been the case because COX-derived metabolites are essential for neonatal development and reproduction. COX-1-deficient mice have delayed maturation, and pups do not survive past postpartum day 1 (11). This phenotype can be rescued by PGF₂ injection at term (11). COX-2-deficient mice develop severe nephropathy linked to impaired nephron maturation (39, 102). These phenotypic consequences of COX enzyme deficiency have precluded studies designed to evaluate renal blood flow and fluid electrolyte homeostasis in COX gene-disrupted mice. Development of organ-targeted, COX gene-disrupted mice should prove more useful in evaluating the influence of COX-1 and COX-2 metabolites on renal vascular and tubular function. Therefore, investigations have relied on pharmacological agents that selectively inhibit either COX-1 or COX-2.

Recently, interactions between COX-2 and neuronal nitric oxide synthase (nNOS) have been observed for afferent arteriolar tubuloglomerular feedback responsiveness (Fig. 3) (62, 63). Nitric oxide is an oxidizing radical that interacts with heme-containing proteins like COX (64). COX-2 and nNOS are constitutively expressed predominantly in and around the macula densa (12, 55, 173, 178). This unique localization suggested that these enzymes could interact and play an important role in tubular flow-dependent responses. Recent studies conducted by Ichihara et al. (62, 63) demonstrated that, during increased tubular flow past the macula densa, increased nNOS-derived nitric oxide dilates the preglomerular vasculature directly and stimulates COX-2 vasodilatory prostaglandin production. Therefore, the increased levels of nitric oxide and prostaglandins buffer the magnitude of the tubular glomerular feedback-mediated afferent arteriolar vasoconstriction.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Cell diagram depicting the pathways by which changes in solute delivery at the macula densa segment lead to renin secretion and tubuloglomerular feedback (TGF)-mediated afferent arteriolar vasoconstriction. nNOS, neuronal NOS.

As previously discussed, COX-derived metabolites interact with and modulate the renal hemodynamic responses to angiotensin II. Metabolites of COX pathways can also modulate the renin-angiotensin system by altering kidney renin production. Numerous studies have suggested a role for COX-derived prostaglandins in the regulation of renal renin secretion. Overall, prostaglandins have been demonstrated to stimulate renin release from kidneys (Fig. 3), and COX inhibition suppresses renin production (70, 110). Prostaglandins appear to mediate renin release during macula densa activation (50, 161, 174). In the isolated perfused rabbit juxtaglomerular apparatus preparation, COX-2 inhibition nearly abolished renin release in response to decreases in luminal NaCl (161). In support of the concept that COX-2-derived metabolites mediated renin secretion, Harding et al. (54) demonstrated that COX-2 inhibition prevented the increase in renal renin mRNA in animals fed a low-salt diet. Renal cortical COX-2 mRNA expression also increases in animals placed on a low-salt diet, and angiotensin-converting enzyme inhibition or angiotensin II type 1 receptor (AT₁) blockade enhances the increase in COX-2 (174). This study suggests that angiotensin II acts as a negative feedback controller that limits the stimulation of COX-2 and renin synthesis during low dietary salt intake. Overall, these studies suggest that an interaction between the renin-angiotensin system and COX-2 may contribute to the regulation of renal hemodynamics in response to salt depletion.

In support of the concept that an interaction between angiotensin II and COX-2 may contribute to renal hemodynamic function, renal blood flow and glomerular filtration rate decreased transiently in salt-depleted individuals given 400 mg/day of the COX-2 selective inhibitor celecoxib (134). The influence of COX-2 on renal hemodynamics in humans depends on salt intake because glomerular filtration rate does not change during COX-2 inhibition in subjects on a normal-salt diet (30). In contrast, acute administration of the COX-2 inhibitor MF-tricyclic did not alter renal blood flow in volume-depleted dogs (18). Although these studies suggest that macula densa COX-2-derived metabolites are essential for the kidneys response to dietary salt, the profile of prostaglandins produced by the macula densa cells is unknown. A recent study provides evidence that renal cortical PGE₂ stimulates renin secretion and maintains renal blood flow during salt depletion (80). Additionally, PGE₂ activation of EP₄ receptors may preserve renal hemodynamic function because EP₄ receptors are upregulated in isolated glomeruli, mesangial cells, and juxtaglomerular granular cells in response to low dietary salt (80). Therefore, COX-2-derived metabolite production is regulated and localized to the structure in the kidney that plays an essential role in renal blood flow and fluid-electrolyte homeostasis.

	LIPOXYGENASE METABOLITES

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

The lipoxygenase enzymes metabolize arachidonic acid to form leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and lipoxins (LXs). Lipoxygenase metabolites are primarily produced by leukocytes, mast cells, and macrophages in response to inflammation and injury (70, 110). The 5-lipooxygenase enzyme (5-LO) that synthesizes LTs has limited cellular distribution and has been found in leukocytes, mast cells, and macrophages (35). Although LTs are primarily produced by leukocytes, the glomerular mesangial cells and endothelial cells contain LTA₄-hydrolase that converts LTA₄ to LTB₄ (2, 13, 110). Glomeruli, mesangial cells, cortical tubules, and vessels also produce the 12-lipoxygenase (12-LO) product 12(S)-HETE and the 15-LO product 15-HETE (2, 13, 78). Gene expression of 5-LO, 12-LO, and 15-LO has been detected in cultured glomerular cells (91). Additionally, leukocyte-generated LTA₄ can be transformed to LXs via 12-LO in the glomerulus and endothelial cells (13). These lipoxygenase metabolites have effects on renal hemodynamics and glomerular filtration and have been implicated in inflammatory diseases and glomerulonephritis (70, 110).

The lipoxygenase metabolites, 12(S)-HETE, 15-HETE, LTs, and LXs, are involved in inflammatory responses and have effects on renal hemodynamics (13, 82, 96, 110). The lipoxygenase products 12(S)-HETE and 15-HETE are potent glomerular and renal vascular vasoconstrictors. 12(S)-HETE decreases renal blood flow and glomerular filtration independently of COX activity (82). Although 12(S)-HETE causes depolarization of the vascular smooth muscle cell membrane and may act through activation of PKC (96), the mechanism by which 12(S)-HETE constricts the preglomerular vasculature remains to be identified. In a recent study, the afferent arteriolar vasoconstriction and increase in renal microvascular smooth muscle cell calcium, in response to 12(S)-HETE, were greatly attenuated by diltiazem treatment (73). These results suggest that activation of voltage-gated L-type calcium channels is an important mechanism responsible for the renal vasoconstriction elicited by 12(S)-HETE.

The 12-LO product, 12(S)-HETE, appears to be critically involved in regulating angiogenesis and atherosclerosis. In various endothelial cell lines, 12-LO inhibition reduces cell proliferation and growth factor release, whereas overexpression of 12-LO stimulates cell migration and endothelial tube formation (112). Similarly, disruption of the 12/15-LO gene diminishes atherosclerotic lesions in apo E-deficient mice (38). Elimination of the leukocyte 12-LO enzyme in mice also ameliorates the development of streptozotocin diabetes (19). 15-LO appears to protect renal function during glomerulonephritis because transfection of rat kidney with human 15-LO enzyme suppresses inflammation and preserves glomerular filtration (106). Hence, 12-LO and 15-LO metabolites appear to protect the kidney during various inflammatory diseases.

LXs also increase renal vascular resistance and may act through activation of LT receptors (13, 82). LXA₄, LXB₄, and 7-cis-11-trans-LXA₄ when administered into the renal artery cause vasoconstriction (82). The actions of LXA₄ are COX dependent, whereas the effect of LXB₄ on renal hemodynamics is independent of COX activity (82). LTD₄ receptor blockade abolished the 7-cis-11-trans-LXA₄ renal vasoconstrictor response and demonstrates that the response was due to activation of a LT receptor (82). LTC₄ and LTD₄ also increase renal vascular resistance and decrease glomerular filtration rate when administered into the renal artery (13, 14, 110). The cellular signaling mechanisms utilized by LTs are better understood because LT receptors have been identified and characterized. Activation of the LTC₄ and LTD₄ receptors by their endogenous ligands results in renal and glomerular vasoconstriction (14, 110). In accordance with a role for LT receptors in glomerular inflammatory responses, LTC₄ has been shown to stimulate mesangial cell growth (13, 91). The effects of inhibition of LT receptors to decrease glomerular capillary pressure suggest that LTs constrict the efferent arteriole to a greater extent then the afferent arteriole (14).

Recently, a seven-transmembrane-spanning cysteinyl leukotriene receptor (CysLTR) implicated in inflammatory disorders has been identified and characterized in human embryonic kidney (HEK-293) cells (138). LTs activate the CysLTR expressed in HEK-293 cells, resulting in mobilization of intracellular calcium (138). Two CysLTRs have been found on the vascular smooth muscle and endothelium of the pulmonary vasculature (166). Another LT receptor that has recently been cloned and characterized in the rat is the LTB₄ receptor (160). When expressed in HEK-293 cells, the LTB₄ receptor demonstrated specific and high-affinity binding to LTB₄ (160). The LTB₄ receptor has been implicated in acute renal ischemic-reperfusion injury (113), and LTB₄ receptor blockers ameliroate nephrotoxic nephritis in rats (155). Moreover, polymorphonuclear neutrophil recruitment and reperfusion injury are greatly amplified in transgenic mice overexpressing the LTB₄ receptor (34). The contribution of CysLTRs and LTB₄ receptors to influence renal hemodynamics and their role in pathophysiological states remains to be determined.

Interactions between lipoxygenase metabolites and renal vasoactive autocoids have been investigated. The involvement of the lipoxygenase metabolite 12(S)-HETE in the renal vasoconstrictor response to angiotensin II has been demonstrated in several studies (Fig. 1) (17, 67, 107, 114). A study performed by Bell-Quilley et al. (17) demonstrated that, in the isolated perfused rat kidney, inhibition of the 12-LO pathway attenuated the angiotensin II-mediated decrease in renal blood flow. That angiotensin II, but not norepinephrine, evoked vasoconstriction of the afferent arteriole has also been demonstrated to be attenuated by the lipoxygenase inhibitor baicalein (67). Attenuation of the vasoconstrictor response to angiotensin II, but not norepinephrine, has also been demonstrated for large arteries of the skeletal muscle and pulmonary vasculature (84, 107). In contrast, lipoxygenase inhibition attenuated the renal arcuate artery vasoconstrictor response to norepinephrine and KCl but had no effect on the vascular response to ET-1 (177). Interactions between LTs and the vasodilator nitric oxide have been shown in isolated perfused renal arteries and veins (121, 122). Specifically, LTD₄ vasodilation of renal arteries was demonstrated to be endothelial dependent and mediated by nitric oxide (122). Thus much remains to be learned about the actions of lipoxygenase metabolites on renal hemodynamics even though these metabolites play a key role in glomerular nephritis and inflammatory diseases.

	CYP450 METABOLITES

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

The metabolism of arachidonic acid by CYP450 enzymes in the kidney has been recognized for several years and displays one of the highest organ CYP450 activities (65, 99, 110, 127). The kidney possesses two CYP450 monooxygenase pathways that metabolize arachidonic acid to generate epoxyeicosatrienoic acids (EETs) that are hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) and HETEs (26, 70, 99). CYP450 monooxygenases are mixed-function oxidases that depend on molecular oxygen and NADPH as cofactors (26). EETs and DHETs are formed primarily via CYP450 epoxygenase enzymes, and HETEs are formed primarily via CYP450 hydroxylase enzymes (99, 110). CYP450 enzymes are distributed throughout the kidney vasculature and tubular segments, and arachidonic acid metabolites of the CYP450 pathway have an impact on renal hemodynamics (65, 99, 110, 127).

Production of EET, DHET, and HETE occurs throughout the kidney, but the specific metabolites formed and the amounts produced of each metabolite vary depending on the cell type and CYP450 isoform expressed. The CYP450 4A gene family is the major pathway for synthesis of hydroxylase metabolites (99, 129, 168), whereas the production of epoxygenase metabolites is primarily via the 2C gene family (26, 65). Another epoxygenase-producing enzyme family, CYP450 2J, was initially demonstrated to be present in the lung epithelial and vascular smooth muscle (180). Members of the 2J family actively produce EETs and midchain HETEs, and the transcripts are most abundant in the mouse kidney and present in lower levels in the liver (94). In situ hybridization revealed that the CYP450 2J protein was present in the proximal tubules and collecting ducts of the renal cortex and outer medulla (94). Several other enzymes, including CYP450 2E1 and 2B1/2, are present in proximal and distal tubule cells, but their functional roles remain to be resolved (37). The specialized tissue distribution and expression of various renal CYP450 epoxygenase and hydroxylase isoforms capable of producing EETs, DHETs, and HETEs allow for site-specific regulation and action. The production of epoxygenase and hydroxylase metabolites by the kidney is regulated by hormonal and paracrine factors, including angiotensin II, endothelin, parathyroid hormone, and epidermal growth factor (65, 70, 99, 110, 127, 129). Regulation of renal CYP450 activity is also importantly involved in the kidney response to changes in dietary salt intake (26, 65, 129). Additionally, alterations in the production of CYP450 metabolites occur after uninephrectomy, the induction of diabetes mellitus, and during the development of hypertension (26, 65, 127). Investigations are beginning to describe some of the changes in and regulation of specific CYP450 isoforms that may be associated with alterations in renal hemodynamics that occur during various pathophysiological states.

	CYP450 HYDROXYLASE METABOLITE: 20-HETE

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

Renal arterioles, glomeruli, and pericytes surrouding vasa recta capillaries contain CYP450 4A enzymatic protein that is primarily responsible for the formation of 20-HETE (74, 77, 116, 127, 168). RT-PCR analysis of microdissected rat renal microvessels revealed the presence of CYP450 4A1, CYP450 4A2, and CYP450 4A3 mRNAs (74, 168). Interestingly, intravenous administration of antisense oligonucleotides against CYP450 4A1 or CYP450 4A2/4A3 markedly inhibited renal preglomerular vascular 20-HETE synthesis and implicated CYP450 4A1 as the major 20-HETE-forming isoform (168). Although earlier studies have produced conflicting data concerning the contribution of CYP450 hydroxylase metabolites to the control of renal hemodynamics, many of these conflicts have been recently resolved and a better understanding has emerged. 20-HETE is a potent vasoconstrictor of the preglomerular arterioles and may contribute importantly to the renal blood flow autoregulatory responsiveness of the afferent arteriole (99, 110, 129). The actions of 20-HETE on hemodynamics appear to be paracrine and autocrine in nature rather than by the exertion of its effects humorally or systemically (99, 110, 129).

The CYP450 -hydroxylase product 20-HETE has been demonstrated to be a renal vasoconstrictor, but under certain experimental conditions it causes vasodilation. 20-HETE vasodilated the isolated perfused rabbit kidney preconstricted with phenylephrine, and this vasodilation was determined to be COX dependent (29). In contrast, 20-HETE vasoconstricted the rabbit afferent arteriole preconstricted with norepinephrine (7). 20-HETE also resulted in vasoconstriction of the rabbit renal artery that was partially dependent on an intact endothelium and metabolism via COX (41). Dog renal arteries and rat afferent arterioles of the juxtamedullary region and isolated perfused rabbit afferent arterioles vasoconstrict in response to 20-HETE (74, 95). In the rat, the afferent arteriolar vasoconstriction to 20-HETE is COX independent (74), whereas about one-fourth of the vasoconstriction to 20-HETE in the rabbit afferent arteriole is mediated by an endothelial-dependent COX pathway (7). The COX-dependent responses most likely reflect the transformation of 20-HETE to vasodilatory metabolites 20-OH PGE₂ and 20-OH PGI₂ or vasoconstrictor metabolites 20-OH PGH₂ and 20-OH TxA₂ (99). Thus the direction of the COX-dependent vascular response may depend on the prevailing prostaglandin isomerase that transforms 20-HETE. Recently, stable agonistic analogs of 20-HETE have been synthesized by Dr. J. R. Falck, and these 20-HETE analogs vasoconstrict the rat preglomerular vasculature (4). In the future, these 20-HETE analogs will be valuable investigative tools for determining cellular signaling mechanisms used by 20-HETE and its role in renal hemodynamic function.

To date, no evidence has been presented for the existence of cell membrane-bound 20-HETE receptors. More likely, 20-HETE acts within the cell where it is produced or within a closely associated cell, a manner similar to the way nitric oxide acts. Investigations have evaluated the cellular signaling mechanisms utilized by 20-HETE to vasoconstrict the renal vasculature. The renal arcuate artery vasoconstrictor response to 20-HETE was demonstrated to be associated with membrane depolarization and a sustained rise in intracellular calcium in vascular smooth muscle cells isolated from this artery (95). 20-HETE afferent arteriolar vasoconstriction was abolished by K⁺ channel blockade, and 20-HETE inhibited a renal microvascular smooth muscle cell 250-pS Ca²⁺-activated K⁺ channel (K_Ca) recorded by cell-attached patch-clamp techniques (74, 181). More recently, Sun et al. (153) demonstrated that 20-HETE increased expression of extracellular signal-regulated kinases (ERK) and that tyrosine kinase inhibition greatly attenuated the renal arteriolar vasoconstriction to 20-HETE. These recent data suggest that activation of tyrosine kinase and subsequent closing of K_Ca channels is the cellular mechanism by which 20-HETE vasoconstricts afferent arterioles.

The contribution of the CYP450 metabolite 20-HETE to the renal vasodilator response to nitric oxide was recently evaluated (Fig. 2). In the renal microcirculation, 70-75% of the nitric oxide-mediated vasodilation has been demonstrated to be cGMP independent (3, 5, 152). CYP450 inhibitors 17-octadecynoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) abolished the cGMP-independent renal arteriolar vasodilation to nitric oxide donors, suggesting that nitric oxide increases renal blood flow via inhibition of the vasoconstrictor CYP450 metabolite 20-HETE (3, 5). Nitric oxide inhibits renal microvessel formation of 20-HETE, and this could be due to nitric oxide's ability to bind heme and inhibit CYP450 enzymes (5, 152). Indeed, nitric oxide has been shown to bind the heme moiety of CYP450 4A enzymes and inhibit formation of 20-HETE (152). Similarly, chronic inhibition of nitric oxide with nitro-Larginine methyl ester for 10 days increases 20-HETE formation and CYP450 4A protein levels but had a very slight effect on epoxygenase activity (119). Because 20-HETE vasoconstricts the renal vasculature by inhibiting K_Ca channels (74, 181), a decrease in 20-HETE levels in vascular smooth muscle would lead to activation of K_Ca channels. Thus the nitric oxide-elicited renal vasodilator actions appear to be mediated through its effects to elevate cGMP levels and decrease 20-HETE formation.

	CYP450 EPOXYGENASE METABOLITES

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

The kidney has the ability to produce epoxygenase metabolites primarily via CYP450 enzymes of the 2C gene family (26, 65). CYP450 4A2 and CYP450 4A3 expressed in sF9 insect cells have also been shown to form 11,12-EET, suggesting that these isoforms may contribute to renal vascular epoxygenase production (169). Microsomes prepared from renal microvessels and vessels of other organ beds metabolize arachidonic acid to EETs (53, 74, 130, 169, 183). Although receptors for EETs have not been identified, specific binding of 14,15-EET in monocytes has been demonstrated (175, 176). EETs can also be incorporated into vascular smooth muscle and endothelial cell membranes (162, 163), suggesting that epoxygenase metabolites may have long-lasting effects or can be released after hormonal activation. Like CYP450 hydroxylase metabolites, epoxygenase metabolites of arachidonic acid effect renal blood flow and glomerular filtration rate by acting locally (65, 70, 110). 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 (47, 71, 156). These COX-dependent effects may be the result of 5,6-EET and 8,9-EET conversion to prostaglandin- or thromboxane-like compounds (47, 156).

Even though 11,12-EET is an excellent candidate for being an endothelium-derived hyperpolarizing factor (EDHF) in the renal microcirculation (65, 71), epoxygenase metabolites have also been found to be either renal vasoconstrictive or vasodilatory (65, 110). Infusion of 5,6-EET or 8,9-EET into the renal artery of the rat results in an increase in renal vascular resistance (156). The observed vasoconstriction was found to be COX dependent because 5,6-EET increased renal blood flow during administration of indomethacin (156). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET vasodilated the isolated perfused rabbit kidney preconstricted with phenylephrine, and the vasodilation to 5,6-EET was COX dependent (28, 29). In a study that directly examined the responses of the preglomerular vasculature, it was demonstrated that 11,12-EET and 14,15-EET vasodilated, and 5,6-EET resulted in vasoconstriction of the interlobular artery and afferent arterioles (71). Additionally, it was demonstrated that the preglomerular vasoconstriction to 5,6-EET was COX dependent and required an intact endothelium, whereas the vasodilation to 11,12-EET was the result of direct action of the epoxide on the preglomerular vascular smooth muscle (71). In the dog, 11,12-EET has been shown to dilate renal arteries and activate K_Ca channels (182). The activation of K⁺ channels by 11,12-EET in the renal preglomerular vasculature appears to be mediated by cAMP stimulation of protein kinase A because the afferent arteriolar vasodilation to the sulfonimide analog of 11,12-EET was greatly attenuated by protein kinase A inhibition (69). Furthermore, the action of 11,12-EET on renal vessels and K_Ca channels is consistent with the possibility that this metabolite is an EDHF. 11,12-EET produced by CYP450 2C has recently been identified to be EDHF in the coronary microvasculature (42), and an epoxygenase-derived EDHF appears to mediate a large portion of the afferent arteriolar vasodilatory response to bradykinin in the isolated perfused kidneys (Fig. 2) (48).

	CYP450 METABOLITES: RENAL HEMODYNAMIC REGULATORY INFLUENCE

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

Besides the direct actions of CYP450 metabolites, a more important role for these arachidonic acid products is their contribution to modulate renal vascular responses to hormones and renal blood flow autoregulation. A number of studies have suggested that CYP450 metabolites may participate in some component of renal blood flow autoregulation (99, 110, 129). In isolated canine arcuate arteries treated with indomethacin, arachidonic acid administration enhanced myogenic responsiveness and was blocked by CYP450 inhibition (83). Infusion of 17-ODYA into the renal artery in vivo attenuated renal blood flow autoregulation (110, 129). Additionally, the afferent arteriolar vasoconstriction to increasing renal perfusion pressure was attenuated by CYP450 inhibitors, and this attenuation was associated with impairment of glomerular capillary pressure autoregulation (110, 129). However, selective inhibition of the epoxygenase pathway with the imadazole derivatives miconazole or clotrimazole had no effect on renal blood flow autoregulation in vivo or in the isolated perfused kidney (139, 183). These studies suggest that an endogenous -hydroxylase metabolite of arachidonic acid participates in renal blood flow autoregulatory responses.

The lack of CYP450-selective inhibitors that can block the renal hydroxylation and epoxidation of arachidonic acid has precluded evaluation of specific CYP450 pathways in renal autoregulatory responses. A major breakthrough by Dr. J. R. Falck's group has been the recent development of compounds that selectively inhibit renal microsomal epoxygenase or -hydroxylase enzymatic activity (167). In a recent study, administration of the selective hydroxylase inhibitor DDMS greatly attenuated the afferent arteriolar vasoconstriction to increasing renal perfusion pressure (68). In contrast, the selective epoxygenase inhibitors 6-(2-propargyloxyphenyl)hexanamide (PPOH) and N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) enhanced afferent arteriolar diameter responses to elevations in renal perfusion pressure (68). Taken together, these results provide further support for the concept that 20-HETE is an integral component of the afferent arteriolar autoregulatory adjustment and that release of vasodilatory epoxygenase metabolites in response to increases in renal perfusion pressure attenuates this preglomerular vasoconstriction.

Micropuncture studies have provided evidence that a CYP450 metabolite may be involved in the tubuloglomerular feedback response (44, 70, 129) (Fig. 3). Franco et al. (44) demonstrated that tubular perfusion of arachidonic acid simulated a tubuloglomerular feedback response. Tubular infusion of inhibitors of the lipoxygenase and COX pathways had no affect on tubuloglomerular feedback-mediated reductions in stop-flow pressure (44). Inhibition of the CYP450 pathway was demonstrated to abolish both the tubuloglomerular feedback response and the reduction in stop-flow pressure to tubular perfusion of arachidonic acid (183). Additionally, tubular perfusion of 20-HETE restores the tubuloglomerular feedback response after CYP450 inhibition (183). These studies implicate an important role for metabolites of the CYP450 hydroxylase pathway in the tubuloglomerular feedback response.

In accordance with the evidence that CYP450 metabolites are involved in tubuloglomerular feedback responsiveness, these metabolites have also been implicated in the renal maintenance of fluid and electrolyte homeostasis during changes in dietary salt (70, 99, 110). 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 (184). Because both 20-HETE and EETs inhibit tubular sodium reabsorption (26, 65, 99, 129), the increased sodium excretion to CYP450 blockade appears to be secondary to the increase in renal interstitial hydrostatic pressure and papillary blood flow. An alternative explanation is that these CYP450 inhibitors cannot discriminate between the specific CYP450 4A isoforms expressed in the renal vessels and tubules and the effect of 17-ODYA on the vasculature CYP450 4A predominates. High dietary salt intake increases renal epoxygenase production and urinary excretion of EETs (26, 27). Oyekan and colleagues (119) recently demonstrated that 2% NaCl in the drinking water for 1 wk increased epoxygenase activity in the renal cortex and medulla and reduced 20-HETE formation in the cortex. The increased epoxygenase activity was also associated with upregulation of the CYP450 2C enzymes (27). Besides the direct action of EETs on the kidney to promote sodium excretion (26, 65), part of the excretory response may be attributable to the ability of 14,15-EET to inhibit renin secretion (60). Interestingly, administration of clotrimazole induced hypertension in high-salt-diet animals, suggesting an antihypertensive role for EETs (97). More recently, the production of 20-HETE and EETs by glomeruli has been shown to be decreased in rats fed a diet containing 8% NaCl (78). This study also demonstrated increased 20-HETE and CYP450 4A protein levels in the glomerulus of rats fed a low-salt diet (78). Thus the regulation of renal CYP450 production may act importantly on kidney and glomerular hemodynamics to maintain water and electrolyte homeostasis during alterations in dietary salt intake.

Previous studies have provided evidence that CYP450 metabolites may modulate responses to hormonal and paracrine agents; yet, little is known about the renal vascular interactions between these vasoactive compounds and metabolites of the CYP450 pathway. Vasopressin is one such vasoactive peptide that has been demonstrated to increase renal effluent CYP450 metabolites (164). Renal mesangial cell increases in cytosolic calcium elicited by vasopressin are amplified by epoxygenase metabolites (159). Additionally, CYP450 inhibition reduced the magnitude of the mesangial cell increase in calcium in response to vasopressin (159). 14,15-EET has also been shown to augment the ability of vasopressin to increase cultured mesangial cell thymidine incorporation (147). In the isolated perfused rat kidney, the renal vasoconstrictor response to vasopressin was attenuated by CYP450 inhibition (164). Taken together, these studies demonstrate that CYP450 metabolites contribute to glomerular proliferative and renal hemodynamic responses to vasopressin.

Like COX metabolites, CYP450 metabolites participate in the renal hemodynamic response elicited by the powerful vasoconstrictor ET-1 (72, 117, 118). ET-1 increases renal efflux of 19-HETE and 20-HETE in the isolated perfused kidney, and the ET-1-induced increases in renal vascular resistance are attenuated by CYP450 hydroxylase inhibition (117, 118). More recently, it was shown that administration of the CYP450 hydroxylase inhibitor DDMS significantly attenuated the afferent arteriolar vasoconstriction evoked by ET-1 (72). The results of this study suggest that -hydroxylase metabolites of the CYP450 pathway contribute to the renal vascular actions of endothelin. In contrast, ET-1-mediated afferent arteriolar vasoconstriction was enhanced when the CYP450 epoxygenase pathway was inhibited by MS-PPOH (72). The ability of EETs to oppose the vasoconstrictor effect does not involve calcium regulation at the level of the renal microvascular smooth muscle cell, because MS-PPOH did not change the intracellular calcium response to ET-1 (72). These studies suggest that an endothelial-derived vasodilatory EET opposes the preglomerular vasoconstrictor activity of ET-1.

The contribution of renal microvascular smooth muscle cell signaling mechanisms utilized by CYP450 hydroxylase metabolites to the ET-1-mediated decrease in renal blood flow is beginning to be resolved. In addition to attenuating the afferent arteriolar vasoconstriction, DDMS reduced the rise in renal microvascular muscle cell calcium elicited by ET-1 (Fig. 4) (72). Although ET_A and ET_B receptor stimulation can result in PLA₂ activation and arachidonic acid release (16, 118, 141), the ET-1 increase in calcium in freshly isolated renal microvascular smooth muscle cells is primarily due to ET_A receptor activation (146). These studies demonstrated that a vascular smooth muscle cell-derived CYP450 hydroxylase metabolite importantly contributes to the ET_A receptor-mediated afferent arteriolar vasoconstriction (72, 146). Interestingly, a CYP450 hydroxylase metabolite transformed by cyclooxygenase to a vasoconstrictor PGH₂ analog, 20-OH PGH₂, may contribute to the renal response to ET-1 (99, 117, 118). Nevertheless, the exact nature of the CYP450 hydroxylase metabolites involved in the afferent arteriolar response to ET-1 remains to be defined.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Intracellular calcium response of single microvascular smooth muscle cells exposed to endothelin-1 (ET-1) in the absence and presence of the cytochrome P-450 (CYP450) hydroxylase inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). DDMS decreased peak intracellular calcium response and eliminated the steady-state calcium response to ET-1. Inset: contraction of a preglomerular smooth muscle cell to ET-1. Data are from Ref. 73.

Angiotensin II is another vasoactive peptide that has complex interactions with eicosanoids, including CYP450 metabolites (Fig. 1). Afferent arteriolar responses to angiotensin II, but not norepinephrine, are enhanced by nonselective inhibition of the CYP450 pathway (67). In the presence of AT₁ receptor antagonism, angiotensin II causes an endothelial-dependent vasodilation in rabbit afferent arterioles that is inhibited by AT₂ receptor or CYP450 blockade (6). These studies suggest that AT₂ receptor activation of vasodilatory epoxygenase metabolites oppose the angiotensin II-mediated decrease in renal blood flow. On the other hand, angiotensin II has also been shown to increase 20-HETE release from isolated preglomerular microvessels (154). A preliminary report shows that 20-HETE contributes to one-half of the angiotensin II-mediated decrease in diameter in interlobular arteries with the endothelium removed (154). The CYP450 inhibitor 17-ODYA blocked angiotensin II-mediated inhibition of renal microvascular smooth muscle cell K_Ca channel activity (154). Taken together, these studies support the concept that angiotensin II releases a vasodilatory epoxygenase metabolite from the endothelium and the vasoconstrictor 20-HETE from the preglomerular microvasculature, which participate in the overall renal hemodynamic response.

	ISOPROSTANES

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR... COX METABOLITE: THROMBOXANE COX METABOLITES: RENAL BLOOD... LIPOXYGENASE METABOLITES CYP450 METABOLITES CYP450 HYDROXYLASE METABOLITE:... CYP450 EPOXYGENASE METABOLITES CYP450 METABOLITES: RENAL... ISOPROSTANES SUMMARY REFERENCES

Isoprostanes are generated by peroxidation of arachidonic acid by oxygen radicals (105). Interest in these metabolites has been fueled by studies demonstrating an elevation in the urine isoprostane 8-iso-PGF₂ levels during oxidant stress and cardiovascular disease states (70, 108, 136, 143). A unique property of isoprostanes is that they can be formed while arachidonic acid is still in the cell membrane (103). Esterification of isoprostanes has been demonstrated for vascular smooth muscle cells, mesangial cells, and renal epithelial cells (88, 108, 136). Plasma levels of isoprostanes are increased in spontaneously hypertensive rats and during angiotensin II-infused hypertension (136, 143). Similarly, superoxide dismutase mimetic administration decreases renal isoprostane excretion and lowers blood pressure in the spontaneously hypertensive rat (143). Oxidant injury as a result of carbon tetrachloride poisoning is also associated with tremendous elevations in isoprostane levels (70, 104). Because of their possible contribution to renal and cardiovascular diseases, the renal hemodynamic actions of isoprostanes have begun to be explored.

Isoprostanes decrease renal blood flow and glomerular filtration rate by preferentially constricting the preglomerular vasculature (45, 157). Isoprostanes have also been demonstrated to potentiate the vascular vasoconstrictor responses to angiotensin II and norepinephrine (137). The renal hemodynamic effects of 8-iso-PGF₂ are completely abolished by TP receptor antagonism, suggesting that the action of isoprostanes may be mediated by the TP receptor (157). These effects appear to be mediated via the TPa receptor because when the TPa receptor is expressed in human embryonic kidney (HEK-293) cells the calcium response to U-46619 or 8-epi PGF₂ was similar and blocked by the TP antagonists, SQ-29,548 (86). In contrast, 8-iso-PGF₂ stimulates vascular smooth muscle cell IP₃ generation and DNA synthesis at much lower concentrations than it takes this isoprostane to displace TP receptor ligands (45, 46, 125). The actions of 8-iso-PGF₂ on vascular smooth muscle cells are only partially blocked by TP receptor antagonism (46). Thus, taken together, these studies provide evidence suggesting the possible existence of a novel receptor for isoprostanes.

	SUMMARY

TOP ABSTRACT INTRODUCTION COX METABOLITES: VASODILATOR...