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P-450 Eicosanoids: A Novel Signaling Pathway Regulating Renal Function

Richard J. Roman and Magdalena Alonso-Galicia

R. J. Roman is Professor and M. Alonso-Galicia is Postdoctoral Fellow in the Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226.

	Abstract

 
Cytochrome P-450 enzymes primarily metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney. These compounds serve as second messengers that play a central role in the regulation of renal vascular tone and sodium reabsorption in the proximal tubule and thick ascending loop of Henle.

	Introduction

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 
It has long been recognized that cyclooxygenase and lipoxygenase enzymes metabolize arachidonic acid (AA) in the kidney and that the products formed influence both renal tubular and vascular function (3, 8). However, in the last 10 years, a new pathway for the renal metabolism of AA has emerged. Indeed, recent studies indicate that in the renal vasculature, glomerulus, proximal tubule (PT), and thick ascending limb of the loop of Henle (TALH), AA is primarily metabolized by cytochrome P-450 enzymes to epoxyeicosatrienoic acids (EETs), the corresponding dihydroxyeicosatetraenoic acids (DiHETEs) and 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-HETE). It is now apparent that these metabolites play a central role as second messengers in the regulation of renal vascular tone, tubuloglomerular feedback (TGF) and sodium transport in the PT and TALH (4, 11, 15).

Enzymes of the cytochrome P-450 4A family (CYP 4A) catalyze the formation of 20-HETE. cDNAs encoding for 13 different isoforms in this family of enzymes have been identified in various species. CYP 4A11 and CYP 4F2 are expressed in the human kidney, and CYP 4F2 appears to be the isoform primarily responsible for the formation of 20-HETE (12). Four isoforms, CYP 4A1, 4A2, 4A3, and 4A8, have been cloned from the kidneys of rats. All, except CYP 4A8, produce 20-HETE when incubated with AA. Messages for the CYP 4A2 and 4A3 isoforms are expressed in renal arterioles, glomeruli, proximal tubules, cortical and medullary TALH, and cortical and medullary collecting ducts. CYP 4A8 mRNA is expressed in the glomerulus and in cortical, but not medullary, nephron segments. CYP 4A protein can be detected in renal arterioles, the glomerulus, PT, and cortical and medullary TALH. CYP 4A protein is also expressed in pericytes surrounding vasa recta capillaries. Proximal tubules, glomeruli, and renal arterioles produce 20-HETE, EETs, and DiHETEs when incubated with AA. The primary metabolite of AA produced by the TALH is 20-HETE (5, 8, 11).

Cytochrome P-450 enzymes of the 1A, 2B, 2C, 2E, and 2J families can catalyze the formation of EETs. Protein for each of these isoforms is expressed in the kidney, but it remains to be determined which enzymes are responsible for the formation of EETs in different nephron segments. In endothelial cells isolated from nonrenal vessels, enzymes of the 2C8 and 9 family are thought to synthesize EETs. In the kidney, enzymes of the 2C and 2J families have been reported by different investigators to be the primary renal epoxygenases that catalyze the formation of EETs (7).

Many factors regulate the expression of cytochrome P-450 enzymes. CYP 4A1 and 4A3 mRNA and protein are highly expressed in the kidney of neonatal rats, but the levels decline into adulthood. In contrast, CYP 4A2 protein is not expressed in the kidneys of neonates, but the levels increase with age until it becomes the major isoform expressed in the kidneys of adult rats (9). In the proximal tubule, angiotensin II (ANG II) increases the formation of EETs, whereas epidermal growth factor (EGF), dopamine, and parathyroid hormone (PTH) increase the formation of 20-HETE (6, 10, 11). In the TALH, a variety of peptide hormones increase the formation of 20-HETE (3, 8). The expression of CYP 4A proteins in the kidney and renal vasculature is downregulated in rats fed a high-salt diet, and this can be prevented if circulating ANG II levels are maintained at normal levels by intravenous infusion. In contrast, a high salt intake increases the expression of CYP 2C23 and the formation of EETs in the kidney (7).

Renal CYP 4A activity is increased by glucocorticoids, mineralocorticoids, and progesterone. Antilipidemic agents, such as clofibrate, induce the expression of CYP 4A1 and 3 and the synthesis of 20-HETE in the kidney (8, 12). Other inducers of P-450 enzymes, such as pentobarbital, 3-methylcholanthrene, and 3,4 benzo[a]pyrene, do not alter renal CYP 4A activity (12). Intrarenal infusion of ANG II, vasopressin, and endothelin increases the synthesis and release of 20-HETE by the kidney, and 20-HETE contributes to the renal vasoconstrictor response to these agonists (8).

Renal CYP 4A activity is elevated in diabetes, pregnancy, hepatorenal syndrome, and cyclosporin-induced nephrotoxicity and in various models of hypertension (8, 11). However, a role for 20-HETE in mediating the changes in renal function associated with these conditions has yet to be established. The expression of CYP 4A2 protein and mRNA is also greater in the kidney of male versus female rats (5, 9) but again the functional significance of this observation remains to be determined.

	P-450 metabolites of AA in the control of renal vascular tone

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 
EETs .
Intact renal arterioles produce both EETs and 20-HETE when incubated with AA. EETs are produced by the endothelium and are potent vasodilators. They hyperpolarize renal vascular smooth muscle (VSM) cells by increasing the activity of the large-conductance calcium-activated potassium (K_Ca) channels (Fig. 1). The finding that blockade of K_Ca channels with tetraethylammonium or iberiotoxin prevents the vasodilator response to EETs indicates that activation of these channels plays a prominent role in this response. Because EETs are released by the endothelium and activate the K_Ca channel, several investigators have suggested that they may be the elusive endothelium-derived hyperpolarizing factor (EDHF) that mediates the remaining response to endothelium-dependent vasodilators after blockade of nitric oxide (NO) synthase (4).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Summary of role of P-450 metabolites of arachidonic acid (AA) in control of renal vascular tone. Membrane stretch and vasoactive agonists activate phospholipase C (PLC), and rise in inositol trisphosphate (IP3) triggers release of intracellular Ca2+ stores. Elevations in intracellular Ca2+ activate Ca2+-sensitive phospholipase A (PLA) and diacylglycerol (DAG) lipase to release AA and form 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE then blocks large-conductance calcium-activated potassium (KCa) channels in renal vascular smooth muscle cells, leading to membrane depolarization (Em), enhanced Ca2+ influx, and a prolonged vasoconstrictor response. Epoxyeicosatrienoic acids (EETs) are produced by endothelium and are potent vasodilators that hyperpolarize renal vascular smooth muscle cells by increasing activity of KCa channels. ANG II, angiotensin; NE, norepinephrine.

A summary of the role of 20-HETE in the regulation of renal vascular tone is presented in Fig. 1. Membrane stretch and vasoactive agonists activate phospholipase C, and the subsequent rise in inositol trisphosphate (IP₃) triggers the release of intracellular Ca²⁺ stores. However, elevations in intracellular Ca²⁺ concentration should activate K_Ca channels, leading to membrane hyperpolarization and reduced Ca²⁺ influx through voltage-sensitive channels. This is highly counterproductive and opposes vasoconstriction. Therefore, some mechanism must exist to buffer against activation of K_Ca channels after the release of intracellular Ca²⁺ stores. This is where 20-HETE is thought to play an important role. Elevations in intracellular Ca²⁺ concentration activate Ca²⁺-sensitive phospholipase A and diacylglycerol (DAG) lipase to release AA. AA is then converted to 20-HETE, which blocks K_Ca channels in renal VSM cells, leading to membrane depolarization, enhanced Ca²⁺ influx, and a prolonged vasoconstrictor response.

There is now considerable evidence that 20-HETE plays this role in the regulation of renal vascular tone both in vivo and in vitro. In this regard, AA enhances the myogenic response of isolated perfused renal arterioles to elevations in transmural pressure, whereas inhibitors of the formation of 20-HETE completely block this response in vitro (4). Infusion of inhibitors of the formation of 20-HETE into the renal artery of rats blocks autoregulation of renal blood flow in vivo (15). These inhibitors also attenuate the renal vasoconstrictor responses to vasopressin, ANG II, endothelin, and norepinephrine both in vivo and in vitro.

20-HETE also participates as a mediator or a modulator of tubuloglomerular feedback (TGF) responses in the rat in vivo. This conclusion is based on the findings that the enzyme responsible for the formation of 20-HETE is expressed in the macula densa of the kidney (5) and that 20-HETE is a potent constrictor of the afferent arteriole (14). The observations that addition of AA to the fluid perfusing the loop of Henle potentiates TGF responses in rats in vivo and that TGF responses are blocked by addition of P450 inhibitors to tubular perfusates further support this hypothesis. Moreover, perfusion of the loop of Henle with a solution containing 20-HETE will restore TGF responses after blockade of the endogenous formation of this compound in the rat in vivo (15). These studies suggest that 20-HETE either serves as a mediator of TGF or acts as a second messenger at the level of the afferent arteriole to transduce the vasoconstrictor response to some other mediator released by the macula densa.

	20-HETE-nitric oxide interaction in the control of renal vascular tone

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 
Numerous studies have indicated that the tonic release of NO plays an important role in opposing renal vasoconstrictor and TGF responses. Although it has generally been assumed that the renal vasodilator response to NO is mediated by elevations in cGMP, there is increasing evidence that NO has effects on K⁺ channels and vascular tone that are cGMP independent. We recently reported (1) that NO binds to heme in cytochrome P-450 4A enzymes and inhibits the formation of 20-HETE in renal arterioles. We therefore evaluated whether inhibition of the formation of 20-HETE contributes to the cGMP-independent vasodilator effects of NO in the renal circulation. The results indicate that NO activates K_Ca channels in renal VSM cells and dilates renal arterioles. These effects are cGMP independent because they cannot be blocked by inhibitors of guanylyl cyclase or cGMP-dependent kinase. We also found that the effects of NO on K⁺ channels and vascular tone were completely blocked by preventing the fall in 20-HETE levels by adding exogenous 20-HETE to the bath (1,13). These findings indicate that NO inhibits the formation of 20-HETE and that this mediates the cGMP-independent component of the renal vasodilator response to NO by activating K_Ca channels in renal VSM cells.

	P-450 eicosanoids and the regulation of sodium transport in the proximal tubule

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 
The role of P-450 metabolites of AA as second messengers in the regulation of sodium transport in various nephron segments has also recently emerged as a dynamic new field. A summary of the effects of these compounds on sodium reabsorption in the proximal tubule is presented in Fig. 2. Several laboratories have reported that 20-HETE is the primary metabolite of AA produced by the PT and that 20-HETE inhibits Na-K-ATPase activity in this nephron segment by enhancing protein kinase C-induced phosphorylation of the -subunit of the Na-K-ATPase (10). Subsequent studies have indicated that the inhibitory effects of dopamine and PTH on Na-K-ATPase activity and sodium transport in the proximal tubule involve activation of phospholipase A₂ and enhanced formation of 20-HETE (2, 10). There is also evidence that P-450 inhibitors can block the inhibitory effects of ANG II on sodium transport in the proximal tubule. This effect is associated with an enhanced formation of 5,6-EET, which may affect the translocation of the Na⁺/H⁺ exchanger in the apical membrane of proximal tubule cells (11).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Summary of effects of P-450 metabolites of AA in control of proximal tubule (PT) sodium reabsorption. 20-HETE is primary metabolite of AA in PT. 20-HETE inhibits Na-K-ATPase activity in this nephron segment by enhancing cAMP-dependent protein kinase (PKC)-induced phosphorylation of -subunit of Na-K-ATPase pump. Inhibitory effects of dopamine and parathyroid hormone (PTH) on Na-K-ATPase activity and sodium transport in PT are caused by activation of phospholipase A2 (PLA2), release of AA, and enhanced formation of 20-HETE. The inhibitory effects of ANG II on sodium transport in PT are associated with an enhanced formation of 5,6-EET, which affects translocation of Na+/H+ exchanger in apical membrane of PT cells. CYP 4A, cytochrome P-450 4A; PL, phospholipids; PLC, phospholipase C; R, receptor.

	Effects of P-450 metabolites of AA on sodium transport in the thick ascending loop of Henle

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Summary of role of P-450 metabolites of AA in control of sodium reabsorption in thick ascending loop of Henle (TALH). 20-HETE is major metabolite of AA produced in TALH cells. 20-HETE blocks a 70-pS K+ channel in apical membrane of TALH cells, which limits K+ availability for transport via Na+-K+-2Cl- transporter. This leads to a reduction in lumen positive transepithelial potential that serves as main driving force for passive reabsorption of cations (Na+, K+, Ca2+, and Mg2+). Inhibitors of cytochrome P-450 enzymes block inhibitory effects of ANG II, bradykinin, endothelin, and vasopressin (ADH) on sodium transport in TALH.

	Mitogenic actions of P-450 metabolites of AA

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

	Perspectives

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 
The role of P-450 metabolites of AA in the regulation of renal function is a rapidly expanding field, but some general concepts have already emerged. First, renal VSM cells produce 20-HETE and this substance serves as a second messenger that plays an important role in the myogenic response, TGF, vascular hypertrophy, and the vascular responses to vasoconstrictors and dilators by regulation of K⁺ channel activity. Second, P-450 metabolites of AA are avidly produced in the proximal tubule and the TALH and serve as second messengers in the regulation of sodium transport. Finally, the renal formation of P-450 metabolites of AA is altered in hypertension, diabetes, hepatorenal syndrome, and pregnancy. Given the importance of this pathway in the regulation of renal tubular and vascular function, it is likely that P-450 metabolites of AA may contribute to some of the changes in renal function associated with these diseases.

	References

Top Introduction P-450 metabolites of AA... 20-HETE-nitric oxide interaction... P-450 eicosanoids and the... Effects of P-450 metabolites... Mitogenic actions of P-450... Perspectives References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roman, R. J. Articles by Alonso-Galicia, M. Search for Related Content PubMed PubMed Citation Articles by Roman, R. J. Articles by Alonso-Galicia, M.