Pediatric Nephrology

Pediatr Nephrol (1992) 6: 490- 498 9 IPNA 1992

Physiology review The renal cytochrome P-450 arachidonic acid system Michal Laniado-Schwartzmanl and Nader G. Abraham 2 Departments of 1Pharmacologyand 2Medicine,New YorkMedicalCollege,Valhalla, NY 10595, USA Receivedand acceptedMay 18, 1992 Abstract. In addition to cyclooxygenase and lipoxygenase, arachidonic acid (AA) is metabolized by the cytochrome P-450 monooxygenase system. The kidney is one of the major extrahepatic tissues that display cytochrome P-450 enzyme activities, in particular the cortex, specifically the proximal tubule demonstrate the highest concentration. AA is metabolized by the renal cytochrome P-450 epoxygenase and co/o)-I hydroxylases to epoxyeicosatrienoic acids and o~/c0-1 alcohols (20- and 19-mono-hydroxyeicosatetraenoic acids), respectively. These metabolites possess a broad spectrum of biological and renal effects which include: vasodilation, vasoconstriction, inhibition and stimulation of Na+-K+-ATPase, inhibition of ion transport mechanisms, natriuresis, inhibition of renin release and stimulation of cell growth. These metabolites are endogenous constituents of the kidney and are present in urine with increasing concentration under pathological conditions such as pregnancy-induced hypertension. The cytochrome P-450-dependent metabolism of AA is specifically localized to the proximal tubule and exhibits developmental changes, i.e., renal production of metabolites is very low in the fetus, newborn and up to 3 weeks of age, after which a remarkable increase in enzyme activities is observed. These characteristics call attention to the importance of this enzyme system in producing cellular mediators for regulating renal function in normal and diseased states. Key words: Renal cytochrome P-450 - Arachidonic acid metabolism

Introduction There is a substantial body of literature regarding the cellular functions of arachidonic acid (AA) metabolites derived Correspondence to: M. Laniado-Schwartzman

through the cyclooxygenase and lipoxygenase pathways, namely the prostaglandins and the leukotrienes, respectively. Recently, it has become evident that another branch of the AA cascade, the cytochrome P-450 (P-450) monooxygenase system, must be added to the list. This review will focus on the biochemistry, pharmacology and the potential physiological significance of the P-450 AA metabolism in the mammalian kidney.

The cytochrome P-450 system in mammalian kidney The oxidative transformation of a large number of endogenous and exogenous substrates is catalyzed by the NADPH-dependent mixed function oxidase system. This system comprises three components: (1) a hemoprotein, the cytochrome P-450; (2) a flavoprotein reductase [NADPH P-450 (c) reductase]; (3) phosphatidylcholine. P-450 exists in multiple forms which differ in substrate specificity, positional specificity and stereospecificity [1, 2]. Consequently, the predominance of one oxidation reaction over another may be controlled by the isozyme compostion of a given tissue or cell type. The highest concentration of P-450 isozymes in mammalian tissues is found in the liver. However, several extrahepatic tissues contain significant levels of P-450; among them the kidney displays the highest concentration [3-5]. Zenser et al. [6] demonstrated a cortical-papillary gradient (highest in the cortex and lowest in papilla) for both monooxygenase activity and P-450 content. The P-450 system appears to be particularly abundant in proximal tubules, as revealed by immunocytochemistry of nephron segments [7, 8]. In the kidney, P-450 monooxygenases participate in the metabolism of drugs and endogenous substances, in particular, hydroxylation of fatty acids and lcz-hydroxylation of 25-hydroxyvitamin D3 [9, 10]. Several P-450 forms have been isolated and purified from rat, rabbit and human kindey; some could be detected only after their induction with substances such as clofibrate, 3-methylcholanthrene and tetrachlorodibenzo-p-dioxin [ 11 - 13].

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P-450 AA metabolism in mammalian kidney The P-450 monooxygenase pathway for AA metabolism is depicted in Fig. 1. In the presence of NADPH and molecular oxygen, P-450 metabolizes to several oxygenated metabolites, including: (1) four regioisomeric epoxides [5,6; 8,9; 11,12; 14,15 epoxyeicosatrienoic acids (EETs)], which can be hydrolyzed by epoxide hydrolase to the corresponding diol derivatives [dihydroxyeicosatrienoic acids (DHETs)], (2) six regioisomeric cis-trans-conjugated mono-hydroxyeicosatetraenoic acids (HETEs); (3) 0~ and o1-1 alcohols [14]. Most of these metabolic pathways have been found to be expressed in the kidney of several animal species. The four regioisomeric EETs have been shown to be synthesized in renal microsomes, with 11,12-EET being the predominant epoxide [13, 15]. The o>hydroxylation of AA leading to the formation of 20-HETE was found to constitute a major P-450 arachidonate metabolite in the kidney and to undergo further oxidation to the carboxy derivative, 20-COOH-AA [9, 13, 16]. o>l-Hydroxylation, as well as hydroxylations at carbons 18, 17 and 16 of the AA backbone, also occur [13, 17, 18]. In the human kidney, 20-HETE, 19-HETE, ll,12-EET and 11,12-DHET are the major metabolites [19]. The fact that these compounds are endogenous products of the kidney and their synthesis can be activated by hormones [20-23], suggest their involvement in the regulation of renal function. Epoxygenation of AA can be carried out by several P-450 isozymes controlled by several genes; among them are the rat enzymes P-450 1A1, P-450 1A2, P-450 2B1, P-450 2B2 and P-450 2 C l l [24], and rabbit enzymes P-450 1A1, P-450 1A2 and P-450 2B4 [25, 26]. Dubois et al. [27] reported the isolation and cloning of a salt-sensitive AA epoxygenase in rat kidney. In contrast, the c0-hydroxylation of AA is an activity restricted to the members of the P-450 4 gene family [28, 29]. We have used a cDNA probe for P-450 4A1 and an antibody raised against the P-450

Fig. 1. The three metabolic pathways of arachidonic acid (AA). HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid

4A1 protein to examine the developmental pattern of this protein in the kidneys of Sprague-Dawley (SD) rats. A close correlation between the age-dependent renal cortical 20-HETE production and P-450 4A1 mRNA expression was observed [30]. In addition, the highest expression of the P-450 4A1 protein and mRNA was localized in the proximal tubules, especially in the $3 segments [31], suggesting that P-450 4A1 may be involved in the in vivo synthesis of 20-HETE. To date no specific isozyme of this subfamily has been found to use selectively AA as the substrate. In contrast, all its members are able to hydroxylate a variety of short-chain fatty acids, such as lauric acid, although some of them [32] exhibit a high specificity for AA.

Age-dependent P-450 AA metabolism in the rat kidney Since the P-450 superfamily is made up of many proteins that are inducible by endogenous compounds such as steroid hormones [2], it is reasonable to assume a developmental/hormonal regulation of the expression of some P-450 isozymes, including those involved in AA metabolism. Indeed, age differences as well as sex differences in the expression of several P-450 isozymes have been reported [33, 34]. The transcriptional activation of constitutively expressed P-450 genes occurs for some immediately after birth, and for others at the onset of puberty; sex-specific activation or suppression at the onset of puberty has also been described [2, 33, 34]. Our interest in investigating the age-dependent expression of P-450 AA metabolism stems from the observations in the kidney of the spontaneously hypertensive rat (SHR). In this animal model, P-450 AA metabolism is significantly higher than in the control WKY rats and is increased during the development of hypertension [35]. In addition, a selective depletion of renal P-450 by tin chloride or heme arginate was found to reduce the blood pressure in the

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Fig. 2. Age-dependentchangesin cytochromeP-450 AA: A co-hydroxylase (20-HETE + 20-COOH-AA), B co-l-hydroxylase (19-HETE), C epoxygenase (EETs+DHETs), D 20-HETE dehydrogenase (20COOH-AA) and E epoxidehydrolase(DHETs) activitiesin corticalmicrosomes from spontaneouslyhypertensiverat (SHR 0) and WKY rat (o) kidneys.*P