J. Exp.

Med., 1992,


166, 93-106

of Renal


Cytochrome Acid



in Hypertension

KEN OMATA,KEISHIABE*, HSU-LANGSHEU, KAZUNORI YOSHIDA, EIKATSUTSUTSUMI, KAORUYOSHINAGA, NADER G. ABRAHAM and MICHALLANIADO-SCHWARTZMAN The Second Department of Internal Medicine and *Department of Clinical Biology and Hormonal Regulation, Tohoku University School of Medicine, Sendai 080, and Department of Pharmacology and Medicine,New York Medical College,Valhalla, New York 10595, USA OMATA,K., ABE,K., SHEU,H.-L., YOSHIDA, K., TSUTSUMI, E., YOSHINAGA, K., ABRAHAM,N.G. and LANIADO-SCHWARTZMAN, M. Roles of Renal Cytochrome P450-Dependent Arachidonic Acid Metabolites in Hypertension. Tohoku J. Exp. Med., 1992, 166 (1), 93-106 Cytochrome P450 represents the third metabolic pathway of arachidonic acid giving rise to several biologically active compounds, such as 19-HETE, 20-HETE and EETs and their corresponding DHETs. The kidney is the rich source of these metabolites which have some important biologic actions within the kidney. These metabolites have a wide and contrasting spectrum of biological and renal effects, from vasodilation to vasoconstriction and from inhibition to stimulation of Na-K-ATPase, their relative production rates may influence not only renal hemodynamics but also pro- and anti-hypertensive mechanisms of hypertension. There is increasing evidence that the abnormality of these metabolites in animal models of hypertension. However, sufficient evidence of the physiological and pathophysiological roles of hypertension in man is still lacking. cytochrome P450; arachidonic acid ; spontaneously hypertensive rat ; Dahl rat ; nephron The biologic blood




to its increasing

of arachidonic

to the rapid


in basic


pathway of stimuli




by which and



Abbreviations acid ; 19-HETE, acid ; 19-keto-AA,


is transformed






of arachidonic

to : Ken Omata,

: EET, Epoxyeicosatrienoic 19 Hydroxyeicosatetraenoic 19 keto


acid metabolism cytochrome P450




in the regulation

in our understanding

and clinical

potential pathways for arachidonic cyclooxygenase, lipoxygenase and specific



in hypertension.


have been identified : monooxygenase. The depends

on the tissue,

cytochrome acid


P450 either

system an epox-

M.D. acid ; DHET, Dihydroxyeicosatrienoic acid ; 20-HETE, 20 Hydroxyeicosatetraenoic

acid ; 20 000H-AA, 93


of this area and

1, 20 dioic



K. Omata

et al.

ygenase system or a monooxygenase system. In the present paper, we shall attempt to describe our current understanding of the role played by the cytochrome P450 dependent arachidonate metabolites in the regulation of the blood pressure in hypertension. Metabolism of arachidonic acid by renal cytochromeP450 Cytochrome P450 monooxygenases represent a family of enzymes. The microsomal cytochrome P450 enzyme comprises a family of hemoproteins that serve as the terminal acceptor in the NADPH-dependent mixed function oxidase system. The presence of microsomal NADPH-dependent cytochrome P450 enzyme system in the mammalian kidney is now firmly established. This enzyme system metabolizes arachidonic acid to several oxygenated metabolites including 1) four regioisomeric epoxyeicosatrienoic acids (5, 6 ; 8, 9 ; 11,12 ; 14,15 EETs), which can be hydrolyzed enzymatically by epoxide hydrolase to the corresponding diols (DHETs) ; 2) six radioisomeric cis-trans conjugated mono-hydroxy eicosatetraenoic acids (HETEs) ; and 3) CQ) - and (c -1)-alcohols (20-HETE and 19-HETE) (Fig. 1). 20-HETE is further oxidized to 1, 20 dioic acid(20-COOHAA). 19-HETE is also further hydrolyzed to 19 keto-AA. The pathway for the







.1. Three major pathways of eicosanoids formation are described : (1) cyclooxygenase, (2) lipoxygenase, and (3) cytochrome P450 monooxygenases. Cytochrome P450-dependent metabolites were described as structural formula. The 20- and 19-hydroxyeicosatetraenoic acid (HETE) are formed by w and (w -1)-hydroxylation. Epoxidation results in the formation of four epoxyeicosatrienoic acids (EETs), 5, 6 ; 8, 9 ; 11,12 ; 14,15 EETs, which can be enzymatically hydrolyzed by epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acid (DHETs).





in Hypertension


metabolism of arachidonic acid by renal cytochrome P450 has been identified in cortical and medullary tissues of rabbit and rat kidneys (Morrison and Pascoe 1981; Oliw et al. 1981; Winokur and Morrison 1981; Schwartzman et al. 1985a, b ; Lapuerta et al. 1988; Hirt et al. 1989; Romero et al. 1990; Takahashi et al. 1990), and cytochrome P450 dependent arachidonic acid metabolites have been demonstrated in human urine (Toto et al. 1987; Catella et al. 1990). Oliw et al. (1981) reported the formation of 11,12 DHET and 14,15 DHET as well as 19-HETE, 20-HETE,19keto-AA and 20-COOH-AA by rabbit renal cortical supernatants and microsomes. Morrison and Pascoe (1981) reported the existence of an active cortical NADPH-dependent monooxygenase that converts arachidonate into 19-HETE and 20-HETE as well as 19keto-AA and 20-COOH-AA. Schwartzman et al. (1985a, b) found the cytochrome P450 dependent metabolites in isolated thick ascending limb cells from the rabbit outer medulla which inhibited Na-K-ATPase. Drugge et al. (1989) characterized arachidonic acid metabolism in cultured cells of medullary ascending limb in rabbits. Complete characterization of this pathway in the rat kidney and demonstration of endogenous products generation by gas chromatography or mass spectrometry has been reported recently (Takahashi et al. 1990; Omata et al. 1992). This is not a species specific phenomena as cytochrome P450 arachidonate metabolism has also been documented in human kidneys where EETs, DHETs and w- and (w -1)-hydroxylated compounds, 19-HETE and 20-HETE (Karara et al. 1990; Schwartzman et al. 1990). Localization along the nephron The mammalian kidney is multiform and heterogeneous. The nephron consists of approximately 20 distinct renal tubule segments. Actually, the profile of eicosanoids generation differs by the division of the zone and the structure within the kidney (Dunn 1983). Arachidonic acid metabolism varies longitudinally along the nephron. In regard to cytochrome P450 enzyme, Endou (1983a, b) has described the distribution of this enzyme along the rabbit and rat nephron. Cytochrome P450 has been localized primarily proximal tubules with the highest activity in the S3 segment of the proximal tubules. Koop et al. (1988) reported that isolated proximal tubule cells in rabbits have a high activity of w and (w 1)-hydroxylases to metabolize arachidonic acid. Lapuerta et al. (1988) also showed epoxygenase activity in the pars recta of the rabbit kidney. Cultured rabbit proximal tubule cells have been also reported to metabolize arachidonic acid to 5, 6 EET (Romero et al. 1990). Although renal cortex has been shown to posses the highest activity and content of P450 enzyme, there are documentations that the outer and inner medulla have this system as well. We demonstrated a cytochrome P450 isozymes in the isolated cells from the thick ascending limb of Henle's loop in rabbits (Schwartzman et al. 1985a, b). EETs were reported to present in extracts purified from cortical collecting tubules in rabbits (Hint et al.

K. Omata


et al.

1989). Because of the importance of arachidonic acid metabolism to the regulation of renal function and the heterogenous nature of ion transport and hormonal responsiveness, the pattern of P450 arachidonate metabolism should be related to the specific nephron segment. Thus it is important to localize the P450 isozyme activities to a specific nephron structure in order to clarify the possible regulation on product formation and the functional role of these compounds in regulating the ion transport mechanisms along the nephron. We developed micro-methods to measure P450 arachidonate metabolism in single nephron segments and determined the tubular localization of this activity in spontaneously hypertensive rats (SHR) and Wister-Kyoto rats (WKY) (Omata et al. unpublished). Formation of


2. Localization of (w -1)- (19-HETE formation) and w-(20-HETE formation)-hydroxylase activities along the nephron of WKY (open column) and SHR (hatched column) kidneys. Results are the mean+s.E. (n=5). Microdissected nephron segments were schematically represented. G1, glomerulus ; S1, early portion of the proximal convoluted tubule ; SZ, late portion of the proximal convoluted tubule ; S3, early proximal straight tubule ; MAL, medullary thick ascending limb of Henle's loop ; CAL, cortical thick ascending limb of Henle's loop ; DCT, distal convoluted tubule ; CNT, connecting tubule ; CCT, cortical collecting tubule ; MCT, medullary collecting tubule. Nephron segments were incubated with [14C] arachidonic acid (0.2 pCi, 76 M) in a total volume of 50 pl of PBS containing 1 mM of NADPH for 90 min at 3TC. Arachidonate metabolites were separated by reverse phase HPLC on a C18Ultrasphere ODS column.





in Hypertension


20-HETE, the product of arachidonate c~-hydroxylase was specifically localized in the entire proximal tubules (S1, S2 and S3 segments), whereas formation of 19-HETE, the product of (-1)-hydroxylase was demonstrable through the tubule (Fig. 2). Although distribution patterns were similar in SHR and WKY, formation of 19-HETE and 20-HETE in the proximal tubules was higher in SHR. EETs, products of arachidonate epoxygenase were shown a wide distribution along the entire nephron, and the formation was not different between SHR and WKY. 19-HETE production rate along the entire nephron segment tested was only from a half to one fifth of the production rate of 20-HETE and EETs, suggesting a lower (c~,-1)-hydroxylase activity than w-hydroxylase and epoxygenase activities in the kidney. Cytochrome P450 reductase has been localized the proximal and distal convoluted tubule, the loop of Henle and the collecting tubule in the medulla in man by immunohistochemical study. Our data also suggest the broad distribution of (CR, -1)-hydroxylase and epoxygenase activities along the entire nephron tested, and clearly demonstrated the different distribution profile along the nephron between cytochrome P450 isozymes, such as (w 1)-hydroxylase, epoxygenase and w-hydroxylase. Anatomical and physiological relationship Since 20-HETE is known to cause vasoconstriction in isolated rat aorta, rabbit renal artery and dog renal microvessels (Escalante et al. i989; Omata et al i990; Kauser et al. 1991), the relationship between the localization of whydroxylase and renal vessels is of interest from its functional roles in the kidney. There are several possible roots of 20-HETE to access to the vasculature within the kidney. Proximal tubules are embedded in the dense capillary plexus in the cortical labyrinth, and blood that perfuses the straight proximal tubule (S3 segment) mix with the blood that perfuses the proximal convoluted tubule(Sl and S2 segments). Thus, 20-HETE production localize the most suitable nephron segments to access to the systemic circulation. Moreover, interlobular veins are generally apposed at one side to the periarterial interstitium and these veins are capillaries and permeable. In this root 20-HETE may directly acts on intrarenal arteries. In addition, the renal cortex has a lymphatic drainage and the drainage of the cortical interstitium is effected by the periarterial connective tissue sheath. Since the peritubular interstitium in the cortex freely communicates with these sheath, 20-HETE synthetized by the proximal tubules can gain access to structures at the vascular pole of the glomerulus as well as the renal arteries and veins. It is attractive to assume that 20-HETE is a circulating and/or paracrine autacoids in the kidney, since localization of cQ,-hydroxylase activity is easy to access to the systemic circulation as well 'as to the intrarenal arteries. 20-HETE has been shown to cause a natriuresis when administered into renal artery in the anesthetized rats (Takahashi et al. 1990) and to cause a inhibition of Na-K-2C1 cotransport in the luminal site of the isolated medullary thick ascending


K. Omata et al.

limb cells in rabbits (Escalante et al. 1991). Although 20-HETE was shown to be further metabolized by cyclooxygenase to the vasoactive substances (Schwartzman et al. 1989), cyclooxygenase activity was scarcely localized in the proximal tubules, thin and thick ascending limb of Henle's loop (Smith and Bell 1978). 20-HETE released into the tubular lumen reaches the distal part of the nephron without further metabolism and may inhibit a Na-K-2C1 cotransporter, resulting a natriuresis. 19(S)-HETE has been shown to stimulates renal Na-K-ATPase activity (Escalante et al. 1988), on the contrary 11,12 EET and 11,12 DHET has been shown to inhibit Na-K-ATPase activity (Schwartzman et al. 1985b). Since Na-K-ATPase activity is distributed widely along the entire nephron in rats, mouse and rabbits (Doucet 1988), broad distribution pattern of 19-HETE and EET productions along the nephron seems suitable to affects the Na-K-ATPase activity in each nephron segments as autocrine system. 5, 6 EET dilates isolated blood vessels (Carroll et al. 1987). However, it causes renal vasoconstriction and reduces glomerular filtration rate by the intrarenal infusion in rats (Takahashi et al. 1990). It was shown to inhibit sodium flux and sodium reabsorption in isolated collecting tubules in rabbits (Jacobson et al. 1984), and the inhibitory effect of sodium reabsorption by angiotensin II was mediated via the generation of 5, 6 EET in cultured proximal tubule cells (Romero et al. 1990). It also inhibit Na flux by increased Ca influx through dihydropyridine sensitive Ca channels in proximal tubular cells in culture (Douglas et al. 1990). Three EETs (5, 6 ; 11,12 ; 14,15 EET) inhibited vasopressin stimulated osmotic water flow across the toad urinary bladder (Schlondorff et al. 1987). Both 5, 6 EET and 11, 12, EET prevented the vasopressin induced increase in intracellular c-AMP levels. 11,12 DHET inhibited vasopressin induced stimulation of adenylate cyclase. Four DHETs (5, 6 ; 8, 9 ; 11,12 ; 14,15 DHET) were reported to inhibit AVP-stimulated hydraulic conductivity in rabbit cortical collecting ducts (flirt et al. 1989). In addition, 14, 15 EET was found to inhibit renin secretion in rat renal cortical slices (Henrich et al. 1990) and it decreases blood pressure in SHR and WKY rats by intravenous and intraarterial infusion (Lin et al. 1990). Since EETs and their corresponding diols have a wide spectrum of renal effects, their broad distribution pattern along the entire nephron segments are suitable to reveal their biological effects. Roles in SHR SHR has been extensively studied as an animal model of human essential hypertension. Total renal cytochrome P450 dependent metabolism of arachidonic acid is increased in SHR and a selective depletion of renal cytochrome P450 by tin chrolide or heme arginate reduced the blood pressure in the 7-week old SHR (Sacerdoti et al. 1988, 1989; Levere et al. 1990). Depleting cytochrome P450 in the SHR specifically reduced metabolites which comigrated on HPLC

CytochromeP450 ArachidonateMetabolismin Hypertension


with authentic 19- and 20-HETEs. The production and the release of the cytochrome P450 arachidonate metabolites from the isolated perfused SHR kidney was increased by AVP and inhibited by pretreatment of the kidney with the cytochrome P450 enzyme inhibitor (Omata et al. 1992), suggesting the capacity of the kidney as an intact organ to generate these compounds. We also clarified the chemical identity of the renal cytochrome P450 arachidonate metabolites and the formation of these compounds in cortical microsomes of both SHR and WKY was age dependent (Fig. 3). Although the production pattern with age was similar for EETs, DHETs, 19-HETE and 20-HETE, great qualita-

Fig. 3. Age-dependent changes in cytochrome P450-arachidonic acid whydroxylase (20-HETE +20-COOH-AA) (A), (w -1)-hydroxylase (19HETE) (B), epoxygenase (EET+DHET) (C), alcohol dehydrogenase (20COOH-AA/20-HETE + 20-COOH-AA) (D) and epoxide hydrolase (DHET/ EET+DHET) (E) activities in cortical microsomes from SHR (closed circle) and WKY (open circle). Results are the mean±s.E. (n=3-6). Cytochrome P450 metabolism of arachidonic acid was determined in fetal renal microsomes (3 days before parturition) and from kidney cortexes of 1-, 3-, 5-, 7-, 9-,13-, 20-week old SHR and WKY rats. The incubation mixture contained 0.1-1 mg/ml microsomal protein, [14C1arachidonic acid (0.4 PCi,15,uM), NADPH (1 mM) in a total volume of 0.5 ml. Metabolites were extracted with ethyl acetate and separated by reverse-phase HPLC on a C18 Ultrasphere ODS column. *p

Roles of renal cytochrome P450-dependent arachidonic acid metabolites in hypertension.

Cytochrome P450 represents the third metabolic pathway of arachidonic acid giving rise to several biologically active compounds, such as 19-HETE, 20-H...
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