Localization of cytochromeP-450 4A isoforms along the rat nephron

Abstract

The expression of P-450 4A isoforms responsible for the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) was examined using the reverse transcription and polymerase chain reaction in various nephron segments and preglomerular arterioles microdissected from the kidneys of Sprague-Dawley rats. Expression of cytochrome P-450 4A1, 4A2, 4A3, and 4A8 mRNA could be detected in RNA extracted from the whole kidney. The expression of P-450 4A1, 4A3, and 4A8 mRNA was similar in the kidney of male and female rats, whereas the expression of 4A2 mRNA was fourfold greater in the kidney of male vs. female rats. At the single-nephron level, P-450 4A1 mRNA could not be detected in either preglomerular arterioles or any nephron segments. P-450 4A2 mRNA was readily detected in preglomerular arterioles, glomeruli, proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). P-450 4A3 mRNA was also detected in every nephron segment, but the expression of this isoform was barely detectable in preglomerular arterioles. The expression of P-450 4A8 mRNA was detected in the glomerulus, PCT, PST, CTAL, and CCD. It was not detectable in preglomerular arterioles, MTAL, OMCD, or IMCD. Immunoblot analysis using a P-450 4A antibody exhibited a strong signal for P-450 4A protein in the proximal tubule. Smaller signals were also observed in glomerulus, MTAL, and preglomerular arterioles, but no signal could be detected in the IMCD. A similar pattern of P-450 4A protein expression was seen in kidney sections immunostained with this antibody. These results indicate that the expression of P-450 4A isoforms in the kidney of rats is sex dependent and that different P-450 4A isoforms are expressed throughout various nephron segments and the renal vasculature of rats.

Formation of 20-Hydroxyeicosatetraenoic Acid, a Vasoactive and Natriuretic Eicosanoid, in Human Kidney

“…Interestingly, these two human P450s exhibited the same highly specific pattern of distribution within the nephron and were not expressed in cells comprising the glomeruli, loops of Henle, or collecting tubules. Thus, the proximal tubular pars convoluta and pars recta segments represents the principal site for 20-HETE formation in the human kidney, an observation similar to that made in experimental animals (18,19,26,29). This pattern of CYP4F2 and CYP4A11 distribution in human kidney has, in all likelihood, important implications with regard to effects of AA-derived eicosanoids on integrated renal function.…”

“…Furthermore, since the proximal tubule is where most electrolyte and water reabsorption occurs (1), the capacity of 20-HETE to potently inhibit Na ϩ /K ϩ -ATPase activity in this region of the nephron probably contributes to its known natriuretic and diuretic effects (4,62,74). In rats, CYP4A expression and 20-HETE formation have also been noted in renal microvessels (19,29,75), another proposed site where this eicosanoid influences vascular tone, autoregulation of renal blood flow, and/or tubuloglomerular feedback (9,10). However, the absence of CYP4 enzymes from the human renal vasculature suggests that synthesis of 20-HETE exclusively within the proximal tubules is sufficient for this compound to elicit its potent effects on kidney function.…”

“…5,6-EET and 11,12-EET) in the regulation of renal function has been proposed (3,24,25). With regard to 20-HETE, three different CYP4A P450s capable of AA 20-hydroxylation, namely CYP4A1, CYP4A2, and CYP4A3, are expressed in rat kidney (19, 26 -28), although more recent studies have attributed the bulk of renal 20-HETE formation in this species to CYP4A2 (18,19,29) The predominant catalyst of AA -hydroxylation in rabbit kidney has been identified as CYP4A6 and/or CYP4A7 (30 -32). In contrast, there have been few studies characterizing P450-mediated AA oxygenation in the human kidney (33,34), and the renal P450 enzymes underlying formation of 20-HETE or any other AA metabolite have yet to be identified in humans.…”

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

“…All of the isoforms catalyze the ω-and ω-1 hydroxylation of arachidonic acid to produce 20-HETE [2,5,[25][26][27]. Ito et al [28] used isoform specific primers to amplify 4A isoforms individually and found that the expression of CYP4A1 mRNA was very low but that CYP 4A2 and 4A3 mRNA are constitutively expressed throughout various nephron segments and the renal vessels in male SD rats. Schwartzman and his colleagues [2] also demonstrated, by Northern blot analysis, that CYP 4A2 and 4A3 mRNA are constitutively expressed in rat kidney, whereas the expression of 4A1 mRNA is nearly undetectable in the kidney of 3 to 7-wk-old male SD rats.…”

Renal Renin-Angiotensin System

“…Recent studies have demonstrated the presence of neuronal nitric oxide synthase (nNOS) [30], cyclooxygenase-2 (COX-2) [31], and cytochrome P 450 [32] in macula densa and adjoining ascending loop of Henle cells. Nitric oxide (NO), and arachidonic acid metabolites have been shown to exert modulating roles on the TGF mechanism as shown in figure 1.…”