Distribution of cytochromeP-450 4A and 4F isoforms along the nephron in mice

Abstract

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. 20-HETE production in the kidney has been extensively studied in rats and humans and occurs primarily via the actions of P-450 enzymes of the CYP4A and -4F families. Recent advancements in molecular genetics of the mouse have made it possible to disrupt genes in a cell-type-specific fashion. These advances could help in the creation of models that could distinguish between the vascular and tubular actions of 20-HETE. However, isoforms of the CYP4A and -4F families that may be responsible for the production of 20-HETE in the vascular and tubular segments in the kidney of the mouse are presently unknown. The goal of this study was to identify the isoforms of the CYP4A and -4F families along the nephron by RT-PCR of RNA isolated from microdissected renal blood vessels and nephron segments from 16- to 24-wk-old male and female C57BL/6J mice. CYP4A and -4F isoforms were detected in every segment analyzed, with sex differences only observed in the proximal tubule and glomeruli. In the proximal tubular segments from male mice, the 4A10 and -12 isoforms were present, whereas the 4A10 and -14 isoforms were detected in segments from female mice. In glomeruli, sex differences in the expression pattern of CYP4F isoforms were also observed, with male mice expressing the 4F13, -14, and -15 isoforms, whereas female mice expressed the 4F13, -16, and -18 isoforms. These results demonstrate that isolated nephron and renal vessel segments express multiple isoforms of the CYP4A and -4F families; therefore, elimination of a single CYP4A or -4F isoform may not decrease 20-HETE production in all nephron segments or the renal vasculature of male and female mice. However, the importance of CYP4A vs. -4F isoforms to the production of 20-HETE in each of these renal tubular and vascular segments of the mouse remains to be determined.

Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

“…32 The kidneys were immediately removed, and the cortex and outer medulla were separated under a dissecting microscope. The tissues were then digested in a dissection solution consisting of wash solution with the following additives: 1 mg/ml collagenase (type II, 1.2 U/mg; Sigma Chemical, St. Louis, MO), 1 mg/ml hyaluronidase (359 U/mg; Sigma Chemical, St. Louis, MO), and 1 mg/ml soybean trypsin inhibitor (Sigma Chemical; St. Louis, MO).…”

Fenofibrate Prevents the Development of Angiotensin II–Dependent Hypertension in Mice

“…11 The reactions were terminated by the acidification with formic acid and were then extracted twice with ethyl acetate and dried under N 2 gas. The metabolites were then resuspended in 500 L of 100% ethanol and separated by high-performance liquid chromatography (HPLC).…”

“…The metabolites were monitored using a radioactive flow detector as previously described. 11 The production of each metabolite was calculated and expressed as picomoles formed per minute per milligram of protein.…”

“…Renal vessels were prepared from the kidneys of control and mice treated with FF for 14 days as previously described. 11 Microsomal proteins (10 g) were separated on 7.5% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, Neb) for 2 hours at room temperature and were incubated with a goat anti-rat Cyp4A1 polyclonal (Daiichi Pure Chemicals Co, Tokyo, Japan) antibody and a mouse anti-␤-actin antibody overnight at 4°C.…”

“…10 These studies suggest that a deficiency in the renal production of 20-HETE may promote the development of hypertension. More recently, we have reported that the renal production of 20-HETE is also very low in the kidney of C57BL/6J mice, 11 indicating that they may be a good model to determine the role of CYP metabolites of AA in the regulation of renal function and blood pressure. Thus, the present study examined the effects of induction of the renal production of 20-HETE with FF on the development of Ang II-dependent hypertension in C57BL/6J mice.…”