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Am J Physiol Regul Integr Comp Physiol 276: R1749-R1757, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 6, R1749-R1757, June 1999

Regulation of P-450 4A activity in the glomerulus of the rat

Osamu Ito and Richard J. Roman

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that an enzyme of the cytochrome P-450 4A family is expressed in the glomerulus, but there is no evidence that 20-hydroxyeicosatetraenoic acid (20-HETE) can be produced by this tissue. The purpose of present study was to determine whether glomeruli isolated from the kidney of rats can produce 20-HETE and whether the production of this metabolite is regulated by nitric oxide (NO) and dietary salt intake. Isolated glomeruli produced 20-HETE, dihydroxyeicosatrienoic acids, and 12-hydroxyeicosatetraenoic acid (4.13 ± 0.38, 4.20 ± 0.38, and 2.10 ± 0.20 pmol · min-1 · mg protein-1, respectively) when incubated with arachidonic acid (10 µM). The formation of 20-HETE was dependent on the availability of NADPH and the PO2 of the incubation medium. The formation of 20-HETE was inhibited by NO donors in a concentration-dependent manner. The production of 20-HETE was greater in glomeruli isolated from the kidneys of rats fed a low-salt diet than in kidneys of rats fed a high-salt diet (5.67 ± 0.32 vs. 2.83 ± 0.32 pmol · min-1 · mg protein-1). Immunoblot experiments indicated that the expression of P-450 4A protein in glomeruli from the kidneys of rats fed a low-salt diet was sixfold higher than in kidneys of rats fed a high-salt diet. These results indicate that arachidonic acid is primarily metabolized to 20-HETE and dihydroxyeicosatrienoic acids in glomeruli and that glomerular P-450 activity is modulated by NO and dietary salt intake.

renal hemodynamics; eicosanoids; 20-hydroxyeicosatetraenoic acid; nitric oxide; hypertension; renal disease; 12-hydroxyeicosatetraenoic acid; epoxyeicosatrienoic acids; cytochrome P-450


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RECENT STUDIES have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) is the primary metabolite of arachidonic acid (AA) produced by the kidney (10, 24, 29, 32, 38) and that this substance plays a critical role in the regulation of renal tubular and vascular function. 20-HETE is a potent constrictor of renal arterioles (19, 23). Inhibition of the formation of 20-HETE blocks autoregulation of renal blood flow and tubuloglomerular feedback responses in the rat in vivo (47, 48). 20-HETE is also produced in the proximal tubule (29) and the thick ascending limb of the loop of Henle (TALH) (10), where it inhibits Na+-K+-ATPase (30) and serves as a second messenger regulating loop Cl- transport (11, 44). Recent studies have also suggested an important role for 20-HETE in the long-term control of arterial pressure (29, 31, 36-38). In this regard, the P-450 4A locus on chromosome 5 cosegregates with blood pressure in an F2 population derived from Dahl S, spontaneously hypertensive, and normotensive strains of rats (36, 38).

The formation of 20-HETE is catalyzed by enzymes of the cytochrome P-450 4A family (28). Over 13 isoforms have been reported in various species (28, 43). Four isoforms (P-450 4A1, 4A2, 4A3, and 4A8) are expressed in the kidney of rats (20). P-450 4A1, 4A2, and 4A3 catalyze the omega -hydroxylation of fatty acids and produce 20-HETE when incubated with AA (43). We recently demonstrated that P-450 4A mRNA and protein are avidly expressed in the proximal tubule, TALH, and renal microvessels of the rat (20). These observations are consistent with the results of previous studies indicating that all these structures produce 20-HETE when incubated with AA (10, 19, 29, 47). Glomeruli also express mRNA and protein for P-450 4A enzymes (20). However, there is no evidence that the glomerulus can produce 20-HETE. Rather, all previous studies indicated that isolated glomeruli produce cyclooxygenase (17) and lipoxygenase (5, 35) metabolites when incubated with AA.

Why P-450 4A protein is expressed in glomeruli but is inactive is unknown. One possibility might be that the formation of 20-HETE in glomeruli is modulated by the endogenous formation of nitric oxide (NO). This hypothesis is based on the recent findings that NO inhibits the formation of 20-HETE in the kidney and that this contributes to the vasodilator effects of NO in the renal microcirculation (1, 40). Thus the purpose of the present study was to determine whether glomeruli isolated from the kidney of rats can produce 20-HETE and to examine the effects of NO and dietary salt intake on the metabolism of AA in the glomerulus. Our results indicate that AA is primarily metabolized via the cytochrome P-450 pathway to 20-HETE and dihydroxyeicosatrienoic acids (diHETEs) in isolated glomeruli and that the formation of these products requires exogenous NADPH and is modulated by PO2, NO, and dietary salt intake.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Experiments were performed on male, 9-wk-old Lewis rats (Lew/CrlBR) purchased from Charles River Laboratories (Wilmington, MA). The rats were housed in an animal care facility at the Medical College of Wisconsin that is approved by the American Association for Accreditation of Laboratory Animal Care. Most of the rats (n = 40) were maintained on a normal-salt diet (1% NaCl by weight) and had free access to food and water throughout the study. Some rats were fed a high-salt (8% NaCl by weight, n = 8) or a low-salt (0.4% NaCl by weight, n = 8) diet for 2 wk before the study. All protocols involving animals were reviewed and received prior approval by the Animal Welfare Committee at the Medical College of Wisconsin.

Isolation of glomeruli. Glomeruli were isolated by using a rapid-sieving technique originally described by Spiro (34). The rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the abdominal aorta was cannulated with a PE-50 catheter below the left renal artery. Blood flow to the kidneys was interrupted, and the kidneys were flushed with 10 ml of cold dissection solution (4°C) containing (in mM) 135 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 2 KH2PO4, 5.5 glucose, and 10 HEPES (pH 7.4). The kidneys were rapidly removed and hemisected, and the medulla was excised. Pieces of the renal cortex were forced through a 180-µm stainless steel sieve with use of the barrel of a 30-ml syringe. The material passing through the sieve was flushed through a 100-µm sieve and collected on a 70-µm nylon sieve. The retained tissue was then washed off this sieve and divided into two portions. One-half of the glomeruli were used for the study of AA metabolism. The remainder of the sample was resuspended in 200 µl of cold homogenization buffer containing 100 mM potassium phosphate (pH 7.25), 30% glycerol, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride and homogenized by sonication for 15 s at moderate power. Aliquots of this homogenate were used in immunoblot experiments. The protein concentration of the homogenates was measured using the Bradford method (7) with bovine gamma -globulin (Bio-Rad Laboratories, Hercules, CA) as a standard.

The quality of the glomerular preparations was assessed by examining the preparation at ×50 magnification with use of a stereomicroscope (model SZH10, Olympus, Tokyo, Japan). The number of glomeruli and the total number of particles in the field were counted. The glomeruli were also examined for the presence of afferent or efferent arterioles.

We also measured alkaline phosphatase activity of the glomerular preparations, since this enzyme is highly expressed in the proximal tubule (13) and has been used to measure the degree of contamination of glomerular preparations. Alkaline phosphatase activities of glomeruli and proximal tubules were measured using a spectrophotometric assay (Sigma Chemical, St. Louis, MO) that follows the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol at a wavelength of 405 nm (6). Further documentation of the purity of the glomerular preparations was obtained by comparing the level of gamma -glutamyltransferase protein, which is a specific maker for proximal tubules (15), in glomerular and proximal tubular samples by using immunoblot analysis.

Metabolism of AA in isolated glomeruli. Isolated glomeruli were preincubated with 0.1% Tween 80 in a 100 mM potassium phosphate buffer (pH 7.4) containing 10 mM MgCl2 and 1 mM EDTA for 15 min at 4°C to permeabilize the tissue and ensure free access of exogenous AA and NADPH to the P-450 enzymes located in the endoplasmic reticulum. The glomeruli (1 mg protein) were washed twice, spun down, and incubated with [14C]AA (0.1 µCi/ml, 10 µM; Amersham Life Science, Arlington Heights, IL) in 1 ml of the potassium phosphate buffer containing 1 mM NADPH and an NADPH-regenerating system (10 mM isocitrate and 0.4 U/ml isocitrate dehydrogenase) in a shaking water bath for 60 min at 37°C. This incubation time was based on preliminary experiments indicating that the formation of diHETEs and 20-HETE was linear for up to 75 min. We previously reported that the metabolism of AA by P-450 4A enzymes in renal tissue is O2 dependent with a Michaelis-Menten constant of ~50 Torr (16). Therefore, 100% O2 gas was blown over the surface of the incubations to maintain the PO2 in the medium at a normal physiological level for the renal cortex of a rat (~100 Torr). The reactions were terminated by acidification to pH 4.0 with 0.1 M formic acid, and the glomeruli were homogenized in the incubation medium by sonication for 60 s at moderate power. AA metabolites were extracted twice from this homogenate with 3 ml of ethyl acetate and dried under N2 gas. The metabolites were resuspended in 500 µl of 100% ethanol and separated using an HPLC gradient system (model 655A-1, Hitachi, Tokyo, Japan) equipped with a 2.1 × 250-mm C18 reverse-phase column and a linear elution gradient ranging from acetonitrile-water-acetic acid (50:50:2 vol/vol/vol) to acetonitrile-acetic acid (100:0.2 vol/vol) over a 40-min period. Products were monitored using a radioactive flow detector (model A-120, Radiomatic Instrument, Tampa, FL). The production rate for each metabolite was calculated and expressed as picomoles formed per minute per milligram of protein.

Immunoblot analysis of P-450 4A protein. Proteins were separated by electrophoresis on a 10 × 20-cm, 8.5% SDS polyacrylamide gel for 1.5 h at 150 V. The proteins were transferred to a nitrocellulose membrane at 100 V in a transfer buffer consisting of 25 mM Tris · HCl, 192 mM glycine, and 20% methanol for 1 h at 4°C. The membrane was blocked overnight at 4°C by immersion into a buffer (TBST-20) containing 10 mM Tris · HCl, 150 mM NaCl, 0.08% Tween 20, and 10% nonfat dry milk (Bio-Rad). The membrane was incubated for 2 h with a 1:2,000 dilution of a goat polyclonal antibody raised against rat P-450 4A1 that recognizes other P-450 4A isoforms (20) (Daiichi Pure Chemicals, Tokyo, Japan). The membrane was rinsed several times with TBST-20 buffer and then incubated with a 1:4,000 dilution of a horseradish peroxidase-coupled, anti-goat secondary antibody (Santa Cruz Biolaboratory, Santa Cruz, CA) for 1 h. The blots were washed three to four times for 5 min in TBST-20 developed using an enhanced chemiluminesence kit (ECL, Amersham, Arlington Heights, IL). The relative intensities of the bands in the 50- to 52-kDa range were measured with a densitometer (Personal Densitometer SI, Molecular Dynamics, Sunnyvale, CA).

Statistics. Values are means ± SE. The significance of differences in mean values was evaluated using ANOVA and Duncan's multiple range test. P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Quality of the glomeruli. The preparations contained >98% intact glomeruli, and vascular poles were present on <1% of the glomeruli. Alkaline phosphatase activities of the proximal tubular preparations were 20-fold higher than those measured in sieved glomeruli. There was no significant difference in the alkaline phosphatase activity between bulk-isolated and microdissected glomeruli (Table 1). The immunoblot experiments with gamma -glutamyltransferase also indicated that the levels of this protein were 60-fold greater in proximal tubules than in isolated glomeruli (Fig. 1).

                              
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  		Table 1.   Alkaline phosphatase activity in isolated proximal tubules and glomeruli



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  		Fig. 1.   Expression of gamma -glutamyl transferase protein in glomeruli and proximal tubules isolated from kidneys of rats. Lanes 1 and 2 were loaded with protein (50 µg) from proximal tubules isolated from 2 different rats; lanes 3 and 4 were loaded with lesser amounts of proximal tubular protein (5 µg); lanes 5-8 were loaded with protein (50 µg) from glomeruli isolated from 3 different rats. Values are means ± SE.

Metabolism of AA by glomeruli. A representative reverse-phase HPLC chromatogram depicting the profile of metabolites of AA produced by intact glomeruli isolated from an inbred Lewis rat is presented in Fig. 2A. The products with retention times of 6.5, 7.5, and 8 min coeluted with synthetic 14,15-, 11,12-, and 8,9-diHETE standards, respectively. The products with retention times of 9.5 and 13.5 min coeluted with synthetic 20-HETE and 12-hydroxyeicosatetraenoic acid (12-HETE) standards. The products with retention times of 16.5, 18, and 18.5 min coeluted with synthetic 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acid (EET) standards, respectively. The peaks with retention times of 1-5 min represent cyclooxygenase metabolites of AA and polar breakdown metabolites of the P-450 products.


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  		Fig. 2.   Metabolism of arachidonic acid in isolated rat glomeruli. Glomeruli (1 mg protein) prepared using a rapid-sieving technique were incubated with [14C]arachidonic acid for 60 min at 37°C in presence of 1 mM NADPH. Reactions were superfused with 100% O2 to maintain a PO2 similar to that in renal cortex of rats (~100 Torr). A: representative reverse-phase HPLC chromatogram of products formed by glomeruli. B: profile of products formed by isolated glomeruli incubated without NADPH. C: profile of products formed by isolated glomeruli incubated with media equilibrated with room air alone. Total counts per minute (CPM) are as follows: 16,066 in A, 16,533 in B, and 12,628 in C. 20-HETE, 20-hydroxyeicosatetraenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; diHETEs, dihydroxyeicosatrienoic acids.

The formation of 20-HETE and diHETEs by isolated glomeruli was completely dependent on the addition of exogenous NAPDH to the reactions. There was no detectable formation of these products in samples incubated in the absence of added NADPH (Fig. 2B). Removal of NADPH, however, had no effect on the formation of 12-HETE (13.5-min peak), indicating that it is likely a lipoxygenase product.

Because the formation of 20-HETE by renal tissue is O2 dependent (13), we next examined the effects of altering the PO2 of the incubations on the production of P-450 metabolites of AA by the glomerulus. Glomeruli were incubated with [14C]AA in sealed vials equilibrated with room air or 100% 02 gas. The results presented in Fig. 2C indicate that the production of 20-HETE is 43% lower in glomeruli incubated in the presence of room air than in those superfused with O2. Lowering the PO2 of the incubation medium had much less effect on the formation of diHETEs and 12-HETE. Overall, the mean rate of production of 20-HETE, diHETEs, polar metabolites, and 12-HETE by glomeruli isolated from 10 different Lewis rats fed a normal-salt diet averaged 4.13 ± 0.38, 4.20 ± 0.38, 4.48 ± 0.37, and 2.10 ± 0.20 pmol · min-1 · mg protein-1, respectively.

Additional studies were performed to evaluate the effects of pharmacological inhibitors on the production of P-450 metabolites. Preincubation of isolated glomeruli with 10 µM 17-octadecynoic acid, an inhibitor of P-450 4A enzymes (49), reduced the formation of 20-HETE and diHETEs (Fig. 3B). It had no significant effect on the production of 12-HETE. Miconazole (10 µM), a selective inhibitor of P-450 epoxygenase (8, 49), reduced the formation of diHETEs, but it had no significant effect on the production of 20-HETE (Fig. 3C). Preincubation of glomeruli with a high concentration (20 µM) of indomethacin reduced the formation of products with retention times of 1-5 min (Fig. 4B), indicating that some of these products are cyclooxygenase metabolites. However, this high concentration of indomethacin also reduced the formation of 20-HETE and diHETEs by 50%. This finding is consistent with the previous findings of Capdevila et al. (8) indicating that >10 µM indomethacin inhibits cytochrome P-450 enzymes. Baicalein (0.5 µM), a selective 12-lipoxygenase inhibitor (33), reduced the formation of 12-HETE, but it had little effect on the formation of diHETEs or 20-HETE (Fig. 4C).


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  		Fig. 3.   Characterization of metabolism of arachidonic acid in isolated rat glomeruli. A: a control reverse-phase HPLC chromatogram of products formed by glomeruli incubated with [14C]arachidonic acid for 60 min at 37°C in presence of 1 mM NADPH and superfused with 100% O2 to maintain a PO2 similar to that in renal cortex of rats (~100 Torr). B: profile of products formed by glomeruli after addition of 10 µM 17-octadecynoic acid (17-ODYA). C: profile of products formed by glomeruli after addition of epoxygenase inhibitor miconazole (10 µM). Total counts per minute are as follows: 16,858 in A, 19,640 in B, and 17,152 in C.



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  		Fig. 4.   Characterization of metabolism of arachidonic acid by isolated rat glomeruli. A: a control reverse-phase HPLC chromatogram of products formed by glomeruli incubated with [14C]arachidonic acid for 60 min at 37°C in presence of 1 mM NADPH and superfused with 100% O2 to maintain a PO2 similar to that in renal cortex of rats (~100 Torr). B: profile of products formed by glomeruli after addition of cyclooxygenase inhibitor indomethacin (20 µM). C: profile of products formed by glomeruli after addition of lipoxygenase inhibitor baicalein (0.5 µM). Total counts per minute are as follows: 16,858 in A, 15,368 in B, and 16,226 in C.

Effect of NO donors on glomerular P-450 activity. The results of these experiments are summarized in Fig. 5. Addition of sodium nitroprusside (SNP, 10-3 M) to the incubation completely blocked the production of 20-HETE and diHETEs by isolated glomeruli, but it had no effect on the formation of polar cyclooxygenase metabolites or 12-HETE (Fig. 5B). The inhibitory effects of SNP on the production of 20-HETE by glomeruli were concentration dependent. At 10-5, 10-4, and 10-3 M, SNP reduced the formation of 20-HETE to 67.2 ± 11.2, 28.6 ± 2.1, and 0 ± 0% of control, respectively (Fig. 5C). Similar results were obtained after the addition of the long-acting NO donor 1-propanamine,3-(2-hydroxy-2-nitroso-1-propylhydrazino (PAPA NONOate) to the incubation. At 10-5, 10-4, and 10-3 M, PAPA NONOate reduced the production of 20-HETE to 74.2 ± 6.8, 47.2 ± 3.8, and 13.0 ± 4.1% of control, respectively (Fig. 5C).


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  		Fig. 5.   Effect of nitric oxide donors sodium nitroprusside (SNP) and 1-propanamine,3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) on metabolism of arachidonic acid by isolated rat glomeruli. A and B: representative reverse-phase HPLC chromatograms of products formed by glomeruli incubated with [14C]arachidonic acid under control conditions and after addition of 1 mM SNP to incubation solution. Total counts per minute are as follows: 23,943 in A and 24,523 in B. C: effects of SNP and PAPA NONOate on formation of 20-HETE by glomeruli expressed as percentage of control. Values are means ± SE from >= 4 separate incubations.

Effect of dietary salt intake on glomerular P-450 activity. The metabolism of AA was compared in glomeruli isolated from the kidneys of Lewis rats maintained on low-salt (0.4% NaCl by weight), normal-salt (1% NaCl by weight), or high-salt (8% NaCl by weight) diets for 2 wk. The results of these experiments are presented in Fig. 6. The production of 20-HETE and diHETEs was significantly greater in glomeruli of rats fed a low-salt diet than in those fed a normal- or a high-salt diet. In contrast, production of 12-HETE was about twofold greater in glomeruli isolated from rats fed a high-salt diet than in those fed a low-salt diet.


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  		Fig. 6.   Effect of dietary salt intake on metabolism of arachidonic acid by isolated rat glomeruli. Glomeruli were isolated from rats maintained on a high-salt (8% NaCl by weight, n = 8), normal-salt (1% NaCl by weight, n = 10), or low-salt (0.4% NaCl by weight, n = 8) diet for 2 wk. Values are means ± SE. * Significantly different (P < 0.05) from corresponding value in glomeruli of rats maintained on a high-salt diet. + Significantly different (P < 0.05) from corresponding value in glomeruli of rats maintained a normal-salt diet.

The effect of dietary salt intake on the expression of P-450 4A protein in the glomerulus is presented in Fig. 7. Alignment of the bands with those seen using a clofibrate-treated rat liver standard that expresses P-450 4A1 and 4A3 suggests that the isoform expressed in glomeruli migrates like 4A2. The level of P-450 4A protein was sixfold greater in glomeruli of rats fed a low-salt diet than in rats fed a high-salt diet for 2 wk.


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  		Fig. 7.   Effect of changes in dietary salt intake on expression of P-450 4A protein in isolated glomeruli. Glomeruli were isolated from rats fed a high-salt (8% NaCl by weight) or a low-salt (0.4% NaCl by weight) diet for 2 wk. Lanes 1-4 were loaded with 50 µg of glomerular protein isolated from kidneys of 4 different rats fed a high salt diet, and lanes 6-9 were loaded with 50 µg of glomerular protein isolated from kidneys of 4 different rats fed a low-salt diet. Lane 5 was loaded with microsomes (20 µg) prepared from liver of a clofibrate-treated rat that expresses all three P-450 4A isoforms. Values are means ± SE from 4 rats. * Significantly different (P < 0.05) from value in glomeruli of rats maintained on a high-salt diet.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies have indicated that mRNA and protein for P-450 4A enzymes are expressed in the proximal tubule (20, 29), TALH (10, 20), and renal microvessels (19, 20, 23, 47). All these tissues produce 20-HETE when incubated with AA. Despite the fact that the cytochrome P-450 4A mRNA and protein are also expressed in the glomerulus (20), there is no evidence that 20-HETE can be produced by this tissue. Indeed, all previous studies have reported that isolated glomeruli produce primarily cyclooxygenase and lipoxygenase metabolites when incubated with AA (5, 17, 21, 35). The results of the present study indicate that glomeruli can produce 20-HETE and diHETEs when incubated with AA in the presence of NAPDH at a PO2 typically found in the renal cortex of the rat (75-100 Torr). No cytochrome P-450 metabolites of AA were formed when NADPH was not added to the incubations. The formation of 20-HETE is also markedly reduced when the glomeruli are incubated in medium equilibrated with room air. These observations suggest that the inability to detect the formation of 20-HETE by glomeruli in previous studies was likely because NADPH was not added to the incubations and physiological PO2 levels for glomeruli were not maintained.

Why exogenous NADPH is required to exhibit cytochrome P-450 activity in glomeruli is unknown. We originally thought that the glomeruli were losing NADPH because they were permeablized with Tween 80. However, untreated glomeruli also required NADPH to form P-450 metabolites of AA. Addition of NADPH also enhances the formation of 20-HETE by isolated proximal tubules and renal arterioles, but unlike glomeruli, it is not required. NADPH is an essential cofactor for all cytochrome P-450 reactions. It is generated via the pentose shunt pathway and the citric acid cycle. It is possible that glomeruli lack the enzymatic machinery necessary to maintain adequate intracellular stores of NAPDH when incubated in vitro with glucose as the sole metabolic substrate. Exogenous NADPH may be taken up and replete intracellular stores in isolated glomeruli, or it may prevent the loss of NADPH by limiting diffusion. Regardless of how NADPH enhances the formation of P-450 metabolites of AA, our results suggest that addition of NADPH to the bath and maintaining physiological PO2 levels are important in the study of the contribution of P-450 metabolites of AA to mediation of renal tubular and vascular responses in vitro.

Because cytochrome P-450 4A activity is high in the proximal tubules (29), we were concerned that the production of 20-HETE observed in isolated glomeruli was an artifact due to contamination of the preparations with adherent tubules. However, this possibility seems remote, because alkaline phosphatase activity (a marker for proximal tubules) was 20-fold greater in our proximal tubular than in our glomerular preparations, but the production of 20-HETE by proximal tubules was only twofold higher (8.58 ± 0.73 pmol · min-1 · mg protein-1, n = 6). Thus it seems unlikely that the production of 20-HETE by isolated glomeruli can be explained solely by the synthesis of this compound by adherent proximal tubules. Moreover, we found that the levels of gamma -glutamyltransferase, a more specific marker for proximal tubules (15), were 60-fold greater in our proximal tubular than in our glomerular preparations. These observations further support the conclusion that there was very little contamination of our glomerular preparations.

The results of the present study indicating that glomeruli can avidly produce 20-HETE contrast with the original findings of Omata et al. (29). They reported that the omega -hydroxylase activity of the kidney of rats was primarily localized in proximal tubules. No significant activity could be detected in glomeruli. However, the microdissected nephron segments in that study were homogenized by freezing and thawing, which diminish the activity of P-450 enzymes. In addition, the samples were not incubated in an O2-rich environment. Under these conditions, Omata et al. may have still been able to detect residual activity in proximal tubules, but not in glomerular samples, in which basal P-450 activity is lower.

The present study also explored the possibility that NO modulates the production of P-450 metabolites of AA in the glomerulus, as has been reported in renal microsomes and arterioles (1, 40). This hypothesis is based on the observations that NO inhibits NO synthase (1, 14, 15, 18) and P-450 enzymes of the 1A, 2B (46), 3C (22), and 4A (40) families by forming iron-nitrosyl complexes at the catalytic heme-binding site in these enzymes. The present results indicate that NO also inhibits the formation of 20-HETE in glomeruli in a concentration-dependent manner. However, our finding that glomeruli incubated without NO synthase inhibitors still produce 20-HETE indicates that the inactivation of glomerular P-450 enzymes by endogenously produced NO is not the reason why previous investigators failed to detect formation of P-450 metabolites of AA by isolated glomeruli.

Numerous studies have indicated that NO plays an important role in the regulation of glomerular hemodynamics. Blockade of NO synthesis increases arterial pressure, decreases renal blood flow and glomerular filtration rate, and potentiates tubuloglomerular feedback responses (26, 27, 41, 45). An endothelial form of NO synthase is highly expressed in the glomerulus (42). NO modulates the contractility of the mesangial cells, which regulates glomerular filtration by altering glomerular capillary surface area (25). The results of the present study suggest that NO may modify glomerular hemodynamics, in part by inhibiting the formation of 20-HETE. Our recent finding that preventing the fall in 20-HETE levels attenuates the renal vasodilator effect of NO (1, 40) is consistent with this hypothesis.

Besides playing an important role in regulation of renal vascular tone, there is evidence that NO modulates tubuloglomerular feedback responses. A neuronal isoform of NO synthase is expressed in the macula densa (26). Addition of inhibitors of NO synthase to tubular fluid enhances tubuloglomerular feedback responses, whereas addition of L-arginine to enhance the local formation of NO has the opposite effect (41, 45). We previously reported that 20-HETE plays a critical role as a mediator or a second messenger of tubuloglomerular feedback (48). Thus it is likely that NO may modulate the sensitivity of tubuloglomerular feedback by interfering with the formation of 20-HETE in the macula densa, the glomerulus, and/or the afferent arteriole.

The present study also examined the effects of changes in dietary salt intake on the production of 20-HETE and the levels of P-450 4A protein in the glomerulus. The production of 20-HETE was twofold greater and the expression of P-450 4A protein was sixfold greater in glomeruli isolated from rats fed a low-salt diet than in glomeruli isolated from rats fed a high-salt diet. It is unknown why there is not a better correlation between the changes in protein levels and enzyme activity. It may be that the difference in enzyme activity between the groups was underestimated, since the production of 20-HETE is substrate rather than enzyme limited and intracellular concentration of AA cannot be precisely controlled in incubations in which intact glomeruli are used. Alternatively, it is possible that some of the P-450 4A protein expressed in rats on a low-salt diet is inactive. It could be poisoned by endogenously produced NO or CO or lack association with the essential cofactors, heme and NADPH reductase.

Overall, the present results are consistent with other findings that the production of 20-HETE is greater in microsomes prepared from the kidneys of spontaneously hypertensive, Brown-Norway, Dahl salt-sensitive, and Dahl salt-resistant rats fed a low-salt diet than of those fed a high-salt diet (24, 29, 36, 38). More recently, we found that the expression of P-450 4A proteins in the liver, kidney, and renal microvessels is downregulated in rats fed a high-salt diet and that this can be prevented if circulating ANG II is maintained at normal levels by intravenous infusion (2). These observations suggest that P-450 4A activity is regulated by circulating ANG II levels and that changes in glomerular P-450 4A activity may contribute to the downregulation of tubuloglomerular feedback and renal vascular reactivity associated with the adaptation to a high-salt diet and the upregulation of these responses after activation of the renin-angiotensin system.

The results of the present study also indicate that isolated glomeruli exhibit epoxygenase activity and avidly produce EETs and diHETEs when incubated with AA. The formation of these products is also dependent on the addition of exogenous NAPDH and blocked by miconazole, indicating that the formation of these metabolites is catalyzed by a cytochrome P-450 enzyme. The production of diHETEs was greater in glomeruli isolated from the kidneys of Lewis rats fed a low-salt diet than in those fed a high-salt diet. This observation is consistent with previous reports that renal epoxygenase activity is higher in Dahl salt-sensitive (24, 36) and Brown-Norway rats (38) fed a low-salt diet than in those fed a high-salt diet. On the other hand, changes in salt intake had no effect on renal epoxygenase activity in Dahl salt-resistant or spontaneously hypertensive rats (24, 38). Moreover, Capdevila et al. (9) reported that renal epoxygenase activity increases in Sprague-Dawley rats fed a high-salt diet. This led them to propose that a failure to induce renal epoxygenase activity may contribute to the development of some forms of salt-induced hypertension. The reasons for the lack of consistent results concerning the effects of salt on renal epoxygenase activity are unknown. There are many P-450 isoforms (1A, 2C, 2J, 2E, and 4A), expressed in the kidney, that produce EETs (28). However, little is known about which of these isoforms are expressed in the glomerulus.

The present study also addressed the influence of changes in salt intake on glomerular 12-HETE production. Previous studies have indicated that 12-HETE and 15-hydroxyeicosatetraenoic acid are potent inhibitors of renin secretion (4). There is also considerable evidence indicating that feedback inhibition of renin secretion by ANG II is mediated by increased formation of 12-HETE in the kidney (3, 39). The present finding that the production of 12-HETE increases in glomeruli isolated from rats fed a high-salt diet is consistent with a recent finding of Stern et al. (39) that 12-HETE levels are elevated in renal cortical tissue of rats fed a high-salt diet. This finding is also supported by the view that increases in the formation of 12-HETE contribute to the fall in renin secretion in rats fed a high-salt diet (39).

Perspectives

The present results indicate that 1) the glomerulus produces 20-HETE, diHETEs, and 12-HETE when incubated with AA, 2) the formation of P-450 metabolites of AA by the glomerulus is dependent on NADPH and PO2, and 3) glomerular P-450 activity is modulated by NO and dietary salt intake. These results suggest that changes in the local production 20-HETE may influence glomerular hemodynamics and contribute to the modulation of glomerular function, renal vascular tone, and tubuloglomerular feedback responses produced by NO and changes in dietary salt intake. Given the importance of metabolites of AA in the regulation of glomerular hemodynamics and in mediating glomerular injury in hypertension, diabetes, and other disease states, our findings suggest that P-450 inhibitors and receptor antagonists might offer a new therapeutic approach to limit glomerular injury in a variety of disease states.


    ACKNOWLEDGEMENTS

The authors thank Lisa Henderson for excellent technical assistance with the P-450 assays.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-29587 and HL-36279.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. J. Roman, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: rroman{at}post.its.mcw.edu).

Received 25 August 1998; accepted in final form 9 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alonso-Galicia, M., H. A. Drummond, K. K. Reddy, J. R. Falck, and R. J. Roman. Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide. Hypertension 29: 320-325, 1997[Abstract/Free Full Text].

2.   Alonso-Galicia, M., A. S. Greene, T. M. Kurth, A. W. Cowley, Jr., and R. J. Roman. 20-HETE contributes to the renal vasoconstrictor actions of angiotensin II (Abstract). FASEB J. 13: A389, 1999.

3.   Antonipillai, I., J. Nadler, and R. Horton. Angiotensin feedback inhibition on renin is expressed via the lipoxygenase pathway. Endocrinology 122: 1277-1281, 1988[Abstract].

4.   Antonipillai, I., J. L. Nadler, E. C. Robin, and R. Horton. The inhibitory role of 12- and 15-lipoxygenase products on renin release. Hypertension 10: 61-66, 1987[Abstract/Free Full Text].

5.   Ardaillou, R., L. Baud, and J. Sraer. Leukotrienes and other lipoxygenase products of arachidonic acid synthesized in the kidney. Am. J. Med. 81, Suppl. 2B: 12-22, 1986[Medline].

6.   Bowers, G. N., and R. B. McComb. A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin. Chem. 12: 70-89, 1966[Abstract].

7.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

8.   Capdevila, J., L. Gil, M. Orellanan, N. J. Marnett, J. I. Mason, P. Yadagiri, and J. R. Falck. Inhibitors of cytochrome P-450 dependent arachidonic acid metabolism. Arch. Biochem. Biophys. 261: 257-263, 1988[Medline].

9.   Capdevila, J. H., W. Shouzou, J. Yan, A. Karara, H. R. Jacobson, J. R. Falck, F. P. Guengerich, and R. N. DuBois. Cytochrome P-450 arachidonic acid epoxygenase: regulatory control of the renal epoxygenase by dietary salt loading. J. Biol. Chem. 267: 21720-21726, 1992[Abstract/Free Full Text].

10.   Carroll, M. A., A. Sala, C. E. Dunn, J. C. McGiff, and R. C. Murphy. Structural identification of cytochrome P-450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells. J. Biol. Chem. 266: 12306-12312, 1992[Abstract/Free Full Text].

11.   Escalante, B., D. Erlij, J. R. Falck, and J. C. McGiff. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science 251: 799-802, 1991[Abstract/Free Full Text].

12.   Escalante, B., W. C. Sessa, J. R. Falck, P. Yuclagiri, and M. L. Schwartzman. Vasoactivity of 20-hydroxyeicosatetraenoic acid is dependent on metabolism by cyclooxygenase. J. Pharmacol. Exp. Ther. 248: 229-232, 1989[Abstract/Free Full Text].

13.   Gesek, F. A., D. W. Wolff, and J. W. Standhoy. Improved separation for rat proximal and distal tubules. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F359-F365, 1987.

14.   Griscavage, J. M., A. J. Habbs, and L. J. Ignarro. Negative modulation of nitric oxide synthase by nitric oxide and nitroso compounds. Adv. Pharmacol. 34: 215-234, 1995.

15.   Hanigan, M. H., and H. F. Frierson, Jr. Immunohistochemical detection of gamma -glutamyl transpeptidase in normal human tissue. J. Histochem. Cytochem. 44: 1101-1108, 1996[Abstract].

16.   Harder, D. R., J. Narayanan, E. K. Birks, J. F. Liard, J. D. Imig, J. H. Lombard, A. R. Lange, and R. J. Roman. Identification of a putative microvascular oxygen sensor. Circ. Res. 79: 54-61, 1996[Abstract/Free Full Text].

17.   Hassid, A., M. Konieczkowski, and M. J. Dunn. Prostaglandin synthesis in isolated rat kidney glomeruli. Proc. Natl. Acad. Sci. USA 76: 1155-1159, 1979[Abstract/Free Full Text].

18.   Hurshman, A. R., and M. A. Marletta. Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effects on catalytic activity. Biochemistry 34: 5627-5634, 1995[Medline].

19.   Imig, J. D., A.-P. Zou, D. E. Stec, D. R. Harder, J. R. Falck, and R. J. Roman. Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. Am. J. Physiol. 270 (Regulatory Integrative Comp. Physiol. 39): R217-R227, 1996[Abstract/Free Full Text].

20.   Ito, O., M. Alonso-Galicia, K. Hopp, and R. J. Roman. Localization of cytochrome P-450 4A isoforms along the rat nephron segments. Am. J. Physiol. 274 (Renal Physiol. 43): F395-F404, 1998[Abstract/Free Full Text].

21.   Katoh, T., K. Takahashi, J. Capdevila, A. Karara, J. R. Falck, H. R. Jacobson, and K. F. Badr. Glomerular stereospecific synthesis and hemodynamic actions of 8,9-epoxyeicosatrienoic acid in rat kidney. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F578-F586, 1991[Abstract/Free Full Text].

22.   Khatsenko, O. G., S. S. Gross, A. B. Rifkind, and J. R. Vane. Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants. Proc. Natl. Acad. Sci. USA 90: 11147-11151, 1993[Abstract/Free Full Text].

23.   Ma, Y.-H., D. Gebremedhin, M. L. Schwartzman, J. R. Falck, J. E. Clark, B. S. S. Masters, D. R. Harder, and R. J. Roman. 20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries. Circ. Res. 72: 126-136, 1993[Abstract/Free Full Text].

24.   Ma, Y.-H., M. L. Schwartzman, and R. J. Roman. Altered renal P-450 metabolism of arachidonic acid in Dahl salt-sensitive rats. Am. J. Physiol. 267 (Regulatory Integrative Comp. Physiol. 36): R579-R589, 1994[Abstract/Free Full Text].

25.   Marsden, P. A., T. A. Brock, and B. J. Ballermann. Glomerular endothelial cells respond to calcium-mobilizing agonists with release of EDRF. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1295-F1303, 1990[Abstract/Free Full Text].

26.   Mundel, P., S. Bachmann, M. Bader, A. Fischer, W. Kummer, B. Mayer, and W. Kriz. Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int. 42: 1017-1019, 1992[Medline].

27.   Navar, L. G., E. W. Inscho, D. S. A. Majid, J. D. Imig, L. M. Harrison-Bernard, and K. M. Mitchell. Paracrine regulation of the renal microcirculation. Physiol. Rev. 76: 425-536, 1996[Abstract/Free Full Text].

28.   Nelson, D. R., T. Kamataki, D. J. Waxman, F. P. Guengerich, R. W. Estabrook, R. Feyereisen, F. J. Gonzalez, M. J. Coon, I. C. Gunsalus, O. Gotoh, K. Okuda, and D. W. Nebert. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial name of enzymes, and nomenclature. DNA Cell Biol. 12: 1-51, 1993[Medline].

29.   Omata, K., N. G. Abraham, and M. L. Schwartzman. Renal cytochrome P-450-arachidonic acid metabolism: localization and hormonal regulation in SHR. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F591-F599, 1992[Abstract/Free Full Text].

30.   Pedrosa Ribeiro, C. M., G. R. Dubay, J. R. Falck, and L. J. Mandel. Parathyroid hormone inhibits Na+-K+-ATPase through a cytochrome P-450 pathway. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F497-F505, 1994[Abstract/Free Full Text].

31.   Sacerdoti, D., N. G. Abraham, J. C. McGiff, R. D. Levere, and M. L. Schwartzman. Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science 243: 388-390, 1989[Abstract/Free Full Text].

32.   Schwartzman, M. L., P. Martsek, A. R. Rios, R. D. Levere, K. Solangi, A. I. Goodman, and N. G. Abraham. Cytochrome P450-dependent arachidonic acid metabolism in human kidney. Kidney Int. 37: 94-99, 1990[Medline].

33.   Sekiya, K., and H. Okuda. Selective inhibition of platelet lipoxygenase by baicalein. Biochem. Biophys. Res. Commun. 105: 1090-1095, 1982[Medline].

34.   Spiro, R. G. Studies on the renal glomerular basement membrane. J. Biol. Chem. 242: 1915-1922, 1967[Abstract/Free Full Text].

35.   Sraer, J., M. Rigarud, M. Bens, H. Radinovitch, and R. Ardaillou. Metabolism of arachidonic acid via the lipoxygenase pathway in human and murine glomeruli. J. Biol. Chem. 258: 4325-4330, 1983[Abstract/Free Full Text].

36.   Stec, D. E., A. Y. Deng, J. P. Rapp, and R. J. Roman. Cytochrome P450 4A genotype cosegregates with hypertension in Dahl S rats. Hypertension 27: 564-568, 1996[Abstract/Free Full Text].

37.   Stec, D. E., D. L. Mattson, and R. J. Roman. Inhibition of renal outer medullary 20-HETE production produces hypertension in Lewis rats. Hypertension 29: 315-319, 1997[Abstract/Free Full Text].

38.   Stec, D. E., M. R. Trolliet, J. E. Krieger, H. J. Jacob, and R. J. Roman. Renal cytochrome P450 4A activity and salt sensitivity in spontaneously hypertensive rats. Hypertension 27: 1329-1336, 1996[Abstract/Free Full Text].

39.   Stern, N., K. Nozawa, E. Kisch, M. L. Tuck, M. Golub, P. Eggena, and E. Knoll. Tonic inhibition of renin secretion by the 12-lipoxygenase pathway: augmentation by high salt intake. Endocrinology 137: 1878-1884, 1996[Abstract].

40.   Sun, C. W., M. Alonso-Galicia, R. M. Taheri, J. R. Falck, D. R. Harder, and R. J. Roman. Nitric oxide-20-hydroxyeicosatetraenoic acid interaction in the regulation of K+ channel activity and vascular tone in renal arterioles. Circ. Res. 83: 1-11, 1998[Free Full Text].

41.   Thorup, C., and A. E. G. Persson. Macula densa derived nitric oxide in regulation of glomerular capillary pressure. Kidney Int. 49: 430-436, 1996[Medline].

42.   Ujiie, K., J. Yuen, L. Hogarth, R. Danziger, and R. A. Star. Localization and regulation of endothelial NO synthase mRNA expression in rat kidney. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F296-F302, 1994[Abstract/Free Full Text].

43.   Wang, M.-H., D. E. Stec, M. Balazy, V. Mastyugin, C. S. Yang, R. J. Roman, and M. L. Schwartzman. Cloning, sequencing, and cDNA-directed expression of the rat renal CYP 4A2: arachidonic acid omega -hydroxylation and 11,12-epoxidation by CYP 4A2 protein. Arch. Biochem. Biophys. 336: 240-250, 1996[Medline].

44.   Wang, W. H., and H. Lu. Effect of arachidonic acid on activity of the apical K channel in the thick ascending limb of the rat kidney. J. Gen. Physiol. 106: 727-743, 1995[Abstract/Free Full Text].

45.   Wilcox, C. S., W. J. Welch, F. Murad, S. S. Gross, G. Taylor, R. Levi, and H. H. Schmidt. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc. Natl. Acad. Sci. USA 89: 11993-11997, 1992[Abstract/Free Full Text].

46.   Wink, D. A., Y. Osawa, J. F. Darbyshine, C. R. Jones, S. C. Eshenaus, and R. W. Nims. Inhibition of cytochrome P450 by nitric oxide and a nitric oxide releasing agent. Arch. Biochem. Biophys. 300: 115-123, 1993[Medline].

47.   Zou, A.-P., J. D. Imig, M. Kaldunski, P. R. Ortiz de Montellano, Z.-H. Sui, and R. J. Roman. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F275-F282, 1994[Abstract/Free Full Text].

48.   Zou, A.-P., J. D. Imig, P. R. Ortiz de Montellano, Z.-H. Sui, J. R. Falck, and R. J. Roman. Effect of P-450 omega -hydroxylase metabolites of arachidonic acid on tubuloglomerular feedback. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F934-F941, 1994[Abstract/Free Full Text].

49.   Zou, A.-P., Y.-H. Ma, Z.-H. Sui, P. R. Ortiz de Montellano, J. E. Clark, B. S. Masters, and R. J. Roman. Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome 450 fatty acid omega -hydroxylase, on renal function in rat. J. Pharmacol. Exp. Ther. 268: 474-481, 1993[Abstract/Free Full Text].

Am J Physiol Regul Integr Compar Physiol 276(6):R1749-R1757
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