Induction of P4504A Activity Improves Pressure-Natriuresis in Dahl S Rats
Clofibrate has been reported to prevent the development of hypertension in Dahl S rats, but its mechanism of action remains to be determined. The present study examined the effects of clofibrate on renal P4504A activity and the pressure natriuresis relationship in Dahl S rats. Dahl S and R rats fed a low-salt diet (0.4% NaCl) were given either clofibrate (240 mg/kg/d) or vehicle (20 mmol/L Na2CO3) in their drinking water for 1 week and then switched to a high salt diet (8% NaCl) while continuing drug treatment. After 3 weeks, mean arterial pressure in ketamine-Inactin anesthetized rats averaged 121±2 (n=8) in Dahl R, 173±8 (n=6) in Dahl S, and 139±4 mm Hg (n=7) in clofibrate-treated Dahl S rats. Increasing renal perfusion pressure (RPP) from 100 to 150 mm Hg in Dahl R rats increased sodium excretion (UNaV) from 2.9±0.7 to 9.7±3.2 μmol/min/g kwt. In contrast, the pressure natriuresis relation was blunted in Dahl S rats and UNaV only increased from 2.7±0.9 to 6.1±1.3 μmol/min/g kwt. The pressure natriuresis relation was improved in clofibrate-treated Dahl S rats and UNaV increased from 5.1±1.3 to 16.7±2.6 μmol/min/g kwt. At similar levels of RPP, the fractional excretion of sodium tended to be higher in clofibrate-treated than in vehicle-treated Dahl S rats, but not significantly. Glomerular filtration rate (GFR) was 40% higher in clofibrate-compared to vehicle-treated Dahl S rats (0.9±0.2 versus 0.6±0.2 mL/min/g kwt), and was not significantly different from the values seen in Dahl R rats (0.9±0.1 mL/min/g kwt). Clofibrate induced the expression of P4504A protein in the renal cortex and outer medulla of Dahl S rats. These data suggest that induction of renal P4504A activity with clofibrate improves the pressure natriuresis relation in Dahl S rats by primarily increasing GFR.
- cytochrome P450
- antilipidemic agents
- renal hemodynamics
- arachidonic acid
- AII = angiotensin II
- Dahl R rats = Dahl salt-resistant rats
- Dahl S rats = Dahl salt-sensitive rats
- FENa = fractional sodium excretion
- GFR = glomerular filtration rate
- 20-HETE = 20-hydroxyeicosatetraenoic acid
- P4504A = cytochrome P450 4A enzymes
- PMSF = phenylmethylsulfonyl fluoride
- PPAR = peroxisome proliferator action receptor
- PTH = parathyroid hormone
- RPP = renal perfusion pressure
- SDS = sodium dodecyl sulfate
- TALH = thick ascending limb of the loop of Henle
- TBS = Tris buffered saline
- UNaV = sodium excretion
Renal transplantation studies strongly support the hypothesis that a renal dysfunction underlies the development of hypertension in Dahl salt-sensitive (S) rats;1 however, the factors responsible for altering renal function in this strain of rats are poorly understood. Several investigators have now identified an abnormality in renal function that precedes the development of hypertension in Dahl S rats.2–4⇓⇓ For any given neural and hormonal background to the kidney, Dahl S rats require a higher renal perfusion pressure to achieve the same rate of sodium excretion as Dahl salt-resistant (R) rats. This abnormality appears to be due to an elevation in Cl− transport in the thick ascending limb of the loop of Henle (TALH).5–7⇓⇓ Moreover, several studies have now demonstrated that the TALH metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), through an enzyme of the cytochrome P450 4A family and that 20-HETE serves as an endogenous inhibitor of the Na+, K+, 2Cl− transporter.8–10⇓⇓ In this regard, we have recently reported that the production of 20-HETE is markedly reduced in the outer medulla of Dahl S compared to Dahl R rats.10 This deficiency in the renal production of 20-HETE may contribute to the enhanced loop Cl− transport, the resetting of the pressure natriuresis relationship, and the development of hypertension in Dahl S rats. In order to test this hypothesis, the present study examined the effects of inducing renal P4504A activity with clofibrate on the pressure natriuresis relationship in Dahl S rats.
Experiments were performed on female inbred Dahl S and R/Jr/Hsd/ MCW rats11 that were obtained from a colony maintained at the Medical College of Wisconsin. Female rats were used because our previous studies on the characterization of this animal model of salt sensitivity were performed in female rats.2,5⇓ The rats were studied during different phases of their estrus cycle and housed in an animal care facility at the Medical College of Wisconsin, which is approved by the American Association for the Accreditation of Laboratory Animal Care; the animals had free access to food and water throughout the study. All protocols involving animals received approval by the Animal Care Committee of the Medical College of Wisconsin. The rats were maintained on a low salt diet (0.4% NaCl) until they were 9 weeks of age and then switched to a high salt diet (8% NaCl) for 3 weeks. Rats from each strain were divided into 2 groups during the high salt intake, one group received vehicle (20 mmol/L Na2CO3), while the other group received clofibrate (80 mg/d) in their drinking water.
Characterization of the Pressure-Natriuresis Response
After 3 weeks, the rats were anesthetized with ketamine (40 mg/kg) and thiobutabarbital (Inactin) (30 mg/kg) and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. Cannulas were placed in the carotid and femoral arteries for measurement of arterial pressure above and below the left renal artery. A cannula was placed in the right external jugular vein for intravenous infusions. The left ureter was cannulated for collection of urine. Two adjustable clamps were placed on the aorta, above and below the left renal artery, and ligatures were loosely placed around the superior mesenteric and celiac arteries for manipulation of renal perfusion pressure (RPP).12 After surgery, the rats were given an intravenous infusion of 3% bovine serum albumin in a 0.9% saline solution at a rate of 100 μL/min throughout the experiment. [3H]inulin (2 μCi/mL) was included in the infusion solution to allow for measurement of glomerular filtration rate (GFR). After a 60 minute equilibration period, RPP was lowered to 100 mm Hg by tightening the aortic clamp above the renal arteries. Pressure was only lowered to 120 mm Hg in vehicle-treated Dahl S rats because they exhibited oliguria at lower RPP. After a 15 minute equilibration period, urine and plasma samples were collected during a 10 minute clearance period. The aortic clamp was then released to allow RPP to return to control levels, and urine and plasma samples were collected in an additional 10 minute clearance period. RPP was then increased to 150 and 175 mm Hg by tightening the aortic clamp below the renal arteries and urine and plasma samples were collected in 2 additional 10 minute clearance periods.
Western Blot Analysis of P4504A Isoforms
Microsomes were prepared from the renal cortex and outer medulla of the kidneys of vehicle- and clofibrate-treated Dahl S and R rats. The tissue was homogenized in a 10 mmol/L potassium phosphate buffer at pH 7.7 containing 250 mmol/L sucrose, 1 mmol/L EDTA, 2 μmol/L leupeptinin, 1 μmol/L pepstatin, 2 μg/mL aprotinin, and 0.1 μmol/L phenylmethylsulfonyl fluoride (PMSF). Homogenates were centrifuged at 10 000g for 15 minutes to remove nuclei and mitochondria, and the supernatant was centrifuged again at 100 000g for 60 minutes. The microsomal pellet was resuspended in a 100 mmol/L potassium phosphate buffer at pH 7.25 containing 1 mmol/L EDTA, 1 mmol/L dithiotriol, 0.1 μmol/L PMSF, and 30% glycerol. Protein concentration from microsomal preparations was measured by the Bradford method (Bio-Rad) using gamma globulin as a standard. Twenty micrograms of sample protein were subjected to 8.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on a 20×20 cm gel at 100 volts for 15 hours. The proteins were transferred electrophoretically to a nitrocellulose membrane at 100 volts for 1 hour. After transfer, nonspecific binding was blocked by incubating the membrane for 2 hours at room temperature in TBS-T buffer (6 mmol/L Tris-HCl, 4 mmol/L Tris-Base, 150 mmol/L NaCl, and 0.08% Tween-20, pH 7.5) containing 5% nonfat dry milk. After the blocking solution was discarded, the membrane was washed 3 times with TBS-T buffer and subsequently incubated for 2 hours with a polyclonal antibody raised against rat liver P4504A1 at a 1:2000 dilution in TBS-T buffer containing 2% milk. This polyclonal antibody cross-reacts with 4A1, 4A2, and 4A3 isoforms in the renal cortex of the rat.13,14⇓ The membrane was then washed 3 times with TBS-T buffer and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology) at a 1:1000 dilution in 2% milk for 1 hour. After 3 more washes, the immunoblots were developed using an enhanced chemiluminescence kit (ECL, Amersham).
Data are presented as mean±SEM. Differences in the values measured at different renal perfusion pressures in each group were evaluated using an analysis of variance for repeated measures followed by a Duncan multiple-range test. Between-group differences in values measured at the same level of renal perfusion pressure were evaluated using a two-way analysis of variance for repeated measures with one independent factor followed by a Duncan multiple-range test. A P<.05 was considered statistically significant.
Effects of Clofibrate on the Pressure-Natriuresis Relation in Dahl S and R Rats
The relationships between urine flow and sodium excretion and renal perfusion pressure in vehicle- and clofibrate-treated Dahl S and R rats are compared in Fig 1. Control mean arterial pressures averaged 173±8 (n=6) in vehicle-treated, and 139±4 mm Hg (n=7) in clofibrate-treated Dahl S rats (P<.05). Mean arterial pressures in vehicle-treated (n=8) and clofibrate-treated (n=9) Dahl R rats were not significantly different (121±2 versus 122±3 mm Hg). Baseline plasma sodium concentration (144±1 versus 142±1 in Dahl S, and 143±1 versus 143±2 mmol/L in Dahl R rats, respectively) as well as hematocrit (0.39±0.01 versus 0.36±0.02 in Dahl S, and 0.37±0.02 versus 0.36±0.01 in Dahl R rats) were not significantly different in vehicle or clofibrate-treated Dahl S and R rats, indicating that similar degrees of volume expansion were obtained in the groups of rats studied. Increasing RPP from 101±1 to 147±1 mm Hg in vehicle-treated Dahl R rats produced a significant threefold increase in urine flow (from 17.0±3.7 to 53.4±17.7 μL/min/g kwt) and UNaV (from 2.9±0.7 to 9.7±3.2 μmol/min/g kwt, Fig 1). Similar results were seen in clofibrate-treated Dahl R rats when RPP was increased over the same range; urine flow increased from 22.5±4.4 to 75.7±11.6 μL/min/g kwt, and UNaV increased from 3.9±0.7 to 14.1±2.0 μmol/min/g kwt. The pressure diuresis and natriuresis relations were blunted in vehicle-treated Dahl S rats. Increasing RPP from 123±2 to 152±2 mm Hg in vehicle-treated Dahl S rats produces only a twofold increase in urine flow from 13.5±5.3 to 33.9±5.8 μL/min/g kwt; UNaV rose from 2.7±0.9 to 6.1±1.3 μmol/min/g kwt (Fig 1). Moreover, at equivalent levels of RPP of 125 and 150 mm Hg, urine flow and sodium excretion were significantly lower in Dahl S rats than in vehicle-treated Dahl R rats. The pressure natriuresis and diuresis relations were potentiated in Dahl S rats given clofibrate; however, only at the highest RPP studied (175 mm Hg), were urine flow and sodium excretion significantly greater in clofibrate-treated than in vehicle-treated Dahl S rats (P<.05). Moreover, at similar levels of RPP, there was no significant difference in urine flow and sodium excretion in clofibrate-treated Dahl S and vehicle-treated Dahl R rats.
The relationships between fractional sodium excretion (FENa) and renal perfusion pressure in vehicle- and clofibrate- treated Dahl S and R rats are presented in Fig 2. Increasing RPP from 101±1 to 147±1 mm Hg in vehicle-treated Dahl R rats produced a threefold increase in FENa from 2.4±0.5 to 6.8±1.9% of filtered load (Fig 2). A similar elevation in FENa was observed when RPP was increased over the same range in clofibrate-treated Dahl R rats (4.5±1.0 to 10.0±1.5% of filtered load). At an equivalent RPP of 125 mm Hg, FENa tended to be lower in vehicle-treated Dahl S compared to Dahl R rats (2.8±1.1 versus 4.1±1.1% of filtered load) although this difference was not statistically significant. Clofibrate tended to increase FENa in Dahl S rats, particularly at the highest RPP studied, but this difference was not significant. Also, there was no significant difference in FENa between clofibrate-treated Dahl S rats and vehicle-treated Dahl R rats at equivalent levels of RPP.
Glomerular filtration rate was similar in vehicle- and clofibrate-treated Dahl R rats (Table 1). Baseline GFR was approximately 25% lower in vehicle-treated Dahl S compared to Dahl R rats at all levels of RPP studied. Clofibrate increased GFR in Dahl S rats, and there was no significant difference in GFR in these rats and vehicle-treated Dahl R rats at equivalent levels of RPP.
Effect of Clofibrate on P4504A Protein Expression in the Kidney
Immunoblot experiments were performed using a polyclonal antibody raised against rat liver P4504A1 enzyme that crossreacts with P4504A1, 4A2, and 4A3 isoforms in the renal cortex of the rat.13,14⇓ Typical examples of immunoblots in Dahl S rats are presented in Fig 3. Three immunoreactive bands were detected in the renal cortex of Dahl S rats with molecular weights ranging from 50 to 52 kDa. These bands correspond to the P4504A1, 4A2, and 4A3 isoforms, which have previously been reported to be expressed in the liver and kidney of rats.15
The levels of all three P4504A isoforms were induced in the renal cortex of clofibrate-treated Dahl S rats. In contrast, only one band was detected in the outer medulla of Dahl S. This band migrates like the P4504A2 isoform. After clofibrate treatment, the expression of P4504A protein also increased in the outer medulla of Dahl S rats.
We have previously reported that the lipid-lowering agent clofibrate lowered arterial pressure in Dahl S rats;16 however, its mechanism of action remains to be determined. The present study examined the effects of clofibrate on renal P4504A activity and the pressure natriuresis relationship to determine whether the antihypertensive actions of clofibrate might be due to an enhanced renal production of 20-HETE which promotes sodium excretion. We confirmed previous observations that chronic treatment of Dahl S rats with clofibrate prevents the development of hypertension.16 The effect of clofibrate is not due to a direct antihypertensive action of the drug or a nonspecific toxic effect, because clofibrate has no effect on blood pressure in Dahl S rats once hypertension was already established.16 In addition, we found that clofibrate has no significant effect on arterial pressure in Dahl R rats fed a high salt diet.
In the present study, the pressure natriuresis relationship was blunted in vehicle-treated Dahl S rats. These results are consistent with previous studies2,7,17⇓⇓ and indicate that the kidney requires a higher perfusion pressure to excrete the same amount of sodium as normotensive animals. The resetting in pressure natriuresis is due to a reduction in GFR and enhanced chloride reabsorption in the loop of Henle.7,17⇓ The factors responsible for the elevated chloride reabsorption remained to be determined, but it is associated with an impairment in the production of 20-HETE which is an inhibitor of the Na+,K+,2Cl− transport in this nephron segment.8,9⇓ There is also evidence that NO excretion is decreased in Dahl S rats, and that L-arginine feeding improves pressure natriuresis3 and prevents the development of hypertension in Dahl S rats.18
In the present study we found that chronic treatment of Dahl S rats with clofibrate improved the pressure natriuretic relationship. There was no significant difference in the pressure natriuresis relations between clofibrate-treated Dahl S and vehicle-treated Dahl R rats. The shift in the relationship was primarily due to a 40% rise in GFR and to a lesser extent to inhibition of tubular reabsorption as indicated by the slightly greater fractional excretion of sodium in clofibrate-treated versus vehicle-treated Dahl S rats for a given level of RPP.
The mechanism by which clofibrate enhances sodium excretion may be due to increased production of 20-HETE in the kidney. Clofibrate directly binds to the PPAR transcription element and induces the expression of the P4504A gene that regulates the ω-hydroxylation of fatty acids in the liver and kidney.13,15⇓ Indeed, in the present study, we found that expression of 4A proteins was elevated in the liver, renal cortex, and outer medulla of clofibrate-treated rats; and, in a previous study, we reported that clofibrate increased the production of 20-HETE in the renal cortex and outer medulla by about 40%.16 Recent studies have indicated that 20-HETE inhibits Na+,K+ ATPase activity, and sodium reabsorption in the proximal tubule.19–21⇓⇓ P450 metabolites of arachidonic acid also mediate the inhibitory effects of dopamine,22,23⇓ PTH,24 and AII25,26⇓ on Na+,K+ ATPase activity and/or sodium transport in this portion of the nephron. In addition, 20-HETE also inhibits sodium transport in the thick ascending loop of Henle by blocking the potassium efflux via a 70 pS potassium channel27 that is required for the efficient operation of the Na+,K+,2Cl− cotransporter in this portion of nephron. Thus, it is possible that the antihypertensive effects of clofibrate may be related to enhanced renal production of 20-HETE which in turn inhibits sodium reabsorption, either in the proximal tubule or the loop of Henle.
In summary, the results of the present study indicate that treatment of Dahl S rats with clofibrate induces the expression of renal P4504A enzymes in the kidney and improves the pressure natriuresis relationship in Dahl S rats. The potentiation of the pressure natriuresis relationship is due primarily to an elevation in GFR and, to a lesser extent, to inhibition of tubular sodium transport. Further studies are needed to identify the nephron segments involved and the role of P450 metabolites of arachidonic acid in mediating these responses.
- Received September 17, 1997.
- Revision received October 10, 1997.
- Accepted October 28, 1997.
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