INTRODUCTION

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AMONG THE MULTIPLICITY OF biological effects of endothelins (ETs), the capacity of ET-1 to constrict blood vessels was the first to be described (33). ET can be produced locally, and, in the rat kidney, specific high-affinity binding sites for ET have been localized to intrarenal arterial structures and glomeruli (8). On account of its ability to contract glomerular arteriolar smooth muscle and mesangial cells, a local modulatory role has been proposed for ET in the regulation of renal hemodynamic and excretory function (27). Studies in rats have documented that ET-1 increased glomerular afferent and efferent arteriolar resistance and decreased the ultrafiltration coefficient (1, 12), indicating a potential role for this peptide in the control of renal vascular tone, glomerular filtration rate (GFR), and mesangial cell function.

Intravenous administration of ET reduced GFR and renal blood flow (RBF) in association with an increase in mean arterial pressure (MAP) (1, 12). Intrarenal arterial infusion of ET-1 decreased GFR and RBF (11) as well as single-nephron GFR (14) without changing MAP. ET produces diuresis and natriuresis (12, 25, 26), despite reducing GFR and RBF. Its diuretic and natriuretic effects have been ascribed to inhibition of renal epithelial Na⁺-K⁺-adenosinetriphosphatase activity (25), release of atrial natriuretic peptide (ANP) (26), and inhibition of arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate in the collecting ducts (22). ET effects are not limited to the kidney because ET releases ANP (11, 26) and increases plasma levels of AVP and aldosterone (19). ETs also possess inotropic and chronotropic effects (10), which may secondarily affect renal function.

ET stimulates phospholipases (30), resulting in release of free arachidonic acid (AA) from membrane phospholipid stores and implicating oxygenase products of AA in some of the effects of ET. Cyclooxygenase (COX) products of AA metabolism were reported to mediate bronchoconstrictor activity of ET in guinea pigs (24), and lipoxygenase products were reported to contribute to the diuretic and natriuretic effects of ET-1 in the rat (25). The contribution of cytochrome P-450 (CYP450)-derived eicosanoids to the renal functional effects of ET-1 was unknown until recently, when the renal vasoconstrictor action of ET-1 was shown to be greatly attenuated by inhibitors of the CYP450 pathway of AA metabolism (23). Moreover, there is additional evidence indicating a link between ETs and the CYP450 system. Thus major perturbations of renal function, such as those caused by renal ischemia, are accompanied by enhanced CYP450-dependent AA metabolism (4), as well as by ET-1 mRNA expression and increased renal vascular ET-1 binding affinity and receptor number (7, 13). Eicosanoids derived from CYP450-dependent AA metabolism are produced in renal blood vessels, including preglomerular microvessels (9) and most segments of the nephron (18), where they modulate ion transport (5, 28), subserve renal autoregulation (34), and participate in tubuloglomerular feedback (35). Vasoactive hormones have the capacity to release renal CYP450-dependent AA metabolites. We have reported that angiotensin II released 20-hydroxyeicosatetraenoic acid (20-HETE) from the rabbit kidney (2).

We have recently linked ET-1 to renal production of CYP450-dependent AA metabolites in the rat isolated kidney; namely, ET-1 released 20-HETE associated with renal vasoconstriction. Furthermore, inhibition of CYP450 monooxygenase activity halved the vasoconstrictor activity of ET-1 while decreasing renal efflux of 20-HETE (23). The role of CYP450-dependent AA metabolites in the renal functional effects of ET-1 has not been addressed in vivo, in particular their contribution to ET-1-induced changes in sodium excretion. We have, in the present study, evaluated the tubular and hemodynamic effects of ET-1 in the anesthetized rat in terms of the possible participation of CYP450-dependent AA metabolites. The major renal CYP450 arachidonate product, 20-HETE, has renal functional effects similar to those of ET-1, viz, promotion of salt and water excretion because of direct tubular effects, which are not offset by concomitant depression of renal hemodynamics. The availability of specific mechanism-based inhibitors of CYP450-dependent AA metabolism has greatly facilitated studies designed to link AA metabolites generated by CYP450 to renal function. We now report that the capacity of ET-1 to promote renal CYP450-dependent AA metabolism is mainly responsible for the diuretic-natriuretic action of ET-1 despite peptide-induced reduction of GFR and RBF.

	MATERIALS AND METHODS

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Indomethacin (Sigma, St. Louis, MO) was dissolved in 50% ethanol, inulin (Sigma) and CoCl₂ (Sigma) were dissolved in 0.9% NaCl, and clotrimazole (Sigma) was dissolved in sesame oil (Sigma). Inactin was obtained from Research Biochemicals International (Natick, MA). ET-1 (Peninsula Laboratories, Belmont, CA) was stored in 0.1% acetic acid at 20°C. Dibromododec-11-enoic acid (DBDD; gift from Dr. Camille Falck, University of Texas South Western Medical Center) was stored in ethanol at 70°C.

Experiments were conducted on male Sprague-Dawley rats (Charles River, Wilmington, MA; body weight 320 ± 8 g) according to protocols approved by the Institutional Animal Care and Use Committee. The animals were placed in a room with lighting adjusted to produce a normal day-night cycle (illuminated from 0800-2000). They were maintained on a standard rat food (Purina chow) and were allowed ad libitum access to water and food before the experiments.

Renal functional measurements. Clearance studies were performed on animals that were anesthetized with Inactin (100 mg/kg ip; Research Biochemical) and instrumented as detailed below. Polyethylene cannulas were placed in the trachea (PE-205) to allow free breathing, in the bladder (PE-205) to facilitate voiding, in the left external jugular vein (PE-50) for administration of drugs or infusion, and in the right carotid artery (PE-50) for measuring and recording systemic arterial pressure by means of a pressure transducer (model P23 ID; Statham, Oxnard, CA) coupled to a polygraph (model 7D; Grass Instruments, Quincy, MA). A tail vein was also cannulated with a 23-gauge butterfly needle (Abbott Hospitals) for infusion of agents. The catheter in the left jugular vein was used for infusion of 0.5 ml of a saline solution (0.9% NaCl) containing 2% inulin over 2 min as a priming dose, followed by a maintenance infusion at the rate of 2 ml/h. A left laparotomy was performed, and electromagnetic flow probes (Carolina Medical Electronics, NC) were placed over the left renal artery to measure RBF. Chlorisondamine (5 mg/kg sc in 2 divided doses) was given to the rats after surgery to eliminate sympathetic reflexes and reduced MAP from 118 ± 5 to 77 ± 3 mmHg within a 20- to 30-min period.

During the experiments, the animals were placed on a heated table to maintain the body temperature at 37°C. The experiments were started after an equilibration period of at least 60 min or when urine flow was steady. Starting from the beginning of the stabilization period, isotonic saline (0.9% NaCl) was infused at a rate of 2 ml/h. After the stabilization period, urine was collected every 30 min. In the clearance experiments, ~400 µl of arterial blood was withdrawn from the femoral artery during the last 5 min of each clearance period for measurements of GFR. An equal amount of normal saline was infused for volume replacement. Urine volume (UV) was measured by gravimetry, and urinary sodium excretion (U_NaV) was measured with the Ciba Corning Na/K/Cl Analyzer (model 644).

Protocols. In the first set of experiments, rats were divided into groups that received infusions of ET-1 only into the jugular vein (0.3, 1, and 3 pmol · kg¹ · min¹; n = 4-5); indomethacin (5 mg/kg iv) over a period of 3 min, 30 min before ET-1; indomethacin alone (n = 4); DBDD (12.5 µg/min; n = 5) 30 min before ET-1; or DBDD alone (n = 5). DBDD was given by intrarenal arterial infusion (12.5 µl/h) 30 min before the control clearance period until the end of the third clearance period. After the postsurgical equilibration period and a 30-min control period (1st clearance period) during which 50% ethanol, the vehicle for indomethacin, and DBDD were administered (12.5 µl/h; intrarenal arterial for DBDD or 1 ml/kg over 3 min into the jugular vein for indomethacin), ET-1 was infused for 45 min. A 30-min urine collection was made, starting after 15 min of ET-1 infusion (2nd clearance period). ET-1 infusion was then replaced with saline infusion for 30 min, and a recovery clearance was obtained. A second 45-min infusion of another (higher) dose of ET-1 was again initiated, and urine collection was made as before (3rd clearance period). No more than two doses of ET-1 were tested in any experiment; any rat in which the baseline UV after the first ET-1 infusion differed significantly from the pre-ET-1 infusion value was discounted. In rats receiving the combination of ET-1 and indomethacin or DBDD, the administration of these inhibitors was initiated 30 min before infusion of ET-1. In time-control experiments (n = 4), renal functional measurements were made over four 30-min clearance periods in rats that received continuous infusion of normal saline or 50% ethanol (12.5 µl/h intrarenal arterially or 1 ml/kg iv into the jugular vein), the vehicle for indomethacin and DBDD.

In a set of rats, the effects of DBDD on urinary excretion of 20-HETE was evaluated. DBDD (n = 5) or its vehicle (50% ethanol; n = 5) was infused for 75-90 min, after which urine collection was begun. Urine was collected for 30 min via indwelling left ureteral catheter. The amount of 20-HETE excreted was determined by gas chromatography-mass spectrometry (GC-MS) by a modification of the method described previously (23). Briefly, urine samples were acidified with 10% formic acid and supplemented with 20,20-dideutero-20-HETE as internal standard (1 ng/ml). The urine samples were subjected to a two-stage purification process involving thin-layer and high-performance liquid chromatography (TLC and HPLC). After extraction with ethyl acetate and evaporation of the organic extracts to dryness, metabolites were purified initially by TLC using the A9 solvent system (ethyl acetate:isooctane:acetic acid:water, 55:25:10:50). After extraction of scraped silica columns corresponding to HETE and evaporation to dryness, metabolites were further purified by reverse-phase HPLC using Ultrasphere C₁₈ column (250 × 4.6 mm; Alltech, San Jose, CA). Samples were chromatographed using linear solvent gradient of water in acetonitrile (37.5 to 0%, containing 0.01% acetic acid) at 1.875%/min and at a solvent flow rate of 1 ml/min. Fractions were collected every minute, and separate fractions containing 20-HETE were evaporated to dryness and dissolved in 100 µl acetonitrile. For GC-MS analyses, samples were esterified with diazomethane and then derivatized with bis(trimethylsilyl)-fluoroacetamide. Selected ion monitoring was used to record ion abundances using a mass spectrometer (HP 5989A; Hewlett Packard, Palo Alto, CA), at mass-to-charge ratio 393, which corresponds to 20-HETE.

Renal function was evaluated in another group of rats in which we intended to block epoxygenase activity with clotrimazole (80 mg/kg ip for 2 days). This dose regimen was reported to produce marked reductions in epoxides and their hydration products (17). Cobalt chloride (CoCl₂) was administered (24 mg/kg ip for 2 days) to another set of rats to deplete their CYP450 enzymes as described by Maines and Kappas (16). Baseline as well as ET-1-induced changes in UV, U_NaV, GFR, RBF, and renal vascular resistance (RVR) were evaluated in clotrimazole-treated rats (n = 5) or vehicle controls (sesame oil, 1 ml/kg ip; n = 5) and CoCl₂-treated (n = 4) or vehicle controls (0.9% NaCl, n = 4). The same protocol as above was followed.

An arterial sample was obtained in the last 5 min of each clearance period for determination of plasma concentration of inulin, GFR being determined by inulin clearance. RVR was estimated from the following formula: (MAP  5)/RBF; 5 mmHg was used as an estimate of renal venous pressure.

Data analysis. All data are expressed as means ± SE. Data were analyzed by analysis of variance followed by a modified t-test (Newman-Keuls) or by Student's paired t-test; P < 0.05 was considered significant.

	RESULTS

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Renal response to infusion of ET-1. Administration of ET-1 (0.3, 1, and 3 pmol · kg¹ · min¹ iv) to euvolemic rats resulted in dose-dependent increases in U_NaV and UV (Fig. 1). UV increased from baseline values ranging between 2.0 and 2.2 µl · 100 g¹ · min¹ during infusion of normal saline to 2.9 ± 0.4 (0.3 pmol · kg¹ · min¹; P < 0.05; n = 4), 3.6 ± 0.5 (1 pmol · kg¹ · min¹; P < 0.05; n = 5), and 4.5 ± 1.1 µl · 100 g¹ · min¹ (3 pmol · kg¹ · min¹; P < 0.05; n = 4). Similarly, U_NaV increased from a pre-ET value range between 0.5 and 0.6 to 0.9 ± 0.2, 1.1 ± 0.2, and 1.6 ± 0.1 µmol · 100 g¹ · min¹ after the administration of 0.3, 1, and 3 pmol · 100 g¹ · min¹, respectively. In time-control, vehicle (0.9% NaCl)-infused rats (n = 3), UV and U_NaV were not significantly altered when monitored over four clearance periods spanning 2 h; 1st clearance period: UV, 2.1 ± 0.3 µl · 100 g¹ · min¹, and U_NaV, 0.54 ± 0.09 µmol · 100 g¹ · min¹, vs. 4th clearance period: UV, 2.6 ± 0.3 µl · 100 g¹ · min¹, and U_NaV, 0.64 ± 0.12 µmol · 100 g¹ · min¹; P > 0.05. After cessation of ET-1 infusion and replacement with saline infusion, changes in UV and U_NaV usually returned to pre-ET-1 infusion values within 20 min. Changes in RBF and MAP were noted consistently only with the highest dose of ET-1, and this was not until after 15 min of infusion (Fig. 1D). Basal value of MAP was 75 ± 3 mmHg and was not increased during administration of 0.3 and 1 pmol · kg¹ · min¹ ET-1, being 74 ± 3 and 78 ± 3 mmHg, respectively. However, ET-1 (3 pmol · kg¹ · min¹) increased MAP to 84 ± 4 mmHg. Compared with pre-ET-1 (baseline values), ET-1 elicited dose-dependent reductions in GFR and RBF, with attendant dose-dependent increases in RVR (Fig. 1). At the highest dose of ET-1 used in this study (3 pmol · kg¹ · min¹), there were significant reductions in GFR (32 ± 4%; P < 0.05) and RBF (18 ± 3%; P < 0.05) and increased RVR (28 ± 4%; P < 0.05). In time controls (n = 3), no changes were noted in any of these parameters over four clearance periods.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Effects of endothelin (ET)-1 (0.3, 1, and 3 pmol · kg1 · min1; n = 4-5) or vehicle (0.9% NaCl, 2 ml · kg1 · h1) given by intravenous infusion on urine flow rate (UV; A), Na+ excretion (UNaV; B), glomerular filtration rate (GFR; C), and renal blood flow (RBF) and renal vascular resistance (RVR) (D). Changes in renal function parameters during the control clearance period (Pre) or after (Post) the annotated injections of ET-1 (doses in parentheses) are shown. Data are presented as means ± SE. * P < 0.05, Pre vs. Post (A-C) or ET-1 vs. vehicle (D).

Effect of COX inhibition of ET-1-induced renal responses. Because the COX pathway is implicated in ET-1-induced biological effects (20, 24, 26) and a functional COX is required for full expression of the biological effects of some renotropic CYP450-dependent AA metabolites (3, 18), these experiments were conducted to evaluate the role of COX-derived eicosanoids in the renal effects evoked by ET-1. In rats treated with indomethacin (n = 5), basal UV and U_NaV were significantly lower than in rats treated with vehicle (50% ethanol; n = 4) (Fig. 2, A and B). Basal UV and U_NaV in indomethacin-treated rats were 0.9 ± 0.1 µl · 100 g¹ · min¹ and 0.42 ± 0.05 µmol · 100 g¹ · min¹, respectively, compared with corresponding values of 2.4 ± 0.2 µl · 100 g¹ · min¹ and 0.58 ± 0.06 µmol · 100 g¹ · min¹ in vehicle-treated rats (n = 4, P < 0.05). GFR was lower in indomethacin-treated rats: 1.2 ± 0.3 (vehicle) vs. 0.8 ± 0.1 ml/min (indomethacin) (P < 0.05; Fig. 2C), although RBF [8.7 ± 0.6 (vehicle) vs. 8.9 ± 0.3 ml/min (indomethacin)] and RVR [8.6 ± 0.9 (vehicle) vs. 9.1 ± 0.7 mmHg · ml¹ · min¹ (indomethacin)] were not different between indomethacin-treated and control rats. The MAPs were not significantly different between rats that received indomethacin (79 ± 3 mmHg) and those that received vehicle (76 ± 4 mmHg). Because the basal renal responses were not significantly different between vehicle-treated (Fig. 2) and untreated rats that received saline infusion (Fig. 1), the effects of indomethacin on renal responses to ET-1 were evaluated by comparing ET-1-induced renal functional effect in untreated and indomethacin-treated rats. The renal tubular responses to indomethacin accentuated the hemodynamic effects of ET-1 while reducing the effects of the peptide on UV and U_NaV. These changes were noted at all doses of ET-1 employed. Thus, in indomethacin-treated rats, the overall attenuations of ET-1-induced increases in UV and U_NaV were 35 ± 5 and 42 ± 4% (P < 0.05), respectively, whereas the overall accentuations of ET-1 induced reductions in GFR and RBF were 26 ±3 and 43 ± 4% (P < 0.05), respectively. In addition, the overall mean percentage change in ET-1-induced increase in RVR was amplified to 37 ± 6% (P < 0.05) by indomethacin compared with a mean percentage increase of 28 ± 6% in vehicle-treated rats (although, dose for dose, the increase in RVR was only significant at 3 pmol · kg¹ · min¹ ET-1 for vehicle- and indomethacin-treated rats). The changes produced were such that doses of ET-1, which hitherto produced little or no hemodynamic effects in untreated rats, elicited significant reductions in GFR and RVR in the presence of indomethacin (Fig. 2, C and D). For example, at the ET-1 dose of 1 pmol · kg¹ · min¹, the 22 ± 7% reduction in GFR (P > 0.05) in vehicle-treated rats accentuated to 40 ± 4% (P < 0.05) in the presence of indomethacin and the 12 ± 6% elevation in RVR (P > 0.05) in vehicle-treated rats increased to 30 ± 4% (P < 0.05) in rats treated with indomethacin.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effects of ET-1 on renal function in the presence of indomethacin (Indo). Intravenous infusion of ET-1 at 0.3, 1, and 3 pmol · kg1 · min1 (n = 4-5) and changes produced on UV (A), UNaV (B), GFR (C), and RVR (D) are shown. Baseline measurements of renal function [ET-1 (0)] in rats that received vehicle infusion (50% ethanol; n = 4) or indomethacin (5 mg/kg iv) are also shown. Numbers in parentheses represent doses of ET-1 (pmol · kg1 · min1). * P < 0.05 vs. ET-1 (0); @ P < 0.05 vs. vehicle; # P < 0.05 vs. ET-1 (0.3, 1.0, and 3.0).

ET-1-induced renal responses in the presence of DBDD. Products of the -/-1 hydroxylase pathway of CYP450-dependent AA metabolism are synthesized in the kidney (9, 18) and influence renal vascular and excretory function. DBDD, a mechanism-based inhibitor of the -/-1 hydroxylase pathway of CYP450-dependent AA metabolism (32), was used to evaluate the contribution of this pathway to ET-1 induced renal responses (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of dibromododec-11-enoic acid (DBDD) given by renal intra-arterial infusion (12.5 µg/min; n = 5) on UV (A), UNaV (B), GFR (C), and RVR (D) in rats that received ET-1 and DBDD (DBDD group) or vehicle (50% ethanol). Doses of ET-1 (pmol · kg1 · min1) are shown in parentheses. Baseline renal functions in rats that received infusion of vehicle (n = 4) or DBDD are also shown. Data for vehicle are the same as those for rats that received 0.9% NaCl in Fig. 1 (see RESULTS). * P < 0.05 vs. ET-1 (0); @ P < 0.05 vs. vehicle. # P < 0.05 vs. ET-1 (0.3, 1.0, and 3.0).

DBDD elicited significant increases in GFR [1.1 ± 0.1 (vehicle) to 1.4 ± 0.1 ml/min (DBDD), P < 0.05] and in RBF [8.3 ± 0.5 (vehicle) to 9.5 ± 0.4 ml/min (DBDD), P < 0.05]. Increases in RVR in response to the high dose of ET-1 in DBDD-treated rats were attenuated significantly (Fig. 3D), and ET-1 produced a lesser reduction in RBF in DBDD-treated rats, viz, 5 ± 1% (DBDD) vs. 18 ± 3% (control). In rats that received DBDD vehicle (50% ethanol), basal renal responses were not significantly different between the first and fourth clearance periods. Thus basal UV and U_NaV were 2.3 ± 0.7 µl · 100 g¹ · min¹ and 0.6 ± 0.1 µmol · 100 g¹ · min¹, values not significantly different from those obtained during the fourth clearance period, 3.1 ± 0.6 µl · 100 g¹ · min¹ and 0.8 ± 0.1 µmol · 100 g¹ · min¹, respectively. Because basal renal responses were not different between rats treated with DBDD vehicle (50% ethanol) and untreated rats (vehicle in Fig. 1), comparisons of ET-1-induced renal responses were made between untreated rats and those receiving DBDD. DBDD markedly blunted ET-1-induced increases in UV and U_NaV by 36 ± 2 and 37 ± 4%, respectively (P < 0.05), despite preventing the reduction in GFR produced by ET-1 (P < 0.05). For example, DBDD reduced the increase in UV from 4.5 ± 1.1 to 2.9 ± 0.3 µl · 100 g¹ · min¹ (P <0.05) and in U_NaV from 1.6 ± 0.1 to 0.9 ±0.1 µmol · 100 g¹ · min¹, P < 0.05, in response to high-dose ET-1 (Fig. 3, A and B).

We verified in vivo our in vitro findings that DBDD inhibited production of 20-HETE (Fig. 4). In control rats (n = 5) receiving infusion of vehicle (50% ethanol), excretion of 20-HETE was 3.4 ± 0.9 ng/h. DBDD infusion reduced the excretion of 20-HETE to 1.1 ± 0.6 ng/h (P < 0.05; n = 5).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of DBDD (12.5 µg/min at 1 ml/h) on renal excretion of 20-hydroxyeicosatetraenoic acid (20-HETE) in the Inactin-anesthetized rat. DBDD or its vehicle (50% ethanol; control) was given by the renal intra-arterial route to the left kidney. Amount of 20-HETE in the urine was analyzed by gas chromatography-mass spectrometry (see METHODS). * P < 0.05 vs. control.

Renal responses to ET-1 in the presence of clotrimazole. Because epoxygenase metabolites of CYP450-dependent AA metabolism have been shown to participate in the renal regulation of salt and water balance (17), these experiments were conducted to evaluate the role of the epoxygenase pathway in the renal effects of ET-1 in rats treated with clotrimazole by a dosing schedule shown to suppress renal epoxygenase activity in rats (17). When compared with rats treated with vehicle (sesame oil; control), clotrimazole was without effect on baseline renal function parameters evaluated in this study (Fig. 5). Moreover, ET-1-induced increases in UV, U_NaV, and RVR, as well as the reductions in GFR and RBF produced by the peptide, were not different in rats treated with clotrimazole or its vehicle.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of ET-1 (0.3, 1, and 3 pmol · kg1 · min1) on UV (A), UNaV (B), GFR (C), and RVR (D) in the presence of clotrimazole (80 mg/kg for 2 days). Data are presented as means ± SE, and comparisons were made between vehicle (sesame oil, 1 ml/kg ip; n = 4)-treated and clotrimazole (n = 5)-treated rats. Clotrimazole did not modify ET-1-induced renal effects. * P < 0.05 vs. ET-1 (0).

Effects of CoCl₂ on renal tubular effect of ET-1. Renal function was also assessed in rats treated with CoCl₂ according to a schedule that inhibited CYP450-dependent AA metabolism (29). This was done to provide additional support for the results obtained with DBDD because these inhibitors of CYP450 differ in their mechanisms of action. CoCl₂ reduced basal UV from 3.4 ± 0.4 (control) to 1.9 ± 0.3 µl · 100 g¹ · min¹ (P < 0.05) and U_NaV from 0.63 ± 0.1 (control) to 0.42 ± 0.07 µmol · 100 g¹ · min¹ (P < 0.05) (Fig. 6). GFR was also lower in CoCl₂-treated rats relative to controls: 0.72 ± 0.09 vs. 0.95 ± 0.11 ml · 100 g¹ · min¹, respectively (P < 0.05). However, basal RBF was not different between untreated and treated rats (8.6 ± 0.9 vs. 8.3 ± 0.7 ml/min), and neither was RVR (Fig. 6D). The capacity of high-dose ET-1 to produce diuresis and natriuresis was greatly reduced in CoCl₂-treated rats despite an increase in GFR elicited by ET-1 (Fig. 6C). MAP was unaffected by CoCl₂ treatment. The increase in RVR produced by ET-1 was also markedly attenuated by CoCl₂ (Fig. 6D). Thus CoCl₂ and DBDD modified the renal functional response to ET-1 similarly. Each reduced the magnitude of the diuresis and natriuresis produced by ET-1 in the face of an increased GFR compared with untreated rats.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of treatment of rats with cobalt chloride (CoCl2) on baseline renal function (Basal) and ET-1 (3 pmol · kg1 · min1)-induced changes in UV (A), UNaV (B), GFR (C), and RVR (D) in vehicle (0.9% NaCl, control; n = 4)- or CoCl2-treated (n = 4) rats. CoCl2 inhibited baseline water excretion (A), Na+ excretion (B), and GFR (C) and inhibited ET-1-induced diuresis (A) and natriuresis (B). Data are presented as means ± SE. * P < 0.05 vs. control (ET-1); # P < 0.05 vs. control (Basal); @ P < 0.05 vs. Basal.

	DISCUSSION

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The precise role of ET in regulating renal excretory function is uncertain. Harris et al. (8) have reported that low doses of ET have potent natriuretic and diuretic effects, which were often masked by the intense renal vasoconstriction that reduced filtered load and blunted increased excretion. On the other hand, King et al. (12) have provided evidence that ET-1-induced increases in sodium excretion resulted from pressure natriuresis. Thus administration of ET-1 leads to a complex balance involving increased renal perfusion pressure, renal vasoconstriction, reduced glomerular filtration, and possible direct effects on tubular absorption as well as indirect effects related to the release of hormones affecting tubular function. ET peptides have been shown to stimulate production and/or release of a variety of vasoactive agents, e.g., prostaglandins (20, 31), ANP (19, 20), and AVP (19), that may contribute to their renal effects.

ETs stimulate phospholipases (30) to release AA; the liberated AA can undergo oxygenation via the several pathways of AA metabolism. In this study, we assessed the effects of subpressor doses of ET-1 and a dose that elicited both renal hemodynamic and pressor effects to examine the possible participation of CYP450 arachidonate metabolites in the renal excretory response to ET-1. The rationale for the present study was based on our published findings that the renal vasoconstrictor action of ET-1 was greatly attenuated by inhibiting renal CYP450 monooxygenase activity (23) and that ET-1 increased renal efflux of 20-HETE, a CYP450 product that resembles ET-1 in terms of effects on renal function. In particular, 20-HETE inhibits transport in the thick ascending limb of the loop of Henle (5) and constricts preglomerular microvessels (9). CYP450-dependent AA metabolites have been demonstrated to be produced in the renal vascular compartment (9), as well as in various segments of the nephron, and are released in response to hormonal stimulation (2, 5, 29). In the present study, ET-1 increased sodium and water excretion at all doses despite reducing GFR, suggesting that ET-1 had direct tubular effects to promote natriuresis and diuresis. Furthermore, the doses of ET-1 selected were either without effect on MAP, as was the case for low and intermediate infusion rates of ET-1, or produced only small elevations in MAP (<10 mmHg), as was the case for the highest dose. That the tubular effects of ET-1 were independent of renal hemodynamic changes was strikingly evident in untreated rats, in which a dose-dependent natriuretic-diuretic action of ET-1 occurred despite a progressive decline in GFR produced by increasing doses of ET-1 (Figs. 2 and 3) (1). At the highest dose of ET-1, which evoked the greatest reduction in GFR, sodium excretion was increased to the greatest degree. Thus a tubular action of ET-1 that promotes diuresis and natriuresis best accounts for our findings and is in agreement with several studies that reported a natriuretic-diuretic response to ET-1, even in the face of marked reductions in GFR (12, 25, 26).

The excretory effects of ET-1 appeared to be dependent to a significant degree on formation of CYP450 product(s) because the diuretic-natriuretic actions of ET-1 were attenuated by inhibition of the CYP450 pathway of AA metabolism. Thus a tubular action of ET-1, presumably mediated by a CYP450 AA metabolite, was uncovered after inhibition of CYP450 with either DBDD or CoCl₂. We used CYP450 monooxygenase inhibitors that differed radically in terms of their mechanism of action to meet questions concerning specificity of their effects. DBDD, a mechanism-based inhibitor of -hydroxylase, has been shown to have the requisite selectivity for identifying a CYP450-dependent mechanism (32). In contrast, CoCl₂ induces heme oxygenase activity, thereby depleting CYP450 enzymes (29). Both inhibitors had comparable effects on the renal functional effects of ET-1; namely, the natriuretic-diuretic actions of ET-1 were blunted by inhibitors of CYP450 despite either attenuation or abolition of the capacity of ET-1 to reduce GFR (Figs. 3C and 6C). For example, treatment with CoCl₂ prevented reduction of GFR in response to ET-1, yet the natriuretic-diuretic response to ET-1 was reduced by >30% (Fig. 6). In any event, we obtained evidence that DBDD inhibited renal production of 20-HETE because treatment with DBDD resulted in a 70% decline in urinary excretion of 20-HETE, which reflects renal biosynthetic capacity of the eicosanoid (2).

Renal functional responses to ET-1 were also evaluated with clotrimazole, an inhibitor of CYP450-dependent epoxygenase activity, which generates epoxyeicosatrienoic acids (EETs): 5,6, 8,9, 11,12, and 14,15. Of these EETs, the 5,6-EET possesses the highest vasoactivity and interacts with COX (3) and has been shown to inhibit sodium transport in the proximal tubules and cortical collecting ducts (25, 28). Clotrimazole was administered according to a dosing regimen that has been shown to suppress renal CYP450 epoxygenase activity in the rat (17). The negative results obtained with clotrimazole in the present study (clotrimazole did not modify the renal functional response to ET-1) are consistent with a CYP450-dependent mechanism subserved by 20-HETE and/or other HETEs, such as 16-, 17-, 18-, and 19-HETEs, in mediating the renal functional effects of ET-1. This interpretation is in accord with our finding that DBDD, which has a principal inhibitory action on 20-HETE formation, did attenuate both the hemodynamic and excretory effects of ET-1 while reducing the excretion of 20-HETE.

We also evaluated the role of COX in ET-1-induced renal functional effects because ET-1 has been associated with release of renal prostaglandins and a functional COX was a prerequisite for expression of some of the biological effects of certain CYP450-AA metabolites, particularly 20-HETE, which is metabolized via COX resulting in generation of biologically active products (18). Ferrario et al. (6) reported that aspirin partially prevented ET-1-induced reduction in GFR without modifying natriuresis in the rat isolated kidney. We also have used the rat isolated kidney and have obtained findings similar to those of Ferrario et al. (6); namely, inhibition of COX decreased the renal vasoconstrictor response to ET-1 (23). However, in the rat isolated kidney, conditions facilitate expression of prostaglandin (PG) E receptors (EP) that mediate vasoconstriction, such as EP₁ and an isoform of EP₃ (21). On the other hand, under in vivo conditions, inhibition of COX with indomethacin produced a decline in basal GFR and an associated reduction in sodium and water excretion (control, Fig. 2C). Thus elimination of modulatory vasodilator prostanoids by treatment with indomethacin augmented the renal vasoconstrictor action of ET-1 as reflected by increased RVR and reduced GFR, suggesting that the vasodilator prostanoids, PGE₂ and PGI₂, exert a countervailing action on the renal vasoconstrictor action of ET-1 (20). The principal effect of indomethacin, then, was to potentiate the negative renal hemodynamic effects of ET-1. As a consequence, the reductions in sodium and water excretion produced by ET-1 when COX is inhibited appear to be primarily a function of reduced GFR rather than a tubular effect of the peptide. However, a PG-related mechanism that affects the tubular actions of ET-1 cannot be excluded. Indeed, there is evidence indicating that, depending on experimental conditions, ET-1 may affect tubular transport via a PG-dependent mechanism (15). Despite the further depression of GFR evoked by ET-1 in rats treated with indomethacin (Fig. 2C), natriuresis occurred and was greatest in response to the highest dose of ET-1, which caused the greatest reduction in GFR, declining to levels one-half or less the levels of GFR produced by DBDD.

In conclusion, we have demonstrated that eicosanoid products generated by COX and CYP450 monooxygenases contribute to the renal functional effects of ET-1. The capacity of ET-1 to enhance PG production was primarily expressed in terms of effects on GFR. Treatment with indomethacin decreased GFR and potentiated the negative effects on GFR elicited by ET-1. On the other hand, CYP450-dependent AA products depressed renal hemodynamics and promoted sodium excretion. Thus inhibition of CYP450-dependent AA metabolism enhanced GFR and blunted the negative effects of ET-1 on renal hemodynamics while reducing the diuretic-natriuretic actions of ET-1. The effects of ET-1 on renal function correspond to those of 20-HETE, the most prominent of the renal CYP450-dependent AA metabolites; namely, natriuresis and diuresis occur despite decreased GFR and RBF. We emphasize that the proposed role of 20-HETE in mediating the renal functional effects of ET-1 is a working hypothesis that must be tested by future studies to be validated.