Endothelin Antagonists Improve Renal Function in Spontaneously Hypertensive Rats
Abstract Hypertension in the spontaneously hypertensive rat (SHR) is associated with reduced renal excretory function, low renal plasma flow, reduced glomerular filtration rate, and reduced renal interstitial hydrostatic pressure. The mechanisms responsible for these abnormalities in renal function are unknown. The purpose of this study was to determine the role of intrarenal endothelin in altering renal hemodynamic and excretory function in the SHR. Both PD 145065 (an endothelin A and B receptor antagonist) and FR 139317 (a selective endothelin A receptor antagonist) or saline was infused into the renal interstitium of 14- to 16-week-old SHR (n=7) and age-matched Wistar-Kyoto rats (WKY) (n=7). Renal perfusion pressure in some SHR was reduced to that of the WKY by a servocontrol system. At a renal perfusion pressure of 124±4 mm Hg, infusion of PD 145065 (0.03 mg · kg−1 · min−1) and FR 139317 (0.02 mg · kg−1 · min−1) significantly increased glomerular filtration rate (Δ22%), renal plasma flow (Δ37%), and renal interstitial hydrostatic pressure (from 3.2±0.5 to 5.4±0.6 mm Hg) in the SHR. These changes were associated with significant increases in urine flow, absolute sodium excretion, and fractional excretion of sodium. Similar improvements in renal plasma flow, renal interstitial hydrostatic pressure, and renal excretory function were obtained in the SHR whose renal perfusion pressure was not reduced (n=7). Renal interstitial infusion of endothelin receptor antagonists had no effect on renal hemodynamic or excretory function in the WKY. These data demonstrate that endothelin receptor blockade within the kidney improves renal hemodynamic and excretory function in the SHR. These results suggest that intrarenal endothelin may mediate, in part, the abnormal renal function in the SHR.
Renal transplantation studies suggest that an intrinsic defect within the kidney plays a crucial role in the development and maintenance of hypertension in the spontaneously hypertensive rat (SHR).1 Abnormalities in renal function that have been identified in the SHR include reduced renal excretory function, low renal plasma flow (RPF), reduced glomerular filtration rate (GFR), and reduced renal interstitial hydrostatic pressure (RIHP).2 3 4 Although these abnormalities in renal function are well established in the SHR, the factors responsible for these changes in renal hemodynamic and excretory function in this model of hypertension are unclear.
During the past decade, it has become increasingly evident that the endothelium produces a number of factors that can affect vascular function. One of these substances is a contracting factor called endothelin. Endothelin-1 (ET-1) is one of the most potent endogenous vasoconstrictors identified to date.5 Recent studies have suggested that ET-1 may play a role in the pathogenesis and maintenance of hypertension by affecting renal hemodynamic and excretory function.6 The vasoconstrictive effect of ET-1 on isolated renal arteries is greater in the SHR than in the Wistar-Kyoto rat (WKY).7 Despite lower plasma levels of endothelin in the SHR than in the WKY, neutralization of plasma endothelin by endothelin-specific antibodies in the SHR induces decreases in mean arterial pressure and renal vascular resistance, and increases in RPF and GFR.8 Because it has been suggested that endothelin acts as a paracrine factor rather than as an endocrine hormone, we proposed that intrarenal endothelin may, in part, mediate the abnormal renal function in the SHR. To test this hypothesis, we examined whether infusion of endothelin receptor antagonists into the renal interstitium would alter renal hemodynamic and excretory function in the SHR.
All experiments were performed in SHR and WKY implanted with a renal interstitial catheter. Two to 3 weeks after catheter implantation, the rats were anesthetized and prepared for the acute clearance experiment.
Renal Interstitial Catheter Design and Implantation
The interstitial catheters used in the present study were made from a polyethylene matrix material with 60-μm pores (Labpor porous tubes, Bel Art Products) that is impermeable to solid tissue yet communicates with the interstitial fluid compartment.9 Each polyethylene matrix was cut into a cylinder 1.0 mm in diameter and 2.0 mm in length. A hole was drilled into one end of the polyethylene matrix so that tubing (PE-50) could be inserted into the hole. The tubing was permanently attached to the matrix with a combination of cement and Silastic glue.
Eleven- to 12-week-old male SHR and WKY (Harlan Sprague Dawley Inc, Indianapolis, Ind) were fed standard rat chow. The rats were allowed free access to tap water. Surgery and care of the rats were conducted in accordance with National Institutes of Health guidelines using protocols approved by the Animal Care and Use Committee at the University of Mississippi Medical Center. One week after delivery, rats were anesthetized with pentobarbital sodium (30 mg/kg body wt IP) and implanted with the renal interstitial catheter. On the day of the catheter implantation, the average body weights of the SHR and WKY were similar. The left kidney was exposed through a midline incision. One end of a stainless steel guide was inserted into the open end of the PE-50 tubing that was attached to the matrix. A small nick was made on the upper ventral pole of the left kidney, and the stainless steel guide was pushed into the nick and through the kidney. The matrix was pushed into the kidney parenchyma, and the PE-50 tubing leading from the matrix exited the dorsal side of the kidney. The interstitial catheter was flushed with isotonic saline to remove any residual blood from the capsule, and the open end of the PE-50 tubing was sealed with a knot. The PE-50 tubing was coiled and placed into the abdomen of the rat. The right kidney was then removed and the abdominal incision was closed. Rats were allowed to recover from surgery for 2 to 3 weeks. Gross examination of kidneys after completion of the clearance study revealed that the matrix was located in the renal parenchyma of the cortex.
All rats were fasted overnight before experimentation but had continuous access to tap water. For the acute experimental study, the rats were anesthetized with thiobutabarbital sodium (Inactin, Promonto GmbH) (100 mg/kg body wt IP) and placed on a thermostatically controlled warming table to maintain body temperature at 37°C. A tracheotomy was performed and PE-240 tubing 3 cm long was inserted into the trachea to maintain an open airway. PE-50 tubing was placed in the left jugular vein for continuous intravenous infusion and in the left carotid artery for blood sampling and continuous arterial pressure monitoring. The right femoral artery was also cannulated with PE-10 tubing, and the catheter was advanced into the abdominal aorta to the level of the left renal artery for continuous measurement of renal perfusion pressure (RPP). The carotid and femoral arterial catheters were connected to a model P23DC (COBE) strain gauge. Mean arterial pressure and RPP were then recorded on a polygraph (MFE Instruments). An anterior midline incision was made, and to control RPP, an electronic servocontrolled Silastic balloon occluder was placed around the abdominal aorta above the left renal artery and connected to a saline-filled syringe.10 Another small midline incision was made and the bladder was cannulated for urine collection with a flare-tipped PE-90 tubing. The left kidney was exposed through a left flank incision, and the previously implanted interstitial catheter was connected to an infusion pump (Syringe Infusion Pump 22, Harvard Apparatus) for renal interstitial infusion. A 1- to 2-mm hole was made in the left kidney with a small-tipped portable cauterizer (Harvard Apparatus). A PE-50 tubing with a 2-mm-long PE-10 tubing tip was then implanted and sealed with cryoacrylic glue. The PE-50 tubing was connected to a pressure transducer for constant monitoring of RIHP. The catheter was checked for patency and responsiveness by determining response of RIHP to partial renal vein constriction.
After completion of surgery, the rats received a maintenance infusion of isotonic saline solution containing 6.25% albumin, [125I]iothalamate (Isotex Diagnosis; 0.05 μCi · kg−1 · min−1), and [131I]iodohippurate (Syncor International Corporation; 0.10 μCi · kg−1 · min−1) at a rate of 2.5 mL/h. Isotonic saline was also infused into the renal interstitium through a chronically implanted interstitial catheter at a rate of 10 μL/min. This infusion rate of saline does not affect renal hemodynamics or excretory function. Furthermore, we have previously demonstrated that infusion of substances into the cortex results in rapid distribution throughout the kidney in the rat.11
After a 60-minute equilibration period, steady-state measurements of arterial pressure and RIHP were obtained during a 30-minute control clearance. The rats were then infused with PD 145065 (an endothelin A [ETA] and B [ETB] receptor antagonist; 0.03 mg · kg−1 · min−1) and FR 139317 (a selective ETA receptor antagonist; 0.02 mg · kg−1 · min−1) into the renal interstitium at a rate of 10 μL/min. Combined use of PD 145065 and FR 139317 was necessary to effectively abolish the renal hemodynamic effect of exogenous endothelin in the rat.12 Both endothelin receptor antagonists were diluted in isotonic saline. Twenty minutes after interstitial infusion of the endothelin receptor antagonists, a 30-minute experimental clearance period was begun, and urine and plasma were collected again. A 750-μL arterial blood sample was obtained at the midpoint of each clearance period for determination of the plasma concentrations of sodium and radioactivities of 125I and 131I.
Three groups of rats were examined. The first consisted of the WKY (n=7). The second consisted of the SHR (n=7), and the third consisted of the SHR, in which an acute occluder was used to servocontrol RPP at the same level as in the WKY by inflation or deflation of the balloon above the left renal artery (n=7). At the end of the experimental period, the rats were killed with an intravenous injection of concentrated potassium chloride. The left kidney was then removed and weighed.
Sodium concentrations in plasma and urine were measured by flame photometry (IL-943, Instrumentation Laboratories). RPF, GFR, urinary excretion of sodium (UNaV), urine volume (UV), and fractional excretion of sodium (FENa) were calculated from the concentration of sodium and radioactivities of 125I and 131I in plasma and urine.
Results are expressed as mean±SEM. Statistical significance within each group was determined with the paired Student’s t test. Statistical significance between groups was determined with the Student’s t test for unpaired data. Differences were considered significant at a value of P<.05.
At the time of acute studies, mean body weight was 324±10, 315±8, and 309±9 g in the WKY, SHR, and servocontrolled SHR, respectively. Mean left kidney weight was 1.58±0.11, 1.55±0.05, and 1.48±0.06 g in the WKY, SHR, and servocontrolled SHR, respectively.
The Table⇓ illustrates the effects of renal interstitial infusion of endothelin receptor antagonists on renal function. Systemic blood pressure in the SHR and the SHR with servocontrolled RPP was significantly higher than in the WKY group under basal conditions. Although there was a tendency for renal hemodynamics and sodium excretion to be lower in the SHR than in the WKY group, there was no statistical difference. In contrast, RPF, GFR, UNaV, UV, RIHP, and FENa were all significantly lower in the SHR-↓RPP group. Infusion of the endothelin receptor antagonist increased RPF, GFR, RIHP, UNaV, UV, and FENa in the SHR and SHR-↓RPP groups while having no effect in the WKY group.
In Fig 1⇓ are compared the magnitudes of the changes in RPP, RPF, and GFR during renal interstitial infusion of endothelin receptor antagonists in the WKY, servocontrolled SHR, and SHR. Endothelin receptor antagonists had no effect on RPP in any group of animals. RPF in the servocontrolled SHR (Δ1.5 [0.3 mL/min]) and in the SHR (Δ1.2 [0.5 mL/min]) increased significantly during infusion of endothelin receptor antagonists. RPF in the WKY (Δ0.1 [0.4 mL/min]) was unchanged by infusion of endothelin receptor antagonists. GFR in the servocontrolled SHR (Δ0.18 [0.05 mL/min]) increased significantly during infusion of endothelin receptor antagonists. GFR in the WKY (Δ−0.04 [0.05 mL/min]) and SHR (Δ−0.02 [0.04 mL/min]) was not affected by infusion of endothelin receptor antagonists.
In Fig 2⇓ are compared the magnitudes of the changes in RIHP, UV, and UNaV during renal interstitial infusion of endothelin receptor antagonists in the WKY, servocontrolled SHR, and SHR. Endothelin receptor antagonists had no effect on RIHP (Δ0.0 [0.3 mm Hg]), UV (Δ−0.81 [1.98 μL/min]), or UNaV (Δ−0.39 [0.24 μmol/min]) in the WKY. RIHP increased significantly in the servocontrolled SHR and the SHR during infusion of endothelin receptor antagonists. The increase in RIHP in the SHR (Δ0.9 [0.3 mm Hg]) was significantly less than that of the servocontrolled SHR (Δ2.2 [0.2 mm Hg]). UV and UNaV in the servocontrolled SHR (Δ7.76±2.11 μL/min and 1.01±0.38 μmol/min, respectively) and SHR (Δ3.79±0.92 μL/min and 0.75±0.17 μmol/min) increased significantly during intrarenal endothelin receptor blockade. There were no significant differences in the diuretic and natriuretic responses to the endothelin receptor antagonist between the SHR and the servocontrolled SHR group.
The results of the current study demonstrate that renal interstitial infusion of PD 145065 (an ETA and ETB receptor antagonist) and FR 139317 (a selective ETA receptor antagonist) improves renal hemodynamic and excretory function in SHR and not in WKY.
Renal transplantation studies have suggested that an intrinsic defect within the kidney plays a crucial role in the development and maintenance of hypertension in SHR.1 As previously described and confirmed in this study, UNaV, RPF, GFR, and RIHP are significantly lower in SHR than in WKY at a comparable perfusion pressure.2 3 4 However, the factors responsible for the altered renal function in SHR remain unknown.
ET-1 is one of the most powerful endogenous vasoconstrictors identified to date.5 Exogenous ET-1 decreases RPF and GFR and increases renal vascular resistance, thereby reducing sodium and water excretion.13 The vasoconstrictive effect of ET-1 on isolated renal arteries is greater in SHR than in WKY.7 Neutralization of plasma endothelin by endothelin-specific antibodies induces decreases in mean arterial pressure and renal vascular resistance, and increases in GFR and RPF in the SHR.8 Although these findings support a role for endothelin in hypertension, other studies fail to confirm a role.14 Thus, the importance of endothelin in hypertension in SHR is still unclear. By infusing endothelin receptor antagonists PD 145065 and FR 139317 into the renal interstitium, we investigated the role of intrarenal endothelin on altered renal hemodynamic and excretory function in SHR. The endothelin receptor antagonists increased GFR, RPF, and RIHP in servocontrolled SHR, and these changes were associated with increases in UV, UNaV, and FENa. Similar responses to the endothelin receptor antagonists also occurred in the SHR without servocontrolled RPP, except that GFR did not increase. In sharp contrast, the endothelin receptor antagonists had no effect on renal hemodynamic or excretory function in WKY.
In recent studies, we and others have reported that RIHP serves as an important intrarenal mediator of pressure natriuresis.15 Increases in RPP result in increases in RIHP. The increases in RIHP lead to decreases in sodium reabsorption and increases in sodium excretion.15 At comparable levels of RPP, RIHP in SHR was lower than in WKY. Khraibi and Knox16 reported that the transmission of RPP into the renal interstitium is blunted in SHR compared with WKY, and our results support these findings. Renal interstitial infusion of endothelin receptor antagonists increased RIHP in both SHR groups but did not affect RIHP in the WKY. These results suggest that intrarenal endothelin receptor blockade may improve the transmission of RPP into the renal interstitium in SHR. Thus, increases in RIHP during renal interstitial infusion of endothelin receptor antagonists may, in part, account for the improved renal excretory function in both groups of SHR. The improvement in sodium excretion in the SHR group without servocontrol of RPP could also be due to enhanced filtered load of sodium, because GFR increased in response to the endothelin antagonists. The improvement of excretory function in the servocontrolled SHR group, however, occurred without significant increases in GFR.
Though not significant, the magnitudes of the increases in RIHP, UV, UNaV, and FENa in the servocontrolled SHR tended to be greater than in the SHR without servocontrol of RPP. The mechanism for this enhanced effect at lower RPP is unclear. Because previous studies have indicated that endothelin may enhance angiotensin II vasoconstriction, the enhanced effect of the endothelin receptor antagonist at lower RPP could be due to an interaction between angiotensin II and endothelin. Angiotensin II levels would be expected to be higher in the SHR group with reduced RPP. Further studies with angiotensin II receptor antagonists will be necessary to quantitate this possible mechanism.
ET-1 has some direct tubular actions like those of a natriuretic factor. ET-1 inhibits Na+,K+-ATPase activity and reduces arginine vasopressin–stimulated osmotic water permeability in the inner medullary collecting duct (IMCD).17 18 Conceivably, intrarenal infusion of endothelin receptor antagonists might antagonize these actions, and theoretically produced antinatriuretic effects in the IMCD in our study. Because we did not investigate the effects of intrarenal endothelin blockade on nephron segments beyond the proximal tubule, it is unclear whether endothelin receptor antagonists caused antinatriuretic responses in the IMCD. It can also be speculated that intrarenal endothelin receptor blockade produced greater natriuretic effects by increasing GFR and RIHP than antinatriuretic effects by inhibiting sodium and water reabsorption in the IMCD. Further studies must be undertaken to define the relationship between intrarenal endothelin receptor blockade and distal nephron function.
Recently, Hughes et al19 have reported that the production of ET-1 by the inner medulla is markedly reduced in adult SHR compared with WKY, and this reduction is most likely due to decreased synthesis and release of ET-1 by IMCD cells. Furthermore, Larivière et al20 have indicated that vascular content of ET-1 is not increased in young or adult SHR compared with age-matched WKY. Despite these results, in the current study intrarenal endothelin receptor blockade improved renal hemodynamic and excretory function in the SHR with or without reduced RPP. It could be that enhanced endothelin production is isolated in the preglomerular vessels in the SHR. On the other hand, it is possible that the sensitivity of endothelin receptors to endothelin in the renal vasculature may be increased in the SHR. Consequently, endothelin receptor antagonists could have significant effects on renal function in both groups of SHR. In support of this hypothesis, isolated arteries from SHR showed a greater vasoconstrictor response to endothelin than did arteries from WKY.7 21 22 23 However, in experiments in vivo, SHR did not manifest a greater pressor response to a bolus injection of endothelin than WKY.21 23 Furthermore, in a previous study no difference was detected between SHR and WKY in the whole-kidney Km and Bmax for 125I–ET-1.24 Thus, whether the sensitivity of endothelin receptors to endothelin in the SHR kidney is increased remains to be identified.
In summary, endothelin receptor blockade within the kidney significantly improves renal hemodynamics in SHR while having no effect in WKY. These improvements in renal hemodynamics and RIHP were associated with significant increases in sodium and excretion. In conclusion, intrarenal endothelin may mediate, in part, the abnormal renal function in the SHR by increasing renal vascular tone, inhibiting the transmission of RPP into RIHP, and reducing GFR in the SHR.
This study was supported by grants HL-33947 and HL-51971 from the National Institutes of Health, Bethesda, Md, and grant 92-6-45 from the American Heart Association, Mississippi Affiliate. Dr Granger is an Established Investigator of the American Heart Association (grant 89263). We thank Rong Chen and John Stuart Williams for excellent technical assistance and Susie Zuller for expert secretarial skill.
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