Role of Kinins in the Renal Response to Enalaprilat in Normotensive and Hypertensive Rats
Abstract This study examined the role of endogenous kinins in the alteration of renal hemodynamics induced by low-dose converting enzyme inhibition in hydropenic normotensive rats and in the nonclipped kidney of hydropenic two-kidney, one clip hypertensive rats. Infusion of a bradykinin B2 receptor antagonist (d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin, 1 or 10 μg · kg−1 · min−1) did not alter renal function of normotensive rats. In a second series of experiments, infusion of enalaprilat at 0.1 mg · kg−1 · h−1 increased renal blood flow (P<.01) and decreased renal vascular resistance (P<.01). The superimposition of the kinin antagonist at 1 μg · kg−1 · min−1 during the enalaprilat infusion decreased renal blood flow to a value similar to the preenalaprilat baseline and significantly different from the mean of the two enalaprilat periods before and after the addition of the kinin antagonist—the “mean effect of enalaprilat.” The decrease in renal blood flow induced by the kinin antagonist was associated with an increase in renal vascular resistance above the mean effect of enalaprilat (P<.025). In two-kidney, one clip hypertensive rats, systemic infusion of enalaprilat augmented the hemodynamics of the nonclipped kidney by a degree similar to that in normotensive rats. In contrast to normotensive rats, superimposition of the kinin antagonist did not alter the enalaprilat-induced change in blood flow or vascular resistance of the nonclipped kidney. The results of this study suggest that endogenous kinins contribute to the increased renal function induced by low-dose converting enzyme inhibition in hydropenic normotensive rats but appear to contribute less to the enalaprilat-induced alterations of renal function in the nonclipped kidney of two-kidney, one clip hypertensive rats.
- angiotensin-converting enzyme inhibitors
- hypertension, renovascular
Angiotensin-converting enzyme (kininase II) metabolizes the conversion of angiotensin I to angiotensin II and catabolizes kinins to inactive fragments.1 Converting enzyme is the major pathway for degradation of circulating kinins2 and is also an important kininase in renal tissue.3 CEI increase renal tissue kallikrein activity,4 plasma and renal kinin levels,5 and urinary kinin excretion.6 These observations support the possibility that the physiological effects of CEI could be mediated by alterations in kinin metabolism. Although studies using specific antagonists to bradykinin B2 receptors have indicated that kinins participate in the renal actions of CEI,7 8 9 10 the role of kinins in the whole-kidney hemodynamic effects of CEI has not been clarified.
In normotensive rats, the increase in RBF induced by the CEI captopril was not altered by treatment with the bradykinin B2 antagonist d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin.9 The same kinin antagonist partially attenuated the increase in RBF induced by enalaprilat treatment in dogs11 but did not significantly attenuate the increase in RBF induced by captopril or lisinopril in rabbits.12 13 Furthermore, the bradykinin B2 antagonist Hoe 140 did not alter the increase in RBF induced by short-term treatment with either ramiprilat or captopril in rabbits14 or ramiprilat in rats.15
2K1C hypertension is a model of secondary hypertension in which the nonclipped kidney participates in the onset and development of the hypertension.16 The participation of the nonclipped kidney in the development phase (approximately the 4 weeks after placement of the renal artery clip) appears to be highly angiotensin dependent because the responses of the nonclipped kidney to CEI and angiotensin antagonists are qualitatively similar.16 However, a number of findings suggest that the kallikrein-kinin system may also contribute to the CEI-induced alteration in the hemodynamic and excretory functions of the nonclipped kidney of 2K1C hypertensive rats. Renal kallikrein gene expression17 18 and urinary kallikrein19 excretion from the nonclipped kidney are maintained at levels not different from normal levels, although tissue kallikrein level is reduced compared with normal kidneys20 and short-term treatment with CEI increased kinin excretion from the nonclipped kidney. Furthermore, studies using bradykinin B2 receptor antagonists indicate that endogenous kinins contribute to the hypotensive effect of CEI in renovascular hypertensive rats.21 22 23 24
Acute administration of low doses of CEI induce similar significant increases in the hemodynamics and excretory function of hydropenic normotensive rats and in the nonclipped kidney of hydropenic 2K1C hypertensive rats. The purpose of this study was to examine the hypothesis that endogenous kinins contribute to the CEI-induced alterations in hemodynamics and excretory function of kidneys of normotensive rats and of the nonclipped kidney of 2K1C hypertensive rats. We tested this hypothesis by assessing the effects of superimposing kinin receptor blockade during acute infusion of low-dose enalaprilat in hydropenic normotensive and 2K1C hypertensive rats. We also examined the effect of systemic kinin receptor blockade in the absence of enalaprilat on renal hemodynamics and excretory function of hydropenic normotensive rats. Superimposed low-dose B2 bradykinin receptor antagonist d-Arg0,[Hyp3,Thi5,8, d-Phe7]-bradykinin reversed the renal vasodilation induced by enalaprilat in the kidney of hydropenic normotensive rats but not the nonclipped kidney of 2K1C hypertensive rats. These observations suggest that endogenous kinins may play a major role in the renal hemodynamic responses induced by enalaprilat in hydropenic normotensive rats. In contrast, kinins appear to contribute less to the enalaprilat-induced alterations of renal function in the nonclipped kidney of 2K1C hypertensive rats.
General Surgical Procedures
Male Wistar rats weighing 210 to 350 g (Charles River, Wilmington, Mass) were fed standard rat chow (Wayne Rodent Blox [0.31% sodium], Teklad) ad libitum and given free access to tap water. Rats were anesthetized with pentobarbital (50 mg · kg−1 IP) and placed on a heated table for maintenance of body temperature at 37°C. After tracheotomy, a triple-lumen cannula was placed in a jugular vein for infusion of fluids, drugs, and anesthetic. A cannula was placed in the right femoral artery for measurement of blood pressure and for blood sampling. The bladder was cannulated to allow urine collection from the left kidney. The right kidney was exposed by a flank incision, freed of perirenal tissue, placed in a Lucite cup, and bathed in 0.9% NaCl. The ureter was then cannulated. In most rats, the right renal artery was freed from connective tissue, and a flow transducer (Carolina Medical Electronics) with either a 1.5- or 2-mm-circum lumen was fitted. The transducer was connected to a square-wave electromagnetic blood flowmeter (Carolina Medical Electronics). Hydropenic preparations were maintained by an intravenous bolus injection of 0.8 mL of 0.9% NaCl after cannulation of the jugular vein and a maintenance infusion of 1.2 mL · h−1 during the surgical procedures. A bolus injection of 0.4 mL of the 0.9% NaCl was given at the end of the surgery, and the infusion rate was set at 0.6 mL · h−1. The enalaprilat and kinin antagonist were delivered through this line. To allow the determination of GFR and in some rats estimated RPF, a 0.4-mL priming dose of 0.9% NaCl containing 12.5% polyfructosan (Inutest, Laevosan-Gesellschaft) and 1% PAH (Merck Sharp & Dohme) was given as a bolus through the other jugular line, followed by infusion at a rate of 0.6 mL · h−1. Consequently, during the experiment the total infusion rate was maintained at 1.2 mL · h−1. The flow transducers were calibrated to zero flow by momentary complete occlusion of the renal artery. Forty-five minutes was allowed for the preparation to reach a steady state before study. Timed urine collections were obtained, with blood (0.4 mL) collected between pairs of clearance periods. For maintenance of hematocrit, red blood cells from each blood sample were reconstituted to the same volume with 0.9% NaCl and reinjected through the arterial cannula.
Protocol 1: Effect of the KA d-Arg0,[Hyp3,Thi5,8, d-Phe7]-Bradykinin on Renal Function of Hydropenic Normotensive Rats
In group 1 (n=5), two 20-minute baseline clearance periods were obtained. The KA d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin (generously provided by Dr J.M. Stewart, Department of Biochemistry, University of Colorado Health Sciences Center, Denver) was injected as a bolus (20 μg · kg body wt−1) and then infused at a rate of 1 μg · kg body wt−1 · min−1. After 10 minutes, two 20-minute clearance periods were obtained. The KA was then injected as a bolus of 200 μg · kg body wt−1 and infused at a rate of 10 μg · kg body wt−1 · min−1. After another 10-minute stabilization period, two additional clearance periods were taken. The KA infusion was discontinued; 20 minutes was allowed for stabilization; and two further clearance (recovery) periods were obtained.
In group 2 (n=5), similarly prepared rats served as time controls. They received equal volumes of vehicle (0.9% NaCl) instead of the KA.
Protocol 2: Effect of the KA d-Arg0,[Hyp3,Thi5,8, d-Phe7]-Bradykinin on Enalaprilat-Induced Alterations in Renal Hemodynamics and Excretory Function
Normotensive rats. In group 3 rats (n=11), two baseline urine collections were obtained, and enalaprilat (Vasotec, Merck Sharp & Dohme) was then injected as a bolus of 0.3 mg · kg body wt−1 and infused at 0.1 mg · kg body wt−1 · h−1 throughout the rest of the experiment. After 20 minutes of equilibration, two 20-minute urine collections were taken. The KA d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin was injected as a bolus (20 μg · kg body wt−1) and then infused at a rate of 1 μg · kg body wt−1 · min−1. Again, 20 minutes was allowed for stabilization, and two 20-minute urine collections were obtained during the superimposed infusion of the KA. The KA was discontinued, and after 20 minutes of stabilization, two further 20-minute clearance periods were obtained.
Rats in group 4 (n=6) were used as time controls with the same protocol except they received equal volumes of vehicle (0.9% NaCl) instead of the KA.
2K1C hypertensive rats. The effect of the KA on enalaprilat-induced augmentation of renal hemodynamics and excretory function of the nonclipped kidney of 2K1C hypertensive rats (group 5, n=8) was examined 3 to 4 weeks after clip. Rats weighing 100 to 125 g were anesthetized with sodium pentobarbital (50 mg · kg−1 IP), and the left kidney was exposed via a retroperitoneal incision. The renal artery was partially freed from the renal vein, and a silver clip with a gap of 0.2 mm was placed around the artery. The rats were allowed to recover and received water and chow ad libitum during the next 3 to 4 weeks. On the day of the experiment, the rats were prepared as described for the general surgical procedure, with the nonclipped kidney (right) exposed and placed in the kidney cup. Only rats with MAP between 150 and 200 mm Hg were used in this study. The experimental protocol was exactly the same as that for group 3.
A second group of 2K1C hypertensive rats (group 6, n=5) was studied with the same protocol except they received equal volumes of vehicle (0.9% NaCl) instead of the KA.
The effectiveness of kinin blockade was evaluated by the vasodilator effect of bradykinin before and after KA administration. In five of the group 3 and four of the group 5 rats, bradykinin (Sigma Chemical Co) was injected as a bolus (0.05 mL IV) at doses of 5 and 10 ng during enalaprilat treatment before the superimposed KA infusion and then again 20 minutes after the start of the KA infusion.
At the end of each experiment, the kidneys were excised, blotted, and weighed. Urine volume was determined gravimetrically. Plasma and urinary sodium and potassium concentrations were determined by flame photometry (model 943, Instrumentation Laboratories). Polyfructosan and PAH concentrations were determined by modified anthrone and colorimetric methods, respectively.25 26 GFR was determined from the clearance of polyfructosan. To avoid a fixed bias of using only the electromagnetic flowmeter to measure RBF, we determined RPF using the clearance of PAH adjusted for the measured extraction of PAH in a number of rats chosen randomly from each of the groups. There was no difference in the extraction ratios between the normotensive and 2K1C hypertensive rats; consequently, all values were combined and the average value (0.55) was used. For these rats, RBF was calculated from RPF and hematocrit. The values for RBF obtained directly were not significantly different from the values determined with PAH. Filtration fraction and RVR were then calculated with standard formulas. Clearance data were normalized to kidney weight.
Values are reported as mean±SEM. For groups 1 through 4, data from both kidneys were combined. Univariate two-factor repeated measures ANOVA was used to test the group effect (two levels; KA compared with vehicle) and the group by repeated measures interaction. Differences between the two groups, at baseline or each treatment level or in the change from baseline, were tested by the relevant Scheffé contrasts, with the significance level adjusted by the Bonferroni method. Data within each group were analyzed by multivariate one-factor repeated measures ANOVA with the Hunyh-Feldt correction or in a number of cases the Greenhouse-Geisser correction. A limited number of comparisons between pairs of means or combinations of means were established for most variables (except MAP) and analyzed by the appropriate Scheffé contrasts. The significance level for the comparisons was modified with the appropriate Bonferroni adjustment. Differences between single pairs of means were tested with the Wilcoxon signed rank test or Student’s t test, with a significance level at a value of P<.05. Analyses were conducted with either StatView 512+ (Brainpower) or SuperANOVA (Abacus Concepts).
Protocol 1: Effect of the KA on Renal Function (Groups 1 and 2)
Mean body and kidney weights of the rats in groups 1 and 2 are presented in Table 1⇓. The effects of the kinin antagonist (1 and 10 μg · kg−1 · min−1) on arterial blood pressure and the renal hemodynamics of hydropenic rats are shown in Fig 1⇓. There was no difference in the basal values of MAP, GFR, or PAH clearance between the KA-infused rats and vehicle-infused time controls. The MAP of the time control rats remained stable during the experiment. In contrast, the blood pressure of rats that received the KA decreased significantly (P<.017) from baseline during the KA infusion at 10 μg · kg−1 · min−1 and decreased further during the recontrol period. Only during the recontrol period was there a significant (P<.0125) difference in MAP between the two groups. Systemic infusion of the KA or vehicle did not alter either GFR or PAH clearance compared with either baseline or the other group.
KA infusion did not alter UV compared with baseline values or those values obtained for the time control rats (Table 1⇑). UNaV and FENa were not significantly altered by KA infusion. In contrast, UNaV and FENa were significantly less (P<.017) than the baseline value during the recontrol period (Table 1⇑). The decreases in UNaV and FENa from baseline during the recontrol period were significantly larger (P<.0125) in the KA group compared with the time control group. KA infusion abolished the increase in UKV observed in the time control group.
Protocol 2: Effect of the KA on Enalaprilat-Induced Alterations in Renal Hemodynamics and Excretory Function
In a subset of five enalaprilat-infused hydropenic normotensive rats, the superimposition of the KA (20 μg · kg−1 bolus and then infusion at 1 μg · kg−1 · min−1) for 20 minutes partially blocked the vasodepressor effect of bolus systemic injections of 5 ng bradykinin by 39±10% (P<.05) and 10 ng bradykinin by 30±7% (P<.05) (Fig 2⇓). A similar degree of blockade was observed for 5 ng bradykinin (31±9%, P<.05) and 10 ng bradykinin (26±9%, P=.06) in 2K1C hypertensive rats.
Normotensive Rats (Groups 3 [KA] and 4 [Time Control])
Mean body weights and mean combined kidney weights were similar for the rats from groups 3 and 4.
Baseline MAP values did not differ between the normotensive rats that received the KA or those that received vehicle (Fig 3A⇓). Systemic infusion of a low dose of enalaprilat (0.3 mg · kg−1 bolus and infusion at 0.1 mg · kg−1 · h−1) induced significant decreases in blood pressure in rats from both groups 3 and 4 during the course of the experiment (Fig 3A⇓).
This experiment was designed to evaluate the effect of the KA on changes in systemic blood pressure and renal function induced by CEI. Thus, the period of superimposed infusion of the KA or vehicle was bracketed by periods of enalaprilat infusion alone. Data from the two periods of enalaprilat infusion alone were averaged, and the value represented the mean effect of CEI. The effects of superimposed infusion of either KA or vehicle were determined as changes from this mean effect of CEI. For visual clarity, in Figs 3D through 3F and 4D through 4F, the mean effects of CEI for each group are presented as zero. The effects of superimposed infusion of the KA or vehicle are thus presented as changes from zero. This analysis allowed comparison of the changes observed for superimposition of KA or vehicle during CEI treatment within and between the groups. The changes in blood pressure observed during the superimposed infusion of the KA or vehicle compared with the mean effect of CEI are plotted in Fig 3D⇑. Superimposition of the KA did not significantly alter MAP when compared with the effects of either enalaprilat alone or the superimposed infusion of vehicle.
The effects of KA on renal hemodynamics in these enalaprilat-infused normotensive rats are presented in Fig 3⇑. For two of the KA-treated rats, inadequate measurements of either RPF or RBF were made during the experiment; consequently, data from these rats were not used for examination of the effect of KA on RPF, RBF, or RVR. Although the baseline RPF of rats from group 3 (KA) was higher than that of rats from group 4 (time control), the low dose of enalaprilat significantly increased RPF by a similar extent in both groups. In group 3 rats, enalaprilat infusion induced a significant increase in mean effective RPF from a baseline value of 3.76±0.21 to 4.55±0.40 mL · min−1 · g kwt−1 (P<.01, 21%) during the first CEI period. Similarly, in group 4 rats, RPF increased from a baseline of 2.74±0.36 to 3.53±0.39 mL · min−1 · g kwt−1 (P<.01, 31%) during the first CEI period. For group 3 rats, the mean RPF during the periods when enalaprilat was infused without superimposed KA (the mean effect of CEI) was 4.40±0.40 mL · min−1 · g kwt−1. During the superimposition of the KA, RPF decreased significantly from the mean effect of CEI by −0.66±0.20 mL · min−1 · g kwt−1 (P<.01, −15%). In group 4 rats, the mean RPF for the periods when enalaprilat was infused without the superimposed infusion of vehicle was 3.40±0.35 mL · min−1 · g kwt−1. In contrast to the KA group, the effect of enalaprilat on RPF was not altered by the superimposed infusion of the vehicle (the difference in RPF between the vehicle infusion and the mean effect of CEI was 0.22±0.09 mL · min−1·g kwt−1, P>.025). Also, the change in RPF induced by the superimposition of the KA was significantly (P<.01) greater than that observed during the infusion of vehicle.
The effects of KA or vehicle on RBF are presented in Fig 3B⇑ and 3E⇑. The baseline RBF for group 3 rats was significantly higher (P<.01) than that for group 4 rats (7.38±0.33 and 5.38±0.56 mL · min−1 · g kwt−1, respectively). Enalaprilat significantly increased RBF by a similar degree in both groups. The low dose of enalaprilat increased the RBF of group 3 rats by 1.58±0.45 mL · min−1 · g kwt−1 (P<.01, 21%) during the first CEI period. For group 4 rats, RBF increased by 1.61±0.28 mL · min−1 · g kwt−1 (P<.01, 32%) during the first CEI period (Fig 3B⇑). The changes in enalaprilat-induced increased RBF observed during the superimposition of either KA or vehicle are plotted in Fig 3E⇑. The superimposition of the KA but not vehicle significantly decreased RBF. Furthermore, the change in RBF induced by the KA (−1.34±0.37 mL · min−1 · g kwt−1, P<.01, −16%) was significantly (P<.01) different from that observed for the vehicle-infused rats (0.45±0.19 mL · min−1 · g kwt−1, P>.05) (Fig 3E⇑).
Low-dose enalaprilat infusion induced significant renal vasodilation in both groups of hydropenic rats. For group 3 rats, enalaprilat significantly decreased RVR by 23% from a mean baseline value of 17.8±1.1 to 13.7±1.1 mm Hg · mL−1 · min−1 · g kwt−1 during period 2 (P<.01) and for group 4 rats, from a baseline value of 26.5±2.3 to 18.8±1.5 mm Hg · mL−1 · min−1 · g kwt−1 (P<.001) during period 2. Superimposition of the low-dose KA reversed the vasodilator effect of enalaprilat (RVR was increased significantly compared with the mean effect of CEI by 2.8±1.3 mm Hg · mL−1 · min−1 · g−1, P<.025, Fig 3F⇑). In contrast, the superimposition of vehicle did not alter RVR (ΔRVR, −1.2±0.7, P>.05, Fig 3F⇑). Furthermore, the increase in RVR induced by the superimposition of the KA was significantly greater than the change in RVR observed during the superimposed infusion of vehicle (P<.025).
Baseline GFR values obtained from group 3 and 4 rats were not significantly different (Table 2⇓). Enalaprilat infusion initially increased GFR in group 3 from a baseline value of 0.96±0.04 to 1.15±0.04 mL · min−1 · g kwt−1 (P<.001) during period 2 and in group 4 rats from a baseline of 1.00±0.04 to 1.28±0.08 mL · min−1 · g kwt−1 (P<.01). However, despite the continued presence of enalaprilat, the increase in GFR was transitory. For group 4 rats, the decrease was approximately linear; GFR during period 3 was not significantly different from that during period 2, but during period 4, GFR was similar to the baseline value (1.00±0.07 mL · min−1 · g kwt−1). In contrast, superimposition of the KA significantly (P<.01) decreased GFR to a value similar to baseline (GFR was 1.01±0.07 mL · min−1 · g kwt−1 during period 3). Infusion of a low dose of enalaprilat into the hydropenic rats induced significant (P<.01, natural log–transformed data) increases in renal excretory function (Table 2⇓). Superimposition of the KA did not alter UNaV compared with period 2. Similarly, the superimposition of vehicle (group 4) did not alter the enalaprilat-induced natriuresis. Enalaprilat infusion significantly increased UV and UKV of groups 3 and 4 rats (Table 2⇓). These increases were again transitory despite the continued presence of the CEI. The superimposition of the KA resulted in a significant decrease in both UV (P<.01) and UKV (P<.001) compared with period 2. In contrast, UV and UKV were not significantly altered during the superimposition of vehicle.
2K1C Hypertensive Rats (Groups 5 and 6)
Mean body weights were not different between the two groups of hypertensive rats (301±11 and 289±12 g [P>.05], groups 5 [KA] and 6 [time control], respectively).
Baseline MAP was also not different between these two rat groups (172±7 and 168±9 mm Hg, respectively). Enalaprilat infusion resulted in a marked decrease in MAP in both groups (Fig 4A⇓). Superimposition of the KA did not significantly alter MAP compared with the effects of either enalaprilat alone or the superimposed infusion of vehicle (Fig 4D⇓).
Baseline renal hemodynamics were not different between the two groups of hypertensive rats. The low dose of enalaprilat increased RPF of the nonclipped kidney by a similar degree in the two groups, although for group 6 rats, the increase in RPF did not reach significance until period 3. The increase in RPF from baseline to period 2 in group 5 rats was 0.72±0.16 mL · min−1 · g kwt−1 (P<.025, 34%). The superimposition of the KA did not alter RPF compared with either the mean effect of enalaprilat alone or the effect of vehicle infusion. The change in RPF from the mean effect of CEI during the superimposition of the KA was −0.07±0.12 mL · min−1 · g kwt−1 and during the superimposition of vehicle was 0.34±0.24 mL · min−1 · g kwt−1. The effects of superimposition of KA or vehicle during enalaprilat infusion on RBF and RVR of the nonclipped kidney of the 2K1C hypertensive rats are presented in Fig 4⇑. The low dose of enalaprilat increased RBF of the nonclipped kidney by a similar degree in the two groups (Fig 4B⇑). RVR (Fig 4C⇑) was significantly decreased in both groups during period 2 (the mean change in RVR induced by enalaprilat was −10.87±3.02 and −9.05±2.08 mm Hg · mL−1 · min−1 · g kwt−1 for groups 5 [P<.025, −29%] and 6 [P<.025, −31%], respectively). The superimposition of the KA did not alter RBF or RVR (Fig 4E⇑ and 4F⇑).
Baseline values of GFR were not significantly different between groups 5 and 6 (Table 2⇑). For group 6 rats, GFR was not significantly increased by enalaprilat infusion. However, in group 5 rats, enalaprilat increased the GFR of the nonclipped kidneys from a baseline value of 0.63±0.05 to 0.78±0.07 mL · min−1 · g kwt−1 during period 2 (P<.025). As was observed for the normotensive rats, the increase in GFR was transient despite the continued presence of enalaprilat. Consequently, the effect of the superimposition of KA or vehicle was examined by comparing the mean GFR at period 3 (during the superimposition of KA or vehicle) with that obtained during period 2 (enalaprilat alone). Compared with period 2, the GFR of the nonclipped kidneys was not significantly altered by the superimposition of the KA (0.71±0.06 versus 0.78±0.07 mL · min−1 · g kwt−1) or vehicle (0.96±0.18 versus 0.86±0.16 mL · min−1 · g kwt−1). The response to superimposition of the KA was not different to that observed during the superimposition of the vehicle (−0.07±0.04 and 0.1±0.09 mL · min−1 · g kwt−1, for groups 5 and 6, respectively).
Enalaprilat infusion induced significant increases in UV and UKV in both groups of 2K1C rats (Table 2⇑). Superimposition of the KA did not alter the rate of either water or potassium excretion compared with the mean effect of CEI (ΔUV and ΔUKV from the mean effect of CEI, 0.2±0.4 μL · min−1 · g kwt−1 and −26±93 nmol · min−1 · g kwt−1, respectively) or with the excretory rates obtained during the superimposition of vehicle (ΔUV and ΔUKV from the mean effect of CEI, 1.2±1.1 μL · min−1 · g kwt−1 and 249±128 nmol · min−1 · g kwt−1, respectively). Low-dose enalaprilat infusion did not significantly increase UNaV of either group of hypertensive rats (Table 2⇑). UNaV during the superimposition of the KA did not differ from either the mean effect of CEI or UNaV during the superimposition of vehicle.
Studies that used the Stewart-Vavrek KA d-Arg0, [Hyp3,Thi5,8,d-Phe7]-bradykinin to investigate the contribution of endogenous kinins in control vascular tone and renal function in rats used relatively high doses of the antagonist to block kinin receptors. In contrast, Jaffa et al27 reported that a low systemic dose of this KA (at least 10-fold lower than used in other studies) prevented the increase in GFR and RBF induced by acute amino acid infusion, suggesting that renal bradykinin B2 receptors are effectively blocked by low doses of the KA. Consequently, we assessed the effects of superimposing a low dose of the KA during acute infusion of low-dose enalaprilat in hydropenic normotensive and 2K1C hypertensive rats.
Although very high doses of bradykinin receptor antagonists have been previously reported to alter baseline MAP28 29 or to reverse the hypotensive effect of CEI,24 30 basal vascular tone and CEI-induced vasodilation were not altered by lower doses of kinin receptor antagonists.9 10 30 31 In the present study, systemic infusion of two low doses of the KA did not alter the basal MAP of hydropenic normotensive rats. Treatment of normotensive rats with the CEI, enalaprilat, decreased systemic blood pressure and increased RBF. The superimposition of low-dose bradykinin B2 receptor antagonist intravenously during the enalaprilat treatment did not reverse the CEI-induced decrease in MAP. These findings are consistent with the above observations that suggest that in normotensive rats, endogenous kinins do not play a major role in either the maintenance of basal vascular tone or the vasodilation induced by CEI treatment. Although kinins participate at least partly in the renal actions of CEI,7 8 9 10 the role of kinins in the whole-kidney hemodynamic effects of CEI has not be clarified.
We report that a low dose of d-Arg0,[Hyp3,Thi5,8, d-Phe7]-bradykinin given systemically reversed the enalaprilat-induced changes in RVR and RBF in hydropenic normotensive rats. This effect of kinin receptor antagonism on CEI-induced changes in whole-kidney hemodynamics was observed at a KA dose substantially lower than that used by previous investigators to examine the effect of systemic bradykinin B2 receptor blockade on CEI-induced changes in renal hemodynamics and excretory function.8 9 Furthermore, this low systemic dose of the KA was similar to the doses used to examine the effect of intrarenal infusion on CEI-induced increases in RBF.12 13 These previous studies did not find a significant effect of the KA on changes in whole-kidney hemodynamics induced by CEI treatment. Thus, the KA dose used in the present study was markedly lower than the doses used in studies that failed to find an effect of systemic kinin receptor blockade on CEI-induced changes in whole-kidney hemodynamics. In the hydropenic normotensive rats, GFR and urinary excretory function increased after treatment with low-dose enalaprilat. This effect of enalaprilat was transient, as these renal function parameters returned to baseline values by the end of the experiment. Despite the transient effect of enalaprilat treatment, superimposition of the low dose of KA reduced GFR and UV but not UNaV to baseline levels. This finding suggests that blockade of kinin B2 receptors attenuated the CEI-induced changes in GFR and UV and is similar to those previously reported except that higher doses of KA attenuated the increase in UNaV after CEI treatment.7 8 9
Although the rats were housed and fed under the same conditions and had similar hematocrit values (suggesting similar volume status) during the clearance experiments, some baseline parameters of renal function differed between the groups that received KA or vehicle. Although these differences were unexpected, they do not appear to compromise the findings of the study because they were negated by our experimental design. The baseline values were obtained to ensure that the effect of enalaprilat treatment was similar for all groups before the superimposed KA or vehicle treatment. Despite differences in baseline renal function between the two groups of normotensive rats, the systemic infusion of enalaprilat induced similar changes in renal function. Furthermore, the period of superimposed infusion of the KA was bracketed by periods of enalaprilat infusion alone. The effect of the KA was determined by the change observed during the superimposed KA treatment from the periods of enalaprilat infusion alone. Data presented in Fig 3E⇑ and 3F⇑ indicate that the superimposition of KA markedly attenuated enalaprilat-induced alterations in renal hemodynamics within the same group of rats.
Infusion of low-dose KA during treatment with enalaprilat partially blocked the hypotensive effect of injected bradykinin (by 30% to 39%). This finding is consistent with other reports of partial blockade of the hypotensive action of exogenous bradykinin by the first generation of effective kinin antagonists, although most previous studies examined the completeness of kinin blockade in the absence of CEI. High doses of KA attenuated the hypotensive effect of intra-arterial bradykinin by 52%32 and 79%31 in conscious normal rats, 20% to 50% in anesthetized normotensive rats,33 and 39% to 72%23 and 45% to 55%21 in anesthetized 2K1C hypertensive rats. Although only partial blockade of systemically administered bradykinin was achieved for the normotensive rats in the present study, the KA abolished the enalaprilat-induced renal vasodilation. Despite the incomplete inhibition of exogenous bradykinin in the other studies, kinin antagonists reduced baseline RBF31 and UV,32 abolished the renal effects of kininase inhibition,33 and attenuated the hypotensive effects of CEI.21 23 Furthermore, d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin almost completely blocked the renal hemodynamic effects of intrarenally infused bradykinin.12 13 Taken together, these observations suggest that blockade of renal bradykinin B2 receptors could be more complete than blockade of systemic receptors.
Our finding that infusion of low-dose KA reversed the renal hemodynamic effects of enalaprilat suggests that endogenous kinins acting on renal bradykinin B2 receptors control renal hemodynamics in normotensive hydropenic rats acutely treated with CEI. This role of kinins in the changes in renal hemodynamics induced by CEI treatment could simply be the result of increased kinin levels consequent to CEI.5 This possibility is supported by the findings that CEI potentiates the hypotensive action of exogenous systemic bradykinin by prolonging its activity.22 23 34 The present observations in hydropenic rats provide support for an action of CEI to augment kinin activity. Intravenous administration of low-dose bradykinin during enalaprilat infusion induced substantial decreases in systemic blood pressure in both the normotensive control and 2K1C hypertensive rats. These bradykinin-induced decreases in MAP were similar to the decreases in MAP reported for 5 to 10 times higher doses of intra-arterially injected bradykinin in the absence of CEI in normotensive and hypertensive rats.23 31 33
Since CEI results in both blockade of angiotensin formation and kinin degradation, the effects of superimposed KA during enalaprilat treatment on renal hemodynamics could also be due to an altered balance in angiotensin-induced vasoconstriction and kinin-mediated vasodilation. This interaction between angiotensin and kinins may be a major factor associated with the difficulty in fully elucidating the role of kinins in the regulation of renal function. Although the use of the Stewart-Vavrek antagonists and Hoe 140, a more potent antagonist, support a role for kinins in contributing to the regulation of sodium excretion, this role appears to be limited to circumstances when perturbations in the levels of both kinins and angiotensin are expected to occur, such as with deoxycorticosterone acetate–salt treatment32 or after treatment with CEI.7 8 9 Furthermore, the Stewart-Vavrek antagonist11 or Hoe 14035 attenuated CEI-induced increases in RBF in sodium-restricted dogs but not those fed a normal sodium diet.35 Since dietary sodium restriction markedly elevates renal interstitial kinin levels,36 these findings suggest that the renal hemodynamic effects of CEI are at least partly kinin dependent when basal kinin activity is elevated.
In contrast to the responses we observed in normotensive rats, superimposed low-dose KA did not alter the enalaprilat-induced increases in hemodynamics and excretory function of the nonclipped kidney of 2K1C hypertensive rats. Although we had previously observed qualitatively similar effects of CEI and angiotensin receptor antagonism on renal function in 2K1C hypertensive rats,16 we hypothesized that endogenous kinins could contribute to the CEI-induced increased function in the nonclipped kidney of 2K1C hypertensive rats. This hypothesis was derived from the findings that in 2K1C hypertensive rats, the hypotensive response to CEI could be attenuated by kinin antagonists,21 22 23 24 glomerular kinin receptors were upregulated in the nonclipped kidney of 2K1C hypertensive rats compared with kidney from normotensive rats,20 and renal kallikrein mRNA expression in the nonclipped kidney was not downregulated as it was in the clipped kidney.17 18 However, the present findings suggest that endogenous kinins contribute substantially less to the enalaprilat-induced alterations in renal hemodynamics for the nonclipped kidney during the early phase of 2K1C hypertension than for kidneys from normotensive rats.
In the present study, the KA attenuated the hypotensive effect of injected bradykinin similarly in the normotensive and hypertensive rats. Therefore, it would appear that the absence of a renal hemodynamic effect of superimposed KA in the hypertensive rats was not due to decreased blockade of kinin receptors. The same low systemic dose of d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin as used in the present study is effective in elucidating the role of kinins in the altered renal hemodynamics of several rat models.27 37 38 This finding suggests that the low dose of the antagonist effectively blocks renal kinin receptors under a number of physiological and pathophysiological conditions, further supporting comparable kinin receptor blockade between the normotensive and hypertensive rats in the present study.
It seems unlikely that our observation that kinin receptor antagonism did not attenuate enalaprilat-induced renal hemodynamics in the nonclipped kidney of 2K1C hypertensive rats was due to the use of d-Arg0, [Hyp3,Thi5,8,d-Phe7]-bradykinin instead of the newer receptor antagonist Hoe 140. Although studies in vitro have found Hoe 140 to be more specific and more potent than the first-generation B2 receptor antagonists and in vivo to almost completely block the hypotensive effect of systemic bradykinin, both d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin and Hoe 140 have suggested a role for kinins in CEI-induced alterations in renal hemodynamics in normal animals.7 10 11 35 Also, both d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin and Hoe 140 effectively block renal bradykinin B2 receptors in rabbits.12 13 14 Interestingly, in contrast to the present study, Hoe 140 did not attenuate the action of CEI on RBF in rats.10 15 These findings suggest that d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin may be as effective an agent as Hoe 140 in elucidating the role of kinins in CEI-induced renal hemodynamics.
Chronic CEI or increased tissue kinin levels have been reported to result in downregulation of bradykinin B2 receptors.6 However, it seems unlikely that acute CEI would downregulate kinin receptors to a greater extent in the nonclipped kidney of 2K1C hypertensive rats compared with kidneys from normotensive rats in the same short time frame.
Intrarenal angiotensin II levels are significantly elevated in the nonclipped kidneys of 2K1C hypertensive rats 4 weeks after clipping.39 This chronic increase in angiotensin activity may alter the responsiveness of the renal vasculature to kinins. Consequently, the nonclipped kidney of the 2K1C hypertensive rat may represent a situation in which the balance between angiotensin II and kinins is such that blockade of angiotensin II formation is the primary determinant of the action of CEI.
In summary, infusion of low doses of the bradykinin B2 receptor antagonist d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin did not alter blood pressure, renal hemodynamics, or excretory function in hydropenic normotensive rats. Infusion of a low dose of the CEI enalaprilat resulted in decreased MAP, decreased RVR, and augmented excretory function. Superimposition of the KA during low-dose enalaprilat infusion reversed the CEI-induced increase in RBF and attenuated the increased excretory function in hydropenic normotensive rats. Infusion of low-dose enalaprilat resulted in decreased MAP and increased RBF for the nonclipped kidney of 2K1C hypertensive rats similar to that observed in normotensive rats. However, the superimposed infusion of the KA did not alter the CEI-induced increase in RBF or significantly change the CEI-induced alterations in excretory function for the nonclipped kidney in these rats. These observations suggest that endogenous kinins contribute to the increased renal function induced by low-dose CEI in hydropenic normotensive rats but appear to contribute less to the enalaprilat-induced alterations of renal function in the nonclipped kidney of 2K1C hypertensive rats.
Selected Abbreviations and Acronyms
|2K1C||=||two-kidney, one clip|
|CEI||=||converting enzyme inhibitor(s), inhibition|
|FENa||=||fractional excretion of sodium|
|GFR||=||glomerular filtration rate|
|KA||=||kinin antagonist (d-Arg0,[Hyp3,Thi5,8,d-Phe7]-bradykinin)|
|MAP||=||mean arterial pressure|
|RBF||=||renal blood flow|
|RPF||=||renal plasma flow|
|RVR||=||renal vascular resistance|
|UKV||=||absolute urinary potassium excretion|
|UNaV||=||absolute urinary sodium excretion|
|UV||=||urine flow rate|
This investigation was supported by funds from Dialysis Clinics Inc and Merit Review Grants from the Research Service of the Department of Veterans Affairs (D.W.P., R.K.M.). Statistical consultation was provided by Dr Philip Rust, Department of Biometry, Medical University of South Carolina. We acknowledge the technical assistance of Emma S. Murner.
Presented in part at the Southern and National Meetings of the American Federation of Clinical Research, New Orleans, La, January, 1990, and Washington, DC, May, 1990, and the American Society of Nephrology, Baltimore, Md, November, 1991, and published in abstract form (Clin Res. 1990;38:21A, Clin Res. 1990;38:428A, and J Am Soc Nephrol. 1991;2:475).
- Received July 6, 1995.
- Revision received August 2, 1995.
- Accepted September 29, 1995.
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