Dihydropyridine (DHP) calcium channel blocker (CaB) is widely used to treat patients with hypertension. One of the goals of this treatment is to protect the kidney from progressing to end-stage renal failure. In prospective clinical trials, treatment with CaB was almost equally beneficial to the angiotensin-converting enzyme inhibitor for ameliorating the advance of renal failure (1). However, the results of the animal studies were reported to have a favorable (2) or nonfavorable effect (3) on CaB regarding progression of glomerular damage. Furthermore, the in vivo net effect on renal function is inconsistent between animal models used or applied CaBs (4). With respect to the pathophysiology of increased glomerular capillary pressure in the progression of glomerular damage in hypertensive or remnant kidney animal models (5), these diverse effects on the prognosis of the renal damage might reflect different effects of CaBs on glomerular hemodynamics.
More recently synthesized DHP derivatives have pharmacologic characteristics of more gradual and longer effects on blood pressure than do earlier developed DHPs. Moreover, recent in vivo studies on these new CaBs in spontaneously hypertensive rats (SHRs) revealed a significant reduction in glomerular capillary pressure (6) or glomerular transcapillary hydrostatic pressure difference (7). Thus the effects on the renal microcirculation also may differ between the DHP derivatives.
To examine the mechanism of differences in the effects of DHP on glomerular hemodynamics, we performed in vivo micropuncture experiments in SHRs and compared the effects of two different DHPs of different generations, benidipine hydrochloride (BDP) and nifedipine (NDP), on renal and glomerular hemodynamics.
Male spontaneously hypertensive rats (SHRs/Izm; Sankyo Labo, Tokyo, Japan) aged 15-19 weeks were allowed free access to tap water and conventional rat chow containing 0.2% NaCl (Oriental Kobo, Tokyo, Japan) until the experiment, at which time they weighed 240-320 g. They were anesthetized with an i.p. injection of thiobutabarbital sodium (100 mg/kg; Research Biochemicals International, Natick, MA, U.S.A.) and placed on a feedback-controlled operating table maintaining body temperature constant at 37°C. After tracheostomy (PE-260) to allow spontaneous breathing, a catheter (PE-50) was inserted from the right femoral artery into the abdominal aorta to monitor mean arterial pressure (MAP) by using a pressure transducer (Nihon Koden, Tokyo, Japan) connected to an analog/digital converter (MacLab, Melbourne, Australia). MAP in the abdominal aorta was regarded as equivalent to the renal perfusion pressure (RPP). The femoral vein was cannulated with two PE-50 catheters, one for constant infusion of isotonic (0.9%) saline at 1 ml/h/100 g body weight throughout the experiment, and the other for the administration of drugs. The left kidney was exposed through a flank incision and embedded in a Lucite cup mounted on the operating table. The ureter was cannulated with PE-50 tubing for free egress of urine. On completion of surgery, 1 ml of littermate donor SHR plasma was infused intravenously, and a 30-60 min equilibration period was allowed before initiating measurements. The following experiments were done only if MAP remained stable and was >140 mm Hg at the end of the equilibration period. All the data were recorded and analyzed by personal computer on line with an A/D converter.
All CaB solutions were freshly prepared immediately before use in each experiment. NDP (Sigma, St. Louis, MO, U.S.A.) or BDP (Kyowa-Hakko, Tokyo, Japan) was dissolved in polyoxyethylene-(20)-sorbitan monooleate; Tween 80 (Wako, Tokyo, Japan) in 1 mg/ml, and the solution was diluted with saline (0.9%) to the desired concentration (NDP, 250 μg/ml, and BDP, 4 μg/ml, for systemic administration).
For microinfusion into peritubular capillaries of a single nephron, solution was prepared with Tween 80 and colored (1% fast green) Ringer's solution to give the desired concentrations of NDP, 10−3M, or BDP, 10−3M. As a control perfusate, 10% Tween 80 in Ringer's solution was used.
Rats were prepared as previously described. To measure renal blood flow (RBF), the left renal artery was cleared and dissected from the renal vein and fitted with an electromagnetic flow probe connected to a flow meter (both Nihon Koden, Tokyo, Japan). Both RPP and RBF signals were passed to an A/D converter to obtain simultaneous on-line recordings throughout the experiments. After basal measurements, control solution (300 μl) or CaB solution (NDP, 250 μg/kg body wt, or BDP, 4 μg/kg body wt) was injected as an i.v. bolus. These doses were selected on the basis of pilot studies so that the effect of either CaB on MAP was almost equivalent and sufficient (ΔMAP, >20 mm Hg) but not excessively hypotensive (MAP, <100 mm Hg) over 15 min after the bolus injection of micropuncture experiments.
Effects of CaB on Psf were assessed by using an in vivo micropuncture method. In the first series of experiments, Psf was monitored in SHRs prepared as described (n = 6) before and after the systemic administration of NDP (250 μg/kg body wt) or BDP (4 μg/kg body wt).
All micropuncture procedures and measurements were started after stabilization of blood pressure, 15-30 min after the completion of each treatment. The animals were prepared as described. The left kidney was fixed in a Lucite cup with 2% agar, and the kidney surface was immersed with warmed (37°C) saline. Proximal tubular segments were identified by injection of a small amount of saline colored with Lissamine green (Chroma, Köngen, F.R.G.) into a randomly chosen superficial tubular lumen with a micropipette. The middle proximal segments were completely blocked by solid wax (Merck, Darmstadt, F.R.G.) injected by a hydraulic pressure system (Effenberger, Attel, F.R.G.). The most proximal segment upstream from the wax block was gently punctured with a micropipette (o.d., 2 μm) filled with 2 M NaCl solution stained with Lissamine green and mounted in a servo-null micropressure system (WPI, Sarasota, FL, U.S.A.) to monitor stop-flow pressure (Psf). It is known that Psf measured at the most proximal segment is a good index of glomerular capillary pressure; however, the absolute value decreases as the segment for measurement is in the downstream of proximal tubule (8). Thus we compared the alteration of Psf in the same segment of each nephron. During the measurements, any segment revealing any leakage of the tinted solution was discarded.
The second series of experiments was done to assess the effect of CaB on Psf without alterations in systemic conditions. The animal was prepared for Psf measurements as described. The welling point near the obstructed nephron was gently punctured with a pipette with a tip diameter of 8 μm attached to a microperfusion pump system (Effenberger). The perfusion pipette was filled with colored (Lissamine green) Ringer solution containing only the solvent or a solution of either NDP (10−3M, n = 6) or BDP (10−3M, n = 6). With this technique, it is not possible to determine the dose-response relation between the amount of substance perfused and its resulting concentration in the surrounding interstitial space (9). Thus we used the same concentrations of CaBs and fixed the capillary perfusion rate in all experiments at 20 nl/min, which was chosen because a peritubular perfusion at the rate was reported not to obstruct normal peritubular blood flow (10). The perfusion procedure was judged satisfactory if the interstitium surrounding the obstructed proximal segment was colored. Psf was continuously monitored during the peritubular capillary perfusion.
After the stabilization of Psf, the values were examined, and the maximal transforming growth factor (TGF) response also was evaluated with the conventional orthograde loop perfusion (40 nl/min) with artificial tubular fluid (ATF; Na, 140 mM; Cl, 140 mM; K, 4 mM; Ca, 4 mM; HCO3, 8 mM; urea, 7.5 mM) through the micropipette (o.d., 10 μm) attached to a second microperfusion pump. As TGF is known to be attenuated by CaB (10,11), we monitored the TGF response during peritubular capillary perfusion to confirm the effect of the perfusate on the vasomotor action of microvasculature near the glomerulus.
After recovery from the TGF response, alterations of Psf to reductions in RPP were subsequently monitored during simultaneous peritubular capillary infusion of control solution or CaB solutions. RPP was reduced from steady-state pressure in steps by constricting an adjustable manual clamp around the abdominal aorta at a site above the origin of left renal artery. The efficacy of autoregulation of Psf was assessed to compare the mean of autoregulation index (AI) calculated within each subjected nephron as follows: Equation (1) where Psf and RPP are denoted by subscripts, BL (value in baseline condition) and CL (values during subsequent aortic clamp). An AI of 0 indicates perfect autoregulation, with larger values indicating less efficient autoregulation.
All values are expressed as mean ± SEM. Analysis of variance (ANOVA) was used to test for statistical significance of differences among the values observed in each CaB administration. The data obtained from the same kidney or single nephron were compared by using Student's paired t test. The relation between Psf and RPP was assessed by calculating the linear regression for individual tubules and calculating the average linear-regression line. A p < 0.05 was considered to have statistical significance.
Alterations in renal hemodynamics seen with the CaB treatments are depicted in Fig. 1. MAP, RBF, and renal vascular resistance (RVR) under baseline conditions were 160.6 ± 7.5 mm Hg, 6.5 ± 0.4 ml/min, and 25.9 ± 2.9 mm Hg/min/ml in NDP-treated SHRs, and 163.0 ± 7.5 mm Hg, 6.4 ± 1.8 ml/min, and 27.3 ± 3.1 mm Hg × min/ml in BDP-treated SHRs, respectively, values fairly comparable between the two groups. MAPs at 1 and 3 min after CaB injection were 115 ± 8 and 113.0 ± 8.0 mm Hg, respectively with NDP, values significantly lower than corresponding values with BDP, 150.0 ± 5.9 and 141.1 ± 4.2 mm Hg. More than 5 min after CaB administration, MAP was not significantly different between the two groups. Effects of both CaBs on RBF were not significant. However, there was a tendency for NDP to decrease RBF, but BDP increased it, leading to a significant decrease in RVR in the BDP-treated group; NDP elicited no comparable reduction in RVR.
Effect of i.v. CaBs on Psf
Changes in Psf before and after the systemic administration of CaBs are shown in Fig. 2. MAP during Psf measurements was almost comparable between each CaB treatment: baseline, 151 ± 4 mm Hg with BDP and 152 ± 3 mm Hg with NDP; and experimental, 120 ± 8 mm Hg with BDP and 118 ± 2 mm Hg with NDP. However, compared with the respective Psf during baseline conditions, BDP led to significant decrease in Psf (from 33.5 ± 1.2 to 28.6 ± 1.3 mm Hg; p < 0.01), whereas NDP caused a nonsignificant increase in Psf (from 32.8 ± 1.1 to 34.3 ± 2.2 mm Hg). There was a significant difference in Psf between treatments of NDP and BDP (p < 0.05).
Effects of peritubular CaBs administration on Psf
Effects of topical applications of solvent or CaBs on Psf and the maximal TGF responses are shown in Fig. 3. MAP did not significantly change throughout these procedures. Peritubular perfusion with solvent without CaB elicited no significant change in Psf, from 32.7 ± 2.0 to 32.0 ± 1.7 mm Hg in the BDP group or from 34.2 ± 1.3 to 34.0 ± 1.4 mm Hg in the NDP group. During the peritubular capillary infusion of the solvent, loop perfusion with ATF at 40 nl/min significantly decreased Psf, 23.1 ± 1.2 mm Hg in the BDP and 25.4 ± 1.0 mm Hg in the NDP groups. After recovery, Psf showed a slight but nonsignificant increase during the capillary infusion of BDP, from 33.2 ± 1.6 to 35.3 ± 1.8 mm Hg, and a significant increase during NDP infusion, from 33.5 ± 2.2 to 40.6 ± 2.0 mm Hg; p < 0.05. Under these conditions, TGF response to tubular perfusion with ATF (40 nl/min) was almost abolished with BDP (from 35.3 ± 1.8 to 34.5 ± 1.0 mm Hg) and with NDP (from 40.6 ± 2.0 to 39.1 ± 1.6 mm Hg).
Effects of peritubular perfusion with CaB on the relation between Psf and RPP
The change in Psf during alteration of RPP is shown in Fig. 4. The mean slope of the correlation lines in NDP-treated nephron (Psf = 0.292 × RPP − 9.664) is steeper (but not significantly) than seen during peritubular capillary perfusion with solvent (Psf = 0.189 × RPP + 5.665) or BDP (Psf = 0.198 × RPP − 0.029). AI (Fig. 5) in nephrons perfused with NDP (1.828 ± 0.287) is significantly higher than that in those treated with solvent (0.884 ± 0.244) or BDP (1.021 ± 0.125).
Recently developed DHP CaBs have a slower onset and a longer duration of action compared with nifedipine or nisoldipine, which were developed earlier. Such diverse modes of action will elicit different systemic counteractions to CaB-induced antihypertensive effects and presumably lead to the different renal hemodynamic effects. We compared the renal or glomerular hemodynamic effects of two CaBs of different generations, BDP and NDP, in SHRs. We observed distinct effects on blood pressure and renal hemodynamics and a substantially different effect on Psf.
In our experimental settings, the effect of BDP on MAP began gradually and was sustained throughout the experiment (Fig. 1). On the contrary, a bolus injection of NDP elicited a rapid, excessive hypotension followed by gradual recovery. Although RBF did not significantly differ between the groups, the decline of RVR with NDP was almost negligible, whereas that observed with BDP was evident. The systemic administration of CaB accompanying the decrease in blood pressure leads to compensatory reflexes attenuating the renal vasodilatory response (4). Acute administration of CaB could augment sympathetic nerve activity (12) or intrarenal renin-an-giotensin system (13). Although we did not assess the in vivo hormonal or nervous system, we do speculate that the transient excessive hypotension observed after NDP administration evoked a much greater systemic counter-action, which could attenuate the hemodynamic effect of the inhibition of a voltage-gated Ca2+ channel, the target of dihydropyridine CaBs.
The new finding in the first part of our micropuncture study is that the in vivo effect of systemic CaB treatment on the glomerular capillary pressure differed between BDP and NDP. From in vitro studies showing preferential dilation of preglomerular microvessels with CaB (4,14-16), it is reasonable to consider that CaB may increase glomerular capillary pressure, especially in the case of an insufficient reduction in blood pressure. However, there is controversy regarding the in vivo glomerular hemodynamic effect of CaB, and there is little information, especially in the hypertensive condition, that systemic administration of CaB causes no change (7) or a decrease (6) in the Psf of SHRs. As Psf is an indicator of glomerular capillary pressure without the tubular flow (i.e., under no TGF regulation), Psf is not perfectly autoregulated and thus is highly dependent on the RPP (17). Therefore the magnitude of the hypotensive effect in each experimental condition may explain the different results seen in the studies. From findings that MAP during each Psf measurement was almost comparable between the two groups, the different change in Psf measured during systemic administration of the two CaBs (Fig. 2) is probably not the result of the simple reflection of the RPP but of the distinct systemic or intrarenal effects of each CaB.
To elucidate whether the effect of the two CaBs on Psf differs during the minimal alteration of the systemic condition, we compared Psf during the interstitial application of CaBs. The method we used was that reported by Mitchel and Navar (9), in which the perfusate would diffuse into the postglomerular capillary or interstitial space or both to gain access to the responsible glomerular vascular segments. The TGF response was almost completely blocked by the two CaBs applied (Fig. 3). Thus it is conceivable that interstitial immersion with the CaBs with the microperfusion technique would affect the vasomotor action of renal microvasculature, at least in the vicinity of the glomerulus.
Topically applied BDP did not affect Psf, whereas NDP significantly increased Psf(Fig. 3). Furthermore, attenuation of the stability of Psf was much more evident with NDP than with BDP (Fig. 4). Our observations of the NDP-treated nephron in SHRs are fairly comparable to those obtained by Mitchel and Navar (10), who used the same microperfusion technique with NDP (10−3M) or the structurally distinct CaB, verapamil (10−3M), although their study was done on normotensive Sprague-Dawley rats. From the attenuation of TGF with interstitial BDP, dilation of afferent arteriole is probable. Thus the insignificant change in Psf under BDP treatment suggests the simultaneous dilation of postglomerular vessels. Direct observation of the renal microvasculature in the isolated perfused hydronephrotic rat kidney revealed the dilative effect of BDP on glomerular efferent arterioles (18). In the in vitro perfused rabbit vessels (19) or the isolated perfused hydronephrotic rat kidney (20), the diameter of preconstricted efferent arterioles was negligibly reversed by bath application of NDP or nicardipine, whereas efonidipine (20) or manidipine (19,20) elicited sufficient dilation. From these findings, it is presumed that among DHP CaBs, there might be diversity in the mode of action on the renal microvessels. Carmines et al. (21) showed that intracellular Ca2+ levels in the rabbit efferent arteriole are less dependent on the voltage-gated Ca2+ channel, the target channel of CaBs (21). If such is also the case in rat renal microvasculatures, a yet-to-beidentified effect of certain types of DHPs may be responsible for the diversity in the effect of CaB on efferent arterioles.
The last part of our experiment revealed that the autoregulation of Psf also differs between NDP and BDP (Fig. 3). Under our experimental conditions, Psf represents the glomerular capillary pressure without tubuloglomerular feedback action and is considered to be regulated mainly by myogenic responses of preglomerular vessels to alterations in RPP (22). The attenuation of autoregulation (Figs. 4 and 5) and increase in Psf(Fig. 2) with NDP treatment suggests that the site of the effect was predominantly in the preglomerular vessels, which is comparable with other findings (10). Although myogenic mechanisms are exerted through the activation of the voltage-gated Ca channel, this difference in autoregulation may also be explained by the diversity in effect of the CaB on preglomerular vasculature.
In summary, we obtained evidence for the difference in renal hemodynamic effects between the DHP derivatives NDP and BDP in SHRs. Although part of the diverse effects may be explained by the different modes of action on the systemic condition, our micropuncture data suggest that the effect of CaB on pre- and postglomerular vasculature may also differ among DHP derivatives. The mechanism of the diverse effect of CaB on glomerular hemodynamics remains to be examined.
Acknowledgment: Benidipine hydrochloride was a generous gift from Kyowa-Hakko C.O. (Tokyo, Japan). We are deeply grateful to Mariko Obara for her secretarial assistance.
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Keywords:© Lippincott-Raven Publishers
SHRs; Renal hemodynamics; Calcium channel blocker; Dihydropyridine derivatives; Micropuncture; Stop-flow pressure