Since the recognition of the vasodepressor action, the calcium antagonist is widely used as an antihypertensive agent (1). Controversy attends the effect of the calcium antagonist as to whether this agent protects against the renal injury. It has been reported that nifedipine fails to prevent the progression of renal injury in diabetic nephropathy (2). Furthermore, in subtotal nephrectomized rats, an animal model of renal injury, amlodipine aggravates the renal damage (3). In contrast, several recent studies demonstrated favorable effects of the calcium antagonist on renal injury (4). We also have reported that efonidipine, a novel calcium antagonist, potently prevents the development of renal injury in subtotally nephrectomized spontaneously hypertensive rats (SHRs) (5). This agent possesses a unique action on renal microcirculation; in contrast to the predominantly afferent arteriolar vasodilator action of traditional calcium antagonists (6), efonidipine elicits both afferent and efferent arteriolar vasodilator action (7). Teleologically, efferent arteriolar dilation should result in improvement in glomerular hypertension. The role of the efferent arteriolar dilation by calcium antagonists in protecting against the renal injury, however, remains undetermined.
Aranidipine (MPC1304) (Fig. 1, top), a novel dihydropyridine-type calcium antagonist, has been established as a potent antihypertensive agent, and is reported to be more potent than other dihydropyridines, nifedipine, nicardipine, nitrendipine, and nisoldipine in reducing blood pressure in SHRs (8,9). Furthermore, when administered in vivo, aranidipine is metabolized to M-1 (Fig. 1, bottom), which also possesses calcium antagonistic activity. Thus unlike other calcium antagonists, the in vivo action of aranidipine is unique in that both the original and its metabolite exert their action in concert to dilate vascular beds. These two compounds, however, possess different actions on vasomotor mechanisms (10), which may affect the renal microcirculation differently. However, the effect of these compounds on renal microcirculation has not been examined. Furthermore, their renoprotective action has not been assessed.
In this study, we examined the vasodilator action of aranidipine and its active metabolite, M-1, on renal afferent and efferent arterioles, using the isolated perfused hydronephrotic rat kidney, which allowed direct visualization of renal microcirculation. The long-term effects of these compounds on the progression of renal injury were assessed in subtotally nephrectomized SHRs. This study demonstrates differing microvascular action of aranidipine and its active metabolite, which may facilitate the understanding of the role of efferent arteriolar tone in the progression of renal injury.
Isolated perfused hydronephrotic kidney model
Isolated perfused hydronephrotic rat kidneys were used to determine the effects of aranidipine and M-1 on the afferent and efferent arteriolar response to norepinephrine (NE). This model is a modification of the in vivo hydronephrotic kidney preparation (11,12) and lacks in the influence of humoral and neural factors that may modulate the renal microvascular responsiveness to vasoactive stimuli. For the preparation of donor animals with unilateral hydronephrosis, 6-week-old male Wistar-Kyoto rats (Charles-River Japan, Kanagawa, Japan) were anesthetized with ether. The right ureter was ligated through a small midabdominal incision. After 8 to 10 weeks, at which time renal tubular atrophy had progressed to a stage that allowed direct microscopic visualization of the renal microvessels, kidneys were harvested for perfusion study (7,13).
The animals were anesthetized with ether. The renal artery of the hydronephrotic kidney was cannulated in situ by introducing the arterial cannula through the superior mesenteric artery and across the aorta. Perfusion with warm oxygenated media was initiated during the cannulation procedure. The perfused kidney was excised and placed on the stage of an inverted microscope modified to accommodate a heated chamber equipped with a thin glass viewing port on the bottom surface. Kidneys were allowed to equilibrate for ≥30 min before initiation of the experimental protocols.
The perfusion media consisted of a Krebs-Ringer bicarbonate buffer containing 5 mM D-glucose (WAKO, Tokyo, Japan), 7.5 g/dl bovine serum albumin (Sigma, St. Louis, MO, U.S.A.), and a complement of amino acids (14). The perfusion apparatus used in this study was a modification of the one described in previous publications (13). The perfusion medium was saturated with a gas mixture of 95% O2/5% CO2. Perfusion pressure was monitored at the level of the renal artery, and was maintained constant at 80 mm Hg by an adjustable back-pressure type regulator (model 10BP; Fairchild Industrial Products Co., Winston-Salem, NC, U.S.A.).
Video images from a CCD camera (XC-77; Sony, Tokyo, Japan) were recorded with a video recorder. To measure the vessel diameter, the video images were transmitted to an IBM computer equipped with a video acquisition and display board (Targa 16+; True Vision, Indianapolis, IN, U.S.A.). Vessel diameter was estimated with an automated program custom designed to permit determination of the mean distance between parallel edges of the selected microvessel. For determination of the vessel wall, a scanning line was set across the vessel, and the peak density in the gray-scale mode was deemed as vessel walls. Mean vessel diameter was determined by averaging all measurements obtained during the plateau of the response.
The ability of aranidipine (Taiho Pharmaceutical Co., Ltd., Tokyo, Japan) and its active metabolite, M-1 (Taiho Pharmaceutical Co.), to relax renal afferent and efferent arterioles was determined under the NE-induced (Sigma) vasoconstriction. Initially, NE was administered directly to the perfusion medium at a final concentration of 0.3 μM. Subsequently, increasing doses (10−8, 10−7, and 10−6M) of aranidipine and M-1 were added to reverse the NE-induced tone, and the afferent and efferent arteriolar responses to these agents were assessed. The vehicle for aranidipine and M-1 (i.e., DMSO) had no effects on renal microvascular tone.
Subtotally nephrectomized SHR model
Six-week-old male SHRs (Charles River Japan, Kanagawa, Japan) weighing 170-200 g were studied. They were placed in individual metabolic cages, and were initially fed a standard rat chow (15 g/day, 0.38% sodium, 0.97% potassium, and 25.1% protein; Nippon Clea, Tokyo, Japan) and given water ad libitum. Subtotal nephrectomy was performed by removal of two thirds of the left kidney (week 0), followed by total right nephrectomy (week 2) under ether anesthesia (15). The standard chow was replaced with a high-salt chow (15 g/day, 5% NaCl) (week 2). Rats were divided into three groups; aranidipine-treated group (3 mg/kg; n = 8), M-1-treated group (10 mg/kg; n = 8), and control group (n = 11). They were allowed free access to tap water throughout the experiments.
Body weight, systolic blood pressure (SBP; measured by tail-cuff method; KN-210; Natsume, Tokyo, Japan), and 24-h urinary protein excretion were evaluated before initiation of nephrectomy and every 2 weeks throughout the study. Blood pressures were measured by a well-trained investigator to eliminate measuring bias (16). At week 10, rats in each group were decapitated, and blood was collected for measurement of serum total protein, creatinine, and urea nitrogen. After the collection of blood, the remnant kidneys of the rats were harvested and fixed with 10% formaldehyde for 1 min. They were embedded in paraffin. The sections were stained with periodic acid-Schiff reagent for light-microscopic examination.
Renal histopathologic evaluation was conducted by two independent pathological experts, who were not informed of the kinds of antihypertensive agents used. Histopathologic examination was performed semiquantitatively as previously described (17). One hundred glomeruli, all sections of blood vessels, and tubulointerstitium were evaluated. Glomerular alterations including mesangial matrix expansion, segmental to global sclerosis, and hyalinosis were graded 0-3 according to the percentage of surface area of involvement in each glomerulus: grade 1 represented involvement <30% of a sectioned glomerular area, and grade 3 indicated >60%. Final overall score was calculated as the mean of all grade numbers multiplied by 100. All the observed sections of blood vessels were graded 0-4 for arteriolar sclerosis according to the severity of hyalinosis and thickening of the vascular wall, and the final overall score of vascular alteration was calculated as the glomerular alteration. Tubulointerstitial changes, including interstitial broadening and tubular atrophy, were assessed and graded 0-3: grade 1 was involvement of <20% of the cortical interstitium, and grade 3 was involvement of >40%.
All experimental procedures were approved by the Keio University Animal Care and Use Committee.
Data are expressed as mean ± SEM. Data were analyzed by two-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison post hoc tests. Histologic results were analyzed by Mann-Whitney nonparametric test. A value of p < 0.05 was considered statistically significant.
Renal arteriolar action of aranidipine and M-1
The administration of NE caused marked constriction of both afferent (from 14.4 ± 0.6 to 9.8 ± 0.9 μm; p < 0.001; n = 20) and efferent arterioles (from 12.6 ± 0.7 to 8.6 ± 0.6 μm; p < 0.001; n = 11), with 33 ± 3% and 31 ± 4% decrements in diameter, respectively (Fig. 2, left).
Subsequently, aranidipine was added to the perfusate. Aranidipine elicited dose-dependent dilation of afferent arterioles. At 10−6M, aranidipine restored afferent arteriolar diameter to 13.5 ± 0.7 μm (p < 0.001 vs. NE-induced constriction; n = 20; i.e., 83 ± 6% reversal from NE-induced constriction).
Similarly, aranidipine was very potent in inhibiting the NE-induced constriction of the efferent arteriole. At 10−6M, the efferent arteriolar diameter was returned to 12.1 ± 0.6 μm, corresponding to 90 ± 6% reversal from NE-induced constriction (p < 0.001; n = 6).
When afferent and efferent arteriolar responses to aranidipine were compared, it was evident that aranidipine inhibited these microvessels by a similar magnitude. No differences in arteriolar responses were observed between these microvessels (p > 0.05).
In additional series of experiments, the ability of M-1 (an active metabolite of aranidipine) to reverse NE-induced constriction of renal microvessels was assessed (Fig. 2, right). M-1 inhibited the afferent arteriolar constriction at 10−7M (from 8.8 ± 0.5 to 11.8 ± 0.4 μm; p < 0.05; n = 9), and further dilation was observed at 10−6M (14.3 ± 0.5 μm; p < 0.05; n = 9; i.e., 79 ± 4% reversal from NE-induced constriction).
In contrast to the effect on afferent arterioles, M-1 caused diminished dilation of efferent arterioles. At 10−6M, efferent arteriolar diameter was modestly returned to 11.5 ± 1.2 μm (p > 0.05; n = 5; i.e., 44 ± 17% reversal from NE-induced constriction).
Renal protective effects in salt-loaded subtotally nephrectomized SHRs
In the control group, subtotal nephrectomy with salt loading caused prominent hypertension (Fig. 3). SBP was significantly increased at week 4 (221 ± 13 mm Hg; n = 11; p < 0.05 vs. 0 week), and remained progressively elevated, reaching 270 ± 6 mm Hg at week 10. In contrast, aranidipine nearly completely suppressed the elevation in SBP; at week 10, SBP (176 ± 11 mm Hg; n = 8) did not differ from the basal value (i.e., 172 ± 4 mm Hg at week 0). Similarly, the M-1-treated group had significantly lower SBP than the control group at week 4 (165 ± 7 mm Hg; n = 8; p < 0.05), and this hypotensive action persisted throughout the study. No difference in SBP was observed between aranidipine and M-1 groups.
Figure 4 depicts the effect of aranidipine on urinary protein excretion. In the control group, proteinuria was increased progressively from 84 ± 7 mg/day (at week 0, n = 11) to 305 ± 26 mg/day (at week 10, n = 11). In contrast, aranidipine partially prevented the increase in proteinuria. Thus at week 6, urinary protein excretion in the aranidipine-treated group was less than that in the control group (200 ± 2 mg/day; n = 8 vs. 257 ± 14 mg/day, n = 8; p < 0.05), and this protective effect persisted throughout the experimental period (week 8, 221 ± 15 vs. 304 ± 16 mg/day; p < 0.05; week 10, 237 ± 9 vs. 305 ± 26 mg/day; p < 0.05). In analogy, M-1 blunted the increase in proteinuria at week 6, and the proteinuria in the M-1-treated group tended to be less than that in the control group at weeks 8 and 10.
At the end of the study, blood chemistry and renal histology were examined. In both aranidipine- and M-1-treated groups, total protein was higher than that in the control group (Table 1). Similarly, aranidipine and M-1 improved renal functional parameters, including serum creatinine and urea nitrogen level.
Examination of renal histology revealed that both aranidipine and M-1 prominently ameliorated the renal injury scores (Fig. 5). Thus glomerular sclerosis was improved in both aranidipine-treated (0.09 ± 0.01; n = 8) and M-1-treated groups (0.19 ± 0.03; n = 8), compared with that in the control group (0.40 ± 0.09; n = 11). Similarly, arteriolar sclerosis index was markedly less in aranidipine-treated (0.29 ± 0.04; n = 8) and M-1-treated groups (0.29 ± 0.03; n = 8) than in the control (1.18 ± 0.20; n = 11). Furthermore, tubulointerstitial changes were markedly reduced by aranidipine (0.50 ± 0.19 vs. 1.91 ± 0.25 for control; p < 0.01), and tended to be less in the M-1-treated group (1.14 ± 0.2; p = 0.08). When compared with the effect of M-1, aranidipine was more potent in preventing the glomerular sclerosis (p < 0.05) and tubulointerstitial changes (p < 0.05). Either treatment did not improved matrix expansion.
Calcium antagonists, used widely as a therapeutic tool for hypertension, cause prominent vasodilation of the renal microvasculature. A growing body of evidence has been accumulated, however, that the renal vasodilation, particularly at the preglomerular arteriole, would facilitate the transmission of systemic blood pressure to the glomerulus, and may thus elicit glomerular hypertension (18). Such a consequence might lead to the progression of renal injury. Indeed, several calcium antagonists, with predominant action on the afferent arteriole, are reported to aggravate the renal injury in a variety of renal diseases, including diabetic nephropathy (2,19) and subtotal nephrectomy (20). In contrast, there have been developed novel types of calcium antagonists with divergent characteristics of chemical property, duration of action (21), and vasomotor activity distinct from voltage-dependent calcium channels (VDCC) (22,23). Furthermore, a couple of new calcium antagonists are reported to dilate efferent arterioles as well as afferent arterioles (7,24), and these agents are suggested to be beneficial for retarding the progression of renal disease (5,25). Thus the effects of calcium antagonists on renal microvascular hemodynamics and glomerular injury may vary depending on the types of the agents used.
In this study, we have demonstrated that both aranidipine and its active metabolite (M-1) possess marked vasodilator action on the renal microvasculature. The vasodilator action on afferent and efferent arterioles, however, differs between these compounds. Thus the vascular action of aranidipine is nearly the same in afferent and efferent arterioles, whereas its metabolite elicits predominant dilation of the afferent arteriole. Traditionally, calcium antagonists exert their action through VDCC, and these channels predominantly prevail on the afferent arteriole (26). It is anticipated, therefore, that calcium antagonists preferentially inhibit the vasoconstriction of the afferent arteriole. In contrast, we also reported that efonidipine and nilvadipine reverse the efferent arterioles in the isolated perfused hydronephrotic kidney model (7,24) and suggested that there existed the subgroup of calcium antagonists that acted on both afferent and efferent arterioles. As demonstrated in this study, the metabolic conversion of aranidipine to M-1 alters the efferent arteriolar action, suggesting that the metabolic change in chemical structure may constitute a determinant of the efferent arteriolar action of the calcium antagonist. In concert, this study supports our previous observations demonstrating heterogeneity in renal microvascular action of the calcium antagonist (6,7,24). Additionally, the association between the efferent arteriolar action and metabolic changes of the calcium antagonist may provide a clue to the understanding of the undetermined issue why certain types of calcium antagonists exert efferent arteriolar action.
Although aranidipine acts mainly on VDCC, this agent is also reported to affect other vasomotor systems. For example, Okumura et al. (23) reported that aranidipine-induced vasodilator action was diminished in the presence of tetraethyl ammonium. This finding suggests that aranidipine possesses vasodilator mechanisms other than the inhibition of VDCC, which are most likely associated with potassium channel activation. In contrast, M-1 has no such effect on this type of mechanism. In this regard, Reslerova et al. (27) have recently demonstrated that activation of potassium channels causes efferent arteriolar dilation independent of VDCC activation. Whether the efferent arteriolar action of aranidipine, but not M-1, is attributed to the activation of potassium channels needs additional investigation.
The salutary action of the calcium antagonist on renal disease remains a matter of controversy. In contrast to the well-established belief that angiotensin-converting enzyme inhibitors (ACEIs) blunt the progression of renal injury (2,5,15,20), the renal protective effect of the calcium antagonist is divergent (2-5). ACEIs dilate both afferent and efferent arterioles, whereas conventional calcium antagonists relax the afferent arteriole predominantly (6). The different renal microvascular action of these agents would therefore produce contrasting effect on glomerular hemodynamics; ACEIs reduce glomerular capillary pressure, whereas calcium antagonists favor glomerular hypertension. Nevertheless, our study indicates that aranidipine acts on both afferent and efferent arterioles. Indeed, in subtotally nephrectomized rats with hypertension, aranidipine reduces proteinuria and ameliorates glomerular and arteriolar injury as well as tubulointerstitial changes (28). In the same setting, M-1 exhibits a moderate decrease in proteinuria, but only partial improvement in renal injury scores. Because aranidipine and M-1 caused persistent reduction in SBP to the same level, the divergent renoprotective action of these two calcium antagonists could be attributed in part to differing renal microvascular actions of these agents.
Although this study reveals a more favorable effect of aranidipine on renal protection than M-1 in subtotally nephrectomized rats, a caveat is in order to clarify the role of efferent arteriolar action of aranidipine in preventing glomerular injury. Because aranidipine is metabolized mainly to M-1 (8), overall in vivo action of aranidipine should be a mixture of the effects of these compounds. Nevertheless, the half-life of aranidipine in rats is reported to be 5.3 h, whereas that of M-1 is 1.0 h (29). The renal protective effect of aranidipine therefore should be ascribed for the most part to the action of aranidipine per se, when administered in vivo, and a more favorable effect of aranidipine could be attributed to the efferent arteriolar action of this compound. Of note, M-1, with predominant afferent arteriolar action, also exerts renal protective effects, and reduces SBP. Thus our study supports the contention that the calcium antagonist retards the progression of renal injury when the blood pressure is controlled, and additional benefit could be offered when the calcium antagonist possesses efferent arteriolar action.
In conclusion, this study demonstrates that a novel calcium antagonist, aranidipine, causes vasodilation of both afferent and efferent arterioles. In contrast, its active metabolite, M-1, dilates predominantly the afferent arteriole, indicating that metabolic changes in the calcium antagonist alter the efferent arteriolar action of this agent. Furthermore, these two substances exert renal protective action in subtotally nephrectomized SHRs. The renal protective effect of aranidipine, however, exceeds that of M-1. It requires further investigations to clarify whether such a difference in renal protective action is attributable to the divergent action on the efferent arteriole.
Acknowledgement: This study was supported in part by a grant from Taiho Pharmaceutical Co., Ltd.
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