Effects of Amlodipine on Glomerular Filtration, Growth, and Injury in Experimental Hypertension
Abstract The objective of this study was to determine whether the calcium antagonist amlodipine could slow the progression of chronic renal disease. We examined the effects of amlodipine on kidney structure and function in two experimental models of hypertension. In the first study, adult, male Munich Wistar rats underwent uninephrectomy and were given weekly injections of desoxycorticosterone and 1% saline for drinking. Rats ingested normal chow or chow containing amlodipine for 8 weeks. The drug reduced systemic blood pressure, but glomerular filtration rate, kidney weight, proteinuria, and morphological evidence of glomerular injury were not affected. In the second study, male spontaneously hypertensive rats underwent uninephrectomy at 5 weeks of age and were followed for 6 months, during which they received no therapy or amlodipine. The drug dose was determined in preliminary studies to be the highest dose not associated with marked growth retardation. Again, although systemic blood pressure was significantly reduced by amlodipine, proteinuria and the prevalence of glomerulosclerosis were similar in amlodipine-treated and control spontaneously hypertensive rats. Micropuncture studies revealed that glomerular pressure remained elevated in amlodipine-treated spontaneously hypertensive rats. Kidney weight and glomerular volume were also similar in amlodipine-treated and control rats. Amlodipine also failed to inhibit platelet aggregation. Therefore, antihypertensive therapy with amlodipine fails to reduce glomerular pressure in spontaneously hypertensive rats as well as glomerular size and injury in spontaneously hypertension rats and desoxycorticosterone-salt hypertension. Although other dihydropyridine calcium antagonists have been found to reduce experimental glomerular injury, these data suggest that amlodipine may not prevent hypertensive nephrosclerosis.
Because glomerular filtration rate declines progressively in chronic forms of renal disease, most kidney patients eventually require dialysis or transplantation. Recent studies in rats1 2 3 and humans4 5 suggest that the rate of decline in renal function can be significantly reduced by antihypertensive therapy. However, all agents may not be equally effective in preventing kidney damage.6 In particular, controversy exists regarding the effects of calcium antagonists on progressive renal injury, and both positive3 7 and negative8 results have been reported.
In previous studies, we developed the DOC-salt–treated hypertensive rat6 9 and the uninephrectomized SHR as models of progressive renal failure.2 3 10 In both models, rats develop severe systemic hypertension, proteinuria, and progressive glomerulosclerosis. Glomerular injury is associated with increases in PGC9 10 and Vg.3 9 In DOC-salt rats, administration of the calcium antagonist nifedipine reduces Vg and lessens glomerulosclerosis.6 In uninephrectomized SHR,3 nifedipine also reduces PGC, Vg, and morphological evidence of glomerulosclerosis. In the present study, we examined the effect of a new, longer-acting dihydropyridine calcium antagonist, amlodipine, on glomerular injury in DOC-salt rats and uninephrectomized SHR. Our study suggests that even structurally similar calcium blockers such as amlodipine and nifedipine can have markedly different effects on glomerular morphology and function.
All studies were performed in rats in accordance with applicable portions of the Animal Welfare Act (PL 99-158 as amended) and the guidelines prescribed in Department of Health and Human Services publication No. 85-23 Guide for the Care and Use of Laboratory Animals, National Institutes of Health implementing instructions, and were approved by the animal use and care committee of our institution. Laboratory animals were housed in the New York University animal facility, which has an approved General Assurance Statement of Compliance with US Public Health Service guidelines.
Studies in DOC-Salt Hypertensive Rats
Description of Groups
Studies were performed in two groups of male Munich Wistar rats with initial weights of 225 to 250 g. All rats underwent a right nephrectomy under pentobarbital (50 mg/kg IP) anesthesia via a flank incision. Rats were given weekly subcutaneous injections of DOC acetate (25 mg) in peanut oil and 1% saline for drinking. Rats ingested either normal chow (CON, n=9) or chow containing amlodipine (AM, n=9) in a concentration that delivered a daily dose of approximately 10 mg/kg body wt. This dose was identified in preliminary studies as one that significantly reduced systolic BP in this model. Because amlodipine may be light sensitive, rats were housed in covered cages and fresh chow was provided daily.
Rats were followed with weekly determinations of weight and biweekly determinations of awake systolic BP and protein excretion rate. After 8 weeks, glomerular filtration rate was determined by inulin clearance, and the kidney was perfusion fixed, weighed, sectioned, and examined for morphological evidence of glomerular injury.
Studies in SHR
Six groups of SHR that underwent right nephrectomy under pentobarbital (50 mg/kg IP) anesthesia via a flank incision at 6 weeks of age were studied. CON rats were fed standard rat chow (Purina Mills, Inc) containing 23% protein and given tap water to drink. AM rats were fed identical chow, except that amlodipine (50 mg/kg body wt) was added to the chow before pelleting. This dose was derived from preliminary studies in which it was found to be the highest dose not associated with marked growth retardation of the rats. Lower doses failed to reduce BP effectively. All rats had free access to tap water. A CON (n=9) and AM (n=11) group of rats were followed for 6 months, with monthly determinations of 24-hour urinary protein excretion rate and awake systolic BP made during the first 5 months.
Protein Excretion and BP Measurements
Rats were placed in metabolic cages, and urine was collected for 24 hours. Protein concentration was measured by precipitation with 3% sulfosalicylic acid, and turbidity was determined by measurement of absorbance at 595 μm with a spectrophotometer (Spectronic 501, Milton Roy). Awake systolic BP was measured at 4-week intervals with a photoelectric tail-cuff device (model 29, IITC Inc) attached to a recorder (model SE 120, IITC Inc). For each rat, the value recorded represented the mean of four to six measurements obtained at a single time.
Inulin Clearance Study
Rats were anesthetized with thiobutabarbital (100 mg/kg IP). A polyethylene catheter (PE-50) was inserted into the femoral artery, and mean arterial pressure was measured by a Statham P23B pressure transducer connected to a recorder (model 7A, Grass Instruments). A tracheostomy was then performed, and polyethylene catheters were inserted into both jugular veins and the left ureter for infusion of test substances and collection of urine. To compensate for surgical losses and specimens drawn, all rats received an intravenous infusion of isoncotic rat plasma of 10 mL/kg body wt at a rate of 0.1 mL/min, followed by a sustained infusion of plasma at a rate of 0.5 mL/h. The rats also received a 0.5-mL intravenous bolus of inulin (10 g/100 mL) in saline followed by an infusion of 1.0 mL/h. After a 45-minute equilibration period, two timed urine collections of 15 minutes each were made. At the midpoint of each collection, 100 μL blood was collected in microhematocrit tubes.
At the conclusion of the clearance study, the kidneys were fixed in situ by perfusion for 5 minutes at the measured BP with 1.25% glutaraldehyde in 0.1 mol/L cacodylate buffer. The kidney was excised and weighed. Inulin concentrations were determined by the anthrone method,9 and inulin clearance was calculated by the standard formula.
Two coronal sections of tissue were embedded in paraffin for light microscopy. Sections 3 μm thick were stained with hematoxylin-eosin and periodic acid–Schiff. The incidence of glomerulosclerosis was quantitatively assessed in a blinded fashion by one observer (H.F.). In each rat, 150 to 200 glomeruli were examined. The percentage of glomeruli with glomerulosclerosis was calculated for each rat by dividing the number of abnormal glomeruli by the total number examined.
Morphometric measurements of Vg were made in the same kidney sections examined for morphological changes. Slides stained with hematoxylin-eosin were examined with the Zeiss Interactive Digital Analysis System (ZIDAS, Carl Zeiss Inc), and calculations were made based on standard stereological principles, as reviewed by Elias et al.11 For determination of Vg, the mean cross-sectional tuft area was obtained by tracing the outlines of capillary tufts of approximately 75 glomeruli per rat. In this method, it is assumed that the glomerulus is spherical and that the glomerular tuft cross-sectional areas represent random sections through a population of spheres that are distributed in a statistically predictable pattern. The area of each section depends on the diameter of the sphere and the distance of the section from the center of the sphere. From the mean cross-sectional area (Ag), Vg can be calculated by the equation Vg=B/k(Ag)3/2, where B=1.38, the shape coefficient for spheres; and k=1.1, the size distribution coefficient.
Additional groups of CON and AM rats underwent platelet studies 2 weeks after nephrectomy or micropuncture measurements of glomerular pressure 5 weeks after nephrectomy. At this point, sustained hypertension has developed, and the hemodynamic response to nephrectomy is complete; however, neither proteinuria nor morphological evidence of kidney damage is present.10
Studies were performed on platelets obtained from 44 CON and 48 AM uninephrectomized SHR. Platelet aggregation was performed in a four-channel aggregometer (Monitor IV Plus, Helena Laboratories) by the use of minor modifications of methods previously described.12 Rats were anesthetized with thiobutabarbital (100 mg/kg IP). The lower aorta was exposed via a midline incision and cannulated. Arterial blood was drained directly into plastic tubes containing heparin (final concentration, 10 U/mL) in phosphate-buffered saline (pH 7.4) with or without 3.8% sodium citrate (9:1, vol/vol). Platelet-rich and platelet-poor plasmas were prepared by differential centrifugation; platelet counts were determined by hemocytometer; and platelet-rich plasma was diluted with platelet-poor plasma to achieve a final platelet count of 200 000/mL. In each experiment, aggregation was induced by various concentrations in a random order of collagen (0 to 20 mg/mL) or ADP (0 to 200 mmol/L) in an alternating sequence of platelet-rich plasma samples from either CON or AM rats. Aggregation was quantified as previously described by determining the area under the curve of light transmission from 0 to 5 minutes by use of digital planimetry on a ZIDAS digitizing tablet. Each curve was measured three times. To facilitate comparisons between individual experiments, all data were expressed as percentages of the maximal aggregation observed in each experiment.
Micropuncture studies were performed in rats at 11 weeks of age. Rats were anesthetized with 100 mg/kg thiobutabarbital and prepared in the standard fashion for micropuncture.2 A tracheostomy was performed, and polyethylene catheters were inserted into the jugular veins, femoral artery, and ureter for infusion of solutions and collection of samples. The kidney was exposed via a subcostal incision, placed on a Lucite holder, and illuminated with a fiber-optic light. All rats received an initial infusion of isoncotic plasma equal to 10 mL/kg body wt followed by a sustained infusion of plasma at approximately 0.5 mL/h adjusted to maintain a stable hematocrit. Hydraulic pressure in cortical tubules was measured directly with the servo-null micropipette technique.10 Systemic protein concentration was determined by refractometry and plasma oncotic pressure calculated with the Landis-Papenheimer equation. Mean PGC was estimated as the sum of the stop-flow pressure and the plasma oncotic pressure, as described by Allison et al.13
Statistical analysis of glomerulosclerosis data was performed by the Kruskal-Wallis test (χ2 approximation) and for all other data by one-way unpaired t test. Statistical significance was defined at a value of P<.05. Data are reported as mean±SE.
Data from the studies performed in rats with DOC-salt hypertension are shown in Table 1⇓ and Figs 1⇓ and 2⇓. Rats in both groups gained a similar amount of weight from the time of nephrectomy to time of death. As expected, average values for awake systolic BP (Fig 1⇓) and mean arterial BP (Table 1⇓) were elevated in DOC-salt rats and reduced by amlodipine throughout the study. As shown in Fig 2⇓, there was a trend toward greater proteinuria in the AM rats; however, there was marked heterogeneity in protein excretion rates in this group, so the difference was not statistically significant. By the 8th week after nephrectomy, protein excretion rate in the CON rats had increased to a mean that was virtually identical to that seen in CON rats. Despite the reduction in BP, inulin clearance, morphological evidence of glomerular injury, and kidney weight were not altered by amlodipine administration.
Studies in Uninephrectomized SHR
There were no significant differences in body weight between the groups at any time during the study. At the conclusion of the study, CON rats weighed 394±7 g and AM rats 404±9 g. Mean values for awake systolic BP are shown in Fig 3⇓. BP was markedly reduced by amlodipine at every time point throughout the study. The effect of amlodipine on 24-hour urinary protein excretion is shown in Fig 4⇓. Proteinuria increased with time in both groups. Surprisingly, proteinuria in AM rats significantly exceeded that observed in CON rats at 4 and 5 months after uninephrectomy. Inulin clearance was similar in the two groups, averaging 0.033±0.007 mL/s in CON and 0.038±0.004 mL/s in AM rats. These values indicate that glomerular filtration rate had not declined significantly in either group by the time of death.
Morphological and Morphometric Data
Glomerular lesions in uninephrectomized SHR were mainly sclerotic, consisting of increases in both mesangial matrix and basement membrane material. Glomerular lesions were predominantly global in distribution, although segmental lesions were also observed. Both superficial and deep nephrons were affected. A complete description of the morphological changes observed in glomeruli of uninephrectomized SHR has been published elsewhere.2 3 A minority of glomeruli were abnormal in both groups, averaging 2.6±0.8% of glomeruli in CON versus 4.1±0.8% in AM rats. These values were not significantly different. Therefore, as assessed by either protein excretion rate or glomerular morphology, amlodipine failed to reduce the severity of glomerular damage in these rats. Morphometric studies indicated that amlodipine failed to inhibit renal growth in uninephrectomized SHR. Mean values for kidney weight and Vg were 3.14±0.10 g and 2.27±0.14×106 μm3, respectively, in CON rats versus 3.17±0.13 g and 2.86±.013×106 μm3 in AM rats. These values were not significantly different.
Mean values for weight, hematocrit, mean arterial pressure, PGC, proximal tubular pressure, efferent arteriolar pressure, the glomerular transcapillary hydraulic pressure difference, and the plasma protein concentration at the time of micropuncture are shown in Table 2⇓. As was the case for awake systolic pressure, arterial pressure in anesthetized AM rats was significantly reduced compared with CON rats. Despite this 35–mm Hg reduction in perfusion pressure, PGC and ΔP values remained elevated in AM rats.
Amlodipine had no significant effect on platelet aggregation, regardless of whether studies were performed in the presence or absence of extracellular calcium.
Studies in animals have suggested a general hypothesis to explain the progressive nature of renal disease. According to this theory, progressive kidney damage is caused by one or more adaptations of the kidney to a partial loss of function. At first, these adaptations appear to be beneficial because they tend to return total kidney function toward normal; however, in the long term, they cause nephron damage. Then, as additional units are injured secondarily, more extreme adaptations develop in those that remain until all nephrons are destroyed.
Although a number of adaptations of damaged kidneys have been associated with progressive renal failure in animals, a large body of evidence implicates two specific alterations as important in this process: (1) an increase in PGC and (2) compensatory kidney growth. PGC is high in experimental diabetes14 and in models characterized by systemic hypertension and reduced renal mass.9 14 15 Furthermore, maneuvers that reduce PGC lessen damage.1 2 3 14 15 For example, angiotensin-converting enzyme inhibitors have been consistently shown1 2 3 to reduce PGC and glomerular injury in a variety of settings. Less constant effects on glomerular pressure have been observed with other antihypertensive agents, including calcium antagonists. Anderson16 found that these drugs reduced glomerular pressure when given acutely to rats with hypertension consequent to a reduction in renal mass. On the other hand, Pelayo et al17 found no reduction in glomerular pressure when rats with remnant kidneys were chronically treated with verapamil. We examined the effects of chronic administration of the dihydropyridine nifedipine to rats with remnant kidneys,18 DOC-salt hypertensive rats,6 or uninephrectomized SHR.3 Although systemic hypertension was either entirely prevented or markedly ameliorated in all three models, glomerular pressure declined only in uninephrectomized SHR. Nevertheless, administration of the drug was associated with a reduction in glomerulosclerosis in all three studies. These findings suggest that calcium antagonists can reduce renal injury but that this effect does not depend on alterations in glomerular perfusion. Rather, it was linked to inhibition of renal growth, assessed morphometrically by analysis of kidney weight and Vg.
Because both the size and perfusion of surviving nephrons generally increase in damaged kidneys, it has been difficult to assess the independent contributions of glomerular hypertension and hypertrophy to injury. For example, maneuvers such as dietary protein restriction19 20 or antihypertensive therapy with angiotensin-converting enzyme inhibitors,20 which were initially felt to lessen glomerular injury by reducing glomerular pressure, also inhibit renal growth. In fact, it may be that injury is most severe when both glomerular hypertrophy and hypertension are present.
Yoshida et al21 compared two groups of rats in which renal excretory function was diminished by five sixths. Both groups underwent infarction of two thirds of one kidney; however, in one group the contralateral kidney was removed, and in the other it was left in situ but its ureter was diverted to drain into the peritoneal cavity. Glomerular hypertension was present in both groups; however, hypertrophy developed only when one kidney was completely removed. Severe injury was also limited to this group. Meyer and Rennke22 compared the effects of 50% reductions in renal mass produced by uninephrectomy or by segmental infarction. Hypertrophy developed in both groups; however, only infarcted rats had glomerular hypertension. As in the study of Yoshida et al,21 injury was observed only in the group in which glomerular hypertension and hypertrophy coexisted.
Salt intake also has a major effect on kidney size and compensatory renal growth. We23 reported that salt restriction reduced glomerular size and injury in uninephrectomized SHR despite persistent elevation in glomerular pressure. Similar observations have been made in rats with remnant kidneys.24 25 In this model, the beneficial effect of the low salt diet is significantly abrogated when its antihypertrophic effect is blocked by simultaneous administration of an androgen.24 In addition to salt restriction, antihypertensive agents may also inhibit renal growth and lessen glomerular injury by this mechanism. Like nifedipine, verapamil has been found to suppress compensatory renal growth after uninephrectomy26 and glomerulosclerosis7 in the remnant kidney model without reducing glomerular pressure.17 Similarly, Yoshida et al20 suggested that the beneficial effects of enalapril and of the combination of hydralazine, hydrochlorothiazide, and reserpine on glomerular structure in remnant kidney rats resulted from inhibition of glomerular growth. Taken together, these studies provide convincing evidence that glomerular damage is promoted when renal growth is stimulated.
In the present study, amlodipine produced a marked fall in BP in both DOC-salt rats and uninephrectomized SHR. The magnitude of the decline in systemic pressure was significant, albeit slightly less than that achieved in our previous studies3 6 of the effects of nifedipine in these models. Unlike nifedipine, amlodipine failed to reduce glomerular injury in these rats. Consistently, amlodipine also did not reduce glomerular pressure or size in uninephrectomized SHR. Although not compared in a single experiment, many aspects of the nifedipine and amlodipine studies were similar, including the species and models examined, the diets used, and the methods used to assess functional and morphological changes in our rats. Taken together, these findings suggest that structurally similar calcium antagonists can have markedly different effects on renal morphology and function that are not closely correlated with changes in systemic BP. However, because we did not anticipate that amlodipine would be less effective than nifedipine in preventing glomerular injury, we did not design our experiment to evaluate differences between the drugs and they were not directly compared in this study. As a result, other factors could account for the differences between the two studies, including drug dose, degree of BP reduction, food intake, or other unknown variables. These questions can be definitively resolved only by another study in which these two agents are directly compared.
The explanation for the failure of amlodipine to inhibit renal growth and injury was not specifically addressed by this study. One possibility, suggested by the observation that systemic BP is quite labile in some models of hypertension,27 is that amlodipine failed to consistently reduce BP in our rats. In fact, BP was measured intermittently, and it is possible that transient elevations in BP might have been missed. However, to our knowledge, there is no direct evidence and little reason to suspect that significant differences in BP lability would exist between rats treated with amlodipine as opposed to nifedipine. In addition, we routinely vary the time of day at which we measure systemic BP. Although not as sensitive as 24-hour BP recording, we have not observed significant differences between pressures measured during the morning and evening hours. Therefore, it seems unlikely that significant, sustained, and unrecognized elevations in BP existed in our rats.
Alternatively, if one assumes that the degree of reduction in systemic BP we observed was accurate and indicative of the extent of blockade of voltage-dependent calcium channels in vascular smooth muscle, then these channels were inhibited to a similar extent in the present and in the previous nifedipine study.3 Logically, the failure of amlodipine to reduce injury might result from a lesser effect of the drug on other tissues, such as the kidney. Of note, although a member of the dihydropyridine family, amlodipine has unique properties that distinguish it from other agents in this class, including nifedipine. As a result of the unique presence of a basic amino side chain on the dihydropyridine ring, amlodipine is positively charged at physiological pH.28 This alters the interactions between amlodipine and both membrane and circulating lipids,29 affecting the volume of distribution, receptor interactions, and pharmacokinetics of the drug. Receptor studies indicate that amlodipine binds to calcium channels not only at the dihydropyridine site but also at the verapamil30 and diltiazem28 sites. The biological consequences of these pharmacological differences are largely unknown; however, in a recent study, subtle differences in diastolic cardiac function were observed between rats treated with amlodipine and those treated with nifedipine.31 These data are consistent with our hypothesis that these drugs may have divergent actions in some tissues.
One additional factor that has been related to progressive kidney damage is coagulation. In SHR,32 administration of heparin to young rats attenuates the rise in BP, suggesting that intravascular thrombosis contributes to the development of hypertension. Morphological studies in the related remnant kidney model33 reveal platelet thrombi occluding glomerular capillaries. In the remnant kidney model, anticoagulant therapy with heparin,34 warfarin,34 or any of a number of antiplatelet agents29 35 lessens injury. Calcium entry is a key event in the platelet aggregation process, and calcium antagonists suppress aggregation in some settings.36 If amlodipine also inhibited platelet aggregation in our rats, this might have reduced glomerular damage. In fact, amlodipine in the dose we administered failed to alter platelet aggregation in vivo, again consistent with its failure to reduce glomerular injury.
In summary, although systemic BP was significantly reduced in DOC-salt rats and uninephrectomized SHR chronically treated with amlodipine, glomerular injury was not avoided. Micropuncture and morphometric studies indicated that neither glomerular pressure nor compensatory kidney growth was reduced by amlodipine. These findings are consistent with a role for glomerular hypertension and hypertrophy in promoting glomerulosclerosis. They suggest that all calcium antagonists may not be equal in their ability to prevent progressive kidney damage. This issue should be further examined in direct comparisons of various agents in individual models of renal disease.
Selected Abbreviations and Acronyms
|PGC||=||glomerular capillary hydraulic pressure|
|SHR||=||spontaneously hypertensive rat(s)|
|Vg||=||glomerular tuft volume|
This study was supported by a grant from Pfizer Laboratories, Inc.
Reprint requests to Dr Lance D. Dworkin, Division of Renal Diseases, Rhode Island Hospital, 593 Eddy St, Providence, RI 02903.
- Received September 16, 1994.
- Revision received November 11, 1994.
- Accepted October 25, 1995.
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