Previous studies showed that rats fed a high-fructose or high-sucrose diet develop arterial hypertension (1,2). The pathogenetic mechanism has not been explained, but concomitant hyperinsulinemia and hypertriglyceridemia were demonstrated in these animals (3), and the increase in blood pressure can be avoided by preventing the hyperinsulinemia (4). This experimental model of hypertension reproduces a pattern often observed in humans, because insulin resistance and hyperinsulinemia are often associated with hypertension, although a direct causal relation has not been clearly demonstrated (5).
Diabetic nephropathy is one of the main causes of uremia in Western countries (6); hypertension plays a negative prognostic role in the development and the progression of this disease (7). Previous studies demonstrated glomerular changes in rats fed a high-fructose diet (HFD) similar to those in diabetic rats (8). Left ventricular hypertrophy is a well-recognized negative prognostic factor in hypertensive patients (9). Kobayashi et al. (10) reported that myocytes rapidly become hypertrophic in rats fed a high-fructose diet. In a previous study, we showed increased presence of collagen III in the cardiac interstitium of rats after a 4-week high-fructose diet. Therefore both the cellular and interstitial changes of left ventricular hypertrophy have been demonstrated in these animals.
Although many studies evaluated the effect of calcium antagonists on diabetic nephropathy, there is still disagreement as regards the protective effect of these drugs (11-13). Previous studies showed that calcium antagonists are effective in preventing the increase in blood pressure induced by the high-fructose diet in rats (14,15). The aim of this study was therefore to evaluate whether lacidipine, a dihydropyridine-derivative calcium antagonist, is effective in preventing not only the increase in blood pressure but also the renal and cardiac changes induced by HFD in Wistar-Kyoto (WKY) rats.
After a 1-week training period for blood pressure measurements, 40 WKY male rats were divided into four groups. Group I was fed with a 60% fructose diet plus vehicle (0.5% Methocel); group 2 received HFD plus lacidipine, 0.3 mg/kg/day; group 3 received HFD plus lacidipine, 3 mg/kg/day; and group 4 received a normal diet plus vehicle. Protein, lipid, and electrolyte contents of both diets were similar; exact composition of the diets is reported in Table 1. Lacidipine or vehicle was administered daily by gastric gavage. The blood pressure was measured weekly by using a tail cuff with conscious rats. Twenty-four-hour urine collection was performed twice, at the beginning and end of the study, by means of a metabolic cage. Urine samples were measured, filtered, and stored at −18°C. After 4 weeks, and after a 5-h fasting period, the animals were anesthetized, a blood sample was taken from the ocular artery, and they were then killed. Kidneys and heart were removed immediately, and weighed and fixed in 10% neutral buffered formalin.
Twenty-four-hour urinary excretion of NO2−/NO3−, the stable metabolic products of NO, was determined by using the brucine method (16).
Insulin concentration was determined by radioimmunoassay (RIA). Plasma glucose, creatinine, and triglycerides were measured by Autoanalyzer.
After fixation, each kidney was dehydrated by means of ethanol (at increasing concentrations: 50, 70, 95, and 100%), clarified in xylene and embedded in paraffin (60°C) for light microscopy. Sections 7-μm thick were stained with hematoxylin and eosin. The outline of 100 glomeruli from each animal was digitized from the light microscopy with a videocamera and a computer-based analysis system (Zeiss MOP-Videoplan, Zeiss, Oberkochen, Germany). Glomerular sections were displayed on the computer screen, and their area was measured by an interactive procedure with an image-analysis software package (Kontron, Eching, Germany). A calculation was then made of the glomerular area, the area of the glomerular tuft, and that of the Bowman's space.
Immunohistochemistry was performed with the alkaline phosphatase method (Kit; Sigma Chemical Company, St. Louis, MO, U.S.A.). After deparaffinization and trypsinization, sections were permeabilized for 10 min with 0.2% Triton X-100 in phosphate-buffered saline (PBS). After three washes in PBS, the samples were incubated for 1 h at 37°C in 4% bovine serum albumin (BSA)/5% normal goat serum (NGS) to block nonspecific binding sites, and then reacted with the primary antibody, antifibronectin or anticollagen III rabbit polyclonal antibody (Chemicon, Temecula, CA, U.S.A.) at 4°C overnight. For this incubation, isoform-specific antisera were diluted 1:100 in 4% BSA/5% NGS in PBS. Slides were washed 3 times with PBS and then incubated for 1 h at 37°C with a 1:100 dilution of alkaline-phosphatase-conjugated anti-rabbit immunoglobulin G (IgG) in 4% BSA/5% NGS in PBS. After three washes, sections were developed by using a solution containing 0.1 mg/ml P-nitrotetrazole blue, 0.05 mg/ml 5-Br-4-Cl-3-indolylphosphate, 4 mM MgCl2, and 100 mM Tris-HCl (pH 9.6), which was added to 5 mM levamisole to inhibit endogenous alkaline phosphatase. The amount of fibronectin or collagen III deposits was graded by four independent observers in a semiquantitative manner. The scores assigned to each group were compared.
Statistical analysis was performed by using the SPSS\pc software package. All data are expressed as mean ± SD. The results obtained in the four groups were compared by using one-way variance analysis and the Scheffé range. Intragroup analysis was performed with Student's t test for paired data.
Blood pressure and body weight
Tail blood pressure was similar in all groups at the beginning, whereas at the end of the study, it was significantly higher in rats treated with HFD plus placebo and lower in those treated with lacidipine at both the lower and higher dosages, the higher dosage determining a more evident reduction (Table 2). Body weight was similar in all groups at the beginning and at the end of the study (Table 3).
Plasma glucose, insulin, triglycerides, and creatinine
Plasma glucose was significantly higher in rats treated with high-dose lacidipine. Plasma insulin was increased by an HFD, but this effect disappeared in rats treated with lacidipine. Plasma triglycerides were increased in rats treated with HFD and placebo, whereas they were similar in the other groups. Plasma creatinine was slightly lower in rats treated with low-dose lacidipine (Table 4), but this difference was statistically significant.
Twenty-four-hour urinary excretion of NO2−/NO3− was similar at the beginning of the study in all groups. It was significantly higher in rats fed an HFD, and it was unaffected by lacidipine (Fig. 1).
Renal weight, glomerular morphometry, and renal fibronectin deposits
There was a significant increase in renal weight, reported as mg/g body weight, in rats fed an HFD, this change was unaffected by treatment with lacidipine (Fig. 2). Total glomerular area, glomerular tuft, and Bowman's space were significantly higher in the group treated with HFD + placebo. This effect was completely absent in rats receiving lacidipine, both at the higher and lower dosage, although at Bowman's space, level the effect of lacidipine, 3 mg/kg, was statistically greater than that of 0.3 mg (Figs. 2-4).
Heart weight and collagen deposit
Heart weights were similar in all groups at the end of the study. The semiquantitative collagen III assay showed that in the group receiving an HFD plus placebo, the score was significantly higher than in the other groups, and that low-dose lacidipine significantly reduces but does not normalize collagen III presence in the heart, whereas high-dose lacidipine completely prevents collagen increase (Figs. 5 and 6).
Several antihypertensive drugs effectively prevent the increase in blood pressure induced by an HFD (14,15,17,18). In our study, lacidipine prevented this increase in a dose-dependent manner. However, previous studies did not evaluate the effect of antihypertensive treatment on the target organs. The only study of this kind was performed by us; we demonstrated the protective effect of losartan on the kidneys in rats fed an HFD. In those animals, losartan, administered for 4 weeks with HFD, effectively prevented the increase in blood pressure, the glomerular hypertrophy, and the renal deposits of fibronectin induced by the diet (19,20).
An increased glomerular filtration rate was described in the early stages of diabetes and in rats fed an HFD (21,22). Many data indicated that glomerular hypertension plays an important role in the genesis and progression of diabetic glomerular damage (21). Because calcium antagonists cause the vasodilatation of afferent arterioles and have only a minimal effect on efferent arterioles (or none at all), these drugs might be supposed to have no protective effect in diabetic nephropathy. However, the most important factor in preventing the progress of renal failure by hypertensive drug is probably strict control of blood pressure (23). Moreover, other pathways were suggested to support the potential protective role of calcium antagonists in diabetic nephropathy and in chronic renal failure. It was observed that these drugs can reduce glomerular hypertrophy, thus reducing the vessel radius and consequently the tension in the vessel wall (24). Moreover, it was reported that calcium antagonists can reduce mesangial entrapment of macromolecules, modulate platelet-activating factor (PAF) and platelet-derived growth factor (PDGF), counteract the effect of endothelins, or modulate the levels of messenger RNA (mRNA) for these agents (25,26). However, conflicting results have been obtained in tests on the nephroprotective effect of these drugs, both in animal models of renal diseases and in clinical trials (11-13). This study used glomerular hypertrophy and fibronectin deposits as markers of the renal damage induced by an HFD. In previous studies, the renal changes reported in rats after long-term HFDs were indistinguishable from those in diabetic rats (8). We observed that glomerular hypertrophy occurs in WKY rats after a 4-week HFD; this change is similar to that observed in rats rendered diabetic by injection of streptozotocin (20) and was prevented by lacidipine.
Fibronectin is a dimeric glycoprotein found in the extracellular matrix of most tissues and serves as a bridge between cells and the interstitial collagen network. Multiple fibronectin forms arise by the alternative splicing of a primary transcript originating from a single gene. Fibronectin also regulates cell migration and proliferation and extracellular matrix formation during embryogenesis, angiogenesis, and wound healing. It appears to be present in increased amounts during glomerular injury and may play a role in the progression of glomerular injury (27). The results of this study show that lacidipine is effective in preventing the increase of fibronectin deposits in rats fed an HFD.
Because lacidipine seems to prevent glomerular hypertrophy and depositing of fibronectin in the kidney, it is reasonable to argue that effective nephroprotection can be achieved in this animal model of hypertension and hyperinsulinemia not only, as previously reported, by using drugs that work on the renin-angiotensin system, but also by means of a calcium antagonist.
In diabetic rats, increased urinary excretion of NO2−/NO3− was shown to be a consequence of an increase in both systemic and intrarenal production (28). Moreover, the differences in renal hemodynamics between healthy and diabetic rats may be canceled out by administering an NO-synthesis inhibitor (29). For this reason, it was suggested that NO might be responsible for the early changes in renal hemodynamics observed in diabetic rats. We demonstrated in previous studies that the urinary excretion of the stable metabolic products of NO is increased in rats fed an HFD, thereby suggesting a common pathway of renal changes in diabetes and in this model (30). The results of this study confirm that the urinary excretion of NO2−/NO3− is increased by an HFD. Lacidipine did not modify this parameter, which seems to indicate that the nephroprotective effect of the drug is not mediated by an influence on NO metabolism.
In recent years, there has been lively debate about the effect of dihydropyridines on cardiovascular risk, because some authors suggested that these drugs can increase cardiovascular mortality in hypertensive patients (31). Subsequent studies have not confirmed these data, but the question still needs to be resolved. Left ventricular hypertrophy is a well-known negative prognostic factor in hypertensive patients (9). An HFD can induce left ventricular hypertrophy in rats. Kobayashi et al. (10) demonstrated that myocyte hypertrophy can be prevented in rats fed an HFD by blocking AT1 receptors. The collagen network in the heart is largely influenced by the load, and it has been reported that pressure overload increases collagen content within the left ventricle and may lead to the development of increased resting tension (32). Collagen I is the main form of cardiac collagen in the healthy heart, whereas an increase in collagen III is more evident in the early stages of left ventricular hypertrophy (33). The results of this study show that collagen III is increased in the heart of rats fed an HFD, and that lacidipine prevented this change in the interstitial compartment of the heart.
As expected, the results of the laboratory assays performed for evaluating the metabolic changes induced by the diet show an increase in concentrations of plasma insulin and triglycerides. Other authors previously demonstrated that some antihypertensive drugs prevented the metabolic abnormalities caused by an HFD. Navarro-Cid et al. (17) demonstrated that losartan reduced plasma insulin but not hypertriglyceridemia. Rilmenidine improved glucose use during a euglycemic hyperinsulinemic clamp but did not reduce plasma insulin levels (18). Enalapril reduced insulin and triglyceride levels (34). In our study, the increase in plasma insulin and triglycerides was prevented by lacidipine in a dose-dependent manner, whereas plasma glucose concentrations tended to be higher in treated rats. Further studies are needed to explain the effect of lacidipine on the metabolic changes induced by an HFD.
In conclusion, in this animal model of hypertension and hyperinsulinism, lacidipine not only prevented the hypertension induced by an HFD, but it also had a protective effect on the kidney and heart. These encouraging results suggest that further studies are warranted to confirm the protective effect of this drug in hypertensive diabetic patients.
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