Renoprotective Effects Of Amlodipine In Combination With Eplerenone In Hypertensive Dahl Rats

Renoprotective Effects of Amlodipine in Combination with eplerenone in Hypertensive Dahl RatsBackground and ObjectiveHypertension affects around 1 billion people worldwide,and suboptimal control of blood pressure is the number one attributable risk for death throughout the world. Clinical studies have demonstrated that around 50%of hypertensive patients have salt-sensitve hypertension,a well-known independent risk factor for increasing cardiovascular morbidity and mortality,and progressive renal functional impairment. Renal dysfunction and injury have been reported to show better correlations with structural damage to the renal tubuloinsterstitium than with damage to the glomeruli.It has been demonstrated that chronic hypoxia in the tubulointerstitium is a final common pathway to end-stage renal failure.In addition,a number of mechanisms that induce tubulointerstitial hypoxia have been suggested;occurring as a consequence of a reduction in peritubular capillary(PTC) blood flow,i.e.,peritubular ischemia and injury. In hypoxia,ATP depletion causes cellular Ca²⁺ increase,mitochondrial injury and apoptosis in renal tubular cells.Tanaka et al.showed that an L-type Ca²⁺ channel blocker(CCB) attenuates mitochondrial injury and apoptosis in hypoxic renal tubular cells.Furthermore,treatment with CCBs restored ischemic/reperfusion-induced apoptosis and angiotensinâ…¡-induced peritubular ischemia.These data suggest that the reno-protective effects of CCBs reported in clinical studies may be mediated through inhibition of tubulointerstitial hypoxia.However,the mechanisms by which amlodipine elicits reno-protective effects in hypertensive subjects are not clear.Aldosterone is traditionally viewed as a hormone regulating electrolyte and blood pressure homeostasis by acting on the distal nephron.Recently,there has been a paradigm shift regarding the role of aldosterone.Growing evidence suggests that aldosterone plays an important pathogenetic roles in cardiovascular and renal injury.In the kidney, aldosterone promotes proteinuria,glomerulosclerosis,arteriopathy,and renal fibrosis. In aldosterone-infused rats,glomerular expressions of slit diaphragm-associated molecules nephrin and podocin were markedly decreased,whereas expression of a damaged podocyte marker desmin was upregulated.Proteinuria and podocyte damage were completely reversed by selective aldosterone blocker eplerenone.Thus,podocytes can be an important therapeutic target.In addition,the combined effects of amlodipine and eplerenone have not been investigated.In the present study,we examined the effects of amlodipine and eplerenone on the blood pressure and renal function in DS rats.In addition,we evaluated the therapeutic efficacy of the combination of amlodipine and eplerenone.Furthermore,we try to investigate the possible mechanisms of the beneficial effects of amlodipine in combination with eplerenone.MethodsSetting up salt-induced hypertension and renal injury model in Dahl salt-sensive rats:6-week-old Dahl salt-sensitive(DS) rats,weighing 165-180g,received 4% high-salt diet for 10 weeks.SBP was measured in conscious rats by tail-cuff plethysmography,and 24-hour urine samples were collected at every two weeks.At the end of the treatment,DS rats showed severe hypertension(SBP > 170 mmHg) and proteinuria(>70 mg/24h).Histological analysis also found severe glomerularsclerosis and tubulointerstitial fibrosis.These evidences confirmed the success of this animal model.Experimental groups:Male DS rats at 6 weeks of age were randomly selected to receive rat diet containing high salt(HS;4%NaCl;n=32;Oriental Yeast,Osaka,Japan) or low salt(LS;0.4%NaCl;n=8;Oriental Yeast) for 10 wk.The DS rats fed with high-salt diet were then randomly divided into the following 4 groups(8 rats in each group):control(HS);amlodipine 3mg/kg/day by gavage(HS+A);eplerenone 50mg/kg/day by gavage(HS+E) and amlodipine 3mg/kg/day combined with eplerenone 50mg/kg/day(HS+A+E).The treatments lasted from 3 to 10 weeks after the start of high-salt diet feeding.SBP was measured in conscious rats by tail-cuff plethysmography,and 24-hour urine samples were collected at every two weeks.Blood and kidney samples were collected at the end of week 10 under sodium pentobarbital anesthesia.Kidneys were perfused with chilled saline solution,then kidney sections were either fixed in 10% paraformaldehyde(pH 7.4) and embedded in paraffin for histological examination or frozen in Tissue-Tek O.C.T.compound for laser capture microdissection(LCM). Remaining renal tissues were snap-frozen in liquid nitrogen and stored at -80 8888888.1.Urine and blood biochemistryDetermine the renal functional impairment by examining the urinary protein and creatinine excretion,plasma creatinine level and creatinine clearance.We also determine the electrolyte homeostasis by checking the plasma sodium,potassium and aldosterone levels.2.Histological examinationEvaluate the glomerular size and glomerularsclerosis by PAS staining.We also evaluate the tubulointerstitial fibrosis by Azan staining.3.ImmunohistochemistryRenal hypoxia was examined by pimonidazole,which binds to tissues with pO₂ levels below 10 mmHg.PTCs loss was examined by immunohistochemistry of aminopeptidase P.Podocyte injury was examined by immunohistochemistry of desmin.4.LCM and RNA isolationWe microdissected the glomeruli tissues by LCM.Glomerular RNA was extracted using RNAqueous-Micro kits.5.Real-time PCRTotal RNA was reverse transcripted into cDNA.We examined the glomerular expression of podocin,nephrin,MR and Sgk-1 by Real-time PCR.6.Statistical analysisValues are presented as mean±SE.Statistical comparisons of the differences between treatments were performed using one-way analysis of variance(ANOVA) combined with the Bonferoni post hoc test for multiple comparisons.A value of P< 0.05 was considered statistically significant.Results1.Salt-sensitive hypertension and renal injury animal model was established successfully. 2.SBPHypertension developed progressively in the rats fed with high-salt diet,but didn't develop in the rats fed with low-salt diet.The hypotensive effects were comparable among all the treatment groups.3.Urinary Protein/Creatinine,Plasma Creatinine and Creatinine Clearance After 10 wk of treatment with a high-salt diet,rats showed a higher urinary protein/creatinine ratio as compared with rats received low-salt diet.Monotherapy with amlodipine or eplerenone could reduce urinary protein/creatinine ratio.Combination of amlodipine and eplerenone exhibited the best improvement of urinary protein/creatinine ratio.Plasma creatinine level was enhanced in the rats fed with high-salt diet compared with rats received low-salt diet.Monotherapy slightly decreased plasma creatinine.Combination treatment could significantly decrease the plasma creatinine level.In accordance with plasma creatinine,creatinine clearance was decreased in rats fed with high salt.Only the combination of amlodipine and eplerenone could significantly increase creatinine clearance compared with other monotherapy.4.Histological findingsRats that received low-salt diet showed almost normal glomeruli and tubulointerstitium;however,rats that received high-salt diet exhibited injured glomeruli characterized by sclerosis and enlarged glomerular size.In rats that received high-salt diet,severe tubulointerstitial fibrosis were also observed.Monotherapy with eplerenone but not amlodipine significantly inhibited glomerularsclerosis.However,single treatment with amlodipine could provide better improvement of tubulointerstitial fibrosis compared with eplerenone.Surprisingly,the combination of amlodipine and eplerenone dramatically reduced both glomerularsclerosis and tubulointerstitial fibrosis.5.Podocyte Injury caused by MR Upregulation and ActivationIn rats receiving a high-salt diet,glomerular expressions of podocin and nephrin were markedly decreased,whereas expression of a damaged podocyte marker desmin was upregulated.Treatment with eplerenone or combining amlodipine with eplerenone improved the podocyte injury.Furthermore,we found that glomerular MR expression was upregualted by high-salt loading.Although the MR expression can not be suppressed by eplerenone,treatment with eplerenone or eplerenone in combination with amlodipine dramatically decreased the upregulation of Sgk-1.6.Assessment of Renal HypoxiaThe pimonidazole-positive area in the renal cortex was markedly increased in control DS rats compared to low salt-fed rats.Treatment with amlodipine partially attenuated the increase in pimonidazole staining,while eplerenone treatment had no effect.The combination treatment markedly improved the increased pimonidazole positive area in renal cortex.It is proposed that the loss of peri-tubular capillary is one of the causes for renal hypoxia.The area positive for anti-endothelial aminopeptidase P antibody was smaller in the renal cortex of control rats.The decreased aminopeptidase P immunoreactivity was partially restored by amlodipine or the combination treatment,whereas eplerenone treatment had no effect on the loss of immunoreactivity by high salt feeding.Conclusion1.Glomerularsclerosis and tubulointerstitial fibrosis don't always co-exist in renal injury.Any of them could cause renal injury alone.2.Glomerularsclerosis and tubulointerstitial fibrosis can be caused by different pathological factors.For example,activation of MR and podocyte injury may play a important role in glomerularsclerosis.However,tubulointerstitial hypoxia may be crucial for tubulointerstitial fibrosis.3.Amlodipine in combination with eplerenone provide better renoprotective effects characterized by reducing both glomerularsclerosis and tubulointerstitial fibrosis through the improvement of both podocyte injury and tubulointerstital hypoxia.