贝那普利和氯沙坦对老年自发性高血压大鼠肾小球足细胞自噬的影响及作用机制

The effects and mechanisms of benazepril and iosartan on glomerular podocyte autophagy in aged spontaneously hypertensive rats

目的观察贝那普利和氯沙坦对老年自发性高血压大鼠（SHR）肾小球足细胞自噬的影响，探讨血管紧张素转化酶抑制剂（ACEI）／血管紧张素受体拮抗剂（ARB）。肾脏保护作用的新机制。方法Wistar-Kyoto（wKY）大鼠为WKY对照组（生理盐水2ml／d），SHR随机分为4组：SHR对照组（生理盐水2ml／d）、ACEI组（贝那普利10mg·kg^-1·d^-1）、ARB组（氯沙坦30mg·kg^-1·d^-1）、联合用药组（贝那普利10mg·kg^-1·d^-1＋氯沙坦30mg·kg^-1·d^-1），每组均为6只18月龄雄性大鼠。实验干预4个月，定期监测大鼠血压。实验终点检测尿蛋白、尿肌酐、血肌酐、血尿素、血清及肾皮质血管紧张素Ⅱ（AngiotensinⅡ，AngⅡ）水平；观察肾组织光镜和电镜下形态学改变，计数足细胞内自噬体；免疫印迹（Westernblot）法检测肾小球nephrin、LC38Ⅱ、Atg5和p62蛋白表达情况。结果给药后，4组SHR血压、尿蛋白／尿肌酐比值仍高于WKY对照组，但ARB组、联合用药组血压、尿蛋白／尿肌酐比值低于SHR对照组；各组血肌酐、血尿素比较，差异无统计学意义（P〉0．05）；联合用药组血清AngⅡ水平为（0．12±0．01）μg／L，高于SHR对照组（0．08±0．00）μg／L（P〈0．05）；4组SHR肾皮质AngⅡ水平均低于WKY对照组，ARB组肾皮质AngⅡ水平高于SHR对照组（均P〈0．05）；4组SHR均可见。肾小球缺血皱缩，足突融合、消失，灶性小管萎缩及间质纤维化，但以SHR对照组病变较重，联合用药组病变较轻，WKY对照组、联合用药组足细胞内自噬体多于SHR对照组（P〈0．05）；SHR对照组nephrin、LC3BⅡ、Atg5、p62水平低于WKY对照组；ACEI组nephrin、LC38Ⅱ、Atg5水平、ARB组和联合用药组nephrin、LC38Ⅱ、Atg5、p62水平均高于SHR对照组（P〈0．05）。结论ACEI／ARB可下调老年SHR肾小球足细胞自噬活性；ACEI／ARB肾脏保护作用可能与其调控AngⅡ诱导的肾小球足细胞自噬有关。

Objective To examine the effects of benazepril and losartan on glomerular podoeyte autophagy in aged spontaneously hypertensive rats （SHRs） and investigate the underlying mechanisms of renal-protective effects of angiotensin-eonverting enzyme inhibitors （ACEI）/angiotensin receptor blockers （ARB）. Methods Wistar-Kyoto rats （WKYs） were used as the normal control （NORM） group （2 ml physiological saline per day）. SHRs were randomly divided into 4 groups： the CTRL group （2 ml physiological saline per clay）, the ACEI group （10 mg·kg^-1·d^-1 ）, the ARB group （30 mg ·kg^-1·d^-1） and the combined group （10 mg·kg^-1·d^-1 benazepril and 30 mg·kg^-1·d^-1 ）, with six 18-month-old male rats in each group. The experiments were conducted during a 4-month period. Blood pressure was monitored regularly. At the end of the experiments, we measured the levels of urine protein, urine creatinine, serum ereatinine （SCR）, blood urea nitrogen （BUN）, and serum and renal cortex angiotensin Ⅱ （AngⅡ）. Ultrastructural changes in the kidney were examined under light and transmission electron microscopy. The expressions of nephrin, LC3BⅡ, Atg5 and p62in the glomerulus were analyzed by Western blot analysis. Results After treatment, the blood pressure and the urine albumin/creatinine ratio of the four SHR groups were still significantly higher than those of the NORM group, but the blood pressure and the urine albumin/creatinine ratio of the ARB group and the combined group were significantly lower than those of the CTRL group （all P〈0.05）; There were no significant differences in SCR and BUN levels among these five groups （P〉0.05） ; The level of serum AngII of the combined group was significantly higher than that of the CTRL group （CTRL （0.08±0.00） μg/L, Combined （0.12±0.01） μg/L, P〈0. 057 ; The levels of cortex AngⅡ of the four SHR groups were significantly lower than those of the NORM group, while the level of cortex AngⅡ of the ARB group was significantly higher than that of the CTRL group （all P〈0.05）; Renal ultrastructural examination revealed shrunken glomeruli, fused or effaced epithelial cell foot processes, and focal atrophy of renal tubules in the four SHR groups. These pathological changes were more serious in the CTRL group but less so in the combined group. There were significantly more autophagosomes in the NORM group and the combined group than in the CTRL group （P〈0.05）. Compared with the NORM group, the expressions of nephrin, LC3BⅡ, Atg5 and p62 in the CTRL group were suppressed significantly （P〈0.05）. The expressions of nephrin, LC3BⅡ and Atg5 in the ACEI group and the expressions of nephrin, LC3BⅡ, Atg5 and p62 in the ARB group and the combined group were higher than in the CTRL group （P〈0.05）. Conclusions ACEI/ARB can decrease the autophagic activity of glomerular podocytes. The renal-protective effects of ACE1/ARB may be mediated by glomerular podocyte autophagy, which is induced by AngⅡ.