醛固酮对肾小球系膜细胞凋亡的影响

目的 研究醛固酮（Ald）对大鼠肾小球系膜细胞（MC）凋亡的影响并探讨其可能机制。 方法 24只SPF级SD大鼠，腹腔埋置渗透性微泵，随机分为对照组（Con组，生理盐水代替Ald处理28 d；n=8）、Ald输注组（Ald组，1.5 μg/h，28 d；n=8）、依普利酮干预组（Epl组，Ald 1.5 μg/h+依普利酮 100 mg&#8226;kg-1&#8226;d-1，28 d；n=8）。药物处理0 d、7 d、14 d、21 d、28 d时，分别测量大鼠尾动脉收缩压并收集24 h尿液，测定尿白蛋白排泄率（UAER）；于28 d时心脏采血并分离大鼠双侧肾脏，检测血肌酐、血钾及血Ald水平；PAS染色观察肾小球病理改变；TUNEL法检测MC凋亡。体外培养大鼠MC，Annexin V-PI双染流式细胞仪检测MC凋亡；实时定量PCR检测Bcl-2、Bax mRNA表达，Western印迹检测Bad磷酸化水平。 结果 Ald组大鼠血压及UAER自7 d时起显著高于同期Con组（P < 0.05），28 d时Ald组大鼠血浆Ald水平约为Con组的6倍（P < 0.05），血钾显著低于Con组（P < 0.05），血肌酐与Con组差异无统计学意义（P > 0.05）。PAS染色显示Ald组大鼠肾小球系膜区增宽，基质增多，部分出现节段硬化。TUNEL检测发现，Ald组大鼠MC凋亡显著高于Con组及Epl组（均P < 0.05）。体外实验发现，随着Ald作用时间的延长，MC凋亡数明显增加（P < 0.05）；Ald组Bcl-2 mRNA表达下降（P < 0.05），Bax mRNA表达升高（P < 0.05）；Ald组Bad蛋白磷酸化水平显著低于Con组（P < 0.05）。依普利酮能显著逆转Ald诱导的上述作用。 结论 Ald能促进大鼠MC凋亡，依普利酮干预可以明显降低Ald的促凋亡效应。Bad蛋白的去磷酸化作用可能是Ald致MC凋亡过程中的重要环节。

Effect of aldosterone on glomerular mesangial cells apoptosis both in vivo and in vitro

Objective To evaluate the effect of aldosterone (Ald) on glomerular mesangial cells apoptosis and to explore the possible mechanisms.Methods Twenty-four Sprngue-Dawley rats were subcutaneously embedded with osmotic mini-pumps and randomly divided into 3 groups.Aldosterone (1.5 μg/h) was administrated subcutaneouly by osmotic mini-pumps in Ald group,eplerenone (Epl,100 mg·kg-1·d-1) and Ald (1.5 μg/h) was given to Epl group.And normal saline was used in control group (Con group).Systolic blood pressure and urinary albumin excretion rate (UAER) were detected on day 0,7,14,21,28.Blood and kidney samples were harvested on day 28.Plasma creatinine,potassium and aldosterone were measured.Renal paraffin sections were stained by PAS and the morphological changes were evaluated by light microscopy.Apoptosis index of mesangial cells were detected by TUNEL assay.The glomerular mesangial cells (MCs) were cultured in a DMEM-F12 media.MCs apoptosis was evaluated by staining cells with Annexin V and propidium iodide (PI) using flow cytometer.Expression of Bcl-2 and Bax mRNA was examined by RT-PCR.The protein level of Bad or phospho-Bad was measured by Western blotting.Results Ald-infused rats developed hyperaldosteronemia and hypokalemia.Rats in Ald group exhibited significant hypertension and marked albuminuria.Ald group rats showed increased number of TUNEL-positive mesangial cells when compared with control rats (P＜0.05).Aldosterone induced mesangial cells apoptosis in a time-dependent manner.Expression of Bcl-2 mRNA was decreased but Bax mRNA was increased in aldosterone treated MCs compared to that in Con group (P＜0.05).Aldosterone promoted dephosphorylation of cytosolic phospho-Bad compared with vehicle treated cells (P＜ 0.05).However,eplerenone attenuated these effects of aldosterone.Conclusion Aldosterone directly promotes mesangial cells apoptosis,and eplerenone can attenuate this effect of aldosterone.Dephosphorylation of cytosolic phospho-Bad may be the key role in the progression of mesangial cells apoptosis induced by aldosterone.