自噬对肾脏缺血再灌注损伤中尿液浓缩功能障碍的作用和机制

Experimental acute renal failure (ARF) induced by ischemia/reperfusion (I/R) in rats manifests structural alterations in renal tubule epithelia in association with a variety of functional defects, prominent among which is an impairment of tubular reabsorption of sodium and water. Rats with ARF showed significant renal insufficiency, increased urine output, and high fractional excretion of urinary sodium. Consistent with this, immunoblotting and immunocytochemistry revealed that the kidney expression of renal aquaporins (AQPs, AQP1, 2, and 3) and sodium transporters (Na,K-ATPase, NKCC2, NHE3, and NCC) in ARF rats was significantly decreased compared to sham-operated control rats. These results suggested that these proteins at least partly contribute to the urinary concentrating defect in response to acute renal failure. .Autophagy is a catabolic process that degrades damaged proteins and organelles in mammalian cells and has a vital role in maintaining cellular homeostasis. Autophagy has been demonstrated to participate in experimental acute renal failure induced by ischemia/reperfusion, but whether autophagy regulates aquaporins to mediate urinary concentrating defect is unknown. .LC3B and p62 are one of the most important autophagy-related proteins. Our preliminary data demonstrated that p62 was increased in the kidney after reperfusion for 24 hrs with ischemia for 45 min, which LC3B was decreased. TDZD-8 attenuated the increased p62, decreased LC3B, and upregulation of NFkB; and also improved the polyuria and abolished the reduction of AQPs, NKCC2, and Na,K-ATPase in the kidney of renal I/R injury. In the cultured epithelial cells (IMCD3), Rapamycin significantly increased LC3B and p62 protein expression. IMCD3 cells were treated with antimycin and 2-DG rapidly to deplete intracellular ATP and induce hypoxia/reoxygenation cell model. Rapamycin significantly improved hypoxia/reoxygenation induced downregulation of LC3B and AQP1 protein expression in IMCD3 cells. The purpose of the present project is to investigate whether the activation of autophagy prevents downregulation of renal aquaporins and sodium transporters in the kidney and whether activation of autophagy improves the polyuria and sodium loss observed in renal I/R injury. We will investigate 1) whether activation of autophagy prevents polyuria, kidney injuries, and downregulation of renal aquaporins and sodium transporters association with renal I/R; 2) whether blockage of autphagy in ATG5 knockout mice could cause an abnormal regulation of water and sodium in the kidney in renal I/R injury; 3) whether activation of autophagy directly regulates AQP1 and AQP2 expression in cell lines or primary cultured inner medullary collecting duct cells and what relevant cellular pathways are involved? The present project will provide us evidence that activation of autophagy has a therapeutic potential in preventing urinary concentrating defect in association with renal ischemia/reperfusion injury.

肾脏缺血再灌注（I/R）可诱导急性肾脏损伤和肾脏水钠代谢失调，水通道蛋白（AQP）和钠转运体表达下降是其重要的分子机制。自噬反应在肾脏I/R时会出现一系列动态变化，但自噬是否影响AQPs表达进而参与水钠代谢调节未有报道。我们的前期工作表明肾脏I/R时激活自噬可以改善肾功能损伤和炎症反应，缓解水通道蛋白减少；肾脏IMCD3细胞株中自噬激活可以抑制细胞缺氧复氧引起的AQP1表达下调。我们因此提出假设：肾脏I/R损伤中激活自噬有利于炎症相关因子的清除，上调肾脏AQPs和钠转运体蛋白表达，进而改善尿液浓缩功能。本项目拟研究（1）利用Atg5基因敲除小鼠，研究阻断自噬流后是否加重肾脏I/R引起的尿液浓缩功能异常；（2）自噬流激活改善尿液浓缩功能的分子机制；（3）在细胞水平阐明自噬调节AQP1和AQP2表达的信号通路。本研究将为临床治疗急性肾脏损伤肾脏浓缩功能障碍提供新的研究思路。

肾脏缺血再灌注可诱导急性肾脏损伤，表现为肾脏水钠代谢失调，水通道蛋白（aquaporin, AQP）表达下降是其重要的分子机制。当细胞受到饥饿、低氧等应激刺激时，会启动自噬相关基因，进行自噬降解过程，维持机体稳态。自噬反应在肾脏I/R时会出现一系列动态变化。本项目的研究目的是探索自噬调节肾脏缺血再灌注损伤中水通道蛋白表达的作用和机制。我们利用自噬激动剂TDZD-8研究自噬的激活对大鼠肾脏缺血再灌注（I/R）损伤的保护作用。肾脏I/R损伤大鼠尿量明显增多，肌酐清除率显著降低；自噬激动剂TDZD-8（GSK-3β抑制剂）预处理明显缓解I/R大鼠的多尿症状，增加肌酐清除率，降低肾脏损伤因子KIM-1和NGAL的mRNA水平，对肾功能的损伤有一定的保护作用。给予TDZD-8干预能够升高AQP1和2的蛋白及mRNA水平。在分子机制研究中，我们发现TDZD通过激活自噬改善肾脏上皮细胞IMCD3细胞缺氧/复氧引起的AQP1表达下调。敲减细胞中的自噬相关Atg5基因后，可明显阻断TDZD的保护作用。提示自噬对AQP1的上调有密切的联系。细胞缺氧和复氧可以明显增加炎症小体的组分NLRP3和IL-1β的表达，TDZD-8可以明显抑制这二者的上调。同时免疫荧光共定位结果发现增加的自噬标志物LC3B可能参与炎症小体衔接蛋白ASC的清除，从而改善HR引起的炎症小体激活。直接给予细胞炎症因子IL-1β刺激可以抑制自噬流的进展，从而抑制AQP1的清除。因此，我们得出结论：肾脏缺血再灌注诱导的急性肾脏损伤可以抑制自噬，减少对炎症因子IL-1β清除，增多的IL-1β可以下调AQP1的表达，肾脏出现浓缩功能障碍。我们也在原代肾脏内髓集合管上皮细胞缺氧/复氧损伤发现自噬相关蛋白LC-3B 和LAMP1表达降低，AQP2表达下降、以及细胞内活性氧增加。本研究为水通道蛋白的调节机制提出新的理论，为治疗重症肾脏损伤提供新的思路。