溶酶体在尿蛋白所致肾小管上皮细胞损伤的自噬流保护机制中的作用

Proteinuria spread injury from glomerulus to tubules by directly damaging tubular epithelial cells (TEC). Recent studies revealed that autophagy prevent TEC from injury induced by exogenous nephrotoxic drugs. Our previous study also found that proteinuria, a endogenous nephrotoxic factor, induced autophagy through oxidative stress pathway and further induced autophagy by rampamycin attenuated TEC injury. On the other hand, we also found that urianry proteins might block autophagy pathyway by inducing lysosome injury, and further inhibited the function of lysosomes by chroloque aggregated TEC damage. The above results demostrated that autophagy related to TEC injury induced by urinary proteins, and also indicated that autophagic flux was not smooth in this state. The further work is needed to investigate how urianry proteins affect lysosomes and then affect the pretective role of atuophagic flux on TEC. We firstly plan to make clear the effects of proteinuria on lysosomal number, memembrane permeability, enzyme activities in TEC. Second, whether urinary proteins hamper the fusion of autophagosomes to lysosomes and impair the degradation of autophagosomes in lysosmes will be confirmed by a newly reported method. Lastly, we plan to explore whether improving lysosome function to unobstruct autophagy pathway and enhance autophagy flux can exert protective effects on TEC injury induced by urinary proteins. The purpose of the present research is to ascertain the role of lysosomes on autophagy flux protecting TEC injury induced by proteinuria to explore a new therapy target for renal tubulointerstitial lesion related to proteinuria.

蛋白尿通过损伤肾小管上皮细胞（TEC）使肾小球损伤向肾小管蔓延。而自噬可保护外源性肾毒药物所致TEC损伤。我们前期研究也发现：一方面，尿蛋白（一种内源性肾毒因子）可通过氧化应激途径在自噬通路上游诱导自噬，用雷帕霉素进一步诱导自噬可减轻尿蛋白所致TEC损伤；另一方面，尿蛋白同时可能影响溶酶体功能而导致自噬通路下游受阻，用氯喹抑制溶酶体功能则加剧TEC损伤。基于以上发现，下一步有必要明确尿蛋白如何损伤溶酶体，并进而如何影响自噬对尿蛋白所致TEC损伤的自救作用发挥？本项目拟首先明确尿蛋白对TEC溶酶体的数量、完整性以及酶活性等产生哪些影响；进而用更为直观方法进一步验证自噬通路下游是否因溶酶体功能受损而受阻；最后探讨增加和保护溶酶体功能，以通畅自噬通路和提高自噬流量能否减轻尿蛋白对TEC损伤。本研究旨在明确溶酶体在尿蛋白所致TEC损伤的自噬流保护机制中的作用，为防治蛋白尿相关TEC损伤寻找新靶点。

目前我们已经完成全部计划研究内容。为了阐明溶酶体是否参与了尿蛋白所致的肾小管上皮细胞（RTECs）的损伤，我们对尿蛋白对溶酶体的影响做了初步探究。首先我们应用肾活检标本进行溶酶体的免疫荧光及电镜检测，以明确溶酶体数量、形态及分布的变化与患者蛋白尿的关系。通过电镜，我们观察到蛋白尿组患者近端肾小管细胞溶酶体数目和体积均高于正常组。通过免疫荧光我们观察到，不仅蛋白尿患者RTECs内溶酶体的数目多与对照组，且其溶酶体完整性也被破坏。接着我们在体外实验中进一步探讨了尿蛋白对RTECs溶酶体的数目、形态及功能的影响。结果发现，尿蛋白处理RTECs细胞后，溶酶体数目和体积均增加、溶酶体碱化、溶酶体膜透性增加、溶酶体酶组织蛋白B活性下降。以上结果共同提示尿蛋白可以导致RTECs溶酶体数目、形态和功能的改变。由于溶酶体是维持细胞正常功能的重要细胞器，且在细胞的损伤和死亡中起到重要作用，因此调节溶酶体可成为治疗尿蛋白所致的RTECs损伤提供一个新策略。