肾小管上皮细胞损伤过程中溶酶体耗竭发生机制及其修复策略

Previous studies from ours and others found that autophagy induction was ordinarily accompanied by TEC injury in the initial stage, but the consequent “lysosome depletion” in turn blocked the autophagosome degradation and autophagy pathway, leading to so-called “autophagic stress”, which may impair protective autophagy of TEC and exacerbate renal tubule damage. Therefore, future work should focus on identifying the mechanism of lysosome depletion and seeking strategies to restore lysosome functions in injured TEC. We postulated that "old" lysosomes destruction and "new" lysosome generation deficiency are the key mechanism of lysosome depletion. After having confirmed that lysosomal membrane permeability (LMP) inducing by oxidative stress was the important mechanism of"old" lysosome destruction,we will further verify that lysosomal biogenesis insufficient and autophagic lysosomal reformation (ALR) deficiency are the main mechanism of “new” lysosome generation deficiency in injured TEC in the present study. Also, drug screening for "old" lysosome protection or for "new" lysosome generation in TEC from the existing medicine will also be carried out. The study may provide a new approach to alleviate severity of acute or chronic tubular damage by improving damage resistance and self-healing of TEC via restoring autophagic pathway.

我们及同行研究发现：虽然多种肾损伤因素所致肾小管上皮细胞（TEC）损伤起始均有自噬诱导现象，但继之而来的“溶酶体耗竭”却造成已诱导生成的自噬泡降解受阻，导致自噬通路堵塞和所谓“自噬应激”现象，进而削弱TEC的保护性自噬和抗损伤能力。因此，下一步的重点是明确造成TEC溶酶体耗竭的发生机制和寻找恢复溶酶体功能的策略。我们推测旧溶酶体破坏过度以及新溶酶体生成不足是导致TEC溶酶体耗竭的关键，并且已证实尿蛋白等肾损伤因素可通过氧化应激途径导致溶酶体膜透化而破坏TEC旧溶酶体，本研究将进一步验证肾损伤因素是否还可通过影响溶酶体生物合成及自噬性溶酶体再生两种促进溶酶体新生机制而使TEC溶酶体新生不足，加剧溶酶体耗竭，并试图从现有药物中寻找具有溶酶体保护或促进溶酶体新生作用的药物以对抗肾损伤因素所致TEC溶酶体耗竭。研究结果有望为修复自噬通路、提高TEC抗损伤能力，进而减轻急、慢性肾小管损伤提供新思路。

经过四年的努力，课题组已超额完成本项目所设定的全部研究内容。首先，我们证实了肾小管上皮细胞（TEC）溶酶体新生不足是导致该细胞在肾损伤因素作用下溶酶体耗竭的主要机制。我们发现，在糖尿病（或尿蛋白超负荷）状态下，肾小管上皮细胞转录因子TFEB被抑制而不能活化入核导致溶酶体生源不足，而高糖状态下mTOR过度活化是导致TFEB被抑制的重要原因，而且，由于新生溶酶体生源不足，没有足够正常的溶酶体维持自噬通路而进一步导致了TEC细胞内受损的溶酶体堆积并形成恶性循环，通过补救实验，我们证实了激活TFEB可促进溶酶体生源，增加溶酶体清除而修复溶酶体耗竭，减轻TEC损伤。继之，我们进一步证实除了TFEB活化受到抑制之外，TFEB表达下降（也即产生减少）也是导致TEC溶酶体新生不足而耗竭的重要原因；更为重要的是，我们发现糖尿病状态下过度激活的Smad3作为转录抑制因子而抑制TFEB转录。我们的研究结果揭示了尿蛋白和AGEs等肾损伤因素导致TEC溶酶体耗竭的机理。以上研究结果为下一步如何纠正溶酶体耗竭和恢复肾脏细胞保护性自噬找到了重要的干预靶点——TFEB，并且进一步丰富了Smad3导致糖尿病肾病进展的机制。