Nit2在糖尿病肾病肾小管上皮细胞溶酶体耗竭中的作用及机制研究

申请人所在团队既往研究发现，糖尿病肾病（DN）肾小管上皮细胞（TECs）溶酶体耗竭所致自噬通路阻滞是TECs损伤的重要原因；后续又发现转录因子EB（TFEB）主导的溶酶体生物合成不足可致溶酶体耗竭，但合成不足的机制不明。预实验发现：DN中TECs的ω-酰胺酶Nit2表达下降；敲低Nit2加重细胞损伤和溶酶体耗竭；而过表达Nit2减轻细胞损伤，增强自噬活性，改善溶酶体耗竭，且升高TFEB表达。据此推测DN中TECs的Nit2低表达导致TFEB主导的溶酶体生物合成不足，引起溶酶体耗竭。本研究拟进一步验证DN中TECs的Nit2表达与溶酶体耗竭及自噬通路阻滞的关系；并在预实验初步发现的基础上明确Nit2下降是否阻碍TFEB主导的溶酶体生物合成，进而导致溶酶体耗竭；最后探讨Nit2酶促反应产物α-酮戊二酸是否介导对TFEB的调控。研究结果可为纠正DN肾脏固有细胞溶酶体耗竭、延缓DN进展提供新线索。

Previous work in our group has shown that lysosome depletion-induced disruption of autophagy-lysosome pathway (ALP) leads to tubular epithelial cells (TECs) injury in diabetic nephropathy (DN). Moreover, we have demonstrated that lysosome depletion can be caused by impaired lysosome biogenesis, which is mastered by transcription factor EB (TFEB). However, it remains largely unrevealed that how lysosome biogenesis is impeded. Our preliminary data illustrates that the ω-amidase Nit2 is decreased in TECs of DN. Additionally, knockdown of Nit2 exacerbate cell injury and lysosome depletion, whereas overexpression of Nit2 ameliorates cell injury, promotes autophagy, diminishes lysosome depletion and increases expression level of TFEB. Therefore, we hypothesize that Nit2 is reduced in TECs of DN, this further results in the suppression of TFEB-mediated lysosome biogenesis, and consequently contributes to lysosome depletion and ALP retardation. To test this, we propose to determine 1) expression of Nit2 in TECs of DN and its association with lysosome depletion and ALP impairment; 2) whether the attenuation of Nit2 compromises TFEB-mastered lysosome biogenesis thus further brings to lysosome depletion; 3) whether the enzymatic activity of Nit2 is involved in the activation of TFEB, and if the up-regulation is mediated by the enzyme product α-ketoglutarate (AKG). Our results will improve understanding of the role of Nit2 in DN, offer mechanistic insights into lysosome depletion in DN, and provide translational insights into the potential application of AKG in DN prevention and treatment.