肾小管上皮细胞αKlotho减少致FAO障碍及其在AKI向CKD发展中的作用与机制研究

The injury of tubular epithelial cells (TECs) is an important pathogenetic basis of chronic kidney disease (CKD). Recent studies reported that the disorder of fatty acid oxidation (FAO) in TECs plays a key role in the progression of acute kidney injury (AKI) to CKD. However, the mechanisms of AKI-induced FAO disorder in TECs are largely unknown. Our previous study showed that increased lipid droplet deposition and decreased production of ATP were observed in TECs in αKlotho deficient mice. As reported, the expression level of αKlotho remarkably decreased in TECs shortly after AKI. Therefore, we hypothesize that αKlotho reduction may play a key role in AKI-induced FAO disorder in TECs, not only by inhibiting lipid hydrolysis through interfering cAMP-PKA-PPARα/PGC-1α signaling pathways, but also by inhibiting autophagic degradation of lipid droplets via activation of mTOR signaling. To confirm our hypothesis, we plan to investigate the effect and mechanisms of αKlotho reduction on FAO in TECs, αKlotho deficient mice and αKlotho transgene mice after ischemia-reperfusion injury, by using RNA interference, three-dimensional reconstructed images, flow cytometry, etc. Then, we will explore the role of αKlotho reduction-associated FAO disorder in the progression of AKI to CKD. The aim of this study is to deeply elucidate the mechanisms underlying the progression of AKI to CKD, and find effective avenues for inhibiting the progression from AKI to CKD.

肾小管上皮细胞（TECs）损伤是引发慢性肾脏病（CKD）的重要病理基础，新近研究表明，TECs脂肪酸氧化（FAO）障碍是急性肾损伤（AKI）向CKD发展的主要始动因素，但AKI后TECs发生FAO障碍的机制远未阐明。我们前期发现αKlotho缺陷小鼠TECs中脂滴沉积并伴有ATP生成减少，而已知AKI后早期TECs中αKlotho表达即显著下调，推测：AKI后αKlotho减少一方面通过干扰cAMP-PKA通路而抑制脂质水解，另一方面通过活化mTOR而抑制脂质自噬，最终导致FAO障碍和ATP生成减少，引起TECs表型转换和CKD发生。为此，本项目拟以αKlotho缺陷及转基因小鼠、TECs细胞株为研究对象，采用RNA干扰、免疫荧光三维重建、流式等技术，深入探讨AKI后αKlotho减少对TECs FAO的影响与机制。旨在深入阐明AKI向CKD发展的分子机理，并寻找有效干预靶点和防治措施。

急性肾损伤（acute kidney diseases, AKI）导致的肾小管上皮细胞（renal tubular epithelial cells, RTECs）内脂滴（lipid droplet，LD）蓄积及由此产生的脂肪酸氧化（fatty acid (FA) β-oxidation，FAO）障碍，在AKI向CKD进展中起着关键作用，但迄今为止，AKI后TECs中LD蓄积的机制仍不完全清楚。项目研究发现一种主要在肾小管上皮细胞表达的抗衰老蛋白αKlotho，其基因敲除（kl/kl）小鼠TECs中出现明显的脂滴沉积并伴有ATP生成减少，而AKI早期TECs中αKlotho表达即显著下调，为此，本项目拟以αKlotho缺陷及转基因小鼠、TECs细胞株为研究对象，采用RNA干扰、免疫荧光三维重建、流式等技术，探讨AKI后αKlotho减少对TECs FAO的影响与机制，旨在深入阐明AKI向CKD发展的分子机理，并寻找有效干预靶点和防治措施。深入研究发现，αKlotho基因缺陷（KL+/-）小鼠制备的AKI模型LD在TECs中的蓄积和FAO障碍进一步加重。在体外，缺氧/复氧通过导致脂解和脂噬障碍破坏脂肪酸（FA）动员，引起LD在TECs中蓄积，αKlotho缺乏则加重缺氧/复氧诱导的LD蓄积和FAO障碍。αKlotho敲除小鼠或缺血再灌注肾损伤（IRI）制备的AKI模型小鼠，同样观察到TECs脂解和脂噬减弱，LD蓄积和FAO障碍。进一步机制研究表明，ATGL作为调控脂解和脂噬的关键分子，其蛋白表达在kl/kl 小鼠和AKI小鼠TECs中显著降低，而αKlotho缺乏通过促进E3连接酶过氧高氧蛋白2（PEX2）与泛素偶联酶E2 D2（UBC4）结合，导致泛素介导的ATGL降解。相反，αKlotho过表达则通过保护ATGL免受泛素化降解，进而维持FA动员，减轻LD蓄积和FAO障碍，抑制AKI向CKD进展。因此，项目研究首次表明，αKlotho是维持TEC脂质代谢稳态的关键分子，通过外源性补偿或上调αKlotho水平可能是改善AKI后TEC损伤和向CKD进展的有效手段。项目研究工作不仅确定了维持脂质稳态是AKI新的治疗途径，而且再次强调了αKlotho在预防AKI-to-CKD中的肾脏保护作用和必要性，为今后可能实施的临床转化提供了依据。