PPARγ乙酰化在调控Klotho转录及肾脏保护中的作用

发布时间：2018-04-04 08:00

本文选题：Klotho　切入点：慢性肾脏损伤　出处：《南京大学》2017年博士论文

【摘要】：Klotho为新近发现的高表达于肾脏的抗衰老蛋白,具有显著的肾脏保护作用。肾脏损伤导致Klotho表达下降并与慢性肾脏损伤的发生发展密切相关。外源性补充Klotho或逆转内源Klotho下降可以缓解慢性肾脏损伤。因此,Klotho是慢性肾脏损伤的一个重要的干预靶点。PPARγ是重要的肾脏保护性转录因子,其功能受多种翻译后修饰调控。PPARγ乙酰化修饰能够改善脂肪细胞转换和脂质代谢,但在肾脏损伤中的作用尚不清楚。Klotho启动子含有PPARγ的结合位点,是PPARγ的靶基因,但PPARγ乙酰化对Klotho的作用尚未见报道。本研究旨在探讨PPARγ乙酰化对Klotho表达的影响以及在慢性肾脏损伤中的作用。组蛋白去乙酰化酶抑制剂Trichostatin A(TSA)可以通过抑制蛋白质去乙酰化而发挥肾脏保护作用。我们研究发现TSA可以明显缓解腺嘌呤诱导的小鼠慢性肾脏损伤和Klotho表达下降。TSA通过增强PPARγ乙酰化和促进PPARγ与Klotho启动子结合从而上调Klotho表达。通过DNA点突变技术,我们发现PPARγ乙酰化对Klotho的调控作用依赖于PPARγ第240位和第265位赖氨酸乙酰化。TSA对PPARγ敲除小鼠慢性肾脏损伤和Klotho下降的缓解作用明显减弱,表明PPARy是蛋白质乙酰化缓解慢性肾脏损伤和Klotho下降的重要靶点。有趣的是,我们发现腺嘌呤小鼠肾脏中组蛋白去乙酰化酶3(HDAC3)表达明显升高。选择性HDAC3抑制剂RGFP966可以有效缓解慢性肾脏损伤和Klotho下降。尾静脉注射小干扰RNA(siRNA)敲低Klotho表达,消除了 RGFP966的肾脏保护作用,说明HDAC3表达升高和Klotho表达降低是介导腺嘌呤小鼠肾脏损伤的重要因素。骨质异常是慢性肾脏病常见并发症。肾脏Klotho敲除小鼠不仅出现肾脏损伤还伴有骨质疏松改变。因此,本研究还探讨PPARγ乙酰化调控Klotho能否缓解慢性肾脏损伤伴随的骨质异常。我们发现腺嘌呤小鼠出现骨质异常。ELISA、Western Blot和qPCR实验表明多种骨重塑因子出现明显异常改变;骨HE染色提示骨小梁变薄且排列紊乱,骨小梁间隙变大;X线扫描发现股骨远端骨密度明显减低;microCT扫描发现股骨远端微结构异常,表现为骨体积分数(BV/TV)降低、骨小梁数量(Tb.N)减少、骨小梁厚度(Tb.Th)变薄和骨小梁分离度(Tb.Sp)增加。PPARy乙酰化增强Klotho表达可以缓解腺嘌呤小鼠上述骨质异常改变。尾静脉注射小干扰RNA(siRNA)敲低Klotho表达,显著降低了 TSA对骨的保护作用,说明Klotho表达减少是HDAC异常导致慢性肾脏损伤骨质异常的重要原因,而抑制异常表达的HDAC能够对慢性肾脏损伤相关的骨质病变提供有效保护。总之,本研究首次在腺嘌呤诱导的小鼠慢性肾脏损伤模型中对PPARγ乙酰化及其对Klotho调控的作用进行评估。此外,我们的研究结果为肾脏病的治疗提供新的思路,即抑制HDAC3可以增加PPARγ乙酰化及其转录活性,维持Klotho的表达缓解慢性肾脏损伤和骨质异常。
[Abstract]:Klotho is a newly discovered anti-aging protein which is highly expressed in the kidney and has significant renal protection.Renal injury leads to the decrease of Klotho expression and is closely related to the development of chronic renal injury.Exogenous supplementation of Klotho or reversal of endogenous Klotho may alleviate chronic renal injury.Therefore, Klotho is an important intervention target for chronic renal injury. PPAR 纬 is an important protective transcription factor of kidney. The function of Klotho is regulated by many posttranslational modifications. PPAR 纬 acetylation can improve adipocyte turnover and lipid metabolism.However, the role of Klotho promoter in renal injury is not clear. Klotho promoter contains the binding site of PPAR 纬 and is the target gene of PPAR 纬. However, the effect of PPAR 纬 acetylation on Klotho has not been reported.The purpose of this study was to investigate the effect of PPAR 纬 acetylation on Klotho expression and its role in chronic renal injury.Histone deacetylase inhibitor Trichostatin Agna can protect the kidney by inhibiting protein deacetylation.We found that TSA significantly alleviated adenine induced chronic renal injury and decreased Klotho expression in mice. TSA upregulated Klotho expression by enhancing PPAR 纬 acetylation and promoting PPAR 纬 binding to Klotho promoter.By DNA point mutation technique, we found that the regulation of PPAR 纬 acetylation on Klotho was dependent on the remission of chronic renal injury and Klotho decrease in PPAR 纬 knockout mice by Lysine acetylation at PPAR 纬 240th and 265th sites.It is suggested that PPARy is an important target for acetylation of protein to alleviate chronic renal injury and decrease of Klotho.Interestingly, we found a significant increase in histone deacetylase 3 (HDAC3) expression in the kidneys of adenine mice.RGFP966, a selective HDAC3 inhibitor, can effectively alleviate chronic renal injury and decrease Klotho.Caudal vein injection of small interference RNAs (siRNAs) reduced the expression of Klotho and eliminated the renal protection of RGFP966, suggesting that the increase of HDAC3 expression and the decrease of Klotho expression were important factors mediating renal injury in adenine mice.Bone abnormality is a common complication of chronic kidney disease.Kidney Klotho knockout mice have not only kidney damage but also osteoporosis changes.Therefore, we also investigated whether PPAR 纬 acetylation can alleviate bone abnormalities associated with chronic renal injury.We found bone abnormalities in adenine mice. ELISAN Western Blot and qPCR experiments showed that various bone remodeling factors were significantly abnormal, bone HE staining suggested that bone trabeculae became thin and disordered.The bone trabecular space was enlarged. The bone mineral density of the distal femur was obviously decreased. The microCT scan revealed the abnormal microstructure of the distal femur, which was manifested by the decrease of the bone volume fraction (BV / TVV) and the decrease of the number of trabeculae (TB. N).Tb.Th) thinning of trabecular thickness and increase of Tb.Span in bone trabeculae can alleviate the abnormal changes of bone in adenine mice by enhancing the expression of Klotho by acetylation of PPARy.The low expression of Klotho by caudal vein injection of small interference RNA-siRNAs significantly reduced the protective effect of TSA on bone, indicating that the decrease of Klotho expression is an important cause of bone abnormality caused by HDAC abnormality in chronic renal injury.Inhibition of abnormal expression of HDAC can effectively protect bone lesions associated with chronic renal injury.In conclusion, this study was the first time to evaluate the effect of PPAR 纬 acetylation and its regulation on Klotho in adenine induced chronic renal injury model in mice.In addition, our results provide a new approach to the treatment of kidney disease, that inhibition of HDAC3 can increase the acetylation of PPAR 纬 and its transcriptional activity, and maintain the expression of Klotho to alleviate chronic renal injury and bone abnormalities.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692

【相似文献】