本文選題：Klotho　切入點：慢性腎臟損傷　出處：《南京大學》2017年博士論文

【摘要】：Klotho為新近發(fā)現(xiàn)的高表達于腎臟的抗衰老蛋白,具有顯著的腎臟保護作用。腎臟損傷導致Klotho表達下降并與慢性腎臟損傷的發(fā)生發(fā)展密切相關(guān)。外源性補充Klotho或逆轉(zhuǎn)內(nèi)源Klotho下降可以緩解慢性腎臟損傷。因此,Klotho是慢性腎臟損傷的一個重要的干預(yù)靶點。PPARγ是重要的腎臟保護性轉(zhuǎn)錄因子,其功能受多種翻譯后修飾調(diào)控。PPARγ乙�；揎椖軌蚋纳浦炯毎D(zhuǎn)換和脂質(zhì)代謝,但在腎臟損傷中的作用尚不清楚。Klotho啟動子含有PPARγ的結(jié)合位點,是PPARγ的靶基因,但PPARγ乙�；瘜lotho的作用尚未見報道。本研究旨在探討PPARγ乙�；瘜lotho表達的影響以及在慢性腎臟損傷中的作用。組蛋白去乙�；敢种苿㏕richostatin A(TSA)可以通過抑制蛋白質(zhì)去乙酰化而發(fā)揮腎臟保護作用。我們研究發(fā)現(xiàn)TSA可以明顯緩解腺嘌呤誘導的小鼠慢性腎臟損傷和Klotho表達下降。TSA通過增強PPARγ乙酰化和促進PPARγ與Klotho啟動子結(jié)合從而上調(diào)Klotho表達。通過DNA點突變技術(shù),我們發(fā)現(xiàn)PPARγ乙酰化對Klotho的調(diào)控作用依賴于PPARγ第240位和第265位賴氨酸乙酰化。TSA對PPARγ敲除小鼠慢性腎臟損傷和Klotho下降的緩解作用明顯減弱,表明PPARy是蛋白質(zhì)乙�；徑饴阅I臟損傷和Klotho下降的重要靶點。有趣的是,我們發(fā)現(xiàn)腺嘌呤小鼠腎臟中組蛋白去乙�；�3(HDAC3)表達明顯升高。選擇性HDAC3抑制劑RGFP966可以有效緩解慢性腎臟損傷和Klotho下降。尾靜脈注射小干擾RNA(siRNA)敲低Klotho表達,消除了 RGFP966的腎臟保護作用,說明HDAC3表達升高和Klotho表達降低是介導腺嘌呤小鼠腎臟損傷的重要因素。骨質(zhì)異常是慢性腎臟病常見并發(fā)癥。腎臟Klotho敲除小鼠不僅出現(xiàn)腎臟損傷還伴有骨質(zhì)疏松改變。因此,本研究還探討PPARγ乙�；{(diào)控Klotho能否緩解慢性腎臟損傷伴隨的骨質(zhì)異常。我們發(fā)現(xiàn)腺嘌呤小鼠出現(xiàn)骨質(zhì)異常。ELISA、Western Blot和qPCR實驗表明多種骨重塑因子出現(xiàn)明顯異常改變;骨HE染色提示骨小梁變薄且排列紊亂,骨小梁間隙變大;X線掃描發(fā)現(xiàn)股骨遠端骨密度明顯減低;microCT掃描發(fā)現(xiàn)股骨遠端微結(jié)構(gòu)異常,表現(xiàn)為骨體積分數(shù)(BV/TV)降低、骨小梁數(shù)量(Tb.N)減少、骨小梁厚度(Tb.Th)變薄和骨小梁分離度(Tb.Sp)增加。PPARy乙�；鰪奒lotho表達可以緩解腺嘌呤小鼠上述骨質(zhì)異常改變。尾靜脈注射小干擾RNA(siRNA)敲低Klotho表達,顯著降低了 TSA對骨的保護作用,說明Klotho表達減少是HDAC異常導致慢性腎臟損傷骨質(zhì)異常的重要原因,而抑制異常表達的HDAC能夠?qū)β阅I臟損傷相關(guān)的骨質(zhì)病變提供有效保護�？傊�,本研究首次在腺嘌呤誘導的小鼠慢性腎臟損傷模型中對PPARγ乙�；捌鋵lotho調(diào)控的作用進行評估。此外,我們的研究結(jié)果為腎臟病的治療提供新的思路,即抑制HDAC3可以增加PPARγ乙�；捌滢D(zhuǎn)錄活性,維持Klotho的表達緩解慢性腎臟損傷和骨質(zhì)異常。
[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 緯 is an important protective transcription factor of kidney. The function of Klotho is regulated by many posttranslational modifications. PPAR 緯 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 緯 and is the target gene of PPAR 緯. However, the effect of PPAR 緯 acetylation on Klotho has not been reported.The purpose of this study was to investigate the effect of PPAR 緯 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 緯 acetylation and promoting PPAR 緯 binding to Klotho promoter.By DNA point mutation technique, we found that the regulation of PPAR 緯 acetylation on Klotho was dependent on the remission of chronic renal injury and Klotho decrease in PPAR 緯 knockout mice by Lysine acetylation at PPAR 緯 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 緯 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 緯 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 緯 and its transcriptional activity, and maintain the expression of Klotho to alleviate chronic renal injury and bone abnormalities.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R692

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