【作者】 王惠珍；

【导师】 李爱青；

【作者基本信息】 南方医科大学 ， 内科学（肾病）， 2022， 博士

【摘要】 研究背景人第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome 10,PTEN)是迄今发现的首个具有双特异磷酸酶活性的重要肿瘤抑制因子,涉及多种生物学过程。急性肾损伤(acute kidney injury,AKI)是一种以肾功能急剧下降为特征的综合征。值得注意的是,由于急性损伤后适应不良性修复,可出现持续的肾脏损害(最近称为急性肾病),甚至进展为慢性肾脏病(chronic kidney disease,CKD)和终末期肾病。我们既往研究表明足细胞特异性过表达PTEN可减轻糖尿病肾病小鼠尿白蛋白排泄和肾小球硬化。虽然PTEN在肾损伤中的作用逐渐显现,但目前PTEN在急性肾损伤和损伤后适应不良性修复中的作用还未清楚。研究方法体内采用缺血再灌注(ischemia/reperfusion,I/R)诱导的肾损伤构建急性肾损伤和损伤后适应不良性修复模型。将PTENfl-stop-fl小鼠与肾小管特异性表达Cre酶的工具鼠Ggt1-Cre小鼠杂交,通过Cre-loxP基因重组技术构建肾小管特异性PTEN过表达小鼠,并采用质谱分析肾小管特异性PTEN过表达小鼠肾组织在I/R后蛋白质组学的变化。此外,体外采用ATP/葡萄糖耗竭法模拟I/R诱导HK-2损伤模型。研究结果我们发现PTEN在I/R诱导的急性肾损伤及损伤后肾适应不良性修复的小鼠肾小管上皮细胞中表达下调,且PTEN的表达与肾损伤指标NGAL及纤维化指标呈负相关关系。与此相一致的是,PTEN在体外I/R诱导的HK-2损伤中表达下调。I/R导致血清PTEN水平下降,且血清PTEN浓度与血肌酐呈负相关关系。肾小管特异性PTEN过表达减轻了 I/R诱导的肾功能下降、肾小管上皮细胞损伤、凋亡和肾纤维化。蛋白质谱分析显示,在I/R下,肾小管特异性PTEN过表达与阴性对照小鼠间差异表达蛋白在吞噬体、PI3K/Akt和HIF-1信号通路中显著富集,并显示荷电多泡体蛋白 2A(charged multivesicular body protein 2A,CHMP2A)在I/R诱导的肾小管特异性PTEN过表达小鼠肾组织中显著上调。在I/R刺激的HK-2中,同时结合LC3-Ⅱ和P62的自噬体积聚,提示自噬流受阻。敲低PTEN可下调CHMP2A,进而抑制吞噬体闭合和自噬溶酶体形成,导致同时结合LC3-Ⅱ和P62的自噬体进一步积聚,加重细胞损伤和凋亡。PTEN过表达则表现出相反的效果。然而,沉默CHMP2A可以阻断过表达PTEN对自噬流和细胞损伤的影响。研究结论综上所述,我们的研究表明,PTEN在I/R诱导的急性肾损伤及损伤后肾适应不良性修复中表达下调,并与I/R损伤的严重程度相关。血清PTEN浓度可能是评估AKI严重性及AKI向CKD转化的一个有前景的生物标志物。肾小管特异性PTEN过表达减轻了 AKI的严重性及减缓了 CKD的进程。机制上,PTEN通过上调CHMP2A介导的吞噬体闭合,进而促进自噬溶酶体形成和活化自噬流,从而缓解细胞损伤、凋亡和肾纤维化。PTEN/CHMP2A介导的自噬流可能成为减轻AKI损伤和阻断AKI向CKD转化的新的治疗靶点。更多还原

【Abstract】 BackgroundPhosphatase and Tensin Homologue on chromosome Ten(PTEN),the unique tumor suppressor possessing double-specific phosphatase activity so far,involves in a variety of biological processes.Acute kidney injury(AKI)is a syndrome defined by rapid loss of renal function.Notably,AKI can trigger immediate kidney damage(referred to as acute kidney disease,AKD)or long-term damage(in the form of chronic kidney disease,CKD,and end-stage renal disease,ESRD),both due to maladaptive repair.We previously demonstrated that podocyte-specific PTEN knock-in alleviated urinary albumin and glomerular sclerosis in mice subjected to DKD.Although PTEN has emerged as a key protein that governs the response to kidney injury,whether and how PTEN works in renal acute injury and maladaptive repair remains unclear.MethodsIschemia/reperfusion(I/R)was conducted to induce acute kidney injury and renal maladaptive repair in mice.Renal proximal tubular cells(RPTC)-specific PTEN overexpression mice were established by crossing PTENfl-stop-fl mice with Ggtl-Cre mice via Cre-loxP system.Mass spectrometry-based proteomics was performed after subjecting these mice to ischemia/reperfusion(I/R).In addition,ATP/glucose depletion was used to establish I/R-induced cultured HK-2 injury model.ResultsWe found that PTEN was downregulated in renal tubular cells in mice and cultured HK-2 subjected to renal acute injury and maladaptive repair induced by I/R.Renal expression of PTEN negatively correlated with NGAL and fibrotic markers.In addition,the level of serum PTEN was decreased in response to I/R,and was negatively correlated with serum creatinine.RPTC-specific PTEN overexpression relieved I/R-induced maladaptive repair,as indicated by alleviative tubular cell damage,apoptosis,and subsequent renal fibrosis.Mass spectrometry analysis revealed that differentially expressed proteins in RPTC-specific PTEN overexpression mice subjected to I/R were significantly enriched in phagosome,PI3K/Akt,and HIF-1 signaling pathway,and found significant upregulation of CHMP2A,an autophagy-related protein.PTEN deficiency downregulated CHMP2A,and inhibited phagosome closure and autolysosome formation,which aggravated cell injury and apoptosis after I/R.PTEN overexpression had the opposite effect.However,the beneficial effect of PTEN overexpression on autophagy flux and cell damage was abolished when CHMP2A was silenced.ConclusionsCollectively,PTEN was downregulated in kidney exposed to I/R-induced renal acute injury and maladaptive repair,and was associated with the severity of I/R.The serum PTEN concentration was a promising biomarker for assessing the severity of AKI and AKI to CKD transition.RPTC-specific PTEN overexpression alleviated acute renal injury and slowed down the progression of CKD.Innovatively,PTEN relieved renal injury and maladaptive repair in terms of cell damage,apoptosis,and renal fibrosis,by upregulating CHMP2A-mediated phagosome closure,suggesting PTEN/CHMP2A-mediated autophagy flux may serve as a novel therapeutic target for AKI and the AKI to CKD transition.更多还原