在高脂条件下足细胞特异性PTEN敲进对肾脏有保护作用

Podocyte-specific knock-in of PTEN protects kidney against high fat diet

目的观察在高脂饮食（HFD）条件下体内足细胞特异性过表达PTEN对小鼠肾脏的作用，并探讨体外氧化低密度脂蛋白（OX—LDL）刺激环境中足细胞内PTEN调控清道夫受体A（SR—A）介导的脂质摄取的机制。方法构建足细胞特异性PTEN敲进（PPKI）转基因小鼠，HFD诱导转基因小鼠肾损伤。小鼠分为正常饮食（ND）＋对照（Ctrl）组、ND＋PPKI组、HFD＋Ctrl组、HFD＋PPKI组。于饮食干预后第24周，检测4组小鼠临床生化指标，包括血肌酐、尿白蛋白排泄（尿白蛋白与肌酐比）等；油红O染色和肾皮质胆固醇定量观察肾脂质积聚情况；PAS染色和电镜评估肾小球硬化情况。体外OX．LDL刺激诱导足细胞损伤。Western印迹检测siRNA沉默YAP表达（si—YAP）后OX—LDL诱导足细胞SR—A蛋白表达的变化，及在siRNA沉默PTEN表达（si—PTEN）、PTEN抑制剂（Bpv—PTEN）、腺病毒过表达PTEN（ad—PTEN）的作用下OX—LDL诱导足细胞YAP磷酸化的变化。结果与ND＋Ctrl组相比，HFD＋Ctrl组小鼠的血肌酐、尿白蛋白排泄水平上升，足细胞SR—A表达及肾内脂质含量明显增加，系膜基质增生，足突融合增加及基底膜增厚（均P〈0．05）；而与HFD＋Ctrl组相比，HFD＋PPKI组小鼠上述肾损伤指标均减轻（均P〈0．05）。体外OX．LDL刺激能上调足细胞SR．A表达，与正常对照组比较差异有统计学意义（P〈0．05）；敲除YAP可使ox-LDL诱导的SR—A表达下降，si—YAP＋ox—LDL组SR—A表达低于ox—LDL组（P〈0．05）。在ox—LDL刺激下足细胞内磷酸化（p）-YAP表达增加，与正常对照组比较差异有统计学意义（P〈0．05）；上调PTEN的表达可抑制ox—LDL诱导的p-YAP表达上调，ad—PTEN＋ox—LDL组p-YAP表达低于ox—LDL组（P〈0．05）；而敲除PTEN或抑制PTEN磷酸酶活性均能进一步上调OX—LDL诱导的p-YAP的表达，si-PTEN＋ox-LDL组和Bpv—PTEN＋OX—LDL组p-YAP表达均高于OX—LDL组（均P〈0．05）。结论足细胞特异性过表达PTEN对脂质。肾损伤有保护作用，且PTEN可能通过使p-YAP去磷酸化进而抑制sR—A介导的脂质摄取缓解OX—LDL诱导的足细胞损伤。

Objective To evaluate the effect of over-expression of phosphatase and tensin homolog does on chromosome ten （PTEN） in podoeytes on kidney under high fat diet （HFD） in vivo and clarify the mechanism how PTEN regulates scavenger receptor A （SR-A） expression exposed to oxidized low density lipoprotein （ox-LDL） in podocytes in vitro. Methods The podocyte-specific PTEN knockin （PPKI） mice were fed with HFD to establish mouse model of lipid-induced renal injury. Mice were divided into four groups： ND＋Ctrl group, ND＋PPKI group, HFD＋Ctrl group and HFD＋PPKI group. After 24 weeks of dietary intervention, all mice were tested for clinical and biochemical parameters, including serum creatinine （Scr） as well as urine albumin excretion rate （UAER）; renal lipid content was measured by oil red O staining and cholesterol quantitative analysis; the pathological changes of glomeruli were observed by PAS staining and electron microscope. Podocyte injury was induced by ox- LDL in vitro. Western blotting was used to detect the changes of SR- A expression induced by ox-LDL after YAP-siRNA interfering （si- YAP）, as well as YAP phosphorylation induced by ox-LDL after interfering by PTEN-siRNA （si- PTEN） and PTEN phosphatase inhibitor （Bpv-PTEN）, and overexpressing by recombinant adenovirus （ad-PTEN）. Results Compared with ND＋Ctrl group, HFD＋ Ctrl group significantly aggravated the levels of Scr and UAER, the expression of SR-A in podocytes, renal lipid content, mesangial matrix expansion, effacement of podocyte foot processes, and incrassation of glomerular basement membrane （all P 〈 0.05）. Conversely, compared with HFD＋Ctrl group, HFD＋ PPKI group obviously alleviated the above lipid- induced renal damage （all P 〈 0.05）. In vitro, the expression of SR- A in podocytes was upregulated when stimulated with ox- LDL （P 〈 0.05）, and the knockout of YAP significantly down-regulated the expression of SR-A induced by ox-LDL （P 〈 0.05）. Exposed to ox- LDL, the expression of p - YAP increased in podocytes （P 〈 0.05）; over- expression of PTEN inhibited p-YAP up-regulation induced by ox-LDL （P 〈 0.05）, while either knockdown of PTEN or inhibition of PTEN phosphatase activity displayed opposite effect （all P 〈 0.05）. Conclusions Over- expression of PTEN in podocytes protected the kidney against damage from HFD in vivo and PTEN might suppress SR-A mediated lipid uptake via dephosphorylating p-YAP to prevent podocyte injury from ox-LDL.