目的:探讨维生素D受体(VDR)对糖尿病状态下肾小管上皮细胞线粒体功能的调节作用及其机制。
方法:本研究采用链脲佐菌素(STZ)和HK-2细胞在高球蛋白(HG)/转化生长因子β(TGF-β)刺激下诱导的糖尿病大鼠。骨化三醇给药24周。肾小管间质损伤和线粒体功能的一些参数,包括线粒体自噬,线粒体裂变,线粒体ROS,线粒体膜电位(MMP),线粒体ATP,检查了复合物V活性和线粒体相关的ER膜(MAMs)完整性。此外,帕立骨化醇,3-MA(自噬抑制剂),VDR过表达质粒,体外应用VDRsiRNA和Mfn2siRNA。
结果:VDR的表达,Pink1Parkin,Fundc1,LC3II,糖尿病年夜鼠肾小管细胞中的Atg5、Mfn2、Mfn1均显著降低。骨化三醇治疗可降低尿白蛋白水平,血清肌酐和减轻STZ诱导的糖尿病大鼠肾小管间质纤维化。此外,VDR激动剂缓解线粒体自噬功能障碍,MAMs完整性,抑制线粒体裂变,线粒体ROS。免疫共沉淀分析表明VDR与Mfn2直接相互作用。线粒体功能包括线粒体自噬,线粒体膜电位(MMP),线粒体Ca2+,在HG/TGF-β环境下,HK-2细胞的线粒体ATP和复合物V活性显着降低。自噬抑制剂3-MA体外预处理HK-2细胞,VDRsiRNA或Mfn2siRNA否定帕立骨化醇对线粒体功能的激活作用。Pricalcitol和VDR过表达质粒激活了Mfn2,然后部分恢复了MAMs的完整性。此外,通过Fundc1,VDR恢复的线粒体自噬与MAMs完整性部分相关。
结论:激活的VDR可能通过Mfn2-MAMs-Fundc1途径促进肾小管细胞线粒体自噬的恢复。VDR可以恢复线粒体ATP,复杂的V活性和MAMs完整性,抑制线粒体裂变和线粒体ROS。说明VDR激动剂部分通过调节线粒体功能改善糖尿病大鼠肾小管间质纤维化。
OBJECTIVE: To investigate the regulatory effect and mechanism of Vitamin D receptor (VDR) on mitochondrial function in renal tubular epithelial cell under diabetic status.
METHODS: The diabetic rats induced by streptozotocin (STZ) and HK-2 cells under high glocose(HG)/transforming growth factor beta (TGF-β) stimulation were used in this study. Calcitriol was administered for 24 weeks. Renal tubulointerstitial injury and some parameters of mitochondrial function including mitophagy, mitochondrial fission, mitochondrial ROS, mitochondrial membrane potential (MMP), mitochondrial ATP, Complex V activity and mitochondria-associated ER membranes (MAMs) integrity were examined. Additionally, paricalcitol, 3-MA (an autophagy inhibitor), VDR over-expression plasmid, VDR siRNA and Mfn2 siRNA were applied in vitro.
RESULTS: The expression of VDR, Pink1, Parkin, Fundc1, LC3II, Atg5, Mfn2, Mfn1 in renal tubular cell of diabetic rats were decreased significantly. Calcitriol treatment reduced the levels of urinary albumin, serum creatinine and attenuated renal tubulointerstitial fibrosis in STZ induced diabetic rats. In addition, VDR agonist relieved mitophagy dysfunction,
MAMs integrity, and inhibited mitochondrial fission, mitochondrial ROS. Co-immunoprecipitation analysis demonstrated that VDR interacted directly with Mfn2. Mitochondrial function including mitophagy, mitochondrial membrane potential (MMP), mitochondrial Ca2+, mitochondrial ATP and Complex V activity were decreased dramatically in HK-2 cells under HG/TGF-β ambience. In vitro pretreatment of HK-2 cells with autophagy inhibitor 3-MA, VDR siRNA or Mfn2 siRNA negated the activating effects of paricalcitol on mitochondrial function. Pricalcitol and VDR over-expression plasmid activated Mfn2 and then partially restored the
MAMs integrity. Additionally, VDR restored mitophagy was partially associated with
MAMs integrity through Fundc1.
CONCLUSIONS: Activated VDR could contribute to restore mitophagy through Mfn2-
MAMs-Fundc1 pathway in renal tubular cell. VDR could recover mitochondrial ATP, complex V activity and
MAMs integrity, inhibit mitochondrial fission and mitochondrial ROS. It indicating that VDR agonists ameliorate renal tubulointerstitial fibrosis in diabetic rats partially via regulation of mitochondrial function.