HIF-1α活化介导的胆固醇稳态失调加速大鼠的糖尿病肾病进展

HIF-1αactivation induces cholesterol homeostasis dysfunction to accelerate progression of diabetic nephropathy in rats

目的探讨糖尿病慢性缺氧时缺氧诱导因子-1α(HIF-1α)激活在糖尿病肾病(DN)胆固醇稳态失调中的作用及机制。方法将SD大鼠随机分为3组,10只/组:对照组、糖尿病组(DM)、YC-1干预组(DM+YC-1)。DM和DM+YC-1组大鼠接受60 mg/kg链脲佐菌素单次腹腔注射构建糖尿病模型,对照组大鼠接受等量柠檬酸缓冲液腹腔注射。体外研究中,应用氯化钴(CoCl_(2),100μmol/L)刺激有或无葡萄糖(30 mmol/L)条件下培养的人近端小管上皮细胞HK-2细胞24 h。采用24 h尿蛋白定量评估各组大鼠肾损伤情况,过碘酸-雪夫染色观察各组大鼠肾小管病理学损伤;总胆固醇定量检测及Filipin染色观察肾组织及HK-2细胞中胆固醇沉积情况;Westernblotting、免疫组织化学染色检测大鼠肾组织HIF-1α的蛋白表达、细胞免疫荧光检测HK-2细胞中HIF-1α的表达情况,检测大鼠肾组织及HK-2细胞中胆固醇代谢相关分子3-羟基3-甲基戊二酰辅酶A还原酶(HMGCR)、低密度脂蛋白受体(LDLr)、CXC型趋化因子配体16(CXCL16)和纤维化相关分子转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)的蛋白表达。结果与对照组相比,DM组大鼠24 h尿蛋白水平升高(P<0.001),肾小管损伤加重,且肾脏胆固醇含量升高(P<0.05)并伴有肾小管HIF-1α蛋白表达升高(P<0.01),而YC-1干预可降低糖尿病大鼠24 h尿蛋白水平(P<0.05)、明显减轻肾小管损伤、降低肾总胆固醇含量(P<0.05)。体外研究中,氯化钴有效诱导HIF-1α表达(P<0.0001)且显著增加HK-2细胞中的胆固醇含量(P<0.05),同时上调胆固醇代谢相关蛋白HMGCR、LDLr、CXCL16以及纤维化因子TGF-β1、CTGF的表达(P<0.01,P<0.05)。与细胞实验结果相一致,YC-1干预可下调糖尿病大鼠肾小管胆固醇代谢相关蛋白的表达,也减少纤维化因子TGF-β1(P<0.001)和CTGF(P<0.05)的表达。结论HIF-1α活化可通过增加胆固醇从头合成、摄取介导肾小管上皮细胞胆固醇稳态失调,加重细胞脂质沉积和损伤,促进DN进展。

Objective To investigate the effect of hypoxia inducible factor 1α(HIF 1α)activation on cholesterol homeostasis dysfunction in diabetic nephropathy(DN).Methods Rat models of type 1 diabetes established by intraperitoneal STZ injection were treated with intraperitoneal injection of Lificiguat(YC-1,a HIF-1αinhibitor).Human proximal tubular cell line HK-2 was incubated with cobalt chloride(CoCl_(2),100μmol/L)in the presence or absence of 30 mmol/L glucose for 24 h.Renal injury of the rats was assessed by measuring 24-h urinary total protein level and PAS staining of the renal tubules.Cholesterol deposition in rat kidneys and HK-2 cells were observed using a quantitative assay of total cholesterol and Filipin staining,and HIF 1αprotein expression was detected using Western blotting,immunohistochemistry or immunofluorescence assay;the expressions of cholesterol metabolism-related proteins HMGCR,LDLr,CXCL16 and profibrogenic factors including TGF-β1 and CTGF were also analyzed.Results The diabetic rats showed significantly increased 24-h urinary protein level(P<0.001),obvious renal tubular injury,and increased renal cholesterol content(P<0.05)with significantly increased HIF-1αexpression in the renal tubular(P<0.01).YC-1 treatment significantly ameliorated tubulointerstitial injury in the diabetic rats as shown by decreased 24-h urinary total protein(P<0.05)and reduced damage area of the tubules,and effectively decreased renal cholesterol levels and renal expression of HIF 1α(P<0.05).In HK-2 cells,CoCl_(2) stimulation in the presence of high glucose effectively activated HIF-1αexpression(P<0.0001),aggravated cholesterol accumulation(P<0.05),and increased the expressions of HMGCR,LDLr,CXCL16,TGF-β1,and CTGF(P<0.05 or 0.01).Consistent with the in vitro study,YC-1 treatment also significantly decreased the expressions of cholesterol metabolism-related proteins and the profibrogenic factors in the renal tubules of the diabetic rats.Conclusion HIF-1αactivation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol,thereby aggravating tubulointerstitial injury in DN.