南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (10): 1782-1788.doi: 10.12122/j.issn.1673-4254.2023.10.17

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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

王 影，周明俊，朱倩文，张 翠，王 林，李 曙，胡泽波   

  1. 皖南医学院基础医学院病理生理学教研室，临床医学院，安徽 芜湖 241002
  • 出版日期:2023-10-20 发布日期:2023-11-02

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

WANG Ying, ZHOU Mingjun, ZHU Qianwen, ZHANG Cui, WANG Lin, LI Shu, HU Zebo   

  1. Department of Pathophysiology, School of Preclinical Medicine, School of Clinical Medicine, Wannan Medical College, Wuhu 241002, China
  • Online:2023-10-20 Published:2023-11-02

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摘要： 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（CoCl2，100 μmol/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；Western blotting、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。

关键词: 糖尿病肾病；缺氧诱导因子-1α；脂质沉积；胆固醇；肾小管

Abstract: 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-1α inhibitor). Human proximal tubular cell line HK-2 was incubated with cobalt chloride (CoCl2, 100 μmol/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-1α protein 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-1α expression 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, CoCl2 stimulation in the presence of high glucose effectively activated HIF-1α expression (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-1α activation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol, thereby aggravating tubulointerstitial injury in DN.

Key words: diabetic nephropathy; hypoxia inducible factor-1α; lipid accumulation; cholesterol; tubules