Chi Yinmao, Zhang Xiuli, Liang Dan, Wang Yue, Cai Xiaoyi, Dong Jiqiu, Li Lingzhi, Chi Zhihong
Department of Physiology, China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China.
Department of Nephrology, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, Guangdong Province, 518000, People's Republic of China.
Biol Trace Elem Res. 2023 May;201(5):2442-2457. doi: 10.1007/s12011-022-03361-w. Epub 2022 Jul 23.
Apoptosis of kidney tubular epithelial cells contributes to diabetic kidney disease (DKD) pathophysiology, but the mechanisms are not fully understood. Zinc transporter protein member 8 (ZnT8, SLC30A8) is a susceptive gene in diabetes. Here, we aim to investigate whether ZnT8 has effects on pathophysiology of DKD. The animal groups include control, ZnT8KO mice, STZ-induced, and ZnT8-KO-STZ. STZ-induced DKD was performed in male C57BL/6 J mice and in ZnT8-KO mice. High glucose (HG)-induced apoptosis in a normal rat kidney tubular epithelial cell line (NRK-52E cells) was performed in vitro. Transfection of hZnT8-EGFP or TNFAIP3 siRNA was done in NRK-52E cells. Flow cytometry with Annexin V-FITC/PI double staining and TUNEL analysis was performed for the detection of apoptosis. Gene expression at mRNA and protein levels was examined with real-time RT-PCR and Western blot. Urine albumin to creatinine ratio, proinflammatory cytokines, and apoptosis were enhanced in kidneys of STZ and ZnT8-KO-STZ mice compared to control or ZnT8-KO mice. ZnT8 overexpression after hZnT8-EGFP transfection decreased HG-stimulated inflammatory activity and apoptosis in NRK-52E cells. Furthermore, treatment with ZnSO blunted HG-induced apoptosis and NF-κB activation. ZnSO increased the abundance of zinc-finger protein TNF-α-induced protein 3 (TNFAIP3). Also, ZnT8 over-expression after hZnT8-EGFP transfection significantly ameliorates HG-induced NF-κB-dependent transcriptional activity and apoptotic protein expressions in NRK-52E cells, but the inhibitory effect of ZnT8 was significantly abolished with TNFAIP3 siRNA. Our study provides evidence that ZnT8 has protective effects against apoptosis of renal tubular epithelial cells through induction of TNFAIP3 and subsequent suppression of the NF-κB pathway.
肾小管上皮细胞凋亡参与糖尿病肾病（DKD）的病理生理过程，但其机制尚未完全明确。锌转运蛋白8（ZnT8，SLC30A8）是糖尿病的一个易感基因。在此，我们旨在研究ZnT8是否对DKD的病理生理有影响。动物分组包括对照组、ZnT8基因敲除（ZnT8KO）小鼠、链脲佐菌素（STZ）诱导组以及ZnT8基因敲除-STZ诱导组。对雄性C57BL/6 J小鼠和ZnT8基因敲除小鼠进行STZ诱导的DKD造模。在体外对正常大鼠肾小管上皮细胞系（NRK-52E细胞）进行高糖（HG）诱导的凋亡实验。对NRK-52E细胞进行hZnT8-EGFP转染或TNFAIP3小干扰RNA（siRNA）转染。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）双染流式细胞术和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）分析检测细胞凋亡。用实时逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测mRNA和蛋白质水平的基因表达。与对照组或ZnT8基因敲除小鼠相比，STZ诱导组和ZnT8基因敲除-STZ诱导组小鼠肾脏的尿白蛋白肌酐比值、促炎细胞因子水平及细胞凋亡增加。hZnT8-EGFP转染后ZnT8过表达降低了HG刺激的NRK-52E细胞炎症活性和凋亡。此外，硫酸锌（ZnSO）处理可减轻HG诱导的细胞凋亡和核因子κB（NF-κB）激活。ZnSO增加了锌指蛋白肿瘤坏死因子α诱导蛋白3（TNFAIP3）的丰度。而且，hZnT8-EGFP转染后ZnT8过表达显著改善了HG诱导的NRK-52E细胞中NF-κB依赖的转录活性和凋亡蛋白表达，但TNFAIP3 siRNA可显著消除ZnT8的抑制作用。我们的研究提供了证据，表明ZnT8通过诱导TNFAIP3及随后抑制NF-κB途径对肾小管上皮细胞凋亡具有保护作用。
