Lin Ziyan, Chen Ai, Cui Hongwang, Shang Ruihua, Su Tian, Li Xiaoyan, Wang Kekun, Yang Jing, Gao Keli, Lv Jie, Shen Jie, Wang Shanzhi, Qi Yonghui, Guo Minghao, Zhu Yongjun
Department of Nephrology, The First Affiliated Hospital of Hainan Medical University, Haikou, China.
Department of Orthopedics, The First Affiliated Hospital of Hainan Medical University, Haikou, China.
FASEB J. 2022 Dec;36(12):e22625. doi: 10.1096/fj.202200706RR.
Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney disease (CKD), ultimately predisposes patients to end-stage renal disease. However, there is no effective therapy for renal fibrosis. Our earlier studies proved that RIP3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury. Under transmission electron microscopy (TEM), we found morphological changes in the necrosis of human renal tissue, and the percentage of necrotic cells increased significantly in patients with stages 2 and 3a CKD. Immunofluorescence analyses showed that the percentages of TUNEL /RIP3 double-positive and TUNEL /MLKL double-positive tubular epithelial cells in renal tubules of patients with stages 2 and 3a CKD were significantly increased compared to those in control patients without renal disease. Immunohistochemistry analyses of renal biopsy specimens from patients with CKD revealed RIP3, MLKL, and p-MLKL upregulation in patients with stages 2 and 3a CKD, suggesting that necroptosis of renal tubular epithelial cells in CKD patients occurs, and the peak of necroptosis was in stages 2 and 3a CKD. We showed that profibrotic factor proteins (TGF-β1, Smad2 and Smad3) and fibroblast activation markers (α-SMA and Vimentin) were specifically upregulated in stage 2 and 3a CKD patients. In addition, Pearson correlation analysis showed that the percentage of necroptotic renal tubular epithelial cells was positively correlated with TGF-β1 and collagen-I. We also showed that RIP1/3 or MLKL inhibitors decreased the expression of RIP3, MLKL, TGF-β1, and Smad3 in HK-2 cells treated with TNF-α. FGF-2, α-SMA, Vimentin and FN were overexpressed in the hRIFs cultured with the supernatant of necroptotic HK-2 cells, whereas necroptosis blockers (Nec-1s, GSK'872 and NSA) and TGF-β1/Smad3 pathway antagonists (LY364947 and SIS3) reduced FGF-2, α-SMA, Vimentin and FN levels. Collectively, necroptosis of renal tubular epithelial cells in CKD patients occurs, and the peak of necroptosis was in stages 2 and 3a CKD. Renal tubular epithelial cell necroptosis mediates renal tubulointerstitial fibrosis in patients with chronic kidney disease, which is related to the TGF-β1/Smad3 signaling pathway.
肾纤维化是几乎所有类型慢性肾脏病（CKD）的常见病理表现，最终会使患者易发展为终末期肾病。然而，目前尚无有效的肾纤维化治疗方法。我们早期的研究证明，RIP3介导的坏死性凋亡可能是慢性肾损伤大鼠肾小管细胞死亡的一种重要方式。在透射电子显微镜（TEM）下，我们发现了人肾组织坏死的形态学变化，并且在2期和3a期CKD患者中坏死细胞的百分比显著增加。免疫荧光分析显示，与无肾病的对照患者相比，2期和3a期CKD患者肾小管中TUNEL/RIP3双阳性和TUNEL/MLKL双阳性肾小管上皮细胞的百分比显著增加。对CKD患者肾活检标本的免疫组织化学分析显示，2期和3a期CKD患者中RIP3、MLKL和p-MLKL上调，这表明CKD患者肾小管上皮细胞发生了坏死性凋亡，且坏死性凋亡的高峰出现在2期和3a期CKD。我们发现，在2期和3a期CKD患者中，促纤维化因子蛋白（TGF-β1、Smad2和Smad3）和成纤维细胞活化标志物（α-SMA和波形蛋白）特异性上调。此外，Pearson相关性分析表明，坏死性凋亡的肾小管上皮细胞百分比与TGF-β1和I型胶原呈正相关。我们还发现，RIP1/3或MLKL抑制剂可降低用TNF-α处理的HK-2细胞中RIP3、MLKL、TGF-β1和Smad3的表达。在用坏死性凋亡的HK-2细胞上清液培养的人肾小管间质纤维化（hRIFs）中，FGF-2、α-SMA、波形蛋白和纤连蛋白过表达，而坏死性凋亡阻滞剂（Nec-1s、GSK'872和NSA）以及TGF-β1/Smad3信号通路拮抗剂（LY364947和SIS3）可降低FGF-2、α-SMA、波形蛋白和纤连蛋白水平。总体而言，CKD患者肾小管上皮细胞发生坏死性凋亡，且坏死性凋亡的高峰出现在2期和3a期CKD。肾小管上皮细胞坏死性凋亡介导慢性肾脏病患者的肾小管间质纤维化，这与TGF-β1/Smad3信号通路有关。
