2026 年第 8 期 第 21 卷

肾小管上皮细胞中N-钙黏蛋白丢失加重肾纤维化的炎症损伤机制分析

Mechanism analysis of inflammatory injury aggravated by loss of N-cadherin in renal tubular epithelial cells during renal fibrosis

作者：胡玉英1田茂青2张璐1王惠明1

英文作者：Hu Yuying1 Tian Maoqing2 Zhang Lu1 Wang Huiming1

单位：1武汉大学人民医院肾内科，武汉430060；2武汉大学中南医院肾内科，武汉430071

英文单位：1Department of Nephrology Renmin Hospital of Wuhan University Wuhan 430060 China; 2Department of Nephrology Zhongnan Hospital of Wuhan University Wuhan 430071 China

关键词：近端肾小管；肾纤维化；N-钙黏蛋白；炎症；细胞焦亡

英文关键词：Proximalrenaltubule;Renalfibrosis;N-cadherin;Inflammation;Pyroptosis

• 摘要：

• 目的 探讨肾纤维化早中期N-钙黏蛋白表达缺失加重肾纤维化病理损伤的机制。方法 构建单侧输尿管梗阻（UUO）小鼠模型及转化生长因子β1（TGF-β1）诱导的体外人肾小管上皮（HK-2）细胞纤维化反应模型。采用肾小管特异性Cdh2敲除（Cdh2cKO）小鼠及Cdh2 shRNA沉默的HK-2细胞。将小鼠分为假手术组、UUO组、假手术+Cdh2cKO组和UUO+Cdh2cKO组；将HK-2细胞分为对照组、TGF-β1组、Cdh2 shRNA组和Cdh2 shRNA+TGF-β1组。通过蛋白质印迹法、免疫荧光染色法检测炎症介质及核苷酸结合寡聚化结构域样受体3（NLRP3）/半胱氨酸天冬氨酸蛋白酶1（CASP1）/Gasdermin家族蛋白D（GSDMD）细胞焦亡通路相关蛋白表达。结果 小鼠肾组织蛋白质印迹法结果表明，假手术+Cdh2cKO组N-钙黏蛋白水平低于假手术组，纤维连接蛋白（FN1）、α-平滑肌肌动蛋白（α-SMA）、单核细胞趋化蛋白1（MCP-1）、白细胞介素1β（IL-1β）和肿瘤坏死因子α（TNF-α）水平均高于假手术组，且UUO+Cdh2cKO组均高于假手术+Cdh2cKO组（均P＜0.05）。免疫荧光染色结果显示，假手术组、UUO组、假手术+Cdh2cKO组、UUO+Cdh2cKO组IL-1β相对荧光强度分别为（1.00±0.32）、（2.59±0.21）、（2.26±0.41）、（3.58±0.27），其中假手术+Cdh2cKO组高于假手术组，且UUO+Cdh2cKO组高于假手术+Cdh2cKO组（均P＜0.05）。HK-2细胞蛋白质印迹法结果 表明，Cdh2 shRNA组N-钙黏蛋白水平低于对照组，FN1、α-SMA、MCP-1、IL-1β和TNF-α水平均高于对照组，且Cdh2 shRNA+TGF-β1组均高于Cdh2 shRNA组（均P＜0.05）。细胞免疫荧光染色结果表明，Cdh2 shRNA组IL-1β、TNF-α水平均高于对照组，且Cdh2 shRNA+TGF-β1组均高于Cdh2 shRNA组（均P＜0.05）。小鼠肾组织蛋白质印迹法结果表明，假手术+Cdh2cKO组GSDMD、GSDMD-N、NLRP3、前CASP1及CASP1 p20的蛋白表达水平均高于假手术组，且UUO+Cdh2cKO组均高于假手术+Cdh2cKO组（均P＜0.05）。免疫荧光染色结果表明，假手术+Cdh2cKO组NLRP3水平高于假手术组，且UUO+Cdh2cKO组高于假手术+Cdh2cKO组（均P＜0.05）。HK-2细胞蛋白质印迹法结果表明，Cdh2 shRNA组GSDMD、GSDMD-N、NLRP3、前CASP1及CASP1 p20的蛋白表达水平均高于对照组，且Cdh2 shRNA+TGF-β1组均高于Cdh2 shRNA组（均P＜0.05）。细胞免疫荧光染色结果表明，Cdh2 shRNA组NLRP3、CASP1水平均高于对照组，且Cdh2 shRNA+TGF-β1组均高于Cdh2 shRNA组（均P＜0.05）。结论 N-钙黏蛋白丢失可能通过激活NLRP3/Caspase-1/GSDMD介导的细胞焦亡通路加剧TECs炎症反应，进而促进肾纤维化进展。

• Objective To investigate the mechanism by which loss of N-cadherin expression aggravates pathological injury in the early and middle stages of renal fibrosis. Methods A mouse model of unilateral ureteral obstruction (UUO) and an in vitro human renal tubular epithelial (HK-2) cell fibrosis model induced by transforming growth factor-β1 (TGF-β1) were established. Tubule-specific Cdh2 knockout (Cdh2cKO) mice and Cdh2 shRNA-silenced HK-2 cells were utilized. Mice were divided into sham operation group, UUO group, sham operation+Cdh2cKO group and UUO+Cdh2cKO group; HK-2 cells were assigned to control group, TGF-β1 group, Cdh2 shRNA group and Cdh2 shRNA+TGF-β1 group. Western blot and immunofluorescence staining were performed to detect the expression of inflammatory mediators and proteins related to the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3)/caspase-1 (CASP1)/Gasdermin D (GSDMD) pyroptosis pathway. Results Western blot results of mouse renal tissues showed that the level of N-cadherin in the sham operation+Cdh2cKO group was lower than that in the sham operation group, while the levels of fibronectin 1 (FN1), α-smooth muscle actin (α-SMA), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were higher than those in the sham operation group; all indicators in the UUO+Cdh2cKO group were further elevated compared with the sham operation+Cdh2cKO group (all P＜0.05). Immunofluorescence staining revealed that the relative IL-1β fluorescence intensities of the sham operation group, UUO group, sham operation+Cdh2cKO group and UUO+Cdh2cKO group were (1.00±0.32), (2.59±0.21), (2.26±0.41) and (3.58±0.27), respectively. The fluorescence intensity was higher in the sham operation+Cdh2cKO group than in the sham operation group, and higher in the UUO+Cdh2cKO group than in the sham operation+Cdh2cKO group (all P＜0.05). Western blot results of HK-2 cells indicated that N-cadherin expression was decreased in the Cdh2 shRNA group relative to the control group, whereas FN1, α-SMA, MCP-1, IL-1β and TNF-α were upregulated; the Cdh2 shRNA+TGF-β1 group exhibited higher levels of the above proteins than the Cdh2 shRNA group (all P＜0.05). Cellular immunofluorescence staining demonstrated that the expression levels of IL-1β and TNF-α were higher in the Cdh2 shRNA group than in the control group, and further increased in the Cdh2 shRNA+TGF-β1 group compared with the Cdh2 shRNA group (all P＜0.05). Western blot of mouse renal tissues showed that the protein levels of GSDMD, GSDMD-N, NLRP3, pro-CASP1 and CASP1 p20 in the sham operation+Cdh2cKO group were higher than those in the sham operation group, and those in the UUO+Cdh2cKO group were higher than those in the sham operation+Cdh2cKO group (all P＜0.05). Immunofluorescence staining results showed that the NLRP3 level in the sham operation+Cdh2cKO group was higher than that in the sham operation group, and that in the UUO+Cdh2cKO group was higher than that in the sham operation+Cdh2cKO group (both P＜0.05). Western blot of HK-2 cells showed that the expression of GSDMD, GSDMD-N, NLRP3, pro-CASP1 and CASP1 p20 was increased in the Cdh2 shRNA group versus the control group, with further upregulation observed in the Cdh2 shRNA+TGF-β1 group (all P＜0.05). Cellular immunofluorescence staining showed higher NLRP3 and CASP1 levels in the Cdh2 shRNA group than in the control group, and higher levels in the Cdh2 shRNA+TGF-β1 group than in the Cdh2 shRNA group (all P＜0.05). Conclusion Loss of N-cadherin may exacerbate inflammatory responses in renal tubular epithelial cells by activating the NLRP3/Caspase-1/GSDMD-mediated pyroptosis pathway, thereby accelerating the progression of renal fibrosis.