[1]王若霜,冷彦飞,袁飞远.mitoTEMPO 通过调控线粒体自噬对慢性肾脏病模型大鼠足细胞损伤的保护作用研究[J].现代检验医学杂志,2024,39(05):22-29.[doi:10.3969/j.issn.1671-7414.2024.05.005]
　WANG Ruoshuang,LENG Yanfei,YUAN Feiyuan.Study on the Protective Effect of mitoTEMPO on Podocyte Injury in Rat Models with Chronic Kidney Disease by Modulating Mitochondrial Autophagy[J].Journal of Modern Laboratory Medicine,2024,39(05):22-29.[doi:10.3969/j.issn.1671-7414.2024.05.005]
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mitoTEMPO 通过调控线粒体自噬对慢性肾脏病模型大鼠足细胞损伤的保护作用研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年05期
页码:
22-29
栏目:
论著
出版日期:
2024-09-15

文章信息/Info

Title:
Study on the Protective Effect of mitoTEMPO on Podocyte Injury in Rat Models with Chronic Kidney Disease by Modulating Mitochondrial Autophagy
文章编号:
1671-7414（2024）05-022-08
作者:
王若霜; 冷彦飞; 袁飞远
（凉山彝族自治州第一人民医院肾病科，四川凉山 615000）
Author(s):
WANG Ruoshuang;  LENG Yanfei;  YUAN Feiyuan
（Department of Nephrology，the First People’s Hospital of Liangshan Yi Autonomous Prefecture，Sichuan Liangshan 615000, China）
关键词:
线粒体靶向的2;  2;  6;  6- 四甲基哌啶-1- 氧基-4- 氨基-2- 氧基乙基; 慢性肾脏病; 足细胞损伤; PINK1/Parkin 信号通路; Nod 样受体家族成员蛋白3 炎症小体
分类号:
R-332
DOI:
10.3969/j.issn.1671-7414.2024.05.005
文献标志码:
A
摘要:
目的 探讨线粒体靶向的2, 2, 6, 6- 四甲基哌啶-1- 氧基-4- 氨基-2- 氧基乙基（mitochondria-targeted 2, 2, 6, 6-tetramethylpiperidin-1-oxy l-4-ylamino，mitoTEMPO）对慢性肾脏病（chronic kidney disease，CKD）模型大鼠足细胞损伤的影响及其相关分子机制。方法 18 只健康SD 大鼠随机分为对照（Control）组、模型组（CKD 组）和mitoTEMPO组，每组6 只。采用全自动生化分析仪测定肾功能；苏木素- 伊红染色检测肾病理结构；免疫组织化学法检测肾脏肌间质蛋白（desmin）和足突蛋白（podocin）表达；透射电镜检测足细胞超微结构；免疫荧光共染检测足细胞线粒体自噬；实时荧光定量PCR 检测肾脏炎性因子表达；蛋白免疫印迹法检测Nod 样受体家族成员蛋白3（nod-like receptor protein3，NLRP3）炎症小体，自噬和帕金蛋白（Parkin）/PTEN 诱导假定激酶1（PTEN induced putative kinase 1，PINK1）通路相关蛋白表达。结果 与Control 组比较，CKD 组大鼠24h 尿蛋白（84.89±8.98 mg/24h vs 5.79±1.39 mg/24h），血清肌酐（serum creatinine，Scr）（75.10±10.46 μmol/L vs 38.57±4.89 μmol/L），尿素氮（urea nitrogen，BUN）（8.96±1.07mmol/L vs 2.73±0.43mmol/L）水平升高，差异具有统计学意义（t=21.322，7.749，13.233，均P<0.001）；肾小球体积增大、系膜增生、大量炎性细胞浸润；足细胞足突融合，基底膜增厚，线粒体嵴断裂和空泡化； desmin 阳性区增多，podocin 阳性区减少，差异具有统计学意义（t=9.903，7.651，均P<0.001）； p62 和desmin 可见共定位； LC3 II/I，PINK1 和Parkin 蛋白表达降低（t=16.984，15.105，11.390），IL-1β，TNF-α，NLRP3，cleaved caspase-1 和p62 蛋白表达升高（t=5.700 ～ 21.571），差异具有统计学意义（均P<0.001）。与CKD 组比较，mitoTEMPO 组24h 尿蛋白、Scr 和BUN 水平降低，差异具有统计学意义（t=12.508，4.712，7.338，均P<0.001）；肾脏病理损伤和足细胞情况显著改善；desmin 阳性区减少，podocin 阳性区增多（t=6.649，7.686，all P<0.001）；LC3 和COX IV 可见共定位；LC3 II/I，PINK1 和Parkin 蛋白表达升高（t=15.481，20.469，5.801），IL-1β，TNF-α，NLRP3，cleaved caspase-1 和p62 蛋白表达降低（t=3.477 ～ 9.681），差异具有统计学意义（均P<0.001）。结论 mitoTEMPO 对CKD 足细胞损伤可产生保护作用，其机制可能与激活PINK1/parkin 通路介导的线粒体自噬来抑制NLRP3 炎症小体有关。
Abstract:
Objective To investigate the effect of mitochondria-targeted 2, 2, 6, 6-tetramethylpiperidin-1-oxyl-4-ylamino （mitoTEMPO）on podocyte injury in rat models with chronic kidney disease (CKD) and its related molecular mechanisms. Methods A total of 18 healthy SD rats were randomly divided into control group, model group (CKD group), and mitoTEMPO group, with 6 rats in each group. Renal function was measured by a fully automated biochemical analyzer, and hematoxylin eosin staining was used to detect renal pathological structure. Immunohistochemical staining was used to detect the expression of desmin and podocin in the renal muscle. Transmission electron microscopy was used to detect the ultrastructure of foot cells. Immunofluorescence co staining was used to detect mitochondrial autophagy in podocytes. Real time fluorescence quantitative PCR was used to detect the expression of renal inflammatory factors. Protein immunoblotting was used to detect the expression of Nod-like receptor protein 3 (NLRP3) inflammatory bodies, autophagy, and Parkin/PTEN induced putative kinase 1 (PINK1) pathway related proteins. Results Compared with the control group, the levels of 24h urinary protein（84.89±8.98 mg/24h vs 5.79±1.39 mg/24h）, serum creatinine (Scr) （75.10±10.46 μmol/L vs 38.57±4.89 μmol/L）and blood urea nitrogen (BUN) （8.96±1.07 mmol/L vs 2.73±0.43mmol/L）in CKD group were increased, with significant differences (t=21.322，7.749， 13.233，all P<0.001). The CKD group showed an increase in glomerular volume, mesangial proliferation, and extensive infiltration of inflammatory cells in rats. The foot processes of podocytes in the CKD group were fused, the basement membrane was thickened, and mitochondrial cristae were fractured and vacuolated. Compared with the control group, the positive area of desmin in the CKD group was increased, while the positive area of podocin was decreased, with significant differences (t=9.903, 7.651, all P<0.001) . Meanwhile, p62 and desmin were co-localized in the CKD group. In addition, the protein expressions of LC3 II/I，PINK1 and Parkin in the CKD group were decreased (t=16.984，15.105，11.390), while the expression of IL-1β, TNF-α, NLRP3, cleaved caspase-1 and p62 protein was increased (t= 5.700 ～ 21.571)，and the differences were significant（all P<0.001）, respectively. Compared with CKD group, the levels of 24h urinary protein, Scr and BUN in mitoTEMPO group were decreased, and the differences were significant (t=12.508，4.712，7.338，all P<0.001)．Renal pathological damage and podocyte condition of mitoTEMPO group were improved， while mitoTEMPO group showed decreased desmin-positive area and increased podocin-positive area (t=6.649，7.686，all P<0.001)，and colocalization of LC3 and COX IV was observed in mitoTEMPO group. In addition, the protein expressions of LC3 II/I， PINK1 and Parkin in mitoTEMPO group were increased (t=15.481, 20.469, 5.801), while IL-1β, TNF-α, NLRP3, cleaved caspase-1, and p62 protein were decreased (t= 3.477~9.681), and the differences were significant(all P<0.001), respectively. Conclusion The mitoTEMPO can be protective against CKD podocyte injury, and the mechanism may be related to the activation of PINK1/parkin pathway-mediated mitochondrial autophagy to inhibit NLRP3 inflammasome.

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备注/Memo

备注/Memo:
基金项目：四川省卫生健康科研课题（课题编号：19PJ302）。
作者简介：王若霜（1979-），男，硕士研究生，主治医师，研究方向：慢性肾脏病，E-mail：18981561416@163.com。
更新日期/Last Update: 2024-09-15