Down-regulating GSK3β alleviates hypoxia/reoxygenation-induced injury of senescent renal tubular epithelial cells by inhibiting the function of ITPR1-GRP75-VDAC1 complex
10.3969/j.issn.1674-7445.2024018
- VernacularTitle:下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤
- Author:
Haiqiang NI
1
;
Shiqi GU
1
;
Xuan PENG
1
;
Nianqiao GONG
1
Author Information
1. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Organ Transplantation of Ministry of Education, Key Laboratory of Organ Transplantation of National Health Commission of China, Key Laboratory of Organ Transplantation of Chinese Academy of Medical Sciences, Wuhan 430030, China.
- Publication Type:OriginalArticle
- Keywords:
Glycogen synthase kinase 3β;
Kidney transplantation;
Middle aged and elderly donor;
Ischemia-reperfusion injury;
Mitochondrial damage;
Senescence;
Mitochondria-associated endoplasmic reticulum membrane;
Calcium overload;
Apoptosis
- From:
Organ Transplantation
2024;15(3):406-414
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the effect of glycogen synthase kinase 3β (GSK3β) on hypoxia/reoxygenation (H/R)-induced injury of senescent renal tubular epithelial cell (RTEC) in aged mice and its regulatory mechanism. Methods RTEC were divided into the Young group (young RTEC with normal growth), Old group (aged RTEC induced by Etoposide), Old+Ad-shNC+H/R group [aged RTEC induced by Etoposide and then transfected with adenovirus negative control (Ad-shNC) for H/R treatment], and Old+Ad-shGSK3β+H/R group (aged RTEC induced by Etoposide and then transfected with short-hairpin RNA-expressing adenovirus with targeted silencing GSK3β for H/R treatment), respectively. Apoptosis level and mitochondrial reactive oxygen species level were detected by flow cytometry. Calcium ion level was determined by immunofluorescence staining. The expression and phosphorylation levels of GSK3β, mitochondria-associated endoplasmic reticulum membrane (MAM)-related proteins of inositol 1,4,5-trisphosphate receptor1 (ITPR1), voltage dependent anion-selective channel 1(VDAC1) and glucose-regulated protein 75 (GRP75) were detected by Western blot. The interaction between GSK3β and MAM-related proteins was analyzed by immunoprecipitation. Results Compared with the Young group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old group. Compared with the Old group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were lower in the Old+Ad-shGSK3β+H/R group, and the differences were statistically significant (all P<0.05). Compared with the Young group, the expression levels of ITPR1, GRP75 and GSK3β proteins were up-regulated, the phosphorylation levels of ITPR1 and GRP75 were increased, whereas the total protein and phosphorylation levels of VDAC1 were decreased in the Old group. Compared with the Old group, the expression level of GSK3β protein was unchanged, the total protein and phosphorylation levels of ITPR1 and GRP75 were increased, the expression level of total VDAC1 protein remained unchanged and the phosphorylation level was increased in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the expression level of GSK3β protein was decreased, the expression levels of total ITPR1, GRP75 and VDAC1 proteins were unchanged, whereas the phosphorylation levels were decreased in the Old+Ad-shGSK3β+H/R group. Immunoprecipitation showed that GSK3β could interact with ITPR1, GRP75 and VDAC1 proteins. Conclusions The expression level of GSK3β is up-regulated in senescent RTEC. Down-regulating GSK3β expression may reduce the phosphorylation level of ITPR1-GRP75-VDAC1 complex, constrain the overload of mitochondrial calcium ion, protect mitochondrial function and mitigate cell damage during reperfusion.
