Endoplasmic Reticulum Stress Contributes To Cisplatin-induced Chronic Kidney Disease Via The PERK-PKCδ Pathway

BackgroundCisplatin is an effective chemotherapeutic drug,but it may induce both acute and chronic kidney problems.The pathogenesis of chronic kidney disease(CKD)associated with cisplatin chemotherapy remains largely unclear.By establishing a repeated low-dose cisplatin(RLDC)model in vivo and in vitro,we explored the role of the PERK-PKCδpathway in cisplatin-related chronic kidney disease.Methods1.In vivo: To establish a mouse RLDC model: C57BL/6 mice(male,8-10 weeks old)were injected with cisplatin(concentration 8 mg/kg)by intraperitoneal injection once a week,four times in a row,and then measured after one week and one month.The glomerular filtration rate of mice,the weight of mice and kidneys were weighed,blood and kidney tissues were collected to determine the changes in renal function;the degree of kidney damage was observed by hematoxylin-eosin staining;the degree of renal fibrosis was determined by Masson staining;The expressions of PERK,P-PERK,e IF-2α and P-e IF-2α were detected by WB;the expression of P-PERK was detected by immunohistochemical staining.To investigate the role of endoplasmic reticulum stress in cisplatininduced chronic kidney disease,C57BL/6 mice(male,8-10 weeks old)were intraperitoneally injected with cisplatin(concentration 8 mg/kg)once a week,Hit four times in a row.The endoplasmic reticulum stress inhibitor4-PBA(20 mg/kg)and TUDCA(250 mg/kg)were injected daily for one week after the fourth cisplatin.One month later,the glomerular filtration rate of the mice was measured,the weight of the mice and kidneys were weighed,and blood and kidney tissues were collected;The expressions of PERK,P-PERK,e IF-2α,P-e IF-2α and P-PKCδ were detected by WB;PPERK was detected by immunohistochemical staining;the changes of renal function were determined by detecting the glomerular filtration rate,serum creatinine and blood urea nitrogen values;the degree of renal atrophy was observed by hematoxylin-eosin staining;Masson staining,FN and vimentin were detected by WB to determine the degree of renal fibrosis;the changes of inflammatory factors Mcp1 and Cxcl1 were detected by PCR;the inflammatory infiltration was determined by immunohistochemical staining to detect F4/80.2.In vitro: Establishment of repeated low-dose cisplatin(RLDC)model of cells: cultured BUMPT cells were cultured with different concentrations(0,0.5,1.0,2.0 μM)of cisplatin for 7 hours,and then treated with cisplatin-free The medium was cultured for 17 hours for 4 days,and the cells were harvested on the fifth day.The cell morphology was detected by light microscope;the expression of FN,vimentin,PERK,P-PERK,e IF-2α and P-e IF-2α was detected by WB.BUMPT cells were incubated with 2.0 μM cisplatin for 7 hours,then cisplatin-free medium for 17 hours for 4 days,and then treated with the endoplasmic reticulum stress inhibitor 4-PBA and the PERK inhibitor GSK2656157 for 17 hours,respectively.The cell morphology was detected by light microscope;the expression of FN,vimentin,PERK,PPERK,e IF-2α,P-e IF-2α was detected by WB in the collected cells;the inflammatory markers Mcp1 and Cxcl1 were detected by PCR.To examine the role of PKCδ in cisplatin-induced CKD,BUMPT cells transfected with PKCδ sh RNA,PKCδ KD and CF plasmids were cultured with 2.0 μM cisplatin for 7 hours and then with cisplatin-free medium for 17 hours for 4 days,and the cells were harvested on the fifth day to detect the expression of FN,vimentin,PERK,P-PERK,e IF-2α,Pe IF-2α and P-PKCδ by WB;the expression levels of inflammatory markers Mcp1 and Cxcl1 were detected by PCR.Findings(1)In C57BL/6 mice,RLDC induced endoplasmic reticulum(ER)stress activation in renal tubular epithelial cells.ER stress inhibitors given immediately after RLDC attenuated kidney dysfunction,tubular atrophy,kidney fibrosis and inflammation in mice.(2)In BUMPT cells,RLDC induced ER stress activation in proximal tubular epithelial cells.The ER stress inhibitor 4-PBA and the PERK inhibitor GSK2656157 attenuated RLDC-induced fibrosis and the expression of inflammatory cytokines.(3)RLDC induced the activation of protein kinase Cδ(PKCδ)in C57BL/6 mice and BUMPT cells.ER stress inhibitors and PERK inhibitors block PKCδ activation.Inhibition of PKCδ expression by overexpression of PKCδ knockdown plasmid(PKCδ-KD)or transfection of PKCδ-sh RNA plasmid attenuated RLDC-induced fibrosis and inflammation.Furthermore,overexpression of active PKCδ-catalytic fragment(PKCδ-CF)reduced the beneficial effects of PERK inhibitors in RLDC-treated cells.The coimmunoprecipitation results further indicated that PERK and PKCδinteracted with each other.InterpretationThese results indicate that ER stress may contribute to the development of chronic kidney pathologies following cisplatin chemotherapy via the PERK-PKCδ pathway.