The Role And Mechanism Of PFKFB3 In Cisplatin-Induced Acute Kidney Injury

Background:Nephrotoxicity is one of the most common side effects of cisplatin during solid tumors chemotherapy.It is often manifested as acute kidney injury(AKI)characterized by acute decline in the excretion of endogenous metabolites such as blood urea nitrogen(BUN)and serum creatinine(Scr),which seriously limits its wide clinical application.The pathogenesis of cisplatin-induced nephrotoxicity is intricate.Previous studies from our group have found that a variety of cellular stress responses,including cell cycle changes and cell apoptosis,are involved in the pathophysiological process of cisplatin-induced AKI.However,it remains to be further explored which key molecules are involved in regulating these cellular stress responses during cisplatin-induced AKI.6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)is a bifunctional glycolytic enzyme with both kinase and phosphatase activities.In addition to promoting glycolysis,studies have shown that PFKFB3 also has atypical roles in regulating tumor cell cycle and apoptosis.However,few studies have investigated PFKFB3 in the field of kidney.Moreover,the role and mechanism of PFKFB3 in cisplatin-induced AKI remains unclear.Objects:In this study,we observed that PFKFB3 was upregulated in the nucleus of proximal renal tubules of cisplatin-induced AKI model in vivo and in vitro.Further,the promoting role of PFKFB3 on the process of cisplatin-induced AKI was clarified by constructing specific proximal tubules Pfkfb3-knockout mice(Pfkfb3-KO)and stable PFKFB3-knockdown rat proximal tubular cell lines.In addition,the potential molecular mechanism of PFKFB3 on regulation of cyclin-dependent kinase 4/ retinoblastoma tumor suppressor signaling pathway(CDK4/Rb)in cisplatin-induced AKI was explored.Methods:1.Expression and localization of PFKFB3 in cisplatin-induced AKI model in vivo and in vitro:(1)In vivo cisplatin-induced AKI model was induced by intraperitoneal injection of cisplatin(30mg/kg)in C57BL/6 mice.The blood urea nitrogen(BUN)and serum creatinine(Scr)were detected by biochemical method;the pathological changes of kidney were detected by hematoxylin and eosin(HE)staining;western blot,immunohistochemistry and immunofluorescence were used to detect the expression and localization of PFKFB3 in mice kidney.(2)In vitro cisplatin-induced AKI model was established by the treatment of 20μM cisplatin in RPTCs to induce cell apoptosis.Western blot and immunofluorescence were used to detect the expression and localization of PFKFB3 in RPTCs.2.Role of PFKFB3 in cisplatin-induced AKI:(1)In vivo,the Lox P-Cre system was used to generate Pfkfb3-KO mice,single dose of cisplatin 30mg/kg intraperitoneal injection was used to establish cisplatin-induced AKI model of Pfkfb3-KO and wild-type mice.The knockout efficiency of PFKFB3 was detected by PCR,western blot and immunohistochemistry;the survival rate of mice was observed;BUN and Scr were detected by biochemical method;renal pathological changes were detected by HE staining;the severity of renal tubular injury was evaluated by renal tubular injury score;cell apoptosis was detected by terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling(TUNEL).The protein level of cleaved caspase-3 was detected by western blot.(2)In vitro RPTCs were treated with 10μM PFK15(PFKFB3 inhibitor)+ 20μM cisplatin.Hoechst 33342 staining was used to observe the apoptosis rate of RPTCS;caspase-3 activity was measured by fluorescence spectrophotometer,and the protein level of cleaved caspase-3 protein was detected by western blot.Meanwhile,the stable PFKFB3 knockdown RPTCs were constructed by lentivirus transfection technology and then treated with 20μM cisplatin to induce cell apoptosis.Hoechst 33342 staining was used to observe the proportion of apoptotic cells;caspase-3 activity was measured by fluorescence spectrophotometer,and the protein level of cleaved caspase-3 protein was detected by western blot.3.The mechanism of PFKFB3 regulating CDK4/Rb in cisplatininduced AKI:1)The interaction between PFKFB3 and CDK4 in cisplatin-induced AKI model in vivo and in vitro(1)In vivo cisplatin-induced AKI model was induced by intraperitoneal injection of cisplatin(30mg/kg)in C57BL/6 mice.Kidney tissue protein was collected for co-immunoprecipitation and western blot to detect the interaction between PFKFB3 and CDK4 proteins.(2)In vitro cisplatin-induced AKI model was established by the treatment of 20μM cisplatin in RPTCs to induce cell apoptosis.Cell lysates was collected for co-immunoprecipitation and western blot to detect the interaction between PFKFB3 and CDK4 proteins.2)Expression and role of CDK4/Rb in cisplatin-induced AKI model in vivo and in vitro(1)In vivo cisplatin-induced AKI model was induced by intraperitoneal injection of cisplatin(30mg/kg)in C57BL/6 mice.The localization of CDK4 were detected by immunohistochemistry.The expression of CDK4 and phosphorylated retinoblastoma tumor suppressor(p-Rb),the target of CDK4,were detected by western blot.(2)Mice were gavage of CDK4 inhibitor Ribociclib(150mg/kg)and then in vivo cisplatin-induced AKI model was induced by single intraperitoneal injection of cisplatin(30mg/kg).The serum BUN and Scr were detected by biochemical method;the pathological changes of kidney were detected by HE staining;the severity of renal tubular injury was determined by renal tubular injury score.western blot was used to detect the expression of cleaved caspase-3.(3)In vitro cisplatin-induced AKI model was established by the treatment of 20μM cisplatin in RPTCs to induce cell apoptosis.The expression of CDK4 and p-Rb were detected by western blot.(4)RPTCs were treated with 20μM cisplatin and 10μM CDK4 inhibitor Ribociclib.Hoechst 33342 staining was used to observe the proportion of apoptotic cells;caspase-3 activity was measured by fluorescence spectrophotometer,and the protein level of cleaved caspase-3 protein was detected by western blot.3)Effect of PFKFB3 inhibition on CDK4/Rb in cisplatin-induced AKI(1)Pfkfb3-KO mice were intraperitoneally injected with single dose of cisplatin 30mg/kg to establish in vivo cisplatin-induced AKI model.western blot was used to detect the expression of p-Rb.(2)The stable PFKFB3 knockdown RPTCs were treated with 20μM cisplatin to establish in vitro cisplatin-induced AKI model.western blot was used to detect the expression of p-Rb.Results:1.Expression and localization of PFKFB3 in cisplatin-induced AKI model in vivo and in vitro:(1)Cisplatin increased BUN and Scr and caused renal tubular injury in C57BL/6 mice,indicating successful construction of in vivo cisplatininduced cisplatin-induced AKI model.The expression of PFKFB3 was increased in the nucleus of the proximal renal tubules of mice after cisplatin treatment.(2)Cisplatin induced apoptosis of RPTCs,indicating successful construction of in vitro cisplatin-induced AKI model.PFKFB3 expression was enhanced in the nucleus of RPTCs after cisplatin treatment.2.Role of PFKFB3 in cisplatin-induced AKI:(1)Pfkfb3-KO mice were successfully constructed for the first time.PFKFB3 protein depletion in the proximal renal tubules of Pfkfb3-KO mice ameliorated BUN and SCr,reduced renal tissue damage,and decreased the expression of TUNEL-positive cells and cleaved caspase-3 which induced by cisplatin treatment.(2)Both pharmacological inhibition and knockdown of PFKFB3 in RPTCs reduced the apoptosis proportion,inhibited caspase-3 activity,and decreased the expression of cleaved caspase-3 which induced by cisplatin treatment.3.The mechanism of PFKFB3 regulating CDK4/Rb in cisplatininduced AKI:(1)Co-immunoprecipitation results showed that PFKFB3 can directly interact with CDK4 in cisplatin-induced AKI in vitro and in vivo.(2)Blocking the CDK4/Rb signaling pathway by Ribociclib reduced BUN and SCr,alleviated the renal tissue damage and reduced the protein expression of cleaved caspase-3 in the renal tissue of mice which were induced by cisplatin.Blocking the CDK4/Rb signaling pathway of RPTCs by Ribociclib reduced proportion of apoptosis,inhibited the caspase-3activity,and decreased the protein expression of cleaved caspase-3 in RPTCS which were induced by cisplatin.(3)The expression of p-Rb protein in renal tissues and RPTCs was increased after cisplatin treatment.PFKFB3 knockout in proximal tubules in vivo(pfkfb3-KO)and Pfkfb3 knockdown in RPTCs in vitro could reduce the above changes of p-Rb protein.Conclusions:1.PFKFB3 is a key molecule in promoting cisplatin-induced AKI,which mediates tubular damage and cell apoptosis in cisplatin-induced AKI.2.The expression of CDK4/Rb is increased in cisplatin-induced AKI model of mice and RPTCs.Inhibition of CDK4 can alleviate the deterioration of renal function,tubular injury and cell apoptosis induced by cisplatin.3.Mechanistically,PFKFB3 may be involved in cisplatin-induced AKI by regulating the CDK4/Rb pathway.