Protective Effect And Mechanism Of Hepatocyte Nuclear Factor-1β On Renal Injury Induced By Cisplatin Or High Glucose

BackgroundAcute kidney injury(Acute Kidney Injury,AKI)refers to a sudden and sustained decline in renal function,clinically manifested as azotemia,water and electrolyte and acid-base balance disorders.AKI occurs in approximately 10%to 15%of inpatients.The use of nephrotoxic drugs is a common cause of AKI,accounting for 55%-60%of hospitalized AKI cases.The drugs that cause kidney damage mainly include:tumor chemotherapy drugs and non-cholesterol anti-inflammatory analgesics.Cisplatin is a powerful chemotherapeutic agent widely used in cancer treatment,One quarter of patients receiving cisplatin have nephrotoxic renal injury.In addition,high glucose stimulation is also an important factor that causes kidney damage.Elevated glucose levels in the body can cause mitochondrial dysfunction and trigger caspase cascades,eventually activating Caspase-3 mediated apoptosis.Renal function and apoptosis and proliferation of renal tubular cells are tightly regulated by transcription factors.However,the transcriptional regulation mechanism of renal injury caused by cisplatin and high glucose is still unclear.HNF-1β(Hepatocyte Nuclear Factor-1β)is a homologous domain transcription factor,mainly expressed in the proximal tubular epithelial cells of the kidney,which can promote the regeneration and repair of tubular epithelial cells through mitochondrial respiration and cell polarity.The previous results indicate that HNF-1β has a protective effect on cisplatin-induced renal injury.However,the protective role and mechanism of HNF-1β in the regulation of cisplatin and high glucose stimulation-induced renal injury still need further study.Apoptosis can be activated by the endoplasmic reticulum pathway,death receptor pathway and mitochondrial pathway.Increased activation of NF-κB(nuclear factor-activated κ-light chain enhancer of B cells)can trigger the caspase cascade.NF-κB is a protein complex that is an important nuclear transcription factor in the cell.It can participate in the body’s inflammatory response and immune response,and at the same time it can regulate cell apoptosis and stress response.NF-κB molecule is a heterodimer composed of two active subunits,P50 and P65,the N-terminus contains a Rel homology domain,which can be combined with DNA and participate in dimerization,and the C-terminus contains a trans-transcription activation domain,which can regulate inflammation.Under normal conditions,NF-κB and its inhibitory protein IκBα exist in the form of a complex in the cytoplasm to inhibit their own nuclear transcription and are in an inactive state;When the cell is stressed,the IκBα proteasome degrades,and NF-_κB is released from the cytoplasmic NF-κB/IκBα complex,and activates and exposes the nuclear localization domain,quickly translocating into the nucleus,and related gene sequences in the nucleus combination occurs to regulate inflammation,apoptosis,and cell proliferation Therefore,it is speculated that HNF-1β regulates cisplatin and high glucose-induced renal injury through the NF-κB pathway.This subject designed in vivo and in vitro experiments to study the protective mechanism of HNF-1β in cisplatin or high glucose-induced kidney injury,and to provide new strategies for slowing kidney injury。Objective1.To study the protective mechanism of HNF-1β in cisplatin kidney injury.2.To study the role and mechanism of HNF-1β in renal injury induced by high glucose.Methods1.In vitro experiments to analyze the mechanism of HNF-1β on cisplatin-induced RPTC cell apoptosisCisplatin(20 μM)treated RPTC HNF-1β NC(Rat proximal renal tubular epithelial cell hepatocyte nuclear factor-1β control)and RPTC HNF-1β KD(Rat proximal renal tubular epithelial cell hepatocyte nuclear factor-1β knockdown)after 20 hours to extract cellular protein,and Hoechst staining was used to detect apoptosis before extracting cellular protein,Western blot was used to detect the protein expression levels of HNF-1β,Caspase-3 spliceosome,P65,phospho-P65 and phospho-P53.Confocal detection was used to detect the localization of P65 after treatment with Cisplatin(20 μM)for RPTC HNF-1β NC and RPTC HNF-1β KD cells after 20 hours.At the same time,after treating RPTC cells with Cisplatin(20 μM)for 20 hours,the location of HNF-1β was detected by confocal detection.In addition,Cisplatin(20 μM)treated RPTC HNF-1β NC and RPTC HNF-1β KD cells respectively for 20 hours and collected the cells for nucleoplasm separation experiment,Western blot was used to detect the protein expression levels of P65 and phospho-P65 in cytoplasmic protein and nuclear protein after Cisplatin treatment.2.In vitro experiments to analyze the mechanism of HNF-1β on cisplatin-induced apoptosis of NRK cellsFive interfering plasmids,HNF-1β shRNA(1),HNF-1β shRNA(2),HNF-1β shRNA(3),HNF-1β shRNA(4)and HNF-1β shRNA scr,were stably transferred into NRK(Rat normal kidney cells)to construct NRK HNF-1β shRNA interfering cell lines.Western blot detected the protein expression of HNF-1β to determine the transfection effect.Cisplatin(50 μM)treated NRK HNF-1β shRNA scr and NRK HNF-1β shRNA(4)cells after 20 hours to extract cellular protein,and Hoechst staining was used to detect apoptosis before extracting cellular protein,Western blot was used to detect the protein expression levels of HNF-1β,Caspase-3,and Cleaved-caspase3,and Tunel staining was used to detect apoptosis.3.In vitro experiments to analyze the effect of NF-κB inhibitor on cisplatin-induced RPTC cell apoptosisCisplatin(20 μM)alone stimulated or Cisplatin(20 μM)and NF-κB inhibitor(TPCA-1)(50 mM)stimulated RPTC HNF-1βNC and RPTC HNF-1β KD cells to collect cellular proteins after 20 hours,Before extracting cell proteins,Hoechst staining was used to detect apoptosis,Western blot was used to detect the protein expression levels of Cleaved-PARP,Caspase-3,and Cleaved-caspase3,Tunel staining was used to detect apoptosis.4.In vivo experimental analysis of the expression of HNF-1β and Cleaved-caspase3 in mouse renal cortex during cisplatin-induced renal injuryC57BL/6 mice were injected intraperitoneally with Cisplatin to establish an acute kidney injury model.The mice were randomly divided into four groups,namely Control group,Cisplatin 1 d group,Cisplatin 2 d group,Cisplatin 3 d group,Mice in each group were given intraperitoneal injection at a dose of 30 mg/Kg Cisplatin,and the jugular venous blood and left and right kidneys of mice were collected at 24 h,48 h,and 72 h after modeling.Jugular venous blood was used to detect the levels of creatinine and urea nitrogen in mice.The kidney was divided into two parts with surgical scissors:one part was used for Western blot to detect the protein expression levels of HNF-1β,Caspase-3,and Cleaved-caspase3;the remaining part was fixed with 4%paraformaldehyde for HE and IHC staining,HE staining was used to detect the pathological changes of kidney tissue,and IHC staining was used to detect the expression of Cleaved-caspase3 in kidney tissue.5.In vivo experimental analysis of HNF-1β expression in mouse renal cortex in HNF-1βinterference mouse modelC57BL/6 mice were injected with HNF-1β interference plasmid through the tail vein to construct HNF-1β interference model.The mice were randomly divided into three groups,namely:HNF-1β shRNA scr 24 h group,HNF-1β shRNA(3)24 h group,HNF-1β shRNA(3)72 h group,the mice in each group were injected into the tail vein according to the PEI+40 μg HNF-1β interference plasmid,and the left and right kidneys of the mice were extracted 24 h and 72 h after modeling.The surgical scissors were used to divide the kidney into two parts:one part was used for Western blot to detect the expression level of HNF-1β protein;the remaining part was fixed with 4%paraformaldehyde for HE and IHC staining respectively,and HE staining was used to detect the pathological changes of kidney tissue,IHC staining was used to detect the expression of HNF-1β in kidney tissues.6.In vivo experimental analysis of the expression of HNF-1β and Cleaved-caspase3 in the renal cortex in the model of cisplatin-induced HNF-1β interference in miceA model of acute kidney injury induced by cisplatin-induced HNF-1β interference in C57BL/6 mice was established.Mice were randomly divided into four groups:HNF-1β shRNA scr 72 h group,HNF-1β shRNA(3)72 h group,Cisplatin+HNF-1β shRNA scr 72 h group,Cisplatin+HNF-1β shRNA(3)72 h group,The mice in each group were injected with tail vein according to PEI+40μg HNF-1β interference plasmid,and the Cisplatin group was given intraperitoneal injection with a dose of 30 mg/Kg.The left and right kidneys of mice were extracted 72 h after modeling.The kidney tissues were fixed with 4%paraformaldehyde and then stained with HE and IHC respectively.HE staining was used to detect the pathological changes of kidney tissue.IHC staining was used to detect the expression of HNF-1β and Cleaved-caspase3 in kidney tissue7.In vitro experiments to analyze the mechanism of HNF-1β on high glucose-induced apoptosis of RPTC cellsHigh glucose(HG,30 mM)was used to stimulate RPTC cells,and Hoechst staining was used to detect apoptosis at 6 h,12 h,24 h and 48 h.After extracting cellular proteins,Western blot was used to detect the protein expression levels of HNF-1β,Cleaved-PARP,Caspase-3,and Cleaved-caspase3,Then stimulate RPTC HNF-1β NC,RPTC HNF-1β KD cells with low glucose(LG,5.5 mM),HG(30 mM),low glucose+mannitol(LG,5.5 mM+D-mannitol,24.5 mM)for 48 h collect cellular proteins,Western blot was used to detect the protein expression levels of HNF-1β,Cleaved-PARP,Caspase-3,and Cleaved-caspase3,and Tunel staining was used to detect the apoptosis.Results1.HNF-1β can reduce cisplatin-induced RPTC and NRK cell apoptosis and P65 phosphorylationIn RPTC cells,Cisplatin treatment up-regulated the expression of Casepase-3 spliceosome protein and the phosphorylation of P65 and P53,and Cisplatin treatment promoted the nuclear translocation of HNF-1β and P65 in RPTC cells.But compared with cytoplasmic proteins,HNF-1β can reduce the phosphorylation of P65 in the nucleus.At the same time,in NRK cells,Cisplatin treatment promotes increased apoptosis of HNF-1β knockdown cells and can up-regulate the expression of Caspase-3 spliceosome protein.2.NF-κB inhibitor(TPCA-1)reduces cisplatin-induced RPTC cell apoptosisHoechst staining and Tunel staining showed that TPCA-1 significantly reduced Cisplatin-induced RPTC cell apoptosis,Western blot analysis showed that TPCA-1 down-regulated the protein expression levels of Caspase-3 spliceosome and Cleaved-PARP.3.Cisplatin induces acute kidney injury in C57BL/6 mice and down-regulates HNF-1βprotein expressionThe histopathological results of mouse kidney showed that compared with the Control group,the renal tissue of the Cisplatin group had different degrees of renal tubular structure destruction,renal tubular cell shedding,and enlarged renal space.Moreover,with the prolonged treatment time of Cisplatin,the serum creatinine and the urea nitrogen levels of C57BL/6 mice increased significantly.In addition,compared with the Control group,the Cisplatin group:the expression of Cleaved-caspase3 in kidney tissue was significantly increased and HNF-1βprotein could be down-regulated4.In the HNF-1β interference C57BL/6 mouse model,the interference efficiency of HNF-1β in the renal cortex reached 82%,and HNF-1β can reduce cisplatin-induced acute kidney injuryThe histopathological results of the kidney of C57BL/6 mice showed that after the injection of the HNF-1β control plasmid and the interference plasmid,the mouse kidney tissue structure was intact and no obvious damage occurred.The results of IHC and Western blot analysis showed that:compared with HNF-1β shRNA scr group,HNF-1β shRNA(3)group HNF-1β expression in kidney tissue was significantly reduced.After intraperitoneal injection of cisplatin,compared with the control group,the Cisplatin group:Compared with the HNF-1βshRNA scr group,the HNF-1β shRNA(3)group showed obvious renal tubular structure damage and renal tubular cells Phenomenon such as falling off.And compared with the control group,the Cisplatin group:Compared with the HNF-1β shRNA scr group,the expression of Cleaved-caspase3 in the kidney tissue in the HNF-1β shRNA(3)group was significantly increased.5.HNF-1β can reduce high glucose-induced apoptosis of RPTC cellsHoechst staining showed that with the extension of HG treatment time,the apoptosis of RPTC cells gradually increased.Western blotting analysis showed that the expression of HNF-1β protein was up-regulated and then down-regulated when HG(30 mM)stimulated RPTC,and the expression of Cleaved-PARP and Cleaved-caspase3 increased when HG stimulated RPTC for 6 hours,and showed time dependence.In addition,compared with RPTC HNF-1βNC cells,compared with LG group and LG+D-mannitol group,HNF-1β knockdown apoptosis in HG group was significantly increased.Conclusion1.HNF-1β may reduce cisplatin-induced renal injury by regulating the NF-κB pathway.2.HNF-1β protects renal tubular epithelial cell apoptosis induced by high glucose.