Upregulation Of TIPE1 In Tubular Epithelial Cell Aggravates Diabetic Nephropathy By Disrupting PHB2 Mediated Mitophagy

Background:Diabetes Mellitus(DM)has become one of the diseases affecting human health in the world today,which is the most serious complication.Hyperglycemia resulted in various types of renal cells being damaged,more and more studies have identified that renal tubular epithelial cells play an important role in the injury and repair of various renal diseases.Recently,it has been found that the abnormal structure and function of renal tubular epithelial cells are the initial factors of renal unit injury and renal dysfunction in diabetic patients.Due to its unique position and important reabsorption function,renal tubular epithelial cells were exposed to various toxic factors of glomerular filtrates,such as albumin,high glucose,advanced glycation end products(AGEs),inflammatory factors,etc.Renal tubule epithelial cell death,senescence,and EMT lead to renal inflammation and fibrosis.Mitophagy removes damaged mitochondria and then maintains the quality and function of mitochondria,which maintain the stability of the intracellular environment.High glucose induced vacuolation and swelling of mitochondria,rupture of the mitochondrial crest,and inhibition of mitophagy activity in renal tubular epithelial cells,which induced insufficient production of the ATP、accumulation of ROS、accelerated progression of DN.Therefore,searching for a key molecule regulating mitochondrial autophagy activity of renal tubular epithelial cells will provide new experimental data for the treatment of diabetic nephropathy.Tumor necrosis factor-α induced protein 8 like-1(TNFAIP8L1)is a newly discovered regulatory molecule that regulates cell necrosis and apoptosis.These family members share a high degree of homology in amino acid sequence and protein structure and have a unique death effector domain(DED).At present,there are few studies on the biological function of TIPE1,mainly focusing on neoplastic diseases.Currently,there are few studies between TIPE1 and autophagy or autophagy-related pathways.Only one study suggested that TIPE1 and FBXW5 could competitively bind TSC,reduce ubiquitination of TSC,inhibit the mTOR signaling pathway,promote the autophagy of dopamine neurons and accelerate the pathogenesis of Parkinson’s disease.Recent studies have found that TIPE1 is highly expressed in renal tissues,especially in renal tubule epithelial cells at the proximal convoluted tubule and dermal medulla junction,with moderate expression in distal convoluted tubule and collecting tubules.However,the mechanism of TIPE1 regulating the occurrence and development of diabetic nephropathy is still lacking.In this study,a mouse model of diabetic nephropathy was constructed to explore the molecular mechanism of TIPE1 regulating renal tubular epithelial cells to regulate mitophagy,so as to provide a new therapeutic target for diabetic nephropathyResearch Objectives:1)To determine the expression changes of TIPE1 in renal tissues of DN animal models and clinical specimens;2)In vitro and in vivo experiments were conducted to investigate whether TIPE1 was involved in the occurrence and development of diabetic nephropathy by regulating mitophagy in renal tubular epithelial cells;3)To search for key molecules related to TIPE1 regulation of mitophagy in renal tubular epithelial cells and clarify its mechanism.Methods and Results:PartⅠ TIPE1 regulates mitochondrial autophagy and aggravates renal tubular epithelial cell injury1.TIPE1 expression is up-regulated in RTECs of DN patients and mice We first analyzed the expression of TIPE1 in DN patients and healthy donors(HD)using data from the Nephroseq Database.The result showed that TIPE1 expression was significantly increased in the DN group compared to that in the healthy donor group.Immunohistochemical and immunofluorescence staining further confirmed the upregulation of TIPE1 in renal tissues of DN patients.The diabetic nephropathy model was constructed by intraperitoneal injection of STZ,and wild-type WT mice were used as the control group.TIPE1 expression was detected by immunohistochemical staining,immunofluorescence staining,real-time quantitative fluorescence polymerase chain reaction,and western blot.The results showed that TIPE1 expression was upregulated in diabetic nephropathy and mainly located in renal tubules.In vitro,HK-2 cells were stimulated with different concentrations of high glucose medium,and TIPE1 expression was detected at the protein level and mRNA level respectively.2、Tipel deficiency in RTECs alleviates renal injury and fibrosis in DN.To further evaluate the role of TIPE1 in tubular cells in DN progression,we generated proximal tubular cell-specific Tipel knockout mice(T1KO)by crossing Kspcre and Tipelf/f mice,Cre-mice were used as a control group.Both groups were intraperitoneally injected with STZ to build diabetic nephropathy models.The results showed that the ratio of urinary protein creatinine in the T1KO group was significantly lower than that in the control group.WB,RT-qPCR,and histology showed that KIM1 and NGAL,markers of renal tubular injury,were downregulated in T1KO mice.The excessive deposition of extracellular matrix(ECM)proteins in the renal tubulointerstitium is a key event in the progression of DN.The changes in EMT indexes were detected by WB,RT-qPCR,and IH assay.The results showed that the expression of α-SMA,Vimentin,and N-cadherin in renal tissues of the T1KO group was significantly lower than that of the control group,while the expression of E-cadherin,an epithelial cell marker,was higher than that of the control group.The level of renal fibrosis in the T1KO group was lower than that in the WT group via Sirius red staining.These results suggest that Tipel knockout can significantly alleviate renal injury and fibrosis in DN.3.Interference of TIPE1 expression ameliorates renal tubular epithelial cell injury and EMT induced by high glucose In in vitro experiments,we infected human renal tubular epithelial cells HK-2 with shRNA vector TIPE1-interfering lentivirus(Lv-shTIPE1)and NC-negative control virus(Lv-NC)to construct stable interfering TIPE1 cell lines.HK-2 cells were stimulated with normal glucose(5.5mM)and high glucose(40mM)medium for 48h,and the effect of TIPE1 interference was verified by WB,meanwhile,KIM1 expression(a marker of renal tubular injury)was significantly downregulated in Lv-shTIPE1 group than the negative group.In addition,EMT markers of N-cadherin and Slug were also downregulated in HK-2 cells infected with TIPE1-interfering lentivirus.4.Ablation of TIPE1 improves mitochondrial homeostasis and decreases apoptosis To further investigate how TIPE1 modulates HG-induced damage and EMT progression in RTECs,we performed a transcriptomic analysis of the renal cortex of WT and TIKO mice with STZ-induced DN.Transcriptional data analysis showed that the TCA cycle was significantly enriched in the T1KO group,which showed that TIPE1 may regulate the function of mitochondria.Then we detected the ROS level of mouse kidney tissues by DHE staining and ATP level by kit detection.The results showed that the ROS level was lower in T1KO mice than the control group,while the ATP level was higher than the control group.These results indicate that Tipel knockout plays an important protective role in alleviating mitochondrial damage and maintaining mitochondrial function.In vitro experiments,seahorse assay,and flow cytometry were used to detect the oxidative phosphorylation level of cells and the levels of MitoSOX and Mitotracker-Red.The results showed that compared with the negative control group,the oxidative phosphorylation level of the Lv-shTIPE1 group increased,and the mitochondrial ROS level decreased significantly,and mitochondrial membrane potential increased.In addition,interference of TIPE1 reduced the RTECs apoptosis induced by high glucose by flow cytometry.5.Enhanced mitophagy by Tipel deletion protects RTECs from HG-induced damage and EMT KEGG analysis of transcriptomic data and heat map analysis of differential genes showed that the mitophagy signal pathway was significantly changed among differential genes.Mitophagy markers Pink1,Parkin,Atg12,and MFN1/2 were significantly increased,while Sqstml/P62 and Rps27a expression were decreased.This identified that the ablation of Tipel activates mitophagy of renal tubular epithelial cells.Then,TEM analysis showed that the number of mitophagosomes in RTECs of T1KO mice was significantly lower than that in the WT group.Mitophagy markers in renal tissue mitochondrial protein were detected by WB.The results showed that compared with WT-DN mice,the expressions of PINK1,Parkin,and LC3II were significantly increased in renal tissue of T1KO-DN mice,while the expression of P62,an indicator of autophagy substrate degradation,was decreased.In in vitro experiments,in order to further clarify the regulation of TIPE1 in mitophagy of renal tubular epithelial cells,we treated cells with Mdivi-1,a mitophagy inhibitor,on the basis of TIPE1 interference.Renal tubular injury marker KIM1 and EMT markers were also detected by WB and there was no significant difference between Lv-NC and Lv-shTIPE1 groups.These results suggest that TIPE1 mediates cell damage and EMT by regulating mitophagy.Part Ⅱ Mechanism of TIPE1 regulating renal tubular epithelial cell mitophagy1.TIPE1 interacts with PHB2 and promotes proteasome degradation of PHB2 To investigate the key molecular of mitophagy regulated by TIPE1,we performed LC-MS/MS using immunoprecipitants from pcTIPE1-HA transfected cells and found that TIPE1 could interact with PHB2.Next,we detected exogenous and endogenous binding in 293 cells and HK-2 cells by CO-IP assay.PHB2 is widely distributed and highly expressed in mitochondria.It is believed that PHB2 acts as a mitochondrial inner membrane receptor to regulate mitophagy.We found that PHB2 was highly expressed in mitochondria and PHB2 expression was downregulated with high glucose stimulation,while interference with TIPE1 expression significantly promoted PHB2 expression by WB and IHC assays.In Lv-shTIPE1 cells,PHB2 mRNA was comparable with Lv-NC infected cells.This suggested that the regulation of TIPE1 on PHB2 might be regulated at the protein level.Then,Cycloheximide(CHX)chase assay further revealed that TIPE1 knockdown increased the half-life of the PHB2 protein in HK-2 cells.In addition,treatment with the proteasome inhibitor(MG132),but not the autophagy inhibitor chloroquine(CQ),abrogated the upregulation of PHB2 induced by TIPE1 silencing in HK-2 cells,indicating that TIPE1 promotes proteasomal degradation of PHB2.The ubiquitination experiment showed that TIPE1 promoted the ubiquitination degradation of PHB2 in HEK293 cells.2.Knockdown of PHB2 abrogates the improvement of TIPE1 deficiency in mitophagy,damage,and EMT of RTECs To further clarify that the regulation of TIPE1 on mitophagy is mediated by PHB2,we verified it in vitro and in vivo by PHB2 lentivirus.In in vitro experiments,we interfered with PHB2 expression on the basis of interfering TIPE1,and the results showed that compared with the group without interfering PHB2,renal injury markers and EMT markers were not significantly changed by interfering TIPE1.Mitochondrial ROS and mitochondrial membrane potential were detected by flow cytometry,and the results showed that the regulation of TIPE1 on mitochondrial ROS and mitochondrial membrane potential disappeared after PHB2 interference.In the in vivo experiment,Phb2-interfering lentivirus and NC-negative control virus were injected into T1KO mice and WT mice renal and constructed diabetic nephropathy mouse model on this basis.The changes in mitophagy indexes were detected by WB,and the results showed that there were no differences in the expressions of Parkin,PINK1,P62,and LC3Ⅰ/Ⅱbetween WT and T1KO mice after PHB2 interference.In addition,there was no difference in the expression of kidney injury markers NGAL and EMT(E-cadherin,Ncadherin,Vimentin,α-SMA).The same results were obtained by HE staining and immunohistochemical staining.3.Clinical validation of the relationship between TIPE1 expression and renal function,mitochondrial phagocytosis,and fibrosis in DN patients We analyze the Nephroseq database,and patients’ TIPE1 expression and GFR(glomerular filtration rate)were positively correlated(P=0.0033,R=-0.5978).Immunohistochemical staining of TIPE1,PHB2,and α-SMA was performed on paraffin sections of renal tissues of patients diagnosed with diabetic nephropathy(n=17).We found that patients with low fibrosis expressed higher levels of PHB2 and lower levels of TIPE1.Spearman correlation analysis showed that TIPE1 expression was positively correlated with α-SMA expression(P=0.018,R=0.6005),and negatively correlated with PHB2 expression(P=0.0219,R=-0.5510).Conclusion:Based on the above experimental results,we firstly clarified the TIPE1 expression and the molecular mechanism of regulating mitophagy in renal tubular epithelial cells of DN,and drew the following conclusions:1.TIPE1 expression was significantly increased in diabetic nephropathy,which was mainly located in renal tubular epithelial cells.2.Knockdown of TIPE1 can significantly reduce renal tubular epithelial cell injury and renal fibrosis under high glucose ambiance.3.Ablation of TIPE1 enhanced mitophagy in renal tubular epithelial cells.TIPE1 inhibits PHB2 mediated mitophagy by promoting PHB2 ubiquitination and degradation,which aggravated renal tubular epithelial cell injury and renal fibrosis,and accelerated the development of DN.Innovation:In this study,it was firstly clarified that TIPE1 was upregulated in DN mouse models and patients,and knockdown of TIPE1 could reduce proteinuria and kidney damage.High glucose stimulated the increased expression of TIPE1,resulting in the decreased expression of PHB2 in mitochondria and inhibited the mitophagy mediated by PHB2 as the mitochondrial inner membrane receptor,then aggravated the progression of diabetic nephropathy.