The Role Of NDRG2 In Hypoxic-induced Renal Tubular Epithelial Cell Injury

Chronic kidney disease(CKD)refers to chronic kidney structural disorder and dysfunction caused by various reasons lasting more than 3 months.Once diagnosed,if no effective intervention measures are taken in time,the patient will eventually develop into end-stage renal disease(ESRD).In recent years,the morbidity and mortality of CKD have increased year by year.Epidemiological survey showed that the prevalence of CKD was about 13.4%.According to the estimates by the world health organization,the mortality rate of CKD will rise to 14/100,000 by 2030,which has become one of the most important public health issues in the world.Renal fibrosis is an important pathological feature of the occurrence and development of CKD and the only way to the development of CKD to ESRD.Pathologically,renal fibrosis is mainly characterized by glomerular sclerosis,tubulointerstitial fibrosis,renal vascular sclerosis,and excessive deposition of extracellular matrix(ECM).Tubulointerstitial fibrosis is an important part of renal fibrosis,which involves tubular epithelial cell injury,inflammatory response,oxidative stress and other aspects,and is jointly affected by a variety of signal pathways and key molecules.In the process of tubulointerstitial fibrosis,excessive deposition of ECM and epithelial mesenchymal transition(EMT)of renal tubular epithelial cells play an important role.How to control and delay the EMT of renal tubular epithelial cells and reduce the deposition of ECM is the key to slow down the progress of CKD.Nephrotoxic drugs,ischemia,hypoxia infection and other factors can lead to the occurrence and development of CKD.Chronic hypoxia is one of the most common factors leading to renal injury.Although the kidney can get 25%of the cardiac output,the oxygen tension in the renal tissue is very low,which makes the kidney relatively sensitive to the change of oxygen delivery and vulnerable to hypoxia injury.Chronic hypoxia is the key pathological state of various kidney diseases.It can be observed that the kidney of patients with CKD is in the state of hypoxia using blood oxygen level dependent magnetic resonance imaging.According to the latest research progress,there are many pathological mechanisms of renal tubulointerstitial hypoxia in CKD:firstly,glomerulosclerosis leads to a decrease in the number and blood flow of capillaries around the downstream renal tubules;secondly,the deformation and loss of renal tubulocapillaries caused by fibrosis further reduces blood perfusion;thirdly,deposition of ECM widens the distance between capillaries and renal tubules,reducing the efficiency of oxygen diffusion.With the development of the disease,renal hypoxia will continue to occur.In turn,continuous renal hypoxia further promotes renal fibrosis,which eventually leads to the deterioration of disease progression,thus forming a vicious circle.Renal tubular epithelial cells play an important role in the development of renal interstitial fibrosis.Renal tubular epithelial cells play an important role in the development of renal interstitial fibrosis.When renal tubular epithelial cells are exposed to a low-oxygen environment,a complex response occurs,activating a number of signaling pathways that play a key role in the process of renal fibrosis,so as to significantly increase the expression level of mesenchymal markers,promote the accumulation of ECM and inhibit its degradation,and then promote the development of renal tubular interstitial fibrosis.Therefore.we used hypoxia incubator to simulate the renal hypoxia environment to stimulate renal tubular epithelial cells,and explored the molecular mechanism of hypoxia induced renal interstitial fibrosis,so as to provide a research basis for hypoxic-induced renal fibrosis caused by various causes.N-myc downstream regulatory gene 2(NDRG2)belongs to the NDRG family.The human NDRG2 gene is located on chromosome 14q11.1-11.2 and is widely expressed in vivo.It can participate in many biological functions and lead to the occurrence and development of tumor,fibrosis,inflammation and other diseases by regulating a variety of signaling pathways.Some studies have shown that overexpression of NDRG2 can reduce the phenotypic transformation of human peritoneal mesothelial cells induced by high glucose.At the same time,NDRG2 can reduce the deposition of ECM in the rat model of liver fibrosis induced by dimethylnitrosamine,thereby reducing liver fibrosis and improving liver function.Therefore,we speculate that it may be involved in and play a key role in hypoxia-induced renal fibrosis.AKT is a serine/threonine protein kinase,also known as protein kinase B(PKB),which is activated by phosphorylation at Thr308 and Ser473 and is involved in a variety of biological processes,inducing EMT to participate in the fibrogenic process of various cytokines.Studies have shown that phosphorylated AKT(p-AKT)is highly expressed during renal fibrosis and is involved in hypoxia-induced EMT of renal tubular epithelial cells.It has been confirmed that NDRG2 can participate in the regulation of esophageal cancer invasion,metastasis,and EMT by regulating the activation of AKT;overexpression of NDRG2 can inhibit the PI3K l AKT signaling pathway,thereby inhibiting EMT of oral squamous cell carcinoma.Based on current research,we speculate that NDRG2 may be involved in mediating renal interstitial fibrosis by targeting AKT.In this study,we took renal tubular epithelial cells as the research object,and observed the expression changes of NDRG2,AKT,p-AKT,EMT related indexes,I type Collagen and Fibronectin,to investigate whether NDRG2 participated in the EMT induced by renal tubular epithelial cell injury by regulating the phosphorylation level of AKT to accelerate the process of renal fibrosis,thus providing a new approach and target for the clinical treatment of CKD.Part I To clarify the expression of NDRG2 in renal tubular epithelial cells under hypoxia and its influenceObjective:(?)1.To determine whether the hypoxic environment leads to EMT of renal tubular epithelial cells and causes changes in the expression levels of Collagen-I,Fibronectin.2.To clarify the changes in the expression level of NDRG2 under hypoxic environment and further explore the effect of NDRG2 on hypoxia-induced EMT and the expression levels of Collagen-I and Fibronectin of renal tubular epithelial cellsMethods:1.Cell culture:human renal tubular epithelial cells(HK2)were cultured in vitro in 37℃ DMEM high glucose medium containing 10%fetal bovine serum and 1%penicillin streptomycin and were incubated at 37℃ with 5%C02 for continuous subculture.2.To detect the expression change of NDRG2 in renal tubular epithelial cells after hypoxic stimulation:① The renal tubular epithelial cells were cultured in a 37°C,5%CO2 incubator.When the cell density reached 40%-50%,the new medium was replaced,and then the time gradient of hypoxic stimulation was established in a hypoxic incubator at 37℃ and 1%O2.According to the experimental results,the appropriate hypoxic stimulation time was selected as the observation time point for subsequent studies.② Western blot were used to detect the the change of NDRG2 protein expression level.3.To detect the changes of renal tubular epithelial cell damage-related indicators after hypoxia stimulation:The most suitable hypoxia stimulation time was selected.Western blot,Real time PCR and cellular immunofluorescence were used to detect the expression levels of intracellular mesenchymal cell-like phenotypic markers a-SMA,N-cadherin,Vimentin and epithelial markers E-cadherin,Fibronectin and Collagen-I after hypoxia stimulation.4.To detect the effects of regulating NDRG2 on renal tubular epithelial cell damage-related indicators after hypoxia stimulation:①NDRG2 overexpression lentiviral vector,blank control viral vector,NDRG2 shRNA lentiviral vector and Scramble viral vector were constructed and transfectied.Western blot was used to detect the protein expression level of NDRG2.②After overexpressing NDRG2 and knocking out NDRG2,western blot was used to detect the changes in expression levels of mesenchymal cell-like phenotypic markers a-SMA,N-cadherin,Vimentin and epithelial marker E-cadherin Fibronectin and Collagen-Ⅰ after hypoxic stimulation.Result:1.The expression level of NDRG2 was significantly reduced in renal tubular epithelial cells(p<0.05),after 24 and 48 hours of hypoxic stimulation.2.The expression levels of α-SMA,N-cadherin,Vimentin,Fibronectin,and Collagen-Ⅰ were significantly increased(p<0.05);the expression levels of E-cadherin were significantly decreased(p<0.05),after 24 and 48 hours of hypoxic stimulation.3.After transfection with NDRG2 overexpressing lentiviral vector,the expression level of NDRG2 in renal tubular epithelial cells were significantly increased(p<0.01).Compared with the hypoxic group,overexpression of NDRG2 significantly reduced the expression levels of α-SMA,N-cadherin,Vimentin,Fibronectin,and Collagen-I in renal tubular epithelial cells after 48 hours of hypoxic stimulation(p<0.05),and increased expression level of E-cadherin(p<0.05).The expression level of each index in the Null group did not change significantly(p>0.05).4.After transfection with shNDRG2 lentiviral vector,the expression level of NDRG2 in renal tubular epithelial cells was significantly reduced(p<0.01).Compared with the hypoxic group,knocking out NDRG2 significantly increased the expression levels of α-SMA,N-cadherin,Vimentin,Fibronectin,and Collagen-I(p<0.05),reduced the expression level of E-cadherin(p<0.05).The expression levels of various indicators in the Scramble group did not change significantly(p>0.05).Conclusion:1.Hypoxic environment can induce EMT in renal tubular epithelial cell and lead to an increased level of the expression of Fibronectin and Collagen-I.2.Under hypoxic environment,the expression level of NDRG2 in renal tubular epithelial cells decreased.NDRG2 participates in the EMT of renal tubular epithelial cells induced by hypoxia and regulates the expression of Fibronectin and Collagen-I.Part Ⅱ NDRG2 mediates the damage of renal tubular epithelial cells under hypoxic conditions by regulating the phosphorylation level of AKTObjective:(?)To determine whether NDRG2 can mediate EMT of renal tubular epithelial cells under hypoxic conditions by targeting AKT and its effect on the expression level of Fibronectin and Collagen-I.Method:1.Western blot was used to detect the changes in the expression levels of AKT and p-AKT in renal tubular epithelial cells after hypoxic stimulation.2.Construction and transfection of AKT small interfering RNA and blank control small interfering RNA,and identification of gene interference efficiency:the mRNA and protein levels of AKT in renal tubular epithelial cells before and after transfection were detected by real time PCR and western blot,and the gene interference efficiency was identified.3.Interaction between NDRG2 and AKT in renal tubular epithelial cells:①The expression levels of AKT and p-AKT were detected by western blot after transfection with NDRG2-overexpressed lentivirus and NDRG2 shRNA lentivirus,respectively;②Renal tubular epithelial cells were co-transfected with NDRG2 shRNA lentivirus and AKT small interfering RNA.Western blot,real time PCR detection hypoxic stimulus within 48 hours after total transfection cell α-SMA,N-cadherin,Vimentin,E-cadherin and the expression of Fibronectin and Collagen-Ichanges.Result1.After 48 hours of hypoxic stimulation in renal tubular epithelial cells,there was no significant change in the expression level of AKT(p>0.05),and the expression level of p-AKT was significantly increased(p<0.05).2.Transfected with AKT small interfering RNA,the expression of AKT was significantly reduced(p<0.05).After 48 hours of hypoxic stimulation,compared with the hypoxic group,the expression level of E-cadherin in renal tubular epithelial cells of si-AKT group increased,and the expression levels of a-SMA,N-cadherin,Vimentin,Fibronectin,and Collagen-I decreased(p<0.05).There was no significant change in the expression levels of various indicators in the NC group(p>0.05).3.After 48 hours of hypoxic stimulation,compared with the control group,there was no significant change in the expression level of AKT in renal tubular epithelial cells transfected with NDRG2 overexpressing chronic disease vector(p>0.05),but the expression level of p-AKT was significantly decreased(p<0.05).After 48 hours of hypoxic stimulation,there was no significant change in the expression level of AKT in renal tubular epithelial cells transfected with NDRG2 shRNA lentiviral vector compared with the control group(p>0.05),but the expression level of p-AKT was significantly increased(p<0.05).4.After 48 hours of hypoxic stimulation,compared with NDRG2 shRNA group,the expression level of E-cadherin in renal tubular epithelial cells was significantly increased in the co-transfected group;the expression levels ofα-SMA,N-cadherin,Vimentin,Fibronectin and Collagen-I were significantly decreased.Conclusion:1.AKT is involved in mediating EMT of hypoxia-induced renal tubular epithelial cell and regulating the expression of Fibronectin and Collagen-I.2.NDRG2 participates in hypoxia-induced EMT of renal tubular epithelial cells by regulating the phosphorylation level of AKT and regulates the expression of Fibronectin and Collagen-I.