The Role Of Metadherin In Renal Fibrosis And Its Underlying Mechanism

Renal Fibrosis(RF)is the common end point from CKD to ESRD.The mechanism of RF is not yet fully understood.It may be related to inflammatory reactions,epithelial-mesenchymal transition(EMT),and excessive deposition of extracellular matrix(ECM).EMT is mainly characterized by loss of epithelial cell polarity,acquiringability to express α-SMA,structural destruction of the tubular basement membrane(TBM),increasing cell migration and invasiveness,and secreting multiple cytokines to cause inflammation and fibrosis.TGF-β is regarded as the most important growth factor that initiates the development of EMT among numerous fibrogenic factors.It regulates the transcription of ECM generation related genes through activating the Smad signaling pathway.The MAPK(P38/ERK/JNK)signaling pathway can produce a pro-fibrogenic effect through promoting the production and activation of TGF-β1,modulating theSmad-dependent and Smad-independent signaling pathways.Metadherin(MTDH)is an oncogene located on chromosome 8q22.Currently,it is found to be closely related to multiple signaling pathways such as PI3K/Akt,Wnt/β-catenin,NF-κB,and MAPK,to mediate EMT process,metastasis and invasion in tumor cells.Mtdh/SND1 could be activated by TGF-β1 to promote the metastasis in breast cancer;Mtdh could increase the autophagy level of human malignant glioma cells and augment the sensitivity of malignant glioma cells to TGF-β,suggesting that there might be cross-talk between Mtdh and intracellular signal pathways.However,the role of Mtdh in kidney diseases,especially in renal fibrosis,has not yet been clarified.This study established experiments in vivo and in vitro to reveal the role and its underlying mechanism of Mtdh in renal fibrosis.(A)The expression of Mtdh in kidney tissue of renal fibrosis models1.1 The expression of Mtdh in unilateral ureteral obstruction(UUO)model OBJECTIVE:To investigate the phenotype of Mtdh in renal tissue of UUO mice.METHODS:A UUO model was established:12 SPF-grade C57 mice(purchased from the Southern Medical University Experimental Animal Center)randomly divided into sham and UUO groups(n=6).The left ureteral obstruction was performed in the UUO group while the sham group was only dissociated.After 14 days,mice were sacrificed,and the left kidney tissue specimens were taken after perfusion.HE staining and Masson staining were performed to observe the pathology changesin renal structure and fibrous collagen deposition.Immunohistochemistry was used to detect the expression of Mtdh and fibrotic-related proteins Fibronectin,E.cadherin,α-SMA;Western blot was conducted to detect the protein level of Mtdh,Fibronectin,E.cadherin,and α-SMA in kidney tissue.RESULTS:Mtdh immunohistochemistry result showed that Mtdh was mainly expressed in renal tubules and glomeruli,and the abundance of Mtdh in UUO group was higher than sham group.Western blot results showed that,compared with sham group,the expression of Mtdh protein in UUO group increased(P<0.05);immunohistochemistry and Western blot results The expression of Fibronectin and ca-SMA increased in the UUO group,while the expression of E.cadherin decreased in the UUO group(P<0.05).1.2 The expression of Mtdh in Diabetic Kidney Diseases(DKD)fibrosis modelOBJECTIVE:Toinvestigate the expression mode of Mtdh in DKD fibrosis modelMETHODS;An animal model of DKD was established:20 SPF-scale db/db mice and 10db/m mice(purchased fromNanjing University Model Animal Institute).Mice were sacrificed at 21 weeks.The serum was collected to detect of FBG,Scr,and BUN,TC,TG.The kidney tissue was fixed with paraformaldehyde to prepare a paraffin section.Immunohistochemical staining was used to detect the expression of Mtdh and fibrotic markers Fibronectin,E.cadherin and α-SMA.RESULTS:FBG in db/db mice was significantly higher than that in db/m mice(p<0.001);Scr in db/db mice were significantly higher than those in db/m mice(p<0.001);There was no significant difference in Urea level between db/db mice and db/m mice(p>0.05);Total cholesterol(TC)and triglyceride(TG)were significantly higher in db/db mice than db/m mice(p<0.05).The results of immunohistochemistry showed that the expression of Fibronectin and α-SMA in db/db mice increased,while the expression of E.cadherin decreased,indicating that db/db mice had developed fibrotic lesions.Mtdh was significantly increased in db/db renal tissue,especially in renal tubules.SUMMARY:Mtdh was up-regulated in both UUO obstructive fibrotic nephropathy model and DKD fibrotic model,suggesting it might play a role in promoting renal fibrogensis.(B)The role of Mtdh in EMT on renal tubular epithelial cells and its underlying mechanismOBJECTIVE:To investigate the role of Mtdh in TGF-β1-induced EMT and its underlying mechanism.METHODS:Mouse renal tubular epithelial cells(mTECs)were incubated with 5 ng/ml TGF-β1 for 0,12,24,and 48 hours.The lentiviral transfection was used to construct Mtdh stable overexpress cell line(LV-Mtdh),random sequence lentivirus(LV-NC)was used as negative control.Western blot was used to detect Mtdh,to verify LV-Mtdh efficacy.EMT phenotypic proteins Fibronectin,E.cadherin,α-SMA and ERK/P38 were also performed by Western blot.Mtdh Stable down-regulated cell line(sh-Mtdh)were constructed.After stimulated by TGF-β1,Mtdh and EMT phenotypic protein Fibronectin,E.cadherin,α-SMA and ERK/P38 were detected by Western blot.RESULTS:Under TGF-β1 stimulation,Fibronectin and α-SMA increased along with E.cadherin decreased,and became more pronounced in a time-dependent manner,indicating that the TGF-β-induced EMT model was successfully constructed.The expression of Mtdh increased as the time of TGF-β1 stimulation prolonged(P<0.05).The Mtdh-overexpressing mTEC cell line LV-Mtdh was constructed,and the expression of Mtdh protein was significantly higher than that of LV-NC group,indicating that the stable overexpressing cell line was successfully built.The expression of Fibronectin,α-SMA and COL I in LV-Mtdh group were higher than that in LV-NC group,while E.cadherin was down-regulated(P<0.05).The phosphorylation of ERK and P38 in LV-Mtdh group was higher than that in LV-NC group(P<0.05).The construction of Mtdh down-regulated sh-Mtdh in mTEC cell line,the expression of Mtdh protein was significantly decreased compared with sh-NC group,demonstrating that the stable down-regulation of cell line was successfully built.Under TGF-β1stimulation,the expression of Fibronectin and α-SMA was lower than that sh-NCgroup(P<0.05).The expression of E.cadherin was higher than that of sh-NC group(P<0.05);The phosphorylation levels of ERK and P38 in the sh-Mtdh group were significantly lower than those in the sh-NC group(P<0.05).SUMMARY:Mtdh expression was up-regulated in TGF-β-induced EMT model in mTECs.Overexpression of Mtdh could promote the production of ECM protein Fibronectin and α-SMA,inhibit the expression of epithelial marker protein E.cadherin.At the same time,it could also increase the phosphorylation level of P38/ERK;Down-regulated Mtdhdecreasedthe sensitivity of TGF-β1-induced EMT model.Thus,Mtdh might promote EMT process through regulating P38/ERK signaling pathway.CONCLUSION:Mtdh was up-regulated in EMT of mTECs,renal tissues of UUO model and db/db model.Mtdh promotes the expression of Fibronectin and α-SMA,reduces the expression of E.cadherin.The underlying mechanism of Mtdh promoting EMT and fibrogenesis might be activating the P38/ERK signaling pathway.