The Role And Mechanism Of TXNIP In Renal Fibrosis Associated With Aging

PART1:THE EFFECT OF AGING ON THE EXPRESSION OF TXNIP AND RENAL FIBROSISObjective: To investigate the relationship between aging and the expression of TXNIP and renal fibrosis.Methods: C57BL/6J male mice were randomly divided into two groups: normal control group(NC group n=18)and high-fat-glucose diet group(HFGD group n=18).Kidney tissues and blood samples of 3,9 and 18 months were collected from the two groups.The biochemical indexes of kidney function in each age group and the expression of TXNIP,aging-related protein P16,renal fibrosis-related protein ?-SMA and TGF-?1 were detected.Renal fibrosis and pathological changes were observed by HE,Masson and PAS staining of paraffin sections.Results:With the increase of age,the pathological staining of paraffin sections of mouse kidney showed glomerular sclerosis,structural damage of renal tubular structure and renal fibrosis,serum creatinine and urea nitrogen increased gradually(P<0.05),the expressions of TXNIP,P16,?-SMA and TGF-?1 were up-regulated in kidney tissues(P<0.05).In the same agegroup,the glomerular sclerosis,tubular atrophy and renal fibrosis were aggravated,serum creatinine and urea nitrogen were significantly increased(P<0.05),and the expressions of TXNIP,P16,?-SMA and TGF-?1in kidney tissues were also significantly up-regulated(P<0.05).Conclusion: With the increase of age,the aging and fibrosis of kidney tissue promoted the expression of TXNIP in kidney tissue,while the expression of TXNIP in kidney tissue caused by high-fat-glucose aggravated aging,and aggravated senescence-related renal fibrosis and renal function obstacle.Aging promotes the expression of TXNIP in kidneys.Whether the up-regulation of TXNIP aggravates the aging and fibrosis of kidney still needs further experimental study.PART2: THE RELATIONSHIP BETWEEN TXNIP AND AGING-RELATED RENAL FIBROSISObjective: To investigate the role of TXNIP in mediating fibrosis associated with renal aging in miceMethod: C57BL/6J male mice(WT n=18)and TXNIP knockout male mice(TXNIP-/-n=18)were fed with a normal diet.kidney tissues and blood samples were collected at 3,9,and 18 months.The kidney function of each group mice was detected.The expression of TXNIP,aging-related protein P16 and fibrosis-related protein in the kidney were detected by Western-blot and immunofluorescence.Renal fibrosis and pathological changes were observed by HE,Masson and PAS staining of paraffin sections.Results:In the same age mice,knockout of TXNIP gene significantly decreased renal senscence and fibrosis,reduing serum creatinine and urea nitrogen significantly(P<0.05),and significantly decreasing the expression of TXNIP,P16,?-SMA and TGF-?1 in renal tissues(P<0.05).Conclusion: TXNIP promotes kidney aging and accelerates renal fibrosis.Knockout of TXNIP can ameliorate senescence-related renal fibrosis and down-regulate the expression of ?-SMA and TGF-?1.PART3: STUDY ON MECHANISM OF TXNIP MEDIATING AGING-RELATED RENAL FIBROSISObjective: To investigate the role and mechanism of TXNIP in promoting fibrosis in renal tubular epithelial cellsMethods:Human renal tubular epithelial cells(HK-2)were transfected with negative control virus(LV-GFP),TXNIP overexpressing virus(LV-TXNIP)and TXNIP silencing virus(LV-TXNIP-si RNA),and divided into three groups.The expression of TXNIP,aging-associated protein P16 and fibrosis-related proteins were detected by western-blot and immunofluorescence in the kidney.The phosphorylation levels of STAT3 pathway and TGF-?1/SMAD3 signaling pathway were detected by Western-blot,and the interaction between TXNIP and STAT3 was tested by immunoprecipitation.Results:After transfection of TXNIP overexpressing virus,the aging-associated protein P16 and fibrosis protein TGF-?1 in renal tubular epithelial cells were significantly up-regulated compared with the negative control group(P<0.05),and the phosphorylation levels of STAT3 signaling pathway and SAMD3 pathway were significantly up-regulated(P< 0.05),the downstream fibrosis proteins ?-SMA and Collagen I were significantly up-regulated(P<0.05).After transfection of TXNIP silencing virus,the intracellular senescence-associated protein P16 and renal fibrosis protein TGF-?1 were significantly lower than the negative control group(P<0.05),and the phosphorylation levels of STAT3 signaling pathway and SAMD3 pathway were significantly decreased(P<0.05).The downstream proteins?-SMA and Collagen I were significantly down-regulated(P<0.05).Secondly,after the specific inhibitor of STAT3 was applied to the tubular epithelial cells,the expressions of TGF-?1,?-SMA,Collagen I and SAMD3 in the overexpression group were significantly down-regulated(P<0.05).In addition,co-immunoprecipitation showed a certain interaction between TXNIP and STAT3.Conclusion: Overexpression of TXNIP can promote the up-regulation of senescence-associated protein P16 in renal tubular epithelial cells,and may activate STAT3 signaling pathway and up-regulate the expression of TGF-? 1,thereby up-regulating the expression of fibrosis protein and promoting fibrosis changes in renal tubular epithelial cells by activating SMAD3 signaling pathway.Knockdown of TXNIP can inhibit STAT3 phosphorylation,down-regulate TGF-? 1/SMAD3 expression,and inhibit the fibrogenic response of renal tubular epithelial cells.Combined with the result of immunoprecipitation,TXNIP can promote the fibrogenic response of renal tubular epithelial cells,which may be related to the activation of STAT3 signaling pathway and up-regulation of TGF-?1/SMAD3 expression.