Expression Of P45 NF-E2 And Its Effect On CTGF/CCN2 In Diabetic Nephropathy

Diabetic nephropathy(DN)is.a major cause of end-stage renal disease(ESRD).Renal tubulointerstitial fibrosis is a common final pathway leading to chronic renal failure(CRF)during the development and progression of DN.Several related factors,such as chronic hyperglycemia(HG),TGF-?1,advanced glycation end product(AGE),and angiotensin ?(AT-?),etc,contribute to this physiopathologic process.During this process,the expression of CTGF/CCN2 is induced,and plays a crucial role in the development and progression of tubulointerstitial fibrosis.CTGF/CCN2 is an immediate-early product and a master mediator of the profibrotic actions of TGF-?1,and may enhance the profibrotic effect of TGF-?1.Expression of CTGF/CCN2 is increased in diabetic nephropathy,and urinary CTGF/CCN2 level in diabetic patients correlates with the degree of microalbuminuria.Therefore studies targeting on CTGF/CCN2 in order to find new therapeutic strategies of diabetic nephropathy(DN)are very promising.NF-E2(Nuclear Factor Erythroid-derived 2)is a heterodimer consisting of a hematopoietic-specific subunit p45 and a more widely expressed small subunit p18.P45 NF-E2 plays a critical role in erythroid and megakaryocytic gene expression.Up to date,studies about the transcription factor NF-E2 mainly focused on erythroid and megakaryocytic development and differentiation,but less studies on other fields reported.Recently,Fu Y,etc,showed that the aging of the brain of SAMP 10 mice was related to the downregulation of NF-E2 expression,which further illustrated the role of NF-E2 in other research areas.But so far,there is no related reports on NF-E2 in the field of kidney disease in world.This study was designed to detect:1.The expression of p45 NF-E2 and CTGF/CCN2 in TGF-?1-induced human proximal tubular epithelial cells(HK11),and to explore the relationship between p45 NF-E2 and CTGF/CCN2 by cell transfection and drug(MG 132)administration;2.The expression of p45 NF-E2 in the kidneys of control and diabetic mice by in vivo studies,and to explore the degradation pathway of p45 NF-E2 and the relationship between p45 NF-E2 and CTGF/CCN2 by cell transfection and drug(MG 132)administration;In vitro studies:Cultured human proximal tubule epithelial cells(HK11)were treated with TGF-?1 for 24 hours to detect the expression of p45 NF-E2 and CTGF/CCN2;NF-E2 cDNA transient transfection was used to induce p45 NF-E2 upregulation in cultured HK11 cells to detect the expression of p45 NF-E2 and CTGF/CCN2;MG132 was administered to HK11 cells treated with TGF-?1 to detect the expression of p45 NF-E2 and CTGF/CCN2.All these methods were used to investigate the molecular mechanism of p45 NF-E2 in diabetic nephropathy.In vivo studies we used multiple low-dose of STZ injection method to establish type 1 diabetic mice model,and the mice were sacrificed to extract the kidney tissue to detect the expression of p45 NF-E2 after the diabetic mice model was established for 3 and 6 months.To explore the role of proteasome inhibitor MG132 in the expression of p45 NF-E2 in diabetic nephropathy,we administered MG 132(10?g/kg/day)by intraperitoneal injection to three-month-old transgenic type 1 diabetic(OVE26)mice,the three-month-old OVE26 mice have increased urinary protein secretion.Three months later after MG132 treatment,the mice were sacrificed,and the kidneys were collected.Western blot,immunohistochemistry methods were used to detect the expression of p45 NF-E2 and CTGF/CCN2 in renal tissue.In vitro studies showed that TGF-?1(lOng/mL)significantly down-regulated the expression of p45 NF-E2 and upregulated the expression of CTGF/CCN2 in renal proximal tubule epithelial cells(HK11),while MG132 administration and NF-E2 cDNA transfection dramatically upregulated p45 NF-E2 expression and down-regulated CTGF/CCN2 expression in TGF-?1 treated HK11 cells.In vivo studies demonstrated that p45 NF-E2 expression was reduced markedly in the kidneys of STZ-induced diabetic 3-month and 6-month mice and OVE 6-month mice.Immunohistochemical staining showed that p45 NF-E2 mainly located in the lining of kidney tubular epithelial cells in control mice.In STZ-induced diabetic 3-month,6-month mice and OVE 6-month mice,the expression of p45 NF-E2 in renal tubular epithelial cells lining lowered compared with control mice,or p45 NF-E2 granularly deposited in the cytoplasm of renal tubular epithelial cells.MG 132 administration remarkably upregulated the expression of p45 NF-E2 protein and downregulated CTGF/CCN2 expression in the kidneys of OVE 6-month mice.In vitro and in vivo studies showed that:?P45 NF-E2 expressed in renal tubular epithelial cells and kidney tissues.?The expression of p45 NF-E2 decreased in renal tissue and renal tubular epithelial cells in diabetic nephropathy,and negatively correlated with CTGF/CCN2 expression.?MG132 reduced the expression of CTGF/CCN2 expression possibly by reducing the degradation of p45 NF-E2 in the kidney tissue and renal tubular epithelial cells in diabetic nephropathy.Thus,p45 NF-E2 might play a protective role through negatively regulating the expression of CTGF/CCN2 in the development and progression of renal interstitial fibrosis of diabetic nephropathy.