The Role Of Hypoxia Inducible Factor 1α In The Pathogenesis Of Albumin Overload Induced Tubular Epithelial Cells Damage

It's well been accepted by the majority of nephrologists that renal fibrosis could be accelerated by sustained hypoxia. Hypoxia promoted chronic kidney disease (CKD) through Hypoxia inducible factor/hypoxia responsive element (HIF/HRE) pathway. As a specific transcription factor stimulated by low oxygen tension, HIF-1 was composed by two subunits, the oxygen sensitive (HIF-1 a) and the constitutive (HIF-1β) subunit. Under low oxygen condition, stable HIF-1αsubunit translocated into the nucleus, where it dimerized with HIF-1 P subunit, recognized and combined with HRE, and then triggered a number of target genes transcription. It's proved that HIF-1αmight be a new regulator of renal fibrosis by the evidence that interstitial fibrosis was promoted by sustained and stable expression of HIF-1 a in tubular epithelial cells of gene knock-out mice.Early, it's been believed that proteinuria was the key factor accelerating renal fibrosis, and that it had a direct-propotional relationship with CKD patients'kidney survival.Sustained proteinuria induced severe tubulointerstitial damage and disease progression. Albumin was the key protein leading to tubulointerstitial damage as albumin comprises the major portion of proteinuria. Large amount of energy was consumed in the process of increased albumin reabsorption and dissolution in tubular epithelial cells (TEC).Another wise, peritubule low oxygen tension existed because of kidney structure weakness. Thus, low oxygen tension along with albumin overload might have a positive relation with the pathogenesis of renal fibrosis. In fact, many target genes of HIF-1,such as Connective Tissue Growth Factor(CTGF) and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1),played crucial roles in the pathogenesis of tubular damage resulted from albumin overload. CTGF and TIMP-1 overexpression induced by albumin in TEC increased the production of extra cellular matrix (ECM) and decreased their degeneration.Based on these evidences, we assumed that it's through the HIF/HRE pathway that bovine serum albumin (BSA) induced TEC damage, then triggered target genes expression and promoted renal fibrosis. Experiment protocol:the effects of fatty acid free bovine serum albumin (BSA) overload on rat NRK-52E cells HIF/HRE transcription activity was detected by a HRE-Luc reporter plasmid. And then, the effects of BSA overload on NRK-52E cells apoptosis, profibrosis factors expression and epithelial-myofibroblast transdifferentiation (EMT) were observed, and the role of HIF-1αin these pathological phenomenons was explored by silence HIF-la with RNAi technique.Objective To explore the effects of BSA at varying concentration in varying stimulus duration on HIF/HRE transcription activity of NRK-52E cells with HRE-Luc reporter plasmid. Methods Luciferase activity NRK-52E cells incubated in a medium contained BSA in varying concentration (0,5,10 or 20mg/ml) and stimulus duration (24h,48h or 72h) was detected by dual luciferase detecting system based on HRE-Luc reporter plasmid and HIF-la expression was detected by Western Blotting. Results HIF/HRE transcription activity of NRK-52E cells was increased by BSA at a relatively low concentration, and increased depended on BSA concentration and incubating duration. HIF-la expression was also increased depended on BSA concentration and incubating duration. Conclusions Our study showed that albumin increased the HIF/HRE transcription activity of TEC.Objective To explore the role HIF-la in BSA induced NRK-52E cells apoptosis. Methods NRK-52E cells apoptosis induced by BSA at varying concentration (0,5, 10 or 20mg/ml) in varying stimulus duration (24h,48h or 72h) was detected by TUNEL and Annexin V-FITC/PI dual labeling flow cytometry, as well as, Bax expression was detected by Western Blotting. And then, we observed the effects of shRNA-HIF-la transfection on BSA induced NRK-52E cells apoptosis.Results A large proportion of the cells that were positive for albumin staining also displayed positive dUTP-FITC staining of their nuclei.A low concentration of BSA (5mg/ml) had little effects on NRK-52E cells apoptosis, however, BSA at a higher concentration(>10mg/ml) or in a longer duration (>48h) promoted apoptosis. BSA (20mg/ml,72h) had no effects on total Bax expression, but elevated active Bax expression detected by 6A7 monoclone antibody. After incubated in a medium contained BSA at a concentration of 20mg/ml incubated for 72h, apoptosis cell ratio and Bax expression in shRNA-HIF-1αtransfected NRK-52E cells were lower than that in naked plasmid transfected cells. Conclusions Our study showed that a relatively higher concentration and longer incubating duration were needed for BSA induced NRK-52E cells apoptosis, and HIF/HRE transcription pathway took part in BSA induced NRK-52E cells apoptosis.Objective Profibrosis factors, such as MCP-1,CTGF and TIMP-1,played crucial role in BSA induced ECM production in NRK-52E cells. This study was designed to explore the role of HIF-1αin BSA induced profibrosis factors expression in NRK-52E cells.Methods Expressions of Fibronectin(FN), MCP-1,CTGF and TIMP-1 in NRK-52E cells induced by BSA in varying concentration and stimulus duration was detected by Western Blotting, RT-PCR or ELISA. Results MCP-1 production in NRK-52E cells was incerased by BSA at a low concentration of 5mg/ml in a short duration of 24h. BSA increased MCP-1 production in NRK-52E cells in a dosage and duration dependent manner. BSA(10mg/ml,72h) induced higher expression of FN, CTGF and TIMP-1 than BSA(0mg/ml,72h).After incubated in a medium contained BSA at a concentration of 10mg/ml incubated for 72h,FN,MCP-1, CTGF and TIMP-1 expression in shRNA-HIF-la transfected NRK-52E cells were lower than that in naked plasmid transfected cells. Conclusions Our study showed that BSA increased these profibrosis factors expression in NRK-52E cells in a dosage and duration dependent manner, and this effect partially depended on HIF/HRE pathway.Objective To explore the role HIF-1αin BSA induced NRK-52E cells EMT. Methods Expressions of tight junction protein ZO-1,myofibroblast marker a-smooth muscle actin (a-SMA) and transforming growth factorβ1(TGF-(31) expression in NRK-52E cells induced by BSA in varying concentration and stimulus duration was detected by Western Blotting, RT-PCR or ELISA. Results BSA (10mg/ml,72h) induced a significantly higher expression of ZO-1 and a lower expression of a-SMA than BSA (0mg/ml,72h), and these effects were partially reversed by shRNA-HIF-1αtransfection. BSA (10mg/ml) induced a significantly higher production of TGF-β1 than BSA (Omg/ml) in varying duration (24h,48h or 72h), and these effects were partially reversed by shRNA-HIF-1αtransfection.Conclusions Our study showed that NRK-52E cells EMT and TGF-β1 overexpression were induced by BSA, and HIF-1αplayed a crucial role in this pathological phenomenon. Above all, all these study showed that albumin increased the HIF/HRE transcription activity of TEC, and indicated that HIF-1αat least partially involved in the effects of BSA on NRK-52E cells damage.