[Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1].

Abstract

To illuminate the possible role of Prohibitin (PHB) in tubulointerstitial fibrosis. (1) Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis, 26 male and 22 female, aged 7.5 +/- 5.5 (2.5 - 13 years), and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-SMA). The correlation between PHB and degree of tubulointerstitial lesion was compared. (2) Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. (1) PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB (all P < 0.01), and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions (r = -0.802, P < 0.01). (2) Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfection of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-SMA expression in the NRK-49F cells (P < 0.01). PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.