The Epigenetic Mechanisms Of Renal Injury Induced By The Organic Solvent

Part 1. The model of renal injury by inhaling xylene in ratsObjective:To build an organic solvents induced renal injury model in rats by exposure to xylene.Methodolgy:Male Sprague-Dawley rats (n=57), weighing 140-160g,4-6 week old, were used. The rats were randomly divided into two groups:control group (n=7) and xylene exposure group (n=50). The rats were exposed to xylene six hours per day for twelve weeks. The 24h urinary protein excretion and urinary NAG levels were measured. In addition, serum total protein(TP), albumin(Alb), urea(BUN), creatinine(Scr), aspartate aminotransferase(AST) and alanine aminotransferase(ALT) levels were examined. The tubular and glomerular morphology were observed by light microscopy and transmission electronmicroscopy. The change of podocin and synaptopodin expression and distribution was determined by indirect immunofluorescence staining.Results:Compared with the control rats, the proteinuria was observed in rats after exposure of 3 weeks, and the amount of urinary protein excretion was increased to 34.78±2.91mg/24h at 8 weeks. Urinary NAG increased obviously after the exposure of 4 weeks. After exposure of 12 weeks, urinary NAG in experiment and control rats was 25.03±4.88U/gScr and 14.36±2.05U/gScr, respectively. No significant differences were found in TP, Alb, Scr and ALT levels. Urea levels increased in xylene exposed rats at 12 weeks (P<0.05) and AST increased significantly. Histological examination by microscopy showed that remarkable injuries were shown in proximal renal tubule, including loss of brush border and vacuolar degeneration of the epithelial cells. Ultra structural study demonstrated that brush border and cytoplasm of tubular epithelial cell were dropped and some epithelial cells were disintegrated. Mitochondria of tubular epithelial cells were swollen and the electron density decreased in matrix. The results by immunofluorescence staining showed that distribution of podocin and synaptopodin along GBM was discontinuous and granular after exposure of 12 weeks.Conclusion:The present study indicates the renal toxicity by exposure to xylene. The injury of tubular epithelial cell was significantly severer than that of podocytes.Part 2. Xylene induced proximal tubular epithelial cells and podocyte injury in vitroObjective:The incidence of patients with nephrotic syndrome induced by organic solvents increased gradually in recent years after exposure to paints,varnishes, lacquers, adhesives, etc. However, the mechanism of such injury remains unknown. Xylene, predominant chemical in paints, is used in this study to induce injuries on cultured cells, including human podocytes and proximal tubular cells.Methodology:HK-2 cells and podocytes were treated with different concentrations of xylene in vitro. The secretion of NGAL and IL-18 in culture medium were measured by ELISA. The injury of xylene on cytoskeleton of podocyte including F-action and synaptopodin, and on expression of the slit diaphragm proteins, including podocin and nephrin, were observed by immunofluorescence. The apoptosis of HK-2 cells and podocytes was detected by flow cytometry.Results:(1) Xylene could induce apoptosis of HK-2 cells. The proportion of apoptotic cells increased significantly while increasing xylene concentration in the medium.2mm of xylene could result in the apoptosis of 98% HK-2 cells. HK-2 cells were incubated with 0.2,0.6,1.0,1.4,1.8mm of xylene. Xylene induced the most obvious increase of NGAL at the concentration of 1.4mm. The secretion of NGAL was not obviously increased at 6h and 12h, however significant increased at 24h and 48h (p<0.01). No change of IL-18 was observed.(2) Xylene could induce the apoptosis of podocytes. Xylene could induce the apoptosis of 35.6% podocytes at the concentration of 1.8mm for 24h. After incubation for 48h, apoptosis of podocytes increased to 81%. Xylene could induce injury on cytoskeleton structure and the expression of slit diaphragm proteins in podocytes. Xylene could disrupt the action cytoskeleton of podocytes and remarkable decrease synaptopodin expression. The expression of nephrin and podocin were also significantly decreased by xylene.Conclusion:Xylene could induce obvious injury and apoptosis on both renal proximal tubular epithelial cells and podocytes in vitro. Xylene could induce the injury of skeleton structure and slit diaphragm proteins of podocytes.Part3. Epigenetic mechanism of xylene-induced renal tubular epithelial cells and podocyte injury in vitroObjective:To investigate the aberrance of DNA methylation in cultured human podocytes and proximal tubular cells which were injuried by xylene exposing.Methodology:DNA methylation variations in cultured human podocytes and proximal tubular cells were analyzed using human 450,000 CpG DNA methylation microarray. Quantitative real-time PCR (qRT-PCR) was carried out to examine the correlations between the mRNA expression profiles and methylation levels. Bisulfite sequencing PCR and pyrosequencing was used to validate the microarray results.Results:(1) We identified 51 probes that displayed significant methylation level change in xylene exposure podocytes compared with control cells. Among them,25 probes showed increased methylation level and 26 probes had decreased methylation level. There were 33 genes involved in.Quantitative RT-PCR revealed the correlations between the mRNA expressions and the methylation levels of IL-20 and P53.The results of BSP were not well consistent with the microarray data. (2) We identified 243 probes that displayed significant methylation level change in xylene exposure tubular epithelial cells compared with control cells. Among them, 109 probes showed increased methylation level and 134 probes had decreased methylation level, here were 138 genes involved in.Quantitative RT-PCR revealed the correlations between the mRNA expressions and the methylation levels of EIF1AY and KDM5D. The results of pyrosequencing were well consistent with the microarray data.Conclusion:injuried renal tubular epithelial cells and podocyte have significant alterations in DNA methylation, and the genes with aberrant methylation level may provide insights into the pathogenesis of renal disease induced by organic solvent.