Study Of Mechanism Of Podocyte Damage In HBV-assoicated Glomerulonephritis

PartⅠ. Apoptosis of resident renal cells in hepatitis B virus-associated membranous nephropathyObjective:To study apoptosis in renal tissues of children with hepatitis B virus-associated membranous nephropathy (HBV-MN) by using activated caspase-3 as a marker and explore the significance of renal inherent cells apoptosis in the pathogenesis of HBV-MN.Methods:Caspase-3 protein was specifically detected by immunohistochemical staining in 19 children with biopsy-proven HBV-MN and 5 normal patients suffering from nephrolithiasis or kidney trauma (control group).Brownish yellow nuclei were regarded as a marker of positive cells containing activated caspase-3 protein. In glomeruli and tubulointerstitial area, the positive cells were quantitated as glomerular and tubulointersititial apoptotic index respectively. And then both apoptotic indexes were correlated with clinical, serological and pathologic data. To confirm results obtained by activated caspase-3 detection, we used the TUNEL method to recognize apoptosis cells with brownish yellow nuclei in 15 children with HBV-MN.We took same manners to count TUNEL-positive cells and determine TUNEL index in glomeruli and tubulointerstitium, and analyzed the relationship between apoptotic index and TUNEL index. HBx protein was specifically detected by immunohistochemical staining in 14 children with HBV-MN.Results:①Caspase-3 protein expression was found in both cytoplasma and nuclei of glomerular cells and tubulointerstitial cells in HBV-MN,but only in cytoplasma of a few tubulointerstitial cells in controls.In glomeruli and tubulointerstitium, there was good correlation between apoptosis index assessed by activated caspase-3 staining and TUNEL index (r=0.812, P=0.0496 & r=0.958, P=0.0026).②The glomerular and tubulointerstitial apoptosis index were significantly correlated with pathologic severity of HBV-MN.The quantitative expression of activated caspase-3 was significantly higher in patients at phaseⅢorⅣthan those at phaseⅠorⅡ,but was not significantly different between patients with stronger HBsAg deposition (≥++) and those with weak deposition (<++).③Activated caspase-3 positive cells were much more in patients with massive proteinuria (≥50mg/kg) than those with moderate proteinuria (<50mg/kg, P=0.0335).But it wasn't correlated with serum levels of C3.④Tubulointerstitial apoptosis index was significantly correlated with daily urine protein excretion and urine RBP levels.⑤HBx protein was detected within the glomerulus in 10/14 (71%)children with HBV-GN. The expression of HBx protein was localized within cytoplasm of podocytes and along the glomerular basement membrane in a fine linear pattern. Conclusion:All children with HBV-MN showed caspase-3-dependent apoptosis of renal intrinsic cells in glomeruli and tubulointerstitium. Furthermore, the expression level of activated caspase-3 was significantly correlated with renal pathologic severity, proteinuria and uric RBP level. Apoptosis of renal intrinsic cell may play an important role in the pathogenesis of HBV-MN in children. HBx may be an important cause of podocyte apoptosis in HBV-MN.PartⅡ.Construction of recombinant adenovirus containing HBx gene and its effect on podocyte apoptosisObjective Apoptosis has been regarded as an important mechanism of podocyte loss in HBV-associated membranous nephropathy. Since HBx has been demonstrated to be the major HBV protein causing host cell responses like proliferation or apoptosis, we hypothesize that HBx might be involved in the podocytopathy in HBV-MN.Therefore, in present study, hepatitis B virus X protein (HBx) was expressed in cultured mouse podocytes via adenoviral infection to determine its effect on podocyte apoptosis. Methods HBx cDNA was obtained from the plasmid PCI-neo-HBx by enzyme digestion, and inserted into shuttle plasmid pShuttle-CMV(-) to generate a recombinant plasmid pShuttle-HBx.Then the HBx gene was transferred from pShuttle-HBx to Adxsi viral DNA to form a recombinant adenoviral plasmid pAd-HBx by means of an in vitro ligation reaction. Finally, after identification, the pAd-HBx was packaged into infectious adenoviral particles Ad.HBx by transfecting human embryonic kidney (HEK) 293 cells. Cell morphologic changes were investigated by staining with Wright-Giemsa. HBx-induced podocyte apoptosis was demonstrated by flow cytometry. Results The recombinant adenovirus Ad.HBx was generated successfully by an in vitro ligation reaction, which was confirmed by restriction enzyme digestion and DNA sequencing. The HBx-expressing podocytes showed increased percentage of meganuclear and polynuclear cells at 24h, and significant apoptosis at 48h compared with control.In HBx-transfected group, the percentage of apoptotic podocytes (indicated by annexin V-posotive cells) increased 1.2-fold over control at 48h. Conclusions The successful construction of the recombinant adenovirus containing HBx gene has laid a foundation for further study of the involvement of HBx in the podocytopathy in HBV-GN. HBx can directly induce podocyte apoptosis, which may play an important role in the pathogenesis of HBV-GN.PartⅢ. Hepatitis B virus X protein reduces podocyte proliferation in vitroBackground Hepatitis B virus-associated nephritis (HBV-GN), characterized by podocyte injury, is a common secondary glomerular disease in China. Our previous studies have shown that podocyte number was significantly decreased in children with HBV-associated membranous glomerulopathy. Lack of proliferation has been regarded as one of the critical mechanisms contributing to injury-induced podocyte loss. Therefore, in current study, hepatitis B virus X protein (HBx) was expressed in cultured mouse podocytes via adenoviral infection to determine its effect on cell proliferation and its regulation at the level of cell cycle.Methods HBx gene was inserted into an adenovirus-based vector and transfected into mouse podocytes. Cell morphologic changes were investigated by staining with Wright-Giemsa. Cell growth was measured by MTT-assay and CFSE proliferation assay. Cell cycle phase was demonstrated by flow cytometry, and the expression of specific cell cycle regulatory proteins was examined by western blot analysis or flow cytometric bivariate analysis.Results The recombinant adenovirus Ad.HBx was generated successfully, which was confirmed by polymerase chain reaction and DNA sequencing. The expression of HBx protein in the Ad.HBx-infected podocytes was confirmed by western blot. At 5 days post-infection, HBx-expressing podocytes presented with the characteristic of mitotic catastrophe such as binuclei,multinuclei,and polymorphonuclei accumulation. MTT assay showed that the growth curve of Ad-infected podocytes was basically consisten with controls, but both were significantly higher than the growth curve of Ad. HBx-infected podocytes after day 4 post-infection. Furthermore, CFSE-based proliferation assay also showed that at 3 days post-infection, the proliferation rate of HBx-expressing podocytes was significantly slower than Ad-infected podocytes and controls (proliferation index: 11.15 vs.15.4,13.3),and this trend became much obvious with the time (proliferation index: 32.5 vs.61.65,53.96).FACS analysis showed that with the HBx-induced G2/M phase arrest, the levels of cyclin B1 and CDK-inhibitor p21 were significantly increased in HBx-expressing podocytes, but the expression of cyclin A was slightly decreased.Conclusions Our study shows that exogenous expression of HBx decreases the growth of podocytes. This effect is mediated through the G2/M phase arrest associated with an increase in the level of cyclin B1 and CDK-inhibitor p21 accompany with a decrease in the expression of cyclin A. These events may partially explain reduced podocyte number in HBV-GN.