Role Of Heparanase In The Pathogenesis Of Minimal Change Nephrotic Syndrome Induced By Respiratory Syncytial Virus

Objective:The pathogenesis of minimal change nephrotic syndrome(MCNS) remains unclear. Respiratory tract viruses infection could contribute to MCNS, and respiratory syncytial virus (RSV) is the most common one. Theβ-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by selectively degrading the negatively charged side chains of heparan sulfate proteoglycans(HSPG) within the glomerular basement membrane(GBM). A loss of negatively charged heparan sulfate proteoglycans may result in alteration of permselective property of glomerular basement membrane, loss of anionic charge in GBM. In this study, we planned to explore the role of heparanase in the pathogenesis of MCNS induced by RSV.Methods:1. To study the role of heparanase in RSV nephropathy in rats, SD rats were inoculated with 6×10⁶ PFU(plaque-forming unit) RSV, and killed on days 4, 8, 14, 28, 56 and 84 postinoculation (RSV₄, RSV₈, RSV₁₄, RSV₂₈, RSV₅₆ and RSV₈₄. The pmteinuria and serum parameters were measured; renal histology was observed by fight microscopy and transmission electron microscopy; RSV mRNA in the kidney and lung was confirmed by in situ hybridization. Anionic sites in GBM were quantitatively evaluated through polyethyleneimine(PEI) staining under transmission electron microscopy. Renal heparanase protein and mRNA expression level were determined by immunohistochemical staining and real-time quantitative reverse transcriptase polymerase chain reaction(real-time quantitative RT-PCR).2. To investigate the effect of heparin on the anionic sites in GBM and heparanase expression, rat were inoculated with 6×10⁶ PFU RSV and heparin 500U/kg. Group A: RSV was given in the first 3 days, then heparin was given in the following 11 days. Group B: RSV and heparin were simultaneously given in the first 3days, then heparin was given in the following 11 days. Group C: heparin was given throughout the experiment, and RSV was given on the 4th, 5th and 6th day. Group D: RSV was given in the first 3days, then no heparin was given in the following 11 days. Normal control group: RSV and heparin were not given throughout the experiment. At the same time, the renal histology, proteinuria excretion, RSV mRNA, heparanase expression and anionic sites in GBM were measured as above.3. To explore the cytopathogenic effect of RSV on rat renal cells, primary rat glomerular epithelial cell were cultured in vitro. Glomerular epithelial cell, HBZY-1 cells, and NRK52E cells infected with RSV were observed through electron microscpy. RSV mRNA was detected with in situ hybridization, MTT colorimetric assay was used to measure the A value of HBZY-1 cells and NRK52E cells at 6 hours, 12 hours, 24 hours and 48 hours after being infected with RSV.4. To determine the expression of heparanase and its signaling pathway, immunohistochernical assay, real-time quantitative RT-PCR, Western blot and immunofluorescent detection were used after inoculated with 6×10¹ PFU RSV. Primary glomerular epithelial cells were isolated from rats and grown monolayers. Glomerular epithelial cells, HBZY-1cells and NRK52E cells were infected at 70% confluency, with 6×10¹ PFU RSV. After 90 min of incubation with RSV at 37℃, the cells was incubated for indicated times before harvesting with viral sustainable medium. The heparanase expression was analyzed in glomerular epithelial cells, HBZY-1 cells and NRK52E cells by immunohistochemistry at 6 hours, 12 hours, 24 hours and 48 hours after being infected with RSV. The heparanase gene and protein expression were measured by real-time quantitative RT-PCR and Western blot, respectively. To detect activation of ERK, p38MAPK and NF-κB, immunofluorescene staining was employed with specific anti-phospho-ERK, anti-phospho- p38MAPK and anti-NF-κB p65. Additionally, HZBY-1 cells and NRK52E cels were pretreated with SB203580 or PD98059, then the cells were infected with 6×10¹ PFU RSV. Twelve hours later, the protein expression level of heparanse was determined with Western blot. Results:1. After inoculation of RSV, the urinary protein increased, foot processes of glomerular epithelial cells were fusional and accompanied with hypoalbuminemia, particularly in rats inoculated with 6×10⁶ PFU RSV on days 14-28 postinoculation when the changes of renal tissue under lightmicroscope and electron microscope (the fusion of foot processes were extensive) bear similarity to human MCNS essentially. Compared with those of normal control, RSV₅₆ and RSV₈₄ groups, anionic sites in GBM significantly from RSV₄, RSV₈, RSV₁₄ groups decreased, and heparanase expression in RSV₄, RSV₈, RSV₁₄ group were elevated. There were a positive correlation between the expression level of heparanase and proteinuria, and a negative correlation between anionic sites and proteinuria.2.There was no significant difference between heparin treated groups(group A, group B and group C) and normal control group in proteinuria and anionic sites in GBM. However, there were weak RSV RNA, and hearanase protein and mRNA expression.3. RSV could directly infect glomerular epithelial cell, HBZY-1 and NRK52E cell. 24 hours after infection of RSV, injury of cell organ and virus-like particles could be observed. The A value of normal cell samples was greater than that of cells infected with RSV. And the A value of HZRY-1 cells infected with RSV were lower than that of NRK52E cells infected with RSV.4. RSV could induce the expression of heparanase. Phosphoyrlated ERK protein, phosphoyrlated p38 MAPK and NF-κB p65 existed in the nucleus in HBZY-1 cell and NRK52E infected with RSV. In addition, inhibition of ERK and p38 MAPK can significantly reduce the protein expression of heparanase in HBZY-1 cells and NRK52E cells.Conclusion:Heparanase expression contributes to the pathogenesis of proteinuria in RSV nephropathy in rats. Heparin, which can compete with heparan sulfate to combine with RSV to keep renal cells from being infected with RSV, can reduce the proteinuria through down-regulating the heparanase expression and decreasing the heparanase activity. RSV can directly infect renal cells and up-regulate heparanase expression, which is involved in the loss of anionic sites in GBM. Activation of ERK and p38MAPK signaling pathway is associated with induction of heparanase. Overall, heparanase may play a pivotal role in the pathogenesis of MCNS induced by RSV.