Role Of M-type PLA2R In SPLA2 IB Induced Podocyte Apoptosis

Objective:The M-type phospholipase A2 receptor (PLA2R), which is a transmembrane receptor, is a receptor for secretory phospholipase A2 (sPLA2). The PLA2R is detected in the lung, placenta and kidney in human. Podocytes are terminally differentiated epithelial cells that are crucial components of the glomerular filtration barrier. Injury or depletion of podocytes plays a vital role in proteinuria onset and glomerular disease progression. Podocytes express the PLA2R in normal human glomeruli but not in rabbit, rat and mouse. However, PLA2R expression is accentuated in the glomeruli of some patients with idiopathic membranous nephropathy (IMN). sPLA2 IB mediates cell proliferation, cell migration, hormone release and eicosanoid production in peripheral tissues via its receptor. Additionally, sPLA2 IB has been reported to induce apoptosis in neuronal cells and in macrophages. Interestingly, sPLA2 IB levels were also found to be 10-fold higher in patients with chronic renal failure when compared with controls. However, the contributory role of sPLA2 IB in maintaining glomerular homeostasis under physiological or pathological conditions remains unclear. Here, we explored the role of PLA2R in sPLA2 IB-induced podocyte apoptosis and its molecular mechanism.Methods:Part 1:Twenty-seven patients with IMN were enrolled in this study. All of the patients showed MN characteristics on renal biopsy. IMN was diagnosed after the exclusion of secondary causes. Twenty- seven patients were divided into two groups according to the sPLA2 IB level in their sera and PLA2R expression in kidney. We defined the serum sPLA2 IB level less than 10 ng/ml with weak PLA2R expression as the low sPLA2 IB expression group (Group ?,10 cases) and more than 10 ng/ml with enhanced PLA2R expression as the high sPLA2 IB expression group (Group II,17 cases). Clinical data of each group of patients with IMN were collected before immunosuppressive treatment. Serum sPLA2 IB level was measured by ELISA. Podocyte apoptosis in kidney tissue was determined by TUNEL assay. PLA2R in kidney tissue was detected by immunohistochemistry. Correlation analysis was performed by Pearson's correlation test.Part 2:Conditionally immortalized human podocytes were cultured in vitro. Differentiated human podocytes were serum-free for 12 hours prior to treatment. (1) Podocytes were exposed to sPLA2 IB at 10-6M for variable incubation time (0h?0.5h? 1h?2h?6h) and at different concentrations (0M?10-8M?10-7M?10-6M?10-5M) for 2h. (2) Podocyte apoptosis was assessed by flow cytometry and Hoechst-33342 staining. (3) Expression of PLA2R was analyzed by Western blotting. (4) PLA2R plasmid and PLA2R siRNA was introduced to investigate the role of PLA2R in sPLA2 IB-induced podocyte apoptosis.Part 3:Conditionally immortalized human podocytes were cultured in vitro. Differentiated human podocytes were serum-free for 12 hours prior to treatment. (1) The role of sPLA2 IB in ERK1/2 and cPLA2a phosphorylation was evaluated and AA level of podocytes was measured respectively. (2) PLA2R plasmid and siRNA was introduced to investigate role of sPLA2 IB in ERK1/2 and cPLA2a phosphorylation and AA level of podocytes. (3) ERKl/2 and cPLA2a inhibitors were introduced to investigate the effects of ERKl/2 and cPLA2a signalings on the expression of PLA2R and sPLA2 IB -induced podocyte apoptosis. (4) Co-immunoprecipitation was used to evaluate the interaction between ERK1/2 and cPLA2a.Part 4:Conditionally immortalized human podocytes were cultured in vitro. Differentiated human podocytes were serum-free for 12 hours prior to treatment. (1) Podocytes were exposed to AA at 10-6M for variable incubation time (0h?0.5h?1h? 2h?6h) and at different concentrations (0M?10-8M?10-7M?10-6M?10-5M) for 2h. (2) Podocyte apoptosis was assessed by flow cytometry and Hoechst-33342 staining. (3) Expression of p53 was analyzed by Western blotting.Results:Part 1:(1) There were no differences between two groups, including age, gender, BP, leukocyte, hemoglobin, serum creatinine, cholesterol and triglyceride values. (2) Compared with the Group I patients, the Group ? patients presented a significantly lower level of serum albumin and a higher level of urinary protein excretion (P<0.05). (3) Podocyte apoptosis in the glomerulus of Group II was increased compared with group ? (P<0.05). (4) serum sPLA2 IB and kidney PLA2R level were correlated positively to kidney podocyte apoptosis (r=0.744, P< 0.05 and r=0.527, P< 0.01 respectively).Part 2:(1) Human podocytes expressed the PLA2R in vitro but mouse podocytes did not express the PLA2R. (2) sPLA2 IB promoted podocyte apoptosis in a time- and dose-dependent manner. (3) Upregulated podocyte PLA2R expression through plasmid transfection obviously increased sPLA2 IB-induced apoptosis of podocytes(P<0.05). (4) Down-regulattion of podocyte PLA2R expression through siRNA transfection obviously prevented sPLA2 IB-induced apoptosis of podocytes (P<0.05)Part 3:(1) sPLA2 IB enhanced podocyte cPLA2a and ERK1/2 phosphorylation and AA level in a time- and dose-dependent manner. (2) Up-regulattion of podocyte PLA2R expression through plasmid transfection obviously increased sPLA2 IB-induced phosphorylation of cPLA2a and ERK1/2 and AA level of podocytes (P<0.05). (3) Down-regulation of podocyte PLA2R expression through siRNA transfection obviously decreased phosphorylation of cPLA2a and ERK1/2 and AA level of podocytes (P<0.05). (4) Pretreatment with MAFP or U0126 dramatically prevented sPLA2 IB-induced podocyte apoptosis respectively (P<0.05)Part 4:(1) AA promoted p53 expression of podocyte in a time and dose-dependent manners (P<0.05). (2) AA induced podocyte apoptosis in a time and dose-dependent manners (P<0.05) Conclusion:(1) sPLA2 IB promotes podocyte apoptosis in vivo and in vitro. (2) sPLA2 IB induces podocyte apoptosis through M-type PLA2R. (3) sPLA2 IB induces podocyte apoptosis may occur via the ERK1/2-cPLA2a-AA-p53 signalling pathway.