sPLA2 IB induces human podocyte apoptosis via the M-type phospholipase A2 receptor

Abstract

The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content.

Figure 1. The levels of podocyte apoptosis and PLA2R expression in kidneys of different IMN patient groups.

a,d Negative control group; b,e Group I: the group with low levels of serum sPLA2 IB; c,f Group II: the group with high levels of serum sPLA2 IB (original magnification, ×400). * p < 0.05 vs. Group I.

Figure 2. The levels of PLA2R expression in mouse podocytes and in human podocytes.

A. Immunofluorescence staining of mouse podocyte and human podocyte PLA2R expression (original magnification, ×400). a Negative control group: PBS instead of the PLA2R antibody. b Mouse podocytes. c Human podocytes. B. Representative Western blotting of the levels of PLA2R in mouse podocytes and in human podocytes. * p < 0.05 vs. mouse podocytes.

Figure 3. sPLA2 IB induced podocyte apoptosis in a time-dependent manner.

A. Representative Western blotting results for cPLA2α, ERK1/2 and their phosphorylation and for the PLA2R at different time points (n = 3). All of the groups were treated with a 10⁻⁶ M concentration of sPLA2 IB. B. Representative Hoechst 33342 staining of apoptotic podocytes stimulated by 10⁻⁶ M sPLA2 IB at various time points (original magnification, ×400). C. HPLC of podocyte AA production at different time points (n = 3). All of the groups were treated with a 10⁻⁶ M concentration of sPLA2 IB. D. Flow cytometry analysis of apoptosis in differentiated podocytes at different time points (n = 3). All of the groups were treated with a 10⁻⁶ M concentration of sPLA2 IB. * p < 0.05 vs. 0 h group, # p < 0.01 vs. 0 h group.

Figure 4. sPLA2 IB induced podocyte apoptosis in a dose-dependent manner.

A. Representative Western blotting results for cPLA2α, ERK1/2 and their phosphorylation and for the PLA2R at different concentrations of sPLA2 IB (n = 3). All of the groups were treated with sPLA2 IB for 2 h. B. Representative Hoechst 33342 staining of apoptotic podocytes stimulated by different concentrations of sPLA2 IB for 2 h (original magnification, ×400). C. HPLC of podocyte AA production at different concentrations of sPLA2 IB (n = 3). All of the groups were treated with sPLA2 IB for 2 h. D. Flow cytometry analysis of apoptosis in differentiated podocytes stimulated by different concentrations of sPLA2 IB (n = 3). All of the groups were treated with sPLA2 IB for 2 h. * p < 0.05 vs. 0 M group, # p < 0.01 vs. 0 M group.

Figure 5. Effects of the PLA2R plasmid and PLA2R siRNA on sPLA2 IB-induced podocyte apoptosis.

A. Western blotting analysis of cPLA2α, ERK and their phosphorylation and for the PLA2R in cultured podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and treated with no plasmid, scrambled plasmid, PLA2R plasmid, scrambled siRNA or PLA2R siRNA (n = 3). B. Representative immunofluorescence staining of PLA2R and sPLA2 IB expression in cultured podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and treated with no plasmid, PLA2R plasmid or PLA2R siRNA (original magnification, ×400). C. HPLC of podocyte AA production in cultured podocytes stimulated with 10⁻⁶ M sPLA2 IB for 2 h and treated with no plasmid, scrambled plasmid, PLA2R plasmid, scrambled siRNA or PLA2R siRNA (n = 3). D. Flow cytometry analysis of apoptosis in differentiated podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and treated with no plasmid, scrambled plasmid, PLA2R plasmid, scrambled siRNA or PLA2R siRNA (n = 3). * p < 0.05 vs. control group, # p < 0.05 vs. scrambled plasmid group or scrambled siRNA group.

Figure 6. The cPLA2α and ERK interaction in podocytes and its effect on podocyte apoptosis.

A. Western blotting analysis of cPLA2α, ERK and their phosphorylation in cultured podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and pretreated with 10 μM MAFP or 10 μM U0126 for 1 h (specific inhibitors of cPLA2α and ERK, respectively; n = 3). B. cPLA2α was immunoprecipitated from normal podocytes and from sPLA2 IB-treated podocytes for 2 h (10⁻⁶ M), which were pretreated with 10 μM U0126 for 1 h. Then, the immunoprecipitates were analysed by Western blotting with ERK and cPLA2α antibodies (n = 3). C. Representative Hoechst 33342 staining of apoptotic podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and pretreated with 10 μM MAFP or 10 μM U0126 for 1 h (original magnification, ×400). D. Flow cytometry analysis of apoptosis in differentiated podocytes stimulated by 10⁻⁶ M sPLA2 IB for 2 h and pretreated with 10 μM MAFP or 10 μM U0126 for 1 h (n = 3). * p < 0.05 vs. control group.

Figure 7. Effects of AA on podocyte apoptosis.

A. Representative Western blotting results of nuclear P53 at different time points (n = 3). All of the groups were treated with a 10⁻⁵ M concentration of AA. B. Representative Hoechst 33342 staining of apoptotic podocytes at different time points. (original magnification, ×400). C. Flow cytometry analysis of apoptosis in differentiated podocytes at different time points (n = 3). All of the groups were treated with a 10⁻⁵ M concentration of AA. * p < 0.05 vs. 0 h group, # p < 0.01 vs. 0 h group.