[Mechanism of podocyte pyroptosis aggravated by up-regulation of phospholipase A receptor by hepatitis B virus X protein].

To explore the effect and underlying mechanism of increased expression of M-type phospholipase A receptor (PLAR) on podocyte membrane induced by hepatitis B virus X protein (HBx) on podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis (HBV-GN). Transfection of the HBx gene into human kidney podocytes was used to mimic the HBV-GN pathogenesis process. Subsequently, podocytes were divided into the following eight groups: normal control plus secretory phospholipase A-ⅠB (sPLA-ⅠB) group, empty plasmid plus sPLA-ⅠB group, HBx group, HBx plus sPLA-ⅠB group, HBx plus sPLA-ⅠB plus PLAR control siRNA group, HBx plus sPLA-ⅠB plus PLAR-siRNA group, HBx plus sPLA-ⅠB plus ROS control siRNA group, and HBx plus sPLA-ⅠB plus ROS-siRNA group. Podocyte morphology was observed under a transmission electron microscope, and PLAR expression was detected under a fluorescence microscope. Podocyte pyroptosis and reactive oxygen species (ROS) expression were analyzed by flow cytometry, and the mRNA and protein expression of PLAR, nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, interleukin (IL)-1β and IL-18 were determined by real-time fluorescence quantitative PCR and Western blot. Compared with the control group, the expression of PLAR on podocyte membrane significantly increased after transfection with HBx plasmid (4.07±0.41 vs 1.01±0.17, <0.001). Transmission electron microscope and fluorochrome-labeled inhibitor of caspases/propidium iodide (FLICA/PI) double staining suggested that overexpressed PLAR combined with sPLA-ⅠB caused aggravated podocyte injury and increased pyroptosis (20.22%±0.36% vs 7.86%±0.28%, <0.001). Moreover, the expression levels of ROS (4 324 515±222 764 vs 12 920±46, <0.001), NLRP3 (48.30±2.73 vs 1.00±0.11, <0.001), ASC (4.02±0.84 vs 1.01±0.15, <0.001), caspase-1 (3.99±0.42 vs 1.00±0.11, <0.001), IL-1β (9.08±0.75 vs 1.00±0.09, <0.001) and IL-18 (19.20±0.70 vs 1.00±0.02, <0.001) increased when PLAR was overexpressed. In contrast, with the addition of PLAR-siRNA or ROS-siRNA to knockdown the expression of related substances, podocyte injury was alleviated and the degree of pyroptosis decreased, and the expressions of genes related to the downstream signaling pathway (NLRP3, ASC, caspase-1, IL-1β and IL-18) decreased (all <0.01). HBx may promote podocyte pyroptosis in HBV-GN by targeting the ROS-NLRP3 signaling pathway via the upregulation of PLAR.