WTAP deficiency in podocytes mediates podocyte injury and contributes to the pathogenesis of FSGS by activating SPP1 signaling

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease (ESRD), with podocyte injury as its core pathological feature. Despite ongoing research efforts, current treatment options remain limited. Genetic factors and the complexity of gene regulatory networks that have yet to be fully resolved. Therefore, this study aims to discover and elucidate the novel pathogenic gene WTAP and its underlying mechanisms in FSGS development.

Immunofluorescence was employed to detect WTAP expression in podocytes of human FSGS patients and adriamycin (ADR)-induced FSGS mouse models. Two distinct podocyte-specific Wtap knockout mouse models were generated using the Cre-loxP system: a rapidly developing constitutive knockout (cKO) model and a tamoxifen-induced conditional knockout (iKO) model. Renal function alterations were assessed via ELISA, while renal pathological changes were examined by using HE and PAS staining. Single-cell RNA sequencing (scRNA-seq) was performed to analyze changes in renal cell types in cKO mice. CellChat was utilized to investigate intercellular communication signaling pathways among various renal cell types in the cKO model. Serum and urine SPP1 levels were measured by ELISA, and cKO mice were treated with intraperitoneal injections of SPP1-neutralizing antibodies for two weeks to evaluate therapeutic effects on renal pathology.

Our findings revealed a significant reduction in WTAP expression in podocytes of both FSGS patients and ADR-induced mouse models. We successfully established two podocyte-specific Wtap knockout mouse models. Both models exhibited progressive FSGS glomerular pathological changes. scRNA-seq results demonstrated a marked decrease in the proportions of podocytes, endothelial cells, and tubular cells in cKO mice, alongside a significant increase in mesangial cells and inflammatory cells, including T cells, macrophages, and fibroblasts. Differential gene expression analysis in podocytes indicated the activation of PANoptotic signaling in cKO mice. Furthermore, in vitro experiments using WTAP-knockout human podocytes (HPCs) confirmed the occurrence of PANoptosis in podocytes. Additionally, we analyzed intricate cell-cell interactions from glomeruli to tubules in the kidneys of control and cKO mice. Cell communication analysis revealed that SPP1 mediates intercellular signaling between glomeruli and tubules in cKO mice. ELISA detected significantly elevated SPP1 levels in serum and urine. Notably, treatment with SPP1-neutralizing antibodies substantially ameliorated renal injury in cKO mice.

In conclusion, we demonstrated that WTAP expression is significantly downregulated in podocytes of FSGS kidneys. Moreover, we successfully constructed two FSGS mouse models that are suitable for studying the pathological mechanisms underlying FSGS or other kidney disorders. We also demonstrated that the SPP1 signaling pathway contributes to the renal pathological changes observed in FSGS. These insights could pave the way for innovative treatments targeting kidney diseases linked to podocyte impairment.