Transcription Factor ZEB2 Determines the Cell Fate of Kidney Stroma Progenitors and Loss of ZEB2 Protects Mice from Proteinuria

Background: ZEB2 is a transcription factor mutated in Mowat-Wilson syndrome, a congenital disorder with renal and urinary tract anomalies. ZEB2 is highly expressed in developing kidney stromal progenitors, and loss of ZEB2 in FOXD1+ developing stromal progenitors leads to abnormal kidney stroma differentiation and kidney fibrosis. Interestingly, no proteinuria can be detected in ZEB2 kidney-specific knockout mice. However, the molecular mechanism is unclear. Methods: We analyzed the single-cell RNA sequencing (scRNA-seq) data generated from the kidney tissues isolated from Zeb2 stroma-specific conditional knockout mice Zeb2flox/flox;Foxd1Cre+ (Zeb2 cKO) and their wild-type (WT) littermate controls. We performed 10x Genomics Chromium single-cell transcriptional profiling of 3-week-old Zeb2 cKO and WT mouse kidney tissues and analyzed the data using our in-house computational pipeline. Immunostaining and RNAscope in situ hybridization (ISH) were performed to confirm the scRNA-seq findings. Results: After removing experimental batch effects and individual variance, our clustering analysis identified nine major cell types. We observed a significant increase in the proportion of podocytes and a decrease in macrophages in the Zeb2 cKO samples. Differential expression analysis revealed upregulation of myofibroblast markers Vimentin and Nestin in the Zeb2 cKO kidney compared to the wild-type control kidney. We also found that interstitial cells expressed podocyte and parietal cell markers such as WT1, TLE4, and claudin 1, suggesting that stroma cell takes podocyte/parietal cell fate. Trajectory and functional enrichment analysis provided further insights into the potential pathways and changes in cell differentiation influenced by Zeb2 deletion in kidney stromal progenitors. Immunostaining and RNAscope ISH results validated the scRNA-seq findings. Conclusion: ZEB2 regulates the cell fate of FOXD1+ kidney stroma progenitors. ZEB2-deficient FOXD1+ stromal progenitors adopt podocyte and parietal-like cell fates that might strengthen the glomerular function barrier and protect mice from proteinuria. Funding: NIDDK Support, Other U.S. Government Support, Private Foundation Support