SnRNA-seq and genome-wide CRISPR screening define the complete transcriptional trajectory and functional drivers of podocyte stress.

Podocyte injury is a central driver of proteinuria and progressive kidney dysfunction. Although podocytes are continuously exposed to diverse stressors in both physiological and pathological contexts, the dynamic processes underlying their adaptation and eventual failure remain poorly defined. Here, we performed integrative single-nucleus RNA sequencing of kidney tissues from patients with six types of representative chronic glomerulonephritis, capturing a spectrum of podocyte injury states. We identified distinct podocyte subpopulations and reconstructed a dynamic trajectory characterized by an initial adaptive activation followed by progressive functional decline. Integration with time-resolved transcriptomics identified 778 candidate genes associated with podocyte stress responses. To distinguish putative functional drivers from secondary transcriptional changes, we integrated these candidates with a genome-wide CRISPR-Cas9 knockout screen, prioritizing genes required for podocyte survival under stress conditions. Subsequent siRNA-mediated validation of five representative candidates-BST1, TALDO1, ATP6V1E1, PPP2R1A and CHL1-showed that knockdown of these genes significantly compromised cell viability and accelerated apoptosis, highlighting a coordinated survival network spanning metabolic regulation, autophagy, and cytoskeletal stability. Our findings define a dynamic framework of podocyte stress adaptation and failure, and suggest that targeting stress-response pathways may prolong podocyte survival, thereby extending the therapeutic window for intervention in chronic kidney disease.