CMAP prediction and experimental validation of Forskolin as a podocyte protective and anti-proteinuric drug for nephrotoxic serum-treated mice.

Abstract

Podocyte injury leads to proteinuria and glomerular diseases. Different podocyte injuries have distinct mechanisms. It is desirable to use a regimen that targets the mechanism of a given podocyte injury for a specific and improved result. However, the mechanisms of the most podocyte injuries are largely elusive, preventing optimal drug choices. Here, we test the feasibility of combining kidney single-cell RNA-seq databases and the Connectivity Map database (CMAP) to predict drugs for a specific podocyte injury. We downloaded glomerular single-cell RNA-seq dataset of nephrotoxic serum (NTS)-treated and control mice from the GEO, and compared their podocyte gene expression, resulting in identification of genes with altered expression in NTS-treated podocytes. GO and KEGG enrichment of them revealed activations of podocyte injurious NFκB, TNFα, AGE-RAGE, apoptosis, cellular senescence, MAPK, and p53 pathways, and dedifferentiation. CMAP analysis of the genes ranked Forskolin top 3. Indeed, we found that NTS-treated mice developed massive proteinuria, which was prevented by Forskolin, accompanied by pathological improvement of podocytes. In treating overdose NTS-induced severe podocyte injury, Forskolin exhibited a comparable efficacy as glucocorticoids (methylprednisolone). In vitro, Forskolin prevented NTS-induced cellular injury in cultured podocytes as shown by cell viability and cytoskeletal integrity assays. Mechanistically, Forskolin inhibited STAT3, p53, NFκB, FAK, and TGF-β pathways, while upregulated podocyte essential genes, WT1, SYNPO, and VEGFA, independently of NTS. In conclusion, Forskolin protects podocytes by directly inhibiting harmful pathways and the associated genes while enhancing podocyte essential gene expression independently of insults, resulting in an efficacy comparable with that of glucocorticoids in NTS-treated mice.