Multiple transforming growth factor (TGF)-beta-induced fibrogenic signals have been described in vitro. To evaluate mechanisms in vivo, we used an adriamycin nephropathy model in 129x1/Svj mice that display massive proteinuria by days 5 to 7 and pathological findings similar to human focal segmental glomerulosclerosis by day 14. TGF-beta mRNA expression increased after day 7 along with nuclear translocation of the TGF-beta receptor-specific transcription factor Smad3. Inhibiting TGF-beta prevented both pathological changes and type-I collagen and fibronectin mRNA expression, but proteinuria persisted. Renal Akt was phosphorylated in adriamycin-treated mice, suggesting PI3-kinase activation. Expression of mRNA for the p110gamma isozyme of PI3-kinase was specifically increased and p110gamma colocalized with nephrin by immunohistochemistry early in disease. Nephrin levels subsequently decreased. Inhibition of p110gamma by AS605240 preserved nephrin expression and prevented proteinuria. In cultured podocytes, adriamycin stimulated p110gamma expression. AS605240, but not a TGF-beta receptor kinase inhibitor, prevented adriamycin-induced cytoskeletal disorganization and apoptosis, supporting a role for p110gamma in podocyte injury. AS605240, at a dose that decreased proteinuria, prevented renal collagen mRNA expression in vivo but did not affect TGF-beta-stimulated collagen induction in vitro. Thus, PI3-kinase p110gamma mediates initial podocyte injury and proteinuria, both of which precede TGF-beta-mediated glomerular scarring.