Depletion of Flot1 attenuates macropinosome-dependent mTORC1 activation in podocytes

Podocytes are terminally differentiated renal epithelial cells that play a crucial role in kidney filtration. Given this essential function, podocyte dysfunction results in kidney diseases known as podocytopathies. Previous studies have demonstrated that maintaining the activation-deactivation balance of mechanistic target of rapamycin complex 1 (mTORC1) is vital for podocyte function. Podocyte-specific knockout (KO) mouse models revealed that abnormal mTORC1 activation leads to severe podocytopathy. Therefore, elucidating the mechanism underlying mTORC1 activation in podocytes may contribute to the development of treatments for certain podocytopathies. In our previous study, we showed that macropinocytosis-large-scale endocytosis-is involved in the molecular mechanism of mTORC1 activation in podocytes. Growth factor (GF) stimulation induces circular dorsal ruffles (CDRs), which are large membrane protrusions on the dorsal surface of podocytes. CDRs serve as precursors to macropinocytosis, generating vesicles called macropinosomes, which transport extracellular nutrients to lysosomes, thereby activating mTORC1. These findings suggest that CDRs-derived macropinosomes modulate the mTORC1 pathway. In the present study, we investigated the molecular mechanism underlying macropinosome formation in podocytes, focusing on flotillin-1 (Flot1), a protein enriched in lipid microdomains. Imaging analysis revealed the localization of Flot1 at CDRs, and Flot1 depletion reduced macropinosome formation. Biochemical analysis further demonstrated impaired GF-stimulated mTORC1 activation in Flot1-KO cells, which exhibited slower growth than control cells. Notably, immuno-staining analysis showed that Flot1 is expressed specifically in podocytes but not in other renal cells. These findings indicate that Flot1 participates in the formation of CDRs-derived macropinosomes and contributes to macropinosome-dependent mTORC1 activation in podocytes.Key words: Flot1, circular dorsal ruffles, macropinocytosis, mTORC1, podocytes.