Hyperosmotic stress in renal tubular epithelial cells promotes TFEB-dependent autophagy in interstitial fibroblasts by involving extracellular glutamate.

Renal tubular epithelial-interstitial fibroblast communication maintains kidney homeostasis but promotes disease when dysregulated. Tubular epithelial cells undergo large physiological osmotic fluctuations; however, it remains unclear whether epithelial hyperosmotic stress sends signals to neighboring fibroblasts. Herein, we examined how epithelial hyperosmotic stress regulates fibroblast autophagy and transcription factor EB (TFEB) activity. We used a Transwell co-culture system with rat proximal tubular epithelial cells (NRK-52E) and renal interstitial fibroblasts (NRK-49F), which allowed evaluation of the paracrine effect on fibroblasts mediated by tubular epithelial cells. Hyperosmotic stress in epithelial cells induced the robust nuclear translocation of TFEB in fibroblasts, increased the expression of lysosomal and autophagy-related genes, and enhanced autophagic flux without altering fibroblast viability or proliferation. Although fibroblasts internalized epithelial-derived extracellular vesicles, they did not activate TFEB, suggesting an extracellular vesicles-independent mechanism. RNA sequencing revealed that hyperosmotic stress markedly alters the expression of epithelial solute carrier genes, particularly those encoding amino acid transporters. Analysis of amino acid metabolism revealed corresponding changes in conditioned media, including an increase in extracellular glutamate. Glutamate alone induced TFEB nuclear translocation and autophagy activation in fibroblasts. Both epithelial hyperosmotic co-culture and glutamate treatment activated AMP-activated protein kinase and inhibited mTORC1 signaling, thereby enabling TFEB activation. These results identify extracellular glutamate as a metabolite-based paracrine signal that couples epithelial osmotic stress with TFEB-dependent autophagy in interstitial fibroblasts.