Mechanistic Target of Rapamycin Complex 1 (mTORC1) Activity in an In Vitro Model of AKI

Background: Acute Kidney Injury (AKI) results in damage to proximal tubules (PT), altering metabolic profiles, including decreasing branched-chain amino acid (BCAA) catabolism. A potential link between BCAA catabolism and cell outcomes is the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which can be activated by leucine, a BCAA. Many in vitro studies use the human kidney (HK2) cell-line, but this has a different metabolic profile from PT in vivo. The link between BCAA catabolism and mTORC1 has not been elucidated in AKI, thus our aim was to establish an in vitro model of AKI to study BCAA-related mTORC1 signaling in human primary PT cells. Methods: Human renal primary proximal tubule cells (hRPTECs) were treated with 25-100uM aristolochic acid I (AAI) for 24-72 hours to induce injury. Cells were also grown in media supplemented with 10 mM leucine. Transient knockdown of Sestrin-2 (SESN2), the leucine sensing component, was performed using shRNA. Reverse-transcription quantitative PCR (RT-qPCR) was performed for injury markers, genes encoding leucine sensing components, and BCAA catabolic genes. Immunocytochemistry (ICC) for mTOR, p-S6 (a downstream effector of mTOR), and LAMP2 (a lysosomal marker) were performed. Live-cell metabolic assays were performed using a Seahorse bioanalyzer. Results: hRPTEC cells expressed all the leucine-sensing components upstream of mTORC1. Whereas HK2 cells demonstrate injury with 25uM AAI, hRPTEC cells required 100uM AAI to result in increased expression of injury markers and leucine-sensing components, and changes in expression of BCAA catabolic enzymes. ICC staining demonstrated colocalization of mTOR and LAMP2 and upregulation of p-S6 in some cells, but not all, at 24 and 48 hours of AAI treatment suggesting mTORC1 activation. Despite cellular injury, mitochondrial bioenergetics were only minimally changed after AAI. Knockdown of SESN2 reduced expression of cell cycle arrest marker CDKN1A after treatment with AAI. Cells grown in excess leucine alone did not exhibit mTORC1 activation. Conclusion: hRPTECs are more resistant to injury compared to HK2 cells. Our findings indicate that injured hRPTECs exhibit mTORC1 hyperactivation in injury, but leucine alone is insufficient. Further studies will include a wider range of donor hRPTECs to study mTORC1 activity in AKI. Funding: NIDDK Support, Private Foundation Support