Mechanosensitive determinants of podocyte physiology

PROJECT SUMMARY Drug nephrotoxicity is often conflated with tubular injury; however, many nephrotoxic agents target the glomerulus leading to proteinuric kidney disease. Mechanisms of drug-induced glomerular dysfunction are not well understood. We have previously shown that altered cytoskeletal integrity of podocytes leads to changes in their resilience making them vulnerable to nephrotoxic agents that impact the cytoskeleton. Using meta- analyses to analyze reported renal adverse events of FDA-approved drugs, we identified a previously unknown mechanism of protein kinase inhibitor toxicity that targets actin biophysics. In this project, we utilize this link between regulatory kinase activity and the biomechanical resilience of podocytes to uncover the key signaling landscape that impacts glomerular function. We use a combination of in vitro and in vivo models of cell signaling to mechanistically link the clinically-observed glomerular adverse events with cellular and subcellular characteristics in human podocytes. Using high-content image analytics, atomic force microscope elastography, single-cell transcriptomics and phosphoproteomics, we will identify molecular, morphological and biophysical properties associated with protein kinase inhibitor induced impairment of podocyte cytoskeletal and focal adhesion dynamics. Grounded with multiple sets of in vivo proteomic data from the transient puromycin aminonucleoside nephropathy and spontaneously hypertensive rat models, we identified key kinome signatures that are responsible for maintaining glomerular integrity. Integrating the in vitro and in vivo model system results using network-based systems biology methods, we will identify potential drug interaction hubs and binding domains that impact cytoskeletal resilience and test these predictions in vivo in three complementary transgenic mouse models of experimental glomerular disease. This integrative study will have an immediate impact on clinical standard of care by improving renal monitoring of patients and by identifying potential risk factors of nephrotoxicity. Furthermore, our quantitative cellular screening platform could help mitigate risks for future treatment regimens. Finally, our project will lead to a better understanding of glomerular structural integrity, and it may enable identification of key kinases that are responsible for maintaining podocyte cytoskeletal dynamics.