We identified motor protein Myo1c as a novel component of slit diaphragm that interacts with Nephrin and Neph1 and regulates their movement. To study the in vivo function of Myo1c, we generated Myo1c floxed mice that were used to construct homozygous null and podocyte-specific Myo1c null mice. While Myo1c homozygous null mice were reported to die prenatally, complete Myo1c deletion in 12 week old mice increased their sensitivity to adriamycin-induced proteinuria (on a C57BL/6J background), which is in agreement with our zebrafish studies. Interestingly, the analysis of podocyte specific Myo1c knockout mouse showed no proteinuria or functional abnormality when aged to 8 months. However, when bred to an adriamycin-sensitive background, these mice were resistant to adriamycin-induced glomerulopathy; they did not develop proteinuria as compared to the control mice suggesting multiple functions for Myo1c. Although surprising, these results are consistent with loss of proteins in podocytes that are involved in signaling and trafficking such as Rac1 and Crk. Podocytes response to injury is commonly assessed through increased phosphorylation of Nephrin and Neph1 that initiates their redistribution and the assembly of an intracellular signaling cascade leading to podocyte effacement. Impairment of these events may attenuate podocytes ability to respond to injury thus inducing protection. Indeed, our recent study demonstrated that inhibiting Neph1 signaling protected podocytes from injury. Since Myo1c has a tethering function that associates its cargo proteins with membranes and actin, we hypothesized that Myo1c participates in a mechanism that regulates movement of these proteins at the membrane that is critical for directing the assembly of a signaling complex by Nephrin and Neph1 and initiating intracellular signaling and trafficking events. Indeed, loss of Myo1c binding attenuated the dynamic movement of Neph1 at the membrane as demonstrated using live FRAP analysis. Furthermore, we treated cultured podocytes with a Myo1c specific inhibitor pentachloropseudilin (PCIP) that arrested membrane and intracellular vesicles movements suggesting the involvement of Myo1c in actin dependent cellular events that are critical for generating cellular response to injury. This further suggested that these cells will hve impaired injury response. Indeed, these cells resisted injury by protamine sulphate (PS) as measured by actin cytoskeleton reorganization. In the Specific Aim 1, we will investigate the hypothesis that Myo1c due to its membrane and actin binding functions, participates in generating an appropriate injury response by podocytes. This involves regulating injury-induced redistribution of Nephrin and Neph1 proteins to intracellular compartments and the assembly of signaling complexes that drives their intracellular signaling and trafficking. In the second Aim, we will investigate how Myo1c depletion at various stages of mouse development affects glomerular function. In addition, we will investigate if Myo1c is a therapeutic target by determining whether podocyte-specific deletion of Myo1c attenuates the disease phenotype in various acute and chronic glomerular injury models.
Glomerular diseases including diabetic nephropathy cause progressive loss of kidney function that leads to end stage renal disease (ESRD), which is the leading cause of renal failure worldwide. The financial and emotional burden of ESRD is rapidly approaching to enormous proportions (several billions of dollars). Due to our limited understanding of the glomerular biology, the effective therapies to treat these life threatening diseases are lacking. Our goal is to contribute towards the understanding of molecular mechanisms that govern the development and function of podocytes that are critical components of the glomerular filtration barrier, thereby providing the nephrology community with potential disease mechanisms and therapeutic targets.
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