<b>Protease-Activated Receptor 1 Mediated Damage of Podocytes in Diabetic Nephropathy</b>

Abstract

<h4>There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca<sup>2+</sup>]<sub>i</sub> dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of non-specific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion-conductance microscopy, and patch-clamp analysis. Goto-Kakizaki, Wistar, type 2 diabetic nephropathy (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca<sup>2+</sup>]<sub>i</sub> via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca<sup>2+</sup>]<sub>i</sub> compared to controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared to Wistar rats. T2DN<sup>Par1-/-</sup> rats were embryonically lethal. T2DN<sup>Par1+/-</sup> rats had a significant decrease in glomerular damage associated with DN liaisons. Overall, this data provides evidence that during the development of DN, elevated levels of serine proteases promote an excessive [Ca<sup>2+</sup>]<sub>i</sub> influx in podocytes through the PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage.</h4>