figure posted on 2023-09-18, 21:42 authored by Ruslan Bohovyk, Sherif Khedr, Vladislav Levchenko, Mariia Stefanenko, Marharyta Semenikhina, Olha Kravtsova, Elena Isaeva, Aron M. Geurts, Christine A. Klemens, Oleg Palygin, Alexander Staruschenko
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 [Ca2+]i 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 [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i 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. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- 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 [Ca2+]i influx in podocytes through the PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage.
This work was supported by the National Institutes of Health grants R01 DK129227 (to AS and OP), R35 HL135749, R21 DK129882, R01 DK135644 (to AS), R01 DK126720 (to OP), Department of Veteran Affairs grant I01 BX004024 (to AS), endowed funds from the SC SmartState Centers of Excellence (to OP), and the American Physiological Society Postdoctoral Fellowship (to RB).