Abstract P245: Activation Of Protease-activated Receptors 1 Leads To Structural Changes In Immortalized Cultured Human Podocytes.

Abstract

Previous studies have suggested that activation of the protease-activated receptors (PAR) exacerbates the development of diabetic nephropathy. PARs are transmembrane proteins activated by extracellular serine proteases, such as thrombin and trypsin, which are frequently elevated in disease states. We hypothesize that serine protease activation of the PAR1 signaling cascade upregulates calcium channel activity and promotes excessive intracellular Ca 2+ ([Ca 2+ ] i ), leading to podocyte foot retraction, apoptosis, and glomerular filtration barrier (GFB) damage. Immunofluorescent labeling revealed the co-localization of PAR1 and podocyte marker nephrin in freshly isolated human glomeruli. To explore the functional role of PAR1 mediated signaling, we used an immortalized cultured human podocyte (hPod) cell line. Live confocal imaging revealed rapid elevation of [Ca 2+ ] i in response to a PAR1 specific agonist (TFLLR-NH 2 ). Moreover, preincubation with a PAR1 antagonist (RWJ 56100) completely blocked this effect (1616±48 vs. 206±135 a.u., for TFLLR vs. TFLLR+RJW, n<100 cells, p<0.05). Furthermore, we tested potential downstream signaling proteins in hPod cells after PAR1 activation using Western blot analysis. Application of TFLLR-NH 2 led to a decrease in PAR1 expression due to internalization and lysosomal degradation, accompanied by an increase in TRPC6 expression and time-dependent changes in p-ERK1/2 and PLC-γ1 cascades. We further used scanning ion-conductance microscopy (SICM) to detect morphological changes in podocytes, which measures real-time surface topography in hPod cells. This imaging revealed normal protrusion of lamellipodium under control conditions. In contrast, PAR1 activation led to retraction of the lamellipodium, and a significant decrease in surface area (+51±46 vs. -15±8 μm 2 , 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05). Our studies suggested that PAR1 is involved in GPCR-induced calcium influx in podocytes. Furthermore, these data demonstrate that PAR1-mediated signaling is involved in podocyte structural alterations and may increase [Ca 2+ ] i , potentially leading to loss of GFB integrity and podocyte apoptosis.