Mesangial cells provide structural support to the glomerular tuft and modulate the glomerular capillary flow via their contractile properties. The mesangial cells' phenotypic changes to myofibroblast-like cells, such as proliferation, mesangial expansion, abnormal glomerular tuft formation, and reduced numbers of capillary loops, are present in several glomerular diseases, including diabetic nephropathy and glomerulonephritis. In addition, thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling and mechanisms in mesangial cells were not studied. This study aims to investigate protease-activated mechanisms regulating mesangial cell contraction and glomerular capillary flow.

We used the human renal mesangial cell (HRMC) line to determine the signaling mechanisms mediated by PAR1 thrombin-activated receptors. Confocal fluorescent microscopy was utilized to detect changes in intracellular Ca²⁺ response to specific PAR1 modulators. Pharmacology and patch clamp electrophysiology was further applied to reveal downstream signaling mechanisms responsible for intracellular Ca²⁺ oscillations. PAR1-mediated Ca²⁺ response display high sensitivity to specific agonist (TFLLR-NH₂) with EC₅₀ values of 3 and 6.3 nM for male and female cultured cells, respectively (the competition of a ligand for receptor binding fit converged; adj. R²=0.98). The response to PAR1 activation promotes initial cytosolic Ca²⁺ increase followed by synchronized, damped Ca²⁺ oscillations with a lag of 6.74±0.84 min between peaks. The pre-application of a specific inhibitor (RWJ56110) eliminated PAR1-mediated response, and oscillations were blocked by the changes of an extracellular solution to zero Ca²⁺. The specific inhibitors for store-operated calcium (SOCs) (Pyr6) and TRPC3 (GSK 2833503A) channels strongly attenuated oscillation behavior (up to 40% when added separately and up to 65% when applied both; two-way ANOVA, * p<0.0001). In addition, the effect of a specific inhibitor of TRPC6 channels (BI-749327) on Ca²⁺ flux was minimal. Further single-channel electrophysiology experiments in HRMC cells confirmed the involvement of SOC and TRPC3 channels in PAR1-mediated GPCR activation.

Our results indicate that coagulation proteases like thrombin may strongly regulate mesangial cell contraction and corresponding glomerular capillary flow by PAR1 GPCRs-related activation. The contraction mechanism is mediated presumably through SOCs entry and TRPC3 channels. Since high thrombin levels are linked to poor diabetic control, the described signaling may play a crucial role in the development of diabetic glomerular disease.