Protease‐activated Receptor‐1‐driven Intracellular Calcium Dynamics in Rat Glomerular Podocytes

Proteinase‐activated receptors (PARs) are a well‐known family of seven‐transmembrane G‐protein‐coupled receptors whose activation is initiated by serine protease‐mediated irreversible cleavage of the N‐terminus. When activated, ligands remain tethered to the receptors, docking intramolecularly to effect transmembrane signaling. To date, four PARs have been identified whose activation is physiologically initiated by serine proteases such as thrombin (acts on PARs 1, 3 and 4) and trypsin (PAR‐2). Previous studies have shown that human glomerular epithelial and mesangial cells express PAR‐1 and this receptor is involved in the pathogenesis of crescentic glomerulonephritis, glomerular fibrin deposition and macrophage infiltration. Furthermore, it was reported that the knockout of PAR‐1 in mice results in protection from renal injury in accelerated anti‐GBM glomerulonephritis model. The goal of this study was to identify the involvement of PARs in regulation of intracellular calcium signaling in podocytes of the freshly isolated Dahl Salt‐Sensitive (SS) rat glomeruli. For these experiments SS rats were fed a 0.4% NaCl salt diet and glomeruli were isolated by differential sieving and loaded with cell permeable calcium indicators (Fluo4/Fura 2TH) for ratiometric confocal fluorescence measurements. Various PAR receptor agonists such as Thrombin and small peptides (TRAP‐6 or TFLLR‐NH2, SLIGNV‐NH2 and AY‐NH2) were used for selective activation of PAR‐1, PAR‐2 and PAR‐4 receptors, respectively. Pharmacological screening revealed that only PAR‐1‐specific agonists, as well as thrombin, were able to produce detectable increases in intracellular calcium in glomeruli podocytes. The stimulated calcium transient was successfully blocked by pre‐incubating the glomeruli with SCH 79797 dihydrochloride, a specific PAR‐1 receptor antagonist. It was shown that endothelial effects of thrombin have been attributed to NO production through Ca 2+ dependent activation of eNOS. Therefore, in addition to testing effects of PAR receptor agonists on changes in intracellular calcium, we also determined their effects on NO production. To test contribution of NO in this pathway podocytes of freshly isolated glomeruli were loaded with the NO‐labeling fluorophore (DAF‐FM). In contrast to the effects of PAR1 receptor agonists on the changes in intracellular calcium concentrations, podocytes failed to produce NO when stimulated with PAR agonists. Our control experiments with Angiotensin II revealed that this hormone triggers both NO production and intracellular calcium signaling in podocytes. SS rats fed a high salt diet develop salt‐sensitive hypertension, which is accompanied by kidney injury including the glomeruli damage. Further studies are required to define PAR receptor signaling under such pathological conditions and molecular mechanisms determining these effects. Therefore, we provide here evidence that PAR1‐receptor mediated signaling is involved in calcium flux in the podocytes in SS rats fed a 0.4% salt diet. Identification of specific PARs responsible for excessive calcium flux in podocytes and following glomeruli damage opens up promising avenues of research to study the therapeutic modality of PARs inhibition or manipulating downstream signaling pathways leading to activation of calcium flux. Support or Funding Information Supported by National Kidney Foundation Young Investigator Grant and American Diabetes Association grant 1‐15‐BS‐172.