Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent

• Published in: Journal of the American Society of Nephrology 2017-09, Vol.28 (9), p.2618-2630
• Main Authors: Sharma, Ruchika, Waller, Amanda P, Agrawal, Shipra, Wolfgang, Katelyn J, Luu, Hiep, Shahzad, Khurrum, Isermann, Berend, Smoyer, William E, Nieman, Marvin T, Kerlin, Bryce A
• Format: Article
• Language: English
• Subjects: Animals; Antithrombins - pharmacology; Basic Research; Cell Survival; Cells, Cultured; Disease Models, Animal; Gene Expression; Hirudins - pharmacology; Humans; Immunophenotyping; Kidney Glomerulus - metabolism; Nephrosis - complications; Nephrosis - metabolism; Nephrosis - pathology; Nephrosis - urine; Podocytes - metabolism; Podocytes - pathology; Proteinuria - etiology; Rats; Receptor, PAR-1 - genetics; Receptor, PAR-1 - metabolism; Receptors, Thrombin - genetics; Receptors, Thrombin - metabolism; Signal Transduction; Thrombin - antagonists & inhibitors; Thrombin - metabolism; Thrombin - pharmacology; Thrombin - urine
• ISSN: 1046-6673; 1533-3450
• DOI: 10.1681/asn.2016070789

Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.