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      Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent

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          Abstract

          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 ex vivo 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 in vivo 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.

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          Most cited references 58

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          High-resolution crystal structure of human Protease-Activated Receptor 1 bound to the antagonist vorapaxar

          Protease-Activated Receptor-1 (PAR1) is the prototypical member of a family of G protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the N-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the receptor’s heptahelical bundle to effect G protein-activation. Here we report a 2.2Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by PAR1’s tethered ligand. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated GPCRs, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. PARs are important targets for drug development. The structure reported here will aid development of improved PAR1 antagonists and discovery of antagonists to other members of this receptor family.
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            Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis.

            Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.
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              Inducible rodent models of acquired podocyte diseases.

              Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded.
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                Author and article information

                Journal
                Journal of the American Society of Nephrology
                JASN
                American Society of Nephrology (ASN)
                1046-6673
                1533-3450
                August 31 2017
                September 2017
                September 2017
                April 19 2017
                : 28
                : 9
                : 2618-2630
                Article
                10.1681/ASN.2016070789
                5576925
                28424276
                © 2017

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