Hyper-activation of Rac1, a small GTPase, in glomerular podocytes has been implicated in the pathogenesis of familial proteinuric kidney diseases. However, the role of Rac1 in acquired nephrotic syndrome is unknown. To gain direct insights into this, we generated a transgenic mouse model expressing a doxycycline-inducible constitutively active form of Rac1 (CA-Rac1) in podocytes. Regardless of the copy number, proteinuria occurred rapidly within five days, and the histology resembled minimal change disease. The degree and severity of proteinuria were dependent on the transgene copy number. Upon doxycycline withdrawal, proteinuria resolved completely (one copy) or nearly completely (two copy). After one month of doxycycline treatment, two-copy mice developed glomerulosclerosis that resembled focal segmental glomerulosclerosis (FSGS) with urinary shedding of transgene-expressing podocytes. p38 MAPK was activated in podocytes upon CA-Rac1 induction while a p38 inhibitor attenuated proteinuria, podocyte loss, and glomerulosclerosis. Mechanistically, activation of Rac1 in cultured mouse podocytes reduced adhesiveness to laminin and induced redistribution of β1 integrin, and both were partially reversed by the p38 inhibitor. Activation of Rac1 in podocytes was also seen in kidney biopsies from patients with minimal change disease and idiopathic FSGS by immunofluorescence while sera from the same patients activated Rac1 in cultured human podocytes. Thus, activation of Rac1 in podocytes causes a spectrum of disease ranging from minimal change disease to FSGS, due to podocyte detachment from the glomerular basement membrane that is partially dependent on p38 MAPK.

Key Points• The present study shows, for the first time, that Gas6 produced by endothelial cells contributes to venous thrombus formation.• Gas6 is required for the expression of tissue factor in endothelial cells.Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (؊/؊) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5M ADP and 10M of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/ reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6 ؊/؊ mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6 ؊/؊ endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism. (Blood. 2013;121(4):692-699) IntroductionVenous thromboembolism (VTE) is a common cause of morbidity and mortality in clinical medicine. The pathophysiology of VTE was first described by Virchow in 1853 and describes a triad of entities accounting for VTE. VTE could be triggered by alterations in the blood composition (thrombophilia), changes in blood flow (eg, stasis), and/or activation of the endothelium. 1 Under normal conditions, the endothelial surface inhibits coagulation because of the presence of various proteins, such as tissue factor (TF) pathway inhibitor, thrombomodulin, and the endothelial cell protein C receptor. 2 However, physical (eg, vascular damage) or functional (eg, hypoxia) perturbation of the endothelium promotes thrombosis because of reduced expression of anticoagulants and the induction of the expression of the transmembrane procoagulant protein TF. 3 Gas6, the product of growth arrest specific gene 6 (Gas6), is a member of the vitamin K-dependent family of proteins, which includes the procoagulant factors II, VII, IX, and X and the anticoagulant factors, protein C and S, as well as protein Z. 4 Even though Gas6 was discovered as a homolog of protein S more than a decade ago, it plays no role in the generation of fibrin and its role in vivo remains incompletely characterized. 5,6 Originally identified in fibroblasts, Gas6 is expressed in various cell types, including endothelial cells, 7 smooth muscle, 8 and bon...

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Nephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidney's glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.

To cite this article: Bertin FR, Lemari e CA, Robins RS, Blostein MD. Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells. J Thromb Haemost 2015; 13: 2260-72.Summary. Background: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE. Objectives: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression.