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Modulation of vascular permeability by Sphingosine kinase 2 in a mouse model of renal injury
Flavia E. Pereira, PhD1, Shobha D. Thangada, PhD1, Kevin R. Lynch, PhD2, Andrew Bolt, PhD3, Timothy Hla, PhD4, Linda H. Shapiro, PhD1, Fernando Ferrer, MD5.
1University of Connecticut Health Center, Farmington, CT, USA, 2University of Virginia, Charlottesville, VA, USA, 3SphynKx Therapeutics, Charlottesville, VA, USA, 4Weill Cornell Medical College, New York, NY, USA, 5Connecticut Childrens Medical Center, Hartford, CT, USA.

Introduction: Sphingosine kinases(SK) 1 and 2 are the two key enzymes responsible for catalyzing the formation of Sphingosine-1-phosphate(S1P), a potent signaling lipid involved in multiple cellular processes. The two SKs appear to have distinct physiologial roles. Adherens junctions(AJs) in endothelial cells lining the blood vessels in different vascular beds including renal microvasculature play an important role in the regulation of endothelial barrier integrity. AJs are composed of vascular endothelial(VE)-Cadherin and catenins(α,β,γ and p120) that are subject to modulation by tyrosine and serine/threonine kinases and phosphatases. In particular, tyrosine phosphorylation of AJ proteins has been demonstrated to disrupt the endothelial barrier and increase vascular permeability in-vitro. Previous data from our laboratory suggests that SK2 KO and SK2 inhibitor treated mice have reduced inflammation and are protected from renal injury. A potential mechanism of renoprotection might be blocking vascular permeability. Also, the function of SK2 in the regulation of AJs remains unknown. Therefore, our aim was to determine the role of SK2 in regulation of renal vascular leakage after injury. In renal pathological conditions, damage of renal microvasculature as evidenced by disruption/loss of VE-cadherin staining has been shown to result in tissue edema and inflammation contributing to deleterious long-term changes such as fibrosis.
Material and Methods: Using an SK2 specific inhibitor, we evaluated the role of SK2 in the regulation of vascular permeability using unilateral ureteral obstruction(UUO) as an in-vivo model of renal injury. C57Bl6 mice were given a daily dose of vehicle or 3mg/kg SK2 inhibitor (SK2I) intraperitoneally for 7 days. On day 4, animals were subjected to UUO. Permeability assays were conducted 3 days after UUO using FITC dextran. For the adoptive transfer study, PKH dye labeled cells were injected IV on the day of UUO, and cell numbers in the kidneys were analyzed 3 days after UUO by fluorescence microscopy. VE-cadherin localization was analyzed by fluorescence microscopy using human umbilical vein endothelial cells(HUVEC). Tyrosine phosphorylation and pSrc association with VE-cadherin was analyzed by immunoprecipitation followed by western blotting.
Results: Mice treated with SK2I showed significantly reduced permeability as assessed by FITC dextran leakage in the obstructed kidney. Reduced numbers of adoptively transferred labeled bone marrow cells were observed in the obstructed kidney of mice treated with SK2I versus vehicle, implying reduced extravasation of potential inflammatory cells into the injured kidney. The in-vivo permeability results were recapitulated in-vitro where thrombin mediated HUVEC monolayer permeability was reduced with SK2I treatment. Furthermore, in-vitro immunofluorescence analysis of VE-cadherin localization suggested that thrombin treatment of HUVECs resulted in loss of VE-cadherin staining at the cell junctions that was restored upon SK2I treatment, thus maintaining the endothelial barrier integrity. Mechanistically, SK2 inhibition reduced the level of pSrc associated with VE-cadherin, thereby reducing the tyrosine phosphorylation of VE-cadherin and β-catenin induced by thrombin.
Conclusion: SK2 inhibition results in significantly reduced vascular permeability in the kidney after injury largely due to its effects on adherens junctions. Therefore, in renal pathological processes, the anti-inflammatory aspects of SK2 inhibition could be exploited to reduce renal injury.

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