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Sphingosine Kinase-2 inhibition suppresses renal inflammation and attenuates renal injury after obstruction in murine Unilateral Ureteral Obstruction (UUO) model
Shobha Thangada1, Mallika Ghosh2, Rehman Mohammed2, Herald Yamase3, Kevin R Lynch4,5, Andrew Bolt5, Fernando Ferrer 1
1 Department of Vascular Biology, University of Connecticut Health center (UCHC), Farmington, CT and Connecticut Children’s Medical Center (CCMC), Hartford, CT, 2Department of Vascular Biology, 3 Department of pathology, UCHC, Farmington, CT.4Department of Pharmacology, University of Virginia, VA, 5Sphynkxtherapeutics,Charlottesville, VA
Background and Aim: Sphingosine-1-Phosphate (S1P) is a bioactive lipid involved in numerous cellular survival and physiological functions. Previous studies from our lab have shown that sphingosine kinase-2 (SphK2) knock out mice demonstrate diminished renal injury when subjected to unilateral ureteral obstruction (UUO), suggesting that SphK2 may play an important role in regulating renal inflammation and fibrosis. In the present study, we utilize a novel SphK2 inhibitor to asses if pharmacological SphK2 inhibition will similarly protect against renal injury.
Methods: Male C57BL/6 mice (6-7 weeks) were divided into three different groups and pretreated for 3 days with either vehicle or with a novel SphK2 inhibitor (derivative of SLR080811 {(S)-2-[3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl] pyrrolidine-1-carboximidamide}), a guanidine-based compound. Two different doses of SpkK2 inhibitor, 3mg/kg and 10mg/kg were administered by intra-peritoneal injection. Once subjected to complete unilateral UUO, the animals continued treatment for three more days. The final drug dose was administered two hours prior to the harvest of the tissue. Obstructed and unobstructed kidneys were examined for histologic changes and expression of key renal injury markers, such as TGF β and α SMA, by immunohistochemistry and western blotting. Immune cell profile was analyzed by flowcytometry, and inflammatory and fibrotic cytokine expression was assessed by quantitative real-time polymerase chain reaction (QRT PCR) method.
Results: Histopathology, as evaluated by an experienced renal pathologist, revealed that SphK2 inhibitor treatment resulted in diminished renal injury compared to vehicle treated animals. In addition, two fibrotic markers, TGF β and α SMA, were significantly decreased in the treatment group as evidenced by immunohistochemistry and western blot analysis. Flowcytometry results indicated that infiltration of inflammatory monocytes (Gr1 high) and macrophages (F4/80+) were significantly reduced in drug treated groups at both the 3mg and the 10mg doses compared to the vehicle treated group. This observation was further supported by diminished expression of inflammatory cytokines, such as MCP-1, TNF α, CXCL2, IL β1 and fibrotic cytokine TGF β, in the drug treated group as assessed by QRT PCR method.
Conclusions: This study results demonstrates that, in a murine renal obstruction model, pharmacological inhibition of Sphingosine kinase-2 significantly decreased inflammatory and fibrotic responses resulting in diminished renal injury. This data supports our previous findings in genetic SphK2 knock out models, and suggests that this is a viable, novel target for manipulation of renal injury.