Functional characterization of the SLIT2-ROBO2 signaling pathway in the podocyte
Pisarek- Horowitz, Anna Justyna
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Receptor ROBO2 and its ligand SLIT2 are required for ureteric bud outgrowth during early kidney development. However, it is unclear if ROBO2 also plays a role during the late stage of kidney development and in the mature kidney. We performed immunohistochemistry and co-labelling assays and found that ROBO2 is a podocyte protein expressed on the basal cell surface of podocyte foot processes and co-localizes with slit-diaphragm protein nephrin/NPHS1. Biochemical analyses revealed that ROBO2 directly interacts with NCK, the adaptor protein that also binds to nephrin. We found that ROBO2, NCK, and nephrin form a complex that is enhanced by SLIT2 stimulation. Functionally nephrin signaling promotes actin polymerization but SLIT2-ROBO2 signaling inhibits nephrin-induced actin polymerization. Mouse genetic studies showed that knocking out Robo2 specifically in podocytes (Robo2 cKO) does not cause significant kidney defects but alleviates the abnormal podocyte phenotype in Nphs1 null mice. These results suggest that SLIT2-ROBO2 signaling pathway acts as a negative regulator of nephrin signaling to influence podocyte foot process architecture. We hypothesized that SLIT2 and ROBO2 play a role during podocyte injury in the adult kidney. To test this hypothesis, we studied two acute glomerular injury mouse models, protamine sulfate (PS) perfusion and nephrotoxic serum (NTS) injection, and found that loss of Robo2 in podocytes protects mice from acute glomerular injury. Robo2 cKO mice develop less podocyte structural defects and functional kidney impairment as compared with their littermate controls. In addition, the level of nephrin is upregulated in Robo2 cKO mice before and after injury. Interestingly we found that SLIT2 and ROBO2 are upregulated in the human membranous nephropathy kidneys. These results suggest deleterious effects for SLIT2-ROBO2 in acute glomerular injury in mice and chronic kidney disease in human. In conclusion, my thesis research revealed SLIT2-ROBO2 pathway as a novel signaling component in podocytes, defined its cellular and molecular mechanism, and provided new knowledge for its function under normal and pathological conditions.