Slit-Robo Signaling: A Novel Approach to Modulate Vascular Injury in Diabetes

Background: Diabetes is a chronic disease that can lead to life-threatening complications, such as diabetic nephropathy (DN), and is the leading cause of kidney failure in the United States. One of the first manifestations of diabetic kidney disease is the development of hyperfiltration, driven in part by glomerular angiogenesis, an early VEGF-driven process. The severity of early hyperfiltration is directly correlated to poor renal outcomes. Slit2, which has recently emerged as a regulator of endothelial function, can either elicit pro- or anti-angiogenic effects in endothelial cells through its Robo1 and Robo4 receptors respectively. The objective of this study was to determine whether Slit2-Robo signaling regulates high glucose (HG)-induced glomerular endothelial cell (GEC) responses in vitro and in vivo. Methods: In normal or HG conditions, Slit2, Robo1, and Robo4 expression in GECs was examined in vitro and in vivo using two models of diabetic nephropathy (db/db mice, streptozotocin (STZ)-induced diabetic Wistar rats). Using tube formation and migration assays, GEC responsiveness to VEGF was examined under both normal and HG conditions, and following manipulation of Robo1 and Robo4 levels. STZ-induced diabetic mice were then randomized to receive either exogenous Slit2 (2 μg thrice weekly via intraperitoneal injections for 3 weeks) or phosphate-buffered saline to observe the effects on glomerular angiogenesis and hyperfiltration. The effects of Robo4 deficiency (Robo4 knockout mice) and Slit2 administration (2 μg thrice weekly via i.p. injections for 3 weeks) on diabetes-induced glomerular angiogenesis and hyperfiltration were analyzed using STZ-induced diabetic rodent models. Results: Healthy GECs expressed high Robo4 and low Robo1 levels. Under HG conditions, Robo4 levels fell while Robo1 levels remained unchanged, reducing the Robo4:Robo1 ratio. Slit2 protein levels were also reduced in rat glomeruli after 3 weeks of STZ-induced diabetes upon HG treatment. These changes in Slit2 and Robo4 expression were accompanied by glomerular capillary growth and hyperfiltration, which were attenuated by exogenous Slit2 administration. Four weeks post-STZ-induced diabetes, Robo4 knockout mouse kidneys showed increased glomerular endothelial density as compared to wild type controls. Conclusions: We found that reduced anti-angiogenic Slit2-Robo4 signaling promotes early diabetic glomerular angiogenesis and hyperfiltration, and that this change can be reversed with Slit2 therapy. Our results suggest that Slit2 may have therapeutic importance as a novel therapy for early diabetic nephropathy.