Slit2 Prevents Neutrophil Recruitment and Renal Ischemia-Reperfusion Injury

Abstract

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

The Neurorepellent Slit2 Inhibits Postadhesion Stabilization of Monocytes Tethered to Vascular Endothelial Cells

“…Full-length Slit2 and bioactive truncated N-Slit2 were purified as previously described (17,21,23). N-Slit2 was also purchased from PeproTech.…”

“…Primary HUVEC and HAEC were grown to confluence and incubated with TNF-a (20 ng/ml) for 4 h. THP-1 cells were labeled with Calcein, AM, and adhesion assays were performed as described (23). Nonadherent monocytic cells were removed by centrifuging the plates (100 3 g, 1 min) upside down (23).…”

“…Nonadherent monocytic cells were removed by centrifuging the plates (100 3 g, 1 min) upside down (23). A fluorescent plate reader was used to measure the fluorescence intensity of each well.…”

“…Monocyte-accumulation and -detachment assays were performed as previously described (11,23). Leukocytes were incubated with Slit2 (30 nM), latrunculin B (LatB; 2 mM), or control vehicle.…”

“…Monocytic cells (5.0 3 10 6 cells/ml) were infused in microfluidic channels at a wall shear stress of 2 dyn/cm 2 . The number of leukocytes that interacted with the monolayer or that remained stationary for .2 s was determined in real time using images from 303 fields (23). Detachment assays.…”