2 NOVEL PROBES REVEAL TUBULAR AND VASCULAR ARG-GLY-ASP (RGD) BINDING-SITES IN THE ISCHEMIC RAT-KIDNEY

We have previously demonstrated that RGD peptides prevent tubular obst ruction in ischemic acute renal failure (ARF) and suggested that expos ed unoccupied integrin receptors represent the target for such therapy . The present study investigated the topography of RGD binding sites a nd integrin receptors in ischemic rat kidneys. Two RGD peptides were s ynthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD pe ptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjecte d to 45 minutes of renal artery occlusion, kidneys were harvested at d ifferent times post-ischemia, and stained with RGD peptides and a pane l of antibodies to integrins. In control, Bt-RGD staining was undetect able in alkaline phosphatase histochemistry, whereas immunofluorescenc e detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP sta ining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed bin ding to the basolateral and apical aspects of proximal tubules, peritu bular capillaries, and desquamated cells within tubular lumen. The mos t conspicuous staining of ischemic kidneys was obtained with antibodie s to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and t he alpha V (glomeruli, tubular epithelia, intima of blood vessels stai ned faintly, while the obstructing cellular conglomerates showed inten se staining) subunits. Double staining with Bt-RGD and antibodies agai nst the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro at tachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding site s and beta 1 integrin subunits along the apical aspect of tubular epit helia and an the surface of desquamated cells is in concert with the h ypothesis on the pathogenetic role of RGD-recognizing integrins in tub ular obstruction. The expression of RGD binding sites along the intima l surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.