Two 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 obstruction in ischemic acute renal failure (ARF) and suggested that exposed unoccupied integrin receptors represent the target for such therapy. The present study investigated the topography of RGD binding sites and integrin receptors in ischemic rat kidneys. Two RGD peptides were synthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD peptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjected to 45 minutes of renal artery occlusion, kidneys were harvested at different times post-ischemia, and stained with RGD peptides and a panel of antibodies to integrins. In control, Bt-RGD staining was undetectable in alkaline phosphatase histochemistry, whereas immunofluorescence detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP staining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed binding to the basolateral and apical aspects of proximal tubules, peritubular capillaries, and desquamated cells within tubular lumen. The most conspicuous staining of ischemic kidneys was obtained with antibodies to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and the alpha V (glomeruli, tubular epithelia, intima of blood vessels stained faintly, while the obstructing cellular conglomerates showed intense staining) subunits. Double staining with Bt-RGD and antibodies against the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro attachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding sites and beta 1 integrin subunits along the apical aspect of tubular epithelia and an the surface of desquamated cells is in concert with the hypothesis on the pathogenetic role of RGD-recognizing integrins in tubular obstruction. The expression of RGD binding sites along the intimal surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.