Two novel probes reveal tubular and vascular Arg-Gly-Asp (RGD) binding sites in the ischemic rat kidney

Abstract

We have previously demonstrated that RGD peptides prevent tubular obstruction in ischemic acute renal failure (ARF) 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 on 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.

Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

“…Although available data are conflicting about the precise changes that occur in vivo, 17,18 redistribution of tubule cell ␤1 integrins from their normal sites at the basal surface during ischemic acute renal failure and related forms of acute tubule injury has been implicated in loss of cell adhesion and dysfunctional re-aggregation of detached tubule cells. 14 -19,60,61 In the present study, immunostaining for ␤1 integrin was strikingly decreased during hypoxia.…”

Anti-Inflammatory Effects of αv Integrin Antagonism in Acute Kidney Allograft Rejection

“…Increases in ␣ v ␤ 3 expression in acute cellular rejection of heart allografts and coronary graft vasculopathy in humans and in ischemic acute renal failure in rat have been observed. [25][26][27] In ischemic rat kidneys ␤ 1 and ␣ v ␤ 3 were found to be localized in proximal and distal tubules and endothelium. 27 Also different forms of glomerulonephritis were found to be associated with modifications in their ␣ v integrin expression: the distal tubular expression of ␤ 3 was increased, and a de novo appearance of ␣ v ␤ 3 and ␣ v ␤ 5 on proximal tubular cells in comparison to normal kidneys was noticed 24,43 ; ␣ v ␤ 6 , which is exclusively expressed by epithelial cells, was elevated in the kidney epithelium of patients with membranous glomerulonephritis, IgA nephropathy, and in acute and chronic rejection of human kidney allografts.…”

“…[25][26][27] In ischemic rat kidneys ␤ 1 and ␣ v ␤ 3 were found to be localized in proximal and distal tubules and endothelium. 27 Also different forms of glomerulonephritis were found to be associated with modifications in their ␣ v integrin expression: the distal tubular expression of ␤ 3 was increased, and a de novo appearance of ␣ v ␤ 3 and ␣ v ␤ 5 on proximal tubular cells in comparison to normal kidneys was noticed 24,43 ; ␣ v ␤ 6 , which is exclusively expressed by epithelial cells, was elevated in the kidney epithelium of patients with membranous glomerulonephritis, IgA nephropathy, and in acute and chronic rejection of human kidney allografts. 44, 45 By immunostaining we could show the increased expression of ␣ v ␤ 3 in rejecting rat kidney allografts, in peritubular capillaries, on the endothelial surface of preglomerular arteries and in tubular epithelial cells in comparison to normal F344 kidneys as well as its presence in the intragraft perivascular cellular infiltrate.…”

“…24 Increased expression of ␣ v ␤ 3 has been found in acute cellular rejection of human heart transplants and transplant coronary vasculopathy as well as in ischemia-reperfusion injury of rat kidneys; its function was not investigated in these settings. [25][26][27] We hypothesized that the antagonism of ␣ v integrins could inhibit cell recruitment across activated microcirculatory endothelia, thereby limiting rejection phenomena in kidney allografts. The effects of S247, a peptidomimetic RGD-based ␣ v integrin antagonist, were investigated in two models of allotransplantation with different major histocompatibility complex disparity; expression of ␣ v ␤ 3 in kidney allografts was determined; the effects on leukocyte-endothelial interactions (LEIs) were studied in vivo by intravital microscopy and in vitro by a laminar flow system.…”

A multi-domain protein for β1 integrin-targeted DNA delivery

“…Apical expression of ␤ 1 integrins has been observed on epithelial cells during inflammation, wound healing and ischaemia. [15][16][17] Endothelial cells also express apical ␤ 1 integrins, which are involved in interactions with leukocytes when activated during the process of inflammation. 18 In conclusion, ␤ 1 integrins are available to external ligands, especially during clinically relevant conditions.…”