Ischemia/reperfusion injury in human kidney transplantation: an immunohistochemical analysis of changes after reperfusion

Abstract

Organs used for transplantation undergo varying degrees of cold ischemia and reperfusion injury after transplantation. In renal transplantation, prolonged cold ischemia is strongly associated with delayed graft function, an event that contributes to inferior graft survival. At present, the pathophysiological changes associated with ischemia/reperfusion injury in clinical renal transplantation are poorly understood. We have performed an immunohistochemical analysis of pre- and postreperfusion biopsies obtained from cadaver (n = 55) and living/related donor (LRD) (n = 11) renal allografts using antibodies to adhesion molecules and leukocyte markers to investigate the intragraft changes after cold preservation and reperfusion. Neutrophil infiltration and P-selectin expression were detected after reperfusion in 29 of 55 (53%) and 24 of 55 (44%) cadaver renal allografts, respectively. In marked contrast, neutrophil infiltration was not observed in LRD allografts, and only 1 of 11 (9%) had an increased level of P-selectin after reperfusion. Immunofluorescent double-staining demonstrated that P-selectin expression resulted from platelet deposition and not from endothelial activation. No statistically significant association was observed between neutrophil infiltration and P-selectin expression in the glomeruli or intertubular capillaries despite the large number of cadaver renal allografts with postreperfusion changes. Neutrophil infiltration into the glomeruli was significantly associated with long cold ischemia times and delayed graft function. Elevated serum creatinine levels at 3 and 6 months after transplantation were also associated with the presence of neutrophils and platelets after reperfusion. Our results suggest that graft function may be influenced by early inflammatory events after reperfusion, which can be targeted for future therapeutic intervention.

Figure 1.

Percentage of cadaver (n = 55) and LRD (n = 11) renal allografts with an increase in neutrophil infiltration and P-selectin expression after reperfusion.

Figure 2.

Immunohistological changes observed after reperfusion in cadaver renal allografts. a: Indirect immunoperoxidase staining of a prereperfusion biopsy with an antineutrophil elastase mAb demonstrating the absence of neutrophils. b: The subsequent postreperfusion biopsy stained with the same antibody showing neutrophils infiltrating into the glomeruli (a and b, magnification, ×200). c: Postreperfusion biopsy stained by the indirect immunoperoxidase method using an anti-P-selectin antibody, showing a positive signal on the intertubular capillaries. d: A consecutive section from the same postreperfusion biopsy stained with the anti-CD41 platelet-specific antibody showing a pattern of staining similar to that detected for P-selectin (c and d, magnification, ×100).

Figure 3.

Double-immunofluorescent staining to identify the origin of the P-selectin expression in postreperfusion biopsies. a to c: Postreperfusion biopsy double-stained with a directly conjugated anti-CD41_FITC mAb and an anti-ICAM-1 antibody detected with a Texas Red-conjugated secondary antibody. a: ICAM-1 staining (red) of glomerular and intertubular capillaries on the kidney. b: CD41+ platelets (green) detected after reperfusion. c: CD41-positive staining of platelets overlaid on ICAM-1+ endothelial structures, suggesting that platelet deposition on the microvascular endothelium is detectable after reperfusion. (a to c, magnification, ×200.) d to f: Double-immunofluorescent staining of a postreperfusion biopsy with an anti-CD41_FITC mAb and an anti-P-selectin mAb developed with a Texas Red-conjugated secondary antibody. d: P-selectin expression (red) detected in glomeruli after reperfusion. e: CD41+ platelets (green) detected on identical structures in the glomerulus. f: Double-positive (yellow) structures within the glomerulus for CD41- and P-selectin-positive staining indicating that P-selectin expression occurs on CD41+ platelets. (d to f, magnification, ×200.)

Figure 4.

Detection of recipient-derived cells in cadaver renal allografts after reperfusion. Indirect immunoperoxidase staining of prereperfusion biopsy from an HLA-A2/B17 negative donor kidney showing the absence of positive staining with MA2.1, an anti-HLA-A2/B17 antibody (a), and postreperfusion biopsy stained with the same antibody showing the presence of recipient-derived HLA-A2/B17-positive cells within the graft after reperfusion (b). Magnification, ×200.