Activated Tissue-Resident CD47 Is a Proximate Promoter of Renal Ischemia Reperfusion Injury

Abstract

Background: Renal ischemia-reperfusion injury (IRI) contributes to both delayed graft function and allograft rejection in the transplanted organ. Improved understanding of mechanisms responsible for IRI is essential for the development of therapeutic strategies. Thrombospondin-1 (TSP1) is a secreted protein produced in response to cellular stress and is the soluble ligand for the ubiquitously expressed cell-surface receptor CD47. The function of the TSP1/CD47 axis in IRI remains unexplored. This prompted us to evaluate the role of CD47 activation in renal IRI using CD47-/- mice, CD47-blocking antibody (Ab) in wild-type mice, and chimeric mice deficient in leukocyte or renal parenchymal CD47. Methods: Wild type (WT) C57BL/6 and CD47-/- (B6) male mice (8-10 wks old, n=8-12 per group) were subjected to bilateral renal IRI, with pedicle clamping for 22 minutes and analysis at 24 hours reperfusion. Mice were also lethally irradiated and rescued with CD47-/- or CD47+/+ bone marrow (BM). In additional experiments, WT mice were pre-treated with CD47-blocking Ab prior to IRI. Results: IRI significantly upregulated mRNA and protein expression of both TSP1 and its receptor CD47 in WT kidneys only. This was associated with substantially more renal damage as reflected by increased tubular injury scores (72% vs 22% tubular damage, p< 0.001), renal dysfunction (serum creatinine 2.75 vs 1.46mg/dl, p< 0.001), leukocyte infiltration (140±26 vs 53±12 neutrophils per hpf, p< 0.0001) and pro-inflammatory cytokine production (including interleukin-6, interleukin-1β and tumor necrosis factor α) in WT controls compared to CD47-/- mice. Caspase-3 expression and TUNEL staining was negligible following IRI in CD47-/- mice compared to significant upregulation in WT animals. Generation of the reactive oxygen species (ROS) hydrogen peroxide (detected via Amplex Red assay) and ROS-mediated protein modifications (quantified by 3-nitroytrosine levels) were both abrogated in CD47-/- mice, as was the induction of nitric oxide synthase (iNOS) mRNA. In addition, an increase in toll-like receptor-2 (TLR2) protein and mRNA expression following IRI was evident in WT but not in CD47-/- mice; however, upregulation of TLR4 was not significantly different. Analysis of chimeric mice demonstrated that CD47 expression on tubular epithelial cells plays a crucial role in IRI, as CD47-/- mice reconstituted with WT BM were similarly protected against tubular damage and functional renal impairment. Pre-treatment with CD47-blocking Ab down-regulated renal CD47 protein expression and subsequently protected WT mice from renal dysfunction and tubular damage compared to animals treated with isotype control. Conclusion: We have identified renal-associated CD47 as an important facilitator of programmed cell death and inflammation in IRI. These data imply that CD47 is fundamental to promoting renal IRI through multiple mechanisms, and blockade provides a novel therapeutic target to prevent or modify IR-mediated damage.