CD47 blockade reduces ischemia/reperfusion injury in donation after cardiac death rat kidney transplantation

Abstract

Modulation of nitric oxide activity through blockade of CD47 signaling has been shown to reduce ischemia-reperfusion injury (IRI) in various models of tissue ischemia. Here, we evaluate the potential effect of an antibody-mediated CD47 blockade in a syngeneic and an allogeneic DCD rat kidney transplant model. The donor organ was subjected to 1 hour of warm ischemia time after circulatory cessation, then flushed with a CD47 monoclonal antibody (CD47mAb) in the treatment group, or an isotype-matched immunoglobulin in the control group. We found that CD47mAb treatment improved survival rates in both models. Serum markers of renal injury were significantly decreased in the CD47mAb-treated group compared with the control group. Histologically the CD47mAb-treated group had significantly reduced scores of acute tubular injury and acute tubular necrosis. The expression of biomarkers related to mitochondrial stress and apoptosis also were significantly lower in the CD47mAb-treated groups. Overall, the protective effects of CD47 blockade were greater in the syngeneic model. Our data show that CD47mAb blockade decreased the IRI of DCD kidneys in rat transplant models. This therapy has the potential to improve DCD kidney transplant outcomes in the human setting.

Keywords: basic (laboratory) research/science; donation after circulatory death (DCD); donors and donation; ischemia reperfusion injury (IRI); kidney transplantation/nephrology.

Figure 1. Treatment of the kidney graft with CD47mAb improves posttransplant survival rate in two rat DCD renal transplant models

(A) Syngeneic kidney transplant model, Kaplan-Meir survival analysis of the study group showed an increase in CD47mAb group survival. (B) Allogeneic renal transplant model, also showed an increase in CD47mAb group survival.

Figure 2. Serum markers of kidney function were reduced by treatment of donor kidneys with CD47mAb

Serum samples were obtained at 48 hours after renal transplantation from recipients that received IgG-treated kidneys or CD47mAb-treated kidneys. (A) Creatinine, (B) BUN, (C) potassium, and (D) phosphorus were measured using an autoanalyzer. Two days after transplantation, serum creatinine, BUN, potassium and phosphorus were significantly lower in rats that received the CD47mAb-treated kidneys than the IgG-treated control kidneys.

Figure 3. CD47mAb-treated donor kidneys exhibit histological evidence of IRI protection

(A) In the syngenic model and the allogeneic model, CD47mAb-treated donor kidneys showed less histological injury when examined 48 hours after transplantation (magnification ×200) than kidneys receiving control IgG antibody. (B) Histological sections were scored in a blinded manner for ATI and ATN. The CD47mAb-treated groups had significantly decreased scores of ATI and ATN in both models.

Figure 4. CD47mAb treatment decreased the inflammatory response of the DCD renal grafts

(A) CD3⁺ T lymphocytes were visualized by immunofluorescence staining in renal tissue at 48 hours after transplantation (magnification ×400). (B) The number of infiltrating CD3⁺ cells were quantified and found to be significantly decreased in the CD47mAb treated renal grafts versus the control group in both the syngeneic and allogeneic models.

Figure 5. CD47mAb treatment reduced renal tubular cell stress after kidney transplantation

(A) IF histology staining of K18 (magnification x400), colocalized with AQP2 as a marker of the collecting ducts, indicated that CD47mAb treatment reduced expression of K18 in both syngeneic and allogeneic models. (B) Western blot and densitometry analysis found that K18 levels were markedly lower in the CD47mAb treated group in the syngeneic model, while in the allogeneic model, there was no significant difference.

Figure 6. Syngeneic rat DCD renal transplant model, CD47mAb treatment reduced oxidative stress

(A) IF staining of PARP and VDAC1 (magnification ×200) showed lower in the CD47mAb-treated group. (B) Western blot and densitometry analysis showed that expression of activated PARP and VDAC1 were markedly lower in CD47mAb treated group compared to IgG control group.

Figure 7. Allogeneic rat DCD renal transplant model, CD47mAb treatment reduced mitochondrial oxidative stress

(A) IF staining of PARP and VDAC1 (magnification ×200) showed lower in the CD47mAb-treated group. (B) Western blot and densitometry analysis showed that expression of activated PARP was significantly lower in CD47mAb treated group compared to IgG control group, however, there was no significant difference in VDAC1 expression.

Figure 8. Syngeneic rat DCD renal transplant model, CD47mAb treatment reduced cell apoptosis

(A) IF staining of caspase-3 (magnification ×200) showed lower in the CD47mAb-treated group. (B) Western blot and densitometry analysis showed that expression of activated caspase-3, caspase-8, and caspase-9 were significantly lower in CD47mAb treated group compared to IgG control group.

Figure 9. Allogeneic rat DCD renal transplant model, CD47mAb treatment reduced cell apoptosis

(A) IF staining of caspase-3 (magnification ×200) was reduced in the CD47mAb-treated group compared to control. (B) Western blot and densitometry analysis showed that expression of activated caspase-3, caspase-8, and caspase-9 were significantly reduced in CD47mAb treated group compared to IgG control group.