Transgenic overexpression of CD39 protects against renal ischemia-reperfusion and transplant vascular injury

Abstract

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia-reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild-type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation.

Figure 1. hCD39 transgenic mice are protected from renal IRI

Wild-type (WT) and hCD39 transgenic (TG) mice were subjected to 30 min of ischemia by bilateral occlusion of the renal pedicles, followed by reperfusion for 24 or 48 h. (A) Serum creatinine and (B) urea at 24 and 48 h. (C) Representative morphology of kidney sections after 24 h of reperfusion. Left column: H&E; right column: PAS. Magnification 400x. (D) Semiquantitative scoring of tubular damage. Results in A, B and D are presented as mean ± SEM. N.S: not significant.

Figure 2. hCD39 expression in kidneys 24 h after IRI

Immunohistochemistry on frozen sections showing that hCD39 expression is maintained in TG renal tissue post-IRI, particularly on the endothelium (arrows) Magnification: A&B 200×, C&D 400×.

Figure 3. Signaling through the A2A receptor mediates protection

Mice were treated with 1mg/kg CSC (A2A receptor antagonist) 15 min prior to induction of ischemia followed by 24 h of reperfusion. (A) Serum creatinine, (B) semiquantitative tubular injury score in mice pretreated (+) with CSC. (C) Representative histology showing evidence of cast deposition in Cd39 null kidney (arrow); magnification 200×. Results are presented as mean ± SEM. N.S.: not significant.

Figure 4. Apoptosis and CD4⁺ T-cell infiltrate are reduced in hCD39 kidneys after IRI

(A) Sections of renal tissue were analyzed by TUNEL-stain and the number of apoptotic nuclei per high power field (hpf) were counted. Results are presented as mean ± SEM. N.S (not significant). (B) Representative sections of TUNEL-stained renal cortex at 24 and 48 h. Magnification 400×. (C–F) Mice were subjected to 30 min of ischemia and 3 h of reperfusion. Leukocytes were extracted from the kidneys and analyzed by flow cytometry. (C) CD4⁺ T cells. (D) Neutrophils. (E) Macrophages. (F) MPO activity assay was performed following 3 h of reperfusion. Data are representative of two independent experiments; n = 4 for each group in each experiment.

Figure 5. Expression of hCD39 on either bone marrow-derived cells or tissues affords protection against renal IRI

(A) Flow-cytometric analysis of leukocytes and platelets from WT (purple-filled curve), hCD39 transgenic (green line) and hCD39 transgenic mice reconstituted with WT bone marrow (TG_WTBM) (pink line). (B) Flow-cytometric analysis of leukocytes and platelets from WT (purple-filled curve), hCD39 transgenic (green line) and WT mice reconstituted with hCD39 transgenic bone marrow (WT_TGBM) (pink line). Chimeric mice expressing hCD39 only on bone marrow-derived cells (WT_TGBM) or vasculature (TG_WTBM) were subjected to 30 min of ischemia followed by 24 h of reperfusion. (C) Serum creatinine. (D) Semiquantitative tubular injury score. Results are presented as mean ± SEM. N.S., not significant.

Figure 6. Expression of hCD39 on the donor kidney reduces cold ischemic ATN

Bilateral nephrectomized wild-type (WT) mice received a renal transplant from either a WT or hCD39 transgenic donor. Kidney grafts were subjected to 5 h of cold (storage) ischemia followed by reperfusion for 24 h. (A) Representative morphology (PAS) of kidney sections after 24 h of reperfusion. Magnification 40×. (B) Semiquantitative tubular injury score. (C) Serum creatinine. (D) Representative sections of TUNEL-stained renal cortex post transplantation. Magnification 40×. (E) Quantitation of TUNEL-stained apoptotic nuclei per high power field were counted. Results are presented as mean ± SEM.