The vascular ectonucleotidases CD39 [ENTPD1 (ec-tonucleoside 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.

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.

Platelet activation is believed to play an important role in the triggering of thrombosis of human blood by pig islets. We used a transgenic mouse model to investigate whether overexpression of CD39 (ecto nucleoside triphosphate diphosphohydrolase 1 [ENTPD1], EC 3.6.1.5), an ectonucleotidase that degrades the platelet agonists ATP, could interfere with this process. Islets isolated from CD39 transgenic mice showed 2.4-fold higher NTPDase activity than wild-type controls. When incubated with human blood, these islets significantly delayed clotting time compared to wild type islets (7.9 +/- 0.89 min versus 4.3 +/- 0.77 min, P = 0.007). Importantly, expression of human CD39 in the islets of transgenic mice had no deleterious effect on glucose metabolism. These results suggest that transgenic expression of human CD39 does not interfere with islet function and may be a useful strategy to inhibit thrombosis induced by intraportal administration of islet xenografts.

The relative protective effect of CD73 deletion in renal I/RI may reflect an effect of AMP accumulation. Deletion of CD39 showed deleterious effects and administration of soluble CD39 exerted renal protection, which is partially mediated by A2AR. The protective effect conferred by apyrase suggests that supplementing CD39 NTPDase activity may be a useful therapeutic strategy in renal transplantation.

Thrombomodulin (TBM) is an important vascular anticoagulant that has species specific effects. When expressed as a transgene in pigs, human (h)TBM might abrogate thrombotic manifestations of acute vascular rejection (AVR) that occur when GalT-KO and/or complement regulator transgenic pig organs are transplanted to primates. hTBM transgenic mice were generated and characterized to determine whether this approach might show benefit without the development of deleterious hemorrhagic phenotypes. hTBM mice are viable and are not subject to spontaneous hemorrhage, although they have a prolonged bleeding time. They are resistant to intravenous collagen-induced pulmonary thromboembolism, stasis-induced venous thrombosis and pulmonary embolism. Cardiac grafts from hTBM mice to rats treated with cyclosporine in a model of AVR have prolonged survival compared to controls. hTBM reduced the inflammatory reaction in the vein wall in the stasis-induced thrombosis and mouse-to-rat xenograft models and reduced HMGB1 levels in LPS-treated mice. These results indicate that transgenic expression of hTBM has anticoagulant and antiinflammatory effects that are graft-protective in murine models.

Background Adenosine agonists are protective in numerous models of ischemia-reperfusion injury (IRI). Pericellular adenosine is generated by the hydrolysis of extracellular adenosine triphosphate and adenosine diphosphate by the ectonucleotidase CD39 and the subsequent hydrolysis of adenosine monophosphate (AMP) by the ectonucleotidase CD73. CD39 activity is protective in kidney IRI, whereas the role of CD73 remains unclear. Methods Wild-type (WT), CD73-deficient (CD73KO), CD39-transgenic (CD39tg), and hybrid CD39tg.CD73KO mice underwent right nephrectomy and unilateral renal ischemia (18-min ischemia by microvascular pedicle clamp). Renal function (serum creatinine [SCr], micromolar per liter) and histologic renal injury (score 0–9) were assessed after 24-hr reperfusion. Treatments included a CD73 inhibitor and soluble CD73. Results Compared with WT mice (n=33, SCr 81.0, score 4.1), (1) CD73KO mice were protected (n=17, SCr 48.9, score 2.0, P<0.05), (2) CD39tg mice were protected (n=11, SCr 45.6, score 1.3, P<0.05), (3) WT mice treated with CD73 inhibitor were protected (n=9, SCr 43.3, score 1.2, P<0.05), (4) CD73KO mice reconstituted with soluble CD73 lost their protection (n=10, SCr 63.8, score 3.1, P=ns), (5) WT mice treated with soluble CD73 were not protected (n=7, SCr 78.0, score 4.1), and (6) CD39tg.CD73KO mice were protected (n=8, SCr 55.5, score 0.7, P<0.05). Conclusions Deficiency or inhibition of CD73 protects in kidney IRI, and CD39-mediated protection does not seem to be dependent on adenosine generation. These findings suggest that AMP may play a direct protective role in kidney IRI, which could be used in therapeutic development and organ preservation. Investigating the mechanisms by which AMP mediates protection may lead to new targets for research in kidney IRI.

A series of immunological and physiological barriers must be overcome for the successful clinical application of xenotransplantation. The acute phases of xenograft rejection have been prevented or at least attenuated by a variety of interventions including treatment of the recipient and genetic modification of the donor. However, recent data suggest that xenografts have a heightened susceptibility to intravascular thrombosis, a process that is emerging as a major contributor to xenograft loss. Current data strongly suggest that thrombosis is primarily a direct consequence of the rejection process, but it may also be facilitated by the failure of porcine regulators of coagulation to efficiently regulate the primate coagulation cascade. Systemic anticoagulant therapy has met with limited success and poses significant risks. Genetic strategies to express antithrombotic agents on xenograft endothelium appear to be more promising and achievable, with candidate molecules including human and leech anticoagulants and the antiplatelet enzyme CD39. Deletion of porcine procoagulants may also prove to be a useful approach.

BackgroundPhosphate binders’ constituents have alkalotic or acidotic properties and may contribute to acid base balance in haemodialysis patients. This study aimed to investigate the differential effects of phosphate binders on pre-dialysis serum bicarbonate in End Stage Kidney Disease patients on maintenance haemodialysis.MethodsStable out-patients having satellite haemodialysis for at least 3 months were retrospectively studied for 18 months, excluding those with other medical causes for metabolic acidosis. Blood results were censored for inpatient episodes, at the time of death, renal transplant or dialysis modality change. Multivariable multilevel mixed-effects linear regression was used and five groups of phosphate binders were compared: Group A(Calcium (Ca) and/or Aluminium (Al) binders); B(Sevelamer hydrochloride (SH) alone); C(lanthanum carbonate (LC) alone); D(SH and Ca/Al), E(LC and Ca/Al).ResultsOf 320 patients, 292 were eligible for analysis with a mean follow-up of 15.54 (standard deviation, SD 3.98) months. Similar mean pre-dialysis serum levels of bicarbonate were observed at all 6 month-interval analyses. At 18th months, observed mean serum bicarbonate levels in mmol/L were Group B: 21.58 (SD 2.82, P<0.001), C: 23.29 (SD 2.80, P=0.02), D: 21.56 (SD 3.00, P<0.001), and E: 21.29 (SD 3.62, P=0.92) compared with Group A: 22.98 (SD 2.77). Mean serum bicarbonate was related to total SH dose in mmol/L: 22.34 (SD 2.56) for SH <2.5 g/day, 21.61 (SD 2.62) for SH 2.5-4.8 g/day, 21.04 (SD 3.31) for SH >4.8 g/day compared with 22.85 (SD 2.91) for non-users; P-trend<0.001.ConclusionsPhosphate binders’ constituents may contribute to/protect against a predisposition to pre-dialysis metabolic acidosis. This may be dose dependant in patients taking Sevelamer Hydrochloride.