Jennifer L. McRae

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease)

Introduction: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. Subjects: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. Results: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. Conclusion: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4+CD25+CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4+CD25+CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5–7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5–7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.

Liver grafts from CD39-overexpressing rodents are protected from ischemia reperfusion injury due to reduced numbers of resident CD4+ T cells.

Ischemia‐reperfusion injury (IRI) is a major limiting event for successful liver transplantation, and CD4+ T cells and invariant natural killer T (iNKT) cells have been implicated in promoting IRI. We hypothesized that hepatic overexpression of CD39, an ectonucleotidase with antiinflammatory functions, will protect liver grafts after prolonged cold ischemia. CD39‐transgenic (CD39tg) and wildtype (WT) mouse livers were transplanted into WT recipients after 18 hours cold storage and pathological analysis was performed 6 hours after transplantation. Serum levels of alanine aminotransferase and interleukin (IL)‐6 were significantly reduced in recipients of CD39tg livers compared to recipients of WT livers. Furthermore, less severe histopathological injury was demonstrated in the CD39tg grafts. Immune analysis revealed that CD4+ T cells and iNKT cells were significantly decreased in number in the livers of untreated CD39tg mice. This was associated with a peripheral CD4+ T cell lymphopenia due to defective thymocyte maturation. To assess the relative importance of liver‐resident CD4+ T cells and iNKT cells in mediating liver injury following extended cold preservation and transplantation, WT mice depleted of CD4+ T cells or mice genetically deficient in iNKT cells were used as donors. The absence of CD4+ T cells, but not iNKT cells, protected liver grafts from early IRI. Conclusion: Hepatic CD4+ T cells, but not iNKT cells, play a critical role in early IRI following extended cold preservation in a liver transplant model. (HEPATOLOGY 2013)

Regulatory T cells participate in CD39‐mediated protection from renal injury

CD39 is an ecto‐enzyme that degrades extracellular nucleotides, such as ATP, and is highly expressed on by the vasculature and circulating cells including Foxp3+ regulatory T (Treg) cells. To study the role of purinergic regulation in renal disease, we used the adriamycin nephropathy (AN) mouse model of chronic renal injury, using human CD39‐transgenic (hCD39Tg) and wild‐type (WT) BALB/c mice. Effects of CD39 expression by Treg cells were assessed in AN by adoptive transfer of CD4+CD25+ and CD4+CD25− T cells isolated from hCD39Tg and WT mice. hCD39Tg mice were protected from renal injury in AN with decreased urinary protein and serum creatinine, and significantly less renal injury compared with WT mice. While WT CD25+ and hCD39Tg CD25− T cells conferred some protection against AN, hCD39Tg CD25+ Treg cells offered greater protection. In vitro studies showed direct pro‐apoptotic effects of ATP on renal tubular cells. In conclusion, hCD39 expressed by circulating leukocytes and intrinsic renal cells limits innate AN injury. Specifically, CD39 expression by Treg cells contributes to its protective role in renal injury. These findings suggest that extracellular nucleotides mediate AN kidney injury and that CD39, expressed by Treg cells and other cells, is protective in this model.

The Protective Effects of CD39 Overexpression in Multiple Low-Dose Streptozotocin–Induced Diabetes in Mice

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell–mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell–mediated injury.

AMPK couples plasma renin to cellular metabolism by phosphorylation of ACC1

Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the β1-subunit of AMPK (AMPK-β1(-/-) mice) had significantly increased urinary Na(+) excretion on a normal salt diet. This was associated with reduced expression of the β-subunit of the epithelial Na(+) channel (ENaC) and increased subapical tubular expression of kidney-specific Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-β1(-/-) mice fed a salt-deficient diet were able to conserve Na(+), but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)(S79A) mice] were examined. ACC1-KI(S79A) mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na(+)-Cl(-) cotransporter, and ENaC-β were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KI(S79A) mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1.

Location and structure of the human FHR-5 gene

The factor H family of genes has been localised to human chromosome 1q32. This region encodes various proteins involved in complement regulation and is known as the regulators of complement activation (RCA) gene cluster. The factor H genes encode seven known plasma proteins. Using fluorescence in situ hybridisation (FISH), radiation hybrid (RH) mapping and BLAST alignment analysis, we have established that the factor H-related 5 (FHR-5) gene is closely linked to the other factor H gene family members. Analysis of the genomic sequence indicates that the FHR-5 gene is situated between FHR-2 and the non-complement protein factor XIIIb (F13B). Like all members of the factor H family, transcription of FHR-5 is in the telomeric direction. Furthermore, the short consensus repeats (SCRs) of FHR-5 are encoded by individual exons and splicing is of type 1. These data allow the generation of a more complete map of the factor H gene family.

Overexpression of CD39 protects in a mouse model of preeclampsia

CD39 (NTPDase1), a critical immune and vascular ecto‐nucleotidase, hydrolyses pro‐inflammatory and pro‐thrombotic nucleotides (adenosine‐5′‐triphosphate (ATP) and adenosine diphosphate) to adenosine. In humans, CD39 is the dominant ecto‐nucleotidase in placental trophoblastic tissues and modulates ATP‐dependent trophoblastic functions. CD39 is an integral component of regulatory T cells (Treg), which are central to immunological tolerance and maintenance of normal pregnancy. We examined the impact of CD39 overexpression in a mouse model of preeclampsia. Matings were performed between virginal BALB/c female (wild‐type (WT) or CD39 transgenic (CD39TG)) and C57BL/6 male mice. On days 10 and 12 of pregnancy BALB/c Th1‐polarized cells were injected. Systolic blood pressure (SBP) was measured throughout pregnancy. Mice were sacrificed at day 15 of pregnancy. Following transfer of Th1‐polarized cells, SBP of pregnant WT mice increased (118 ± 3 mmHg to 142 ± 5 mmHg). Although ultrastructural changes were evident in the kidney this was not accompanied by significant proteinuria. SBP remained unchanged (115 ± 2 mmHg to 114 ± 3 mmHg) in pregnant CD39TG mice without evidence of renal lesions. We conclude that gestational hypertension can be induced in mice following transfer of maternally derived Th1‐polarized cells and that overexpression of CD39 is protective in this model.

The CD39-adenosinergic axis in the pathogenesis of immune and nonimmune diabetes.

Diabetes mellitus encompasses two distinct disease processes: autoimmune Type 1 (T1D) and nonimmune Type 2 (T2D) diabetes. Despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. In this paper, we discuss the role of the CD39-adenosinergic axis in the pathogenesis of both T1D and T2D, with particular emphasis on the role of CD39 and CD73.