apyrase and Fibrosis

Renal fibrosis, the key histopathological lesion in the development and progression of chronic kidney disease (CKD), has been the focus of much research in recent decades. The growing burden of CKD in both developed and developing nations highlights a need for novel therapies to halt the progression of renal disease. Insights into the pathogenesis of renal fibrosis and the key cellular and molecular mediators have been critical in the process of identifying potential targets of therapy. Adenosine signaling is an innate biological autocrine and paracrine cellular signaling pathway involving several key mediators: ectonucleotidases, adenosine, and adenosine receptors. Short-term activation of the adenosine A2A and A2B receptors decreases inflammation, which precedes renal fibrosis. However, in conditions of persistent, excessive adenosine exposure, such as in patients born with adenosine deaminase (ADA) deficiency, adenosine signaling via A2B receptor promotes renal fibrosis, as seen in chronic inflammation. This review will describe the increasingly recognized complex role of adenosine signaling in the development of renal fibrosis. We will speculate how the knowledge gained may be employed in the search for more effective therapies based on these complex signaling pathways.

Topics: Adenosine; Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Autocrine Communication; Fibroblasts; Fibrosis; Humans; Kidney; Macrophages; Paracrine Communication; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Renal Insufficiency, Chronic; Signal Transduction

Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy.. Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions.. Tonabersat partially negated glucose/TGFβ1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression.. Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFβ1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.

Topics: Adenosine Triphosphate; Apyrase; Communication; Connexin 43; Diabetes Mellitus; Diabetic Nephropathies; Epithelial Cells; Fibroblasts; Fibrosis; Glucose; Humans

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Topics: Adenosine Triphosphate; Animals; Apyrase; CRISPR-Cas Systems; Disease Models, Animal; Dysbiosis; Female; Fibrosis; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Inflammatory Bowel Diseases; Male; Mice; Mice, Inbred C57BL; Probiotics; Receptors, Purinergic P2Y2; Saccharomyces cerevisiae

Renal ischemia-reperfusion injury (IRI) leads to acute kidney injury and renal fibrosis. CD39 is a key purinergic enzyme in the hydrolysis of adenosine triphosphate (ATP) and increased CD39 enzymatic activity protects from acute IRI but its effect on renal fibrosis is not known.. Using a mouse model of unilateral renal IRI, the effects of increased CD39 activity (using soluble apyrase and mice expressing human CD39 transgene) on acute and chronic renal outcomes were examined. Nucleotide (ATP, adenosine diphosphate, adenosine monophosphate) and nucleoside (adenosine and inosine) levels were quantified by high-performance liquid chromatography. Soluble apyrase reduced acute renal injury at 24 hours and renal fibrosis at 4 weeks post-IRI, compared with vehicle-treated mice.. Soluble apyrase reduced renal ATP, adenosine diphosphate, and adenosine monophosphate, but not adenosine levels, during ischemia. In comparison with wild-type littermates, hCD39 transgenic mice were protected from acute renal injury at 24 hours, but had increased renal fibrosis at 4 weeks post-IRI. hCD39 transgene expression was localized to the vascular endothelium at baseline and did not affect total renal nucleotide and nucleoside levels during ischemia. However, hCD39 transgene was more widespread at 4 weeks post-IRI and was associated with higher renal adenosine levels at 4 weeks post-IRI compared with wild-type littermates.. A single dose of apyrase administration before IRI protects from both acute and chronic renal injuries and may have clinical application in protection from ischemic-induced renal injury. Furthermore, transgenic expression of hCD39 is associated with increased renal fibrosis after ischemia.

Topics: Acute Kidney Injury; Adenine Nucleotides; Animals; Antigens, CD; Apyrase; Chronic Disease; Disease Models, Animal; Enzyme Induction; Enzyme Inhibitors; Fibrosis; Genetic Predisposition to Disease; Humans; Hydrolysis; Kidney; Male; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Receptors, Adrenergic, beta-2; Reperfusion Injury; RNA, Messenger; Time Factors

Chronic kidney disease has multiple etiologies, but its single, hallmark lesion is renal fibrosis. CD39 is a key purinergic enzyme in the hydrolysis of ATP and increased CD39 activity on regulatory T cells (Treg) is protective in adriamycin-induced renal fibrosis. We examined the effect of overexpression of human CD39 on the development of renal fibrosis in the unilateral ureteric obstructive (UUO) model, a model widely used to study the molecular and cellular factors involved in renal fibrosis. Mice overexpressing human CD39 (CD39Tg) and their wild-type (WT) littermates were subjected to UUO; renal histology and messenger RNA (mRNA) levels of adenosine receptors and markers of renal fibrosis were examined up to 14 days after UUO. There were no differences between CD39Tg mice and WT mice in the development of renal fibrosis at days 3, 7, and 14 of UUO. Relative mRNA expression of the adenosine A

Topics: Animals; Antigens, CD; Apyrase; Disease Models, Animal; Fibrosis; Kidney; Mice; Mice, Transgenic; Renal Insufficiency, Chronic

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39(+) regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.

Topics: Angiotensin II; Animals; Antigens, CD; Apoptosis; Apyrase; Cells, Cultured; Fibrosis; Hypertension; Immune Tolerance; Kidney; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Neutrophils; Proto-Oncogene Proteins c-vav; Renal Insufficiency; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs.

Topics: Adenosine; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Collagen; Fibroblasts; Fibrosis; Gene Knockdown Techniques; Homeostasis; Hydrolysis; Male; Myocardium; Phenotype; Phosphorylation; Protein Isoforms; Rats; Rats, Sprague-Dawley; Signal Transduction

Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-β1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.

Topics: 5'-Nucleotidase; Adenosine; Animals; Antibiotics, Antineoplastic; Antigens, CD; Apyrase; Bleomycin; Dermis; Fibrosis; Mice; Mice, Knockout; Scleroderma, Systemic; Transforming Growth Factor beta1

Extracellular nucleotides are released from injured cells and bind purinergic-type 2 receptors (P2-Rs) that modulate inflammatory responses. Ectonucleotidases, such as CD39/nucleoside triphosphate diphosphohydrolase-1, hydrolyze extracellular nucleotides to integrate purinergic signaling responses. Because the role of extracellular nucleotides and CD39 in mediating inflammation and fibrosis are understood poorly, we studied the impact of CD39 gene deletion in a model of pancreatic disease.. Pancreatitis was induced by cyclosporine pretreatment, followed by cerulein injections (50 mug/kg, 6 intraperitoneal injections/day, 3 times/wk); mice were killed at day 2, week 3, and week 6. Experimental parameters were correlated with cytokine levels in blood, RNA, and protein expression of purinergic and fibrosis markers in tissues. Immunohistochemistry and pancreatic morphometry of fibrosis were performed in wild-type and CD39-null mice. Effects of CD39 deletion on proliferation of primary pancreatic stellate cells (PSCs) were investigated in vitro.. Wild-type mice developed morphologic features of pancreatitis with the anticipated development of parenchymal atrophy and fibrosis. CD39 and P2-R became overexpressed in vascular and adventitious wild-type tissues. In contrast, CD39-null mice had inflammatory reactions but developed only minor pancreatic atrophy and limited fibrosis. Interferon-gamma became significantly increased in tissues and plasma of CD39-null mice. Wild-type PSCs expressed high levels of CD39 and P2-R. CD39-null PSCs showed decreased rates of proliferation and the expression of procollagen-alpha1 was inhibited significantly in vitro (P < .03).. CD39 deletion decreases fibrogenesis in experimental pancreatitis. Our data implicate extracellular nucleotides as modulators of PSC proliferation and collagen production in pancreatitis.

Topics: Animals; Antigens, CD; Apyrase; Cell Culture Techniques; Cell Proliferation; Disease Models, Animal; Fibrosis; Gene Deletion; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis