Adenosine A(2B) receptor mediates an increase on VEGF-A production in rat kidney glomeruli

Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A₁, A_2A, A_2B and A₃ in isolated rat kidney glomeruli. We localized the expression of A_2BAR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A_2BAR selective antagonist MRS1754 supplementation. Furthermore, we showed that A_2BAR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.     

Reduced Adenosine Uptake and Its Contribution to Signaling that Mediates Profibrotic Activation in Renal Tubular Epithelial Cells: Implication in Diabetic Nephropathy

Altered nucleoside levels may be linked to pathogenic signaling through adenosine receptors. We hypothesized that adenosine dysregulation contributes to fibrosis in diabetic kidney disease. Our findings indicate that high glucose levels and experimental diabetes decreased uptake activity through the equilibrative nucleoside transporter 1 (ENT1) in proximal tubule cells. In addition, a correlation between increased plasma content of adenosine and a marker of renal fibrosis in diabetic rats was evidenced. At the cellular level, exposure of HK2 cells to high glucose, TGF-β and the general adenosine receptor agonist NECA, induced the expression of profibrotic cell activation markers α-SMA and fibronectin. These effects can be avoided by using a selective antagonist of the adenosine A₃ receptor subtype in vitro. Furthermore, induction of fibrosis marker α-SMA was prevented by the A₃ receptor antagonist in diabetic rat kidneys. In conclusion, we evidenced the contribution of purinergic signaling to renal fibrosis in experimental diabetic nephropathy.     

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ET_A and ET_B receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y₁, and adenosine A₁ receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ET_A and ET_B receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.     

Downregulation of A1 and A2B adenosine receptors in human trisomy 21 mesenchymal cells from first-trimester chorionic villi

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A₁ and A_2B expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A₁AR and A_2AAR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A_2B. In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A_2B and A₁ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A₁ and A_2BARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF.     

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A_2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A_2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A_2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.     

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β₁ signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β₁ in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β₁. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.     

Adenosine as an endogenous immunoregulator in cancer pathogenesis: where to go?

Cancer is a chronic disease and its pathogenesis is well correlated with infection and inflammation. Adenosine is a purine nucleoside, which is produced under metabolic stress like hypoxic conditions. Acute or chronic inflammatory conditions lead to the release of precursor adenine nucleotides (adenosine triphosphate (ATP), adenosien diphosphate (ADP) and adenosine monophosphate (AMP)) from cells, which are extracellularly catabolized into adenosine by extracellular ectonucleotidases, i.e., CD39 or nucleoside triphosphate dephosphorylase (NTPD) and CD73 or 5′-ectonucleotidase. It is now well-known that adenosine is secreted by cancer as well as immune cells during tumor pathogenesis under metabolic stress or hypoxia. Once adenosine is released into the extracellular environment, it exerts various immunomodulatory effects via adenosine receptors (A₁, A_2A, A_2B, and A₃) expressed on various immune cells (i.e., macrophages, myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, dendritic cells (DCs), T cells, regulatory T cell (T_regs), etc.), which play very important roles in the pathogenesis of cancer. This review is intended to summarize the role of inflammation and adenosine in the immunopathogenesis of tumor along with regulation of tumor-specific immune response and its modulation as an adjunct approach to tumor immunotherapy.     

Effects of Adenosine Receptor Subtypes on Hippocampal Extracellular Serotonin Level and Serotonin Reuptake Activity

Abstract: To clarify the effects of adenosine receptor subtypes (A₁, A₂, and A₃) on hippocampal serotoninergic function, hippocampal extracellular serotonin (5-HT) levels were determined by in vivo microdialysis in freely moving rats under various conditions. Both adenosine and an adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyladenosine, decreased extracellular 5-HT levels, whereas an adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and caffeine increased these levels. A selective A_2A receptor agonist (CGS-21680), an adenosine A₂ receptor agonist (PD-125944), an adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), and an adenosine A₃ receptor agonist, N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), did not affect extracellular 5-HT levels. When the adenosine A₁ receptor was blocked by CPT, the hippocampal extracellular 5-HT level was increased by adenosine, CGS-21680, and PD-125944, and decreased by caffeine, DMPX, and APNEA. When both adenosine A₁ and A₂ receptors were blocked by CPT and DMPX, the extracellular 5-HT level was decreased by adenosine, caffeine, and APNEA. The hippocampal extracellular 5-HT level was not affected by administration of APNEA alone, but was decreased by this agent when the adenosine A₁ receptor was blocked, irrespective of whether the adenosine A₂ receptor was functional. These inhibitory effects of adenosine, caffeine, and APNEA on extracellular 5-HT levels, during both adenosine A₁ and A₂ receptor blockade, were inhibited by selective 5-HT reuptake inhibitors. These results indicate that the stimulatory effects of the adenosine A₂ receptor and the inhibitory effects of the A₃ receptor on hippocampal extracellular 5-HT levels are masked by the inhibitory effects of the adenosine A₁ receptor.     

Effect of Diabetes/Hyperglycemia on the Rat Retinal Adenosinergic System

The early stages of diabetic retinopathy (DR) are characterized by alterations similar to neurodegenerative and inflammatory conditions such as increased neural apoptosis, microglial cell activation and amplified production of pro-inflammatory cytokines. Adenosine regulates several physiological functions by stimulating four subtypes of receptors, A₁AR, A_2AAR, A_2BAR, and A₃AR. Although the adenosinergic signaling system is affected by diabetes in several tissues, it is unknown whether diabetic conditions in the retina can also affect it. Adenosine delivers potent suppressive effects on virtually all cells of the immune system, but its potential role in the context of DR has yet to be studied in full. In this study, we used primary mixed cultures of rat retinal cells exposed to high glucose conditions, to mimic hyperglycemia, and a streptozotocin rat model of type 1 diabetes to determine the effect diabetes/hyperglycemia have on the expression and protein levels of adenosine receptors and of the enzymes adenosine deaminase and adenosine kinase. We found elevated mRNA and protein levels of A₁AR and A_2AAR, in retinal cell cultures under high glucose conditions and a transient increase in the levels of the same receptors in diabetic retinas. Adenosine deaminase and adenosine kinase expression and protein levels showed a significant decrease in diabetic retinas 30 days after diabetes induction. An enzymatic assay performed in retinal cell cultures revealed a marked decrease in the activity of adenosine deaminase under high glucose conditions. We also found an increase in extracellular adenosine levels accompanied by a decrease in intracellular levels when retinal cells were subjected to high glucose conditions. In conclusion, this study shows that several components of the retinal adenosinergic system are affected by diabetes and high glucose conditions, and the modulation observed may uncover a possible mechanism for the alleviation of the inflammatory and excitotoxic conditions observed in diabetic retinas.     

Adenosine Augmentation Evoked by an ENT1 Inhibitor Improves Memory Impairment and Neuronal Plasticity in the APP/PS1 Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive impairment and synaptic dysfunction. Adenosine is an important homeostatic modulator that controls the bioenergetic network in the brain through regulating receptor-evoked signaling pathways, bioenergetic machineries, and epigenetic-mediated gene regulation. Equilibrative nucleoside transporter 1 (ENT1) is a major adenosine transporter that recycles adenosine from the extracellular space. In the present study, we report that a small adenosine analogue (designated J4) that inhibited ENT1 prevented the decline in spatial memory in an AD mouse model (APP/PS1). Electrophysiological and biochemical analyses further demonstrated that chronic treatment with J4 normalized the impaired basal synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses as well as the aberrant expression of synaptic proteins (e.g., NR2A and NR2B), abnormal neuronal plasticity-related signaling pathways (e.g., PKA and GSK3β), and detrimental elevation in astrocytic A_2AR expression in the hippocampus and cortex of APP/PS1 mice. In conclusion, our findings suggest that modulation of adenosine homeostasis by J4 is beneficial in a mouse model of AD. Our study provides a potential therapeutic strategy to delay the progression of AD.     

Adenosine-induced cell death: evidence for receptor-mediated signalling

Adenosine modulates the proliferation, survival and apoptosis of many different cell types, ranging from epithelial, endothelial and smooth muscle cells, to cells of the immune and neural lineages. In this review, we critically discuss the available in vitro and in vivo data which support a role for adenosine in both development-associated apoptosis, and in diseases characterized by either pathologically increased cell death (e.g., ischemia, trauma and aging-associated neurodegeneration) or abnormally reduced spontaneous apoptosis (e.g., cancer). Particular emphasis is given to the possible role of extracellular adenosine receptors, since these may represent novel and attractive molecular targets for the pharmacological modulation of apoptosis. In some instances, adenosine-induced cell death has been demonstrated to require entry of the nucleoside inside cells; however, in many other cases, activation of specific adenosine extracellular receptors has been demonstrated. Of the four G protein-coupled adenosine receptors so far identified, the A_2A and the A₃ receptors have been specifically implicated in modulation of cell death. For the A₃ receptor, results obtained by exposing both cardiomyocytes and brain astrocytes to graded concentrations of selective agonists suggest induction of both cell protection and cell death. Such opposite effects, which likely depend on the degree of receptor activation, may have important therapeutic implications in the pharmacological modulation of cardiac and brain ischemia. For the A_2A receptor, recent intriguing data suggest a specific role in immune cell death and immunosuppression, which may be relevant to both adenosine-deaminase-immunodeficiency syndrome (a pathology characterized by accumulation of adenosine to toxic levels) and in tumors where induction of apoptosis via activation of specific extracellular receptors may be desirable. Finally, preliminary data suggest that, in a similar way to the adenosine-deaminase-immunodeficiency syndrome, the abnormal accumulation of adenosine in degenerative muscular diseases may contribute to muscle cell death. Although the role of adenosine receptors in this effect still remains to be determined, these data suggest that adenosine-induced apoptosis may also represent a novel pathogenic pathway in muscular dystrophies.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.     

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension

The A_2B adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A_2BAR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A_2BAR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15–20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A_2BAR. Our data indicate varying roles for A_2BAR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.     

Protein kinase Cμ mediates adenosine-stimulated steroidogenesis in primary rat adrenal cells

Adenosine (Ado), an endogenous nucleoside, can stimulate corticosterone synthesis in adrenal cells via the A_2A/A_2B adenosine receptors (ARs). This study evaluated the contribution of protein kinase C (PKC) isoforms in Ado-induced steroidogenesis. The PKC inhibitor calphostin c blocked Ado-induced steroidogenesis, the mitogen-activated protein kinase (MEK)-extracellular signal-related regulated kinase (ERK)-cyclic AMP responsive element-binding protein cascade, and the mRNA expression of steroidogenic acute regulatory protein and CYP11B1. Further analyses revealed that PKCμ was indeed activated by Ado. Moreover, downregulation of PKCμ by small interfering RNA (siRNA) inhibited Ado-stimulated steroidogenesis and ERK phosphorylation. Finally, inhibition of either A_2AAR or A_2BAR led to the suppression of PKCμ phosphorylation. Together, these findings suggest that A₂AR-PKCμ-MEK signaling mediates Ado-stimulated adrenal steroidogenesis.     

Resveratrol ameliorates early diabetic nephropathy associated with suppression of augmented TGF-β/smad and ERK1/2 signaling in streptozotocin-induced diabetic rats

Diabetic nephropathy (DN) is the major cause of end-stage renal disease. The early changes in DN are characterized by an increased in kidney size, glomerular volume, and kidney function, followed by the accumulation of glomerular extracellular matrix, increased urinary albumin excretion (UAE), glomerular sclerosis, and tubular fibrosis. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial effects of RSV on DN and explored the possible mechanism of RSV action.Male Sprague–Dawley rats were injected with streptozotocin at 65 mg/kg body weight. The induction of diabetes mellitus (DM) was confirmed by a fasting plasma glucose level ≥300 mg/dL and symptoms of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75 mg/kg body weight 3 times a day for 8 weeks. Animals were sacrificed and kidney histology was examined by microscopy. Urinary albumin excretion, glomerular hypertrophy and expressions of fibronectin, collagen IV, and TGF-β in the glomeruli were alleviated in RSV-treated DM rats, but not in untreated DM rats. In addition, RSV treatment reduced the thickness of the glomerular basement membrane (GBM) to the original thickness and increased nephrin expressions to normal levels in DM rats. Moreover, RSV inhibited phosphorylation of smad2, smad3 and ERK1/2 in diabetic rat kidneys. This is the first report showing that RSV alleviates early glomerulosclerosis in DN through TGF-β/smad and ERK1/2 inhibition. In addition, podocyte injuries of diabetic kidneys are lessened by RSV.     

All-trans retinoic acid ameliorates inflammatory response mediated by TLR4/NF-κB during initiation of diabetic nephropathy

Diabetic nephropathy (DN) is the leading cause of renal failure worldwide and its complications have become a public health problem. Inflammation, oxidative stress and fibrosis play central roles in the progression of DN that lead to renal failure. Potential deleterious effect of inflammation in early evolution of DN is not fully disclosed. Therefore, it is relevant to explore therapies that might modulate this process in order to reduce DN progression. We explored the beneficial effect of all-trans retinoic acid (ATRA) in early inflammation in glomeruli, proximal and distal tubules in streptozotocin (STZ)-induced diabetes. ATRA was administered (1 mg/kg daily by gavage) on days 3 to 21 after STZ administration. It was found that 21 days after STZ injection, diabetic rats exhibited proteinuria, increased natriuresis and loss of body weight. Besides, diabetes induced an increase in interleukins [IL-1β, IL-1α, IL-16, IL-13, IL-2; tumor necrosis factor alpha (TNF-α)] and transforming growth factor-beta 1 (TGF-β1), chemokines (CCL2, CCL20, CXCL5 and CXCL7), adhesion molecules (ICAM-1 and L-selectin) and growth factors (GM-CSF, VEGF, PDGF) in glomeruli and proximal tubules, whereas ATRA treatment remarkably ameliorated these alterations. To further explore the mechanisms through which ATRA decreased inflammatory response, the NF-κB/p65 signaling mediated by TLR4 was studied. We found that ATRA administration attenuates the TLR4/NF-κB inflammatory signaling and prevents NF-κB nuclear translocation in glomeruli and proximal tubules.     

AMP and adenosine are both ligands for adenosine 2B receptor signaling

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A_2BR). A_2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A_2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A_2BR.The computational modeling suggested that AMP interacts with more favorable energy to A_2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A_2BR, indicating preferential signaling via the G_q pathway. Therefore, a putative AMP-A_2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G_q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.