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For more than two decades, research groups in hydrology, ecology, soil science, and biogeochemistry have performed cryogenic water extractions (CWEs) for the analysis of δ2H and δ18O of soil water. Recent studies have shown that extraction conditions (time, temperature, and vacuum) along with physicochemical soil properties may affect extracted soil water isotope composition. Here we present results from the first worldwide round robin laboratory intercomparison. We test the null hypothesis that, with identical soils, standards, extraction protocols, and isotope analyses, cryogenic extractions across all laboratories are identical. Two standard soils with different physicochemical characteristics along with deionized (DI) reference water of known isotopic composition were shipped to 16 participating laboratories. Participants oven-dried and rewetted the soils to 8 and 20 % gravimetric water content (WC), using the deionized reference water. One batch of soil samples was extracted via predefined extraction conditions (time, temperature, and vacuum) identical to all laboratories; the second batch was extracted via conditions considered routine in the respective laboratory. All extracted water samples were analyzed for δ18O and δ2H by the lead laboratory (Global Institute for Water Security, GIWS, Saskatoon, Canada) using both a laser and an isotope ratio mass spectrometer (OA-ICOS and IRMS, respectively). We rejected the null hypothesis. Our results showed large differences in retrieved isotopic signatures among participating laboratories linked to soil type and soil water content with mean differences compared to the reference water ranging from +18.1 to −108.4 ‰ for δ2H and +11.8 to −14.9 ‰ for δ18O across all laboratories. In addition, differences were observed between OA-ICOS and IRMS isotope data. These were related to spectral interferences during OA-ICOS analysis that are especially problematic for the clayey loam soils used. While the types of cryogenic extraction lab construction varied from manifold systems to single chambers, no clear trends between system construction, applied extraction conditions, and extraction results were found. Rather, observed differences in the isotope data were influenced by interactions between multiple factors (soil type and properties, soil water content, system setup, extraction efficiency, extraction system leaks, and each lab's internal accuracy). Our results question the usefulness of cryogenic extraction as a standard for water extraction since results are not comparable across laboratories. This suggests that defining any sort of standard extraction procedure applicable across laboratories is challenging. Laboratories might have to establish calibration functions for their specific extraction system for each natural soil type, individually.

Extreme sensitivity of soil organic carbon (SOC) to climate and land use change warrants further research in different terrestrial ecosystems. The aim of this study was to investigate the link between aggregate and SOC dynamics in a chronosequence of three different land uses of a south Chilean Andisol: a second growth Nothofagus obliqua forest (SGFOR), a grassland (GRASS) and a Pinus radiata plantation (PINUS). Total carbon content of the 0-10cm soil layer was higher for GRASS (6.7 kg C m-2) than for PINUS (4.3 kg C m-2, while TC content of SGFOR (5.8 kg C m-2) was not significantly different from either one. High extractable oxalate and pyrophosphate Al concentrations (varying from 20.3-24.4 g kg-1, and 3.9-11.1 g kg-1, respectively) were found in all sites. In this study, SOC and aggregate dynamics were studied using size and density fractionation experiments of the SOC, δ13C and total carbon analysis of the different SOC fractions, and C mineralization experiments. The results showed that electrostatic sorption between and among amorphous Al components and clay minerals is mainly responsible for the formation of metal-humus-clay complexes and the stabilization of soil aggregates. The process of ligand exchange between SOC and Al would be of minor importance resulting in the absence of aggregate hierarchy in this soil type. Whole soil C mineralization rate constants were highest for SGFOR and PINUS, followed by GRASS (respectively 0.495, 0.266 and 0.196 g CO2-Cm-2d-1 for the top soil layer). In contrast, incubation experiments of isolated macro organic matter fractions gave opposite results, showing that the recalcitrance of the SOC decreased in another order: PINUS>SGFOR>GRASS. We deduced that electrostatic sorption processes and physical protection of SOC in soil aggregates were the main processes determining SOC stabilization. As a result, high aggregate carbon concentrations, varying from 148 till 48 g kg-1, were encountered for all land use sites. Al availability and electrostatic charges are dependent on pH, resulting in an important influence of soil pH on aggregate stability. Recalcitrance of the SOC did not appear to largely affect SOC stabilization. Statistical correlations between extractable amorphous Al contents, aggregate stability and C mineralization rate constants were encountered, supporting this hypothesis. Land use changes affected SOC dynamics and aggregate stability by modifying soil pH (and thus electrostatic charges and available Al content), root SOC input and management practices (such as ploughing and accompanying drying of the soil).

Anchovy Engraulis ringens and common sardine Strangomera bentincki are two small pelagic fish that co-occur in the same reproductive habitat off central and southern Chile and have a similar reproductive strategy. Interannual and spatial changes in the batch fecundity of both fish were analyzed with the purpose of detecting variability in the parameters and inferring different reproductive tactics that could affect the reproductive potential. Samples were obtained from adult surveys carried out to estimate spawning biomass by the Daily Egg Production Method, from 2002 to 2007, off Chile. The study area was divided into two zones: central and south. The batch fecundity was linearly related to ovary-free female body weight for each year. For anchovy, the slope was not different across years and zones, while for sardine, changes in intercept and slopes varied significantly among years, but not between zones. The average weight of mature females tended to be higher in the south zone for both species, and average batch fecundity ranged from 4,283 to 8,903 eggs per mature female anchovy, and from 5,901 to 10,414 eggs per mature female sardine. Although the anchovy and sardine reproductive strategy is similar, differences in batch fecundity seemed to be the main distinction between the species in the central and southern spawning sites off Chile, with implications for the reproductive potential.

The domestic cat has evolved in various aspects in its journey from original domestication to the present day. Many domestic cats today lead a sedentary indoor lifestyle with low environmental stimulation. In addition, cats have changed their eating habits, transitioning from being carnivorous hunters to animals that eat commercial processed foods. Eating patterns have also changed since cats no longer need to hunt for food but instead have access to several portions throughout the day. All these variables, known as extrinsic epidemiological factors, added to animal-specific or intrinsic epidemiological factors (genetics, sex and breed, among others), predispose cats to the development of overweight and obesity (O&O). The prevalence of O&O has significantly increased in the global cat population, making them a growing clinical concern for companion animals. In turn, O&O contribute to the onset of other pathologies by either increasing susceptibility or creating conditions that allow existing or incipient pathologies to manifest or worsen. Some of these diseases correspond to changes in the musculoskeletal tissue, insulin resistance, type 2 diabetes, skin disorders, and kidney and urinary tract diseases, among others. Therefore, the aim of this review was to analyze the epidemiological variables and diseases associated with O&O in the domestic cat., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Induction of the adenosine receptor A 2B (A 2B AR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A 2B AR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A 2B AR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A 2B AR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A 2B AR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A 2B AR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

Aim: Progression of diabetic nephropathy (DN) is linked to the dysregulated increase of adenosine and altered signaling properties. A major contribution to the maintenance of physiological extracellular adenosine levels relies on cellular uptake activity through plasma membrane nucleoside transporters. Because kidney cells are responsive to insulin, this study aims to determine how DN affects insulin regulation of the equilibrative nucleoside transporter-2 (ENT2)., Methods: Human Podocytes and rat glomeruli were used to study ENT2 regulation. The effects of diabetes and insulin on ENT2 mediated transport activity were determined measuring the fraction of total adenosine uptake in sodium-free medium which is inhibitable by hypoxanthine. Alterations in ENT2 subcellular distribution were assessed in the kidney of people affected with DN and diabetic rats. The consequences of impaired ENT2 activity on the kidney were evaluated using dipyridamole in an animal model., Results: Insulin upregulates ENT2 uptake activity by increasing the V max , thus counteracting decreased adenosine uptake due to high d-glucose and achieving extracellular adenosine homeostasis. Insulin promoted ENT2 translocation to the plasma membrane dependent on PI3-kinase/Akt signaling and actin cytoskeleton integrity. However, in diabetic rats, the insulin-mediated induction of ENT2 activity was lost. Additionally, reduced Akt activation in response to insulin correlated with decreased ENT2 distribution at the plasma membrane. Kidney tissues from diabetic rats and human DN biopsies showed ENT2 redistribution to an intracellular pattern, evidencing dysfunctional adenosine uptake. Through ENT inhibition, we evidenced increased proteinuria and induced alpha-smooth muscle actin as a result of profibrotic activation of cells in the kidney., Conclusion: Deficient insulin regulation of ENT2 activity contributes to chronically high adenosine levels and glomerular alterations that underline diabetic kidney disease progression., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Dissotichus eleginoides has a discontinuous circumpolar geographic distribution restricted to mountains and platforms, mainly in Subantarctic and Antarctic waters of the southern hemisphere, including the Southeast Pacific, Atlantic and Indian oceans and in areas surrounding the peninsular platforms of subantarctic islands. The aim of this work was to determine and characterize the gastrointestinal parasitic and microbial fauna of specimens of D. eleginoides captured in waters of the south-central zone of Chile. The magnitude of parasitism in D. eleginoides captured in waters of the south-central zone of Chile is variable, and the parasite richness is different from that reported in specimens from subantarctic environments. Next-generation sequencing (NGS) of the microbial community associated to intestine showed a high diversity, where Proteobacteria, Firmicutes , and Bacteriodetes were the dominant phyla. However, both parasitic and microbial structures can vary between fish from different geographic regions.

Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-β and adenosinergic signaling contributes to the phenotypic transition of cells and to renal fibrosis in DN models. We evaluated the role of TGF-β on NT5E gene expression coding for the ecto-5`-nucleotidase CD73, the limiting enzyme in extracellular adenosine production. We showed that high d-glucose may predispose HK-2 cells towards active transcription of the proximal promoter region of the NT5E gene while additional TGF-β results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-β with occupancy by the p300 co-activator and the phosphorylated forms of the Smad2/3 complex and RNA Pol II. Transcriptional induction at NT5E in response to TGF-β was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-β in HK-2 cells. Interestingly, we found increased CD73 content in urinary extracellular vesicles only in diabetic patients with renal repercussions. Further, CD73-mediated AMPase activity was increased in the urinary sediment of DN patients. We conclude that the NT5E gene is a target of the profibrotic TGF-β cascade and is a traceable marker of progressive DN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacterial kidney disease (BKD) is widespread in many areas of the world and can cause substantial economic losses for the salmon aquaculture industry. The objective of this study was to investigate the pathophysiological response and gene expression profiles related to the immune response at different water temperatures and to identify the best immunopathological biomarkers to define a phenotype of resistance to BKD. The abundance of msa transcripts of R. salmoninarum in the head kidney was significantly higher in infected fish at 11°C. R. salmoninarum induced significantly more severe kidney lesions, anemia and impaired renal function at 11°C. In addition, the expression pattern of the genes related to humoral and cell-mediated immune responses in infected fish at 11 and 15°C was very similar, although R. salmoninarum induced a significantly greater downregulation of the adaptive immune response genes at the lower water temperature. These results could be due to a suppressed host response directly related to the lowest water temperature and/or associated with a delayed host response related to the lowest water temperature. Although no significant differences in survival rate were observed, fish infected at the lowest temperature showed a higher probability of death and delayed the mortality curve during the late stage of infection (35 days after infection). Thirty-three immunopathological biomarkers were identified for potential use in the search for a resistance phenotype for BKD, and eight were genes related specifically to the adaptive cell-mediated immune response., (Copyright © 2020 Rozas-Serri, Lobos, Correa, Ildefonso, Vásquez, Muñoz, Maldonado, Jaramillo, Coñuecar, Oyarzún, Walker, Navarrete, Gayosa, Mancilla, Peña, Senn and Schwerter.)

Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage-myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A 2B adenosine receptor (A 2B AR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A 2B AR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A 2B AR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1β and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1β, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1β and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

CaCO₃ nanoparticles of around 60 nm were obtained by a co-precipitation method and used as filler to prepare low-density polyethylene (LDPE) composites by melt blending. The nanoparticles were also organically modified with oleic acid (O-CaCO₃) in order to improve their interaction with the LDPE matrix. By adding 3 and 5 wt% of nanofillers, the mechanical properties under tensile conditions of the polymer matrix improved around 29%. The pure LDPE sample and the nanocomposites with 5 wt% CaCO₃ were photoaged by ultraviolet (UV) irradiation during 35 days and the carbonyl index (CI), degree of crystallinity (χ c ), and Young's modulus were measured at different times. After photoaging, the LDPE/CaCO₃ nanocomposites increased the percent crystallinity (χ c ), the CI, and Young's modulus as compared to the pure polymer. Moreover, the viscosity of the photoaged nanocomposite was lower than that of photoaged pure LDPE, while scanning electron microscopy (SEM) analysis showed that after photoaging the nanocomposites presented cavities around the nanoparticles. These difference showed that the presence of CaCO₃ nanoparticles accelerate the photo-degradation of the polymer matrix. Our results show that the addition of CaCO₃ nanoparticles into an LDPE polymer matrix allows future developments of more sustainable polyethylene materials that could be applied as films in agriculture. These LDPE-CaCO₃ nanocomposites open the opportunity to improve the low degradation of the LDPE without sacrificing the polymer's behavior, allowing future development of novel eco-friendly polymers., Competing Interests: The authors declare no conflict of interest.

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

Deficient insulin signaling is a key event mediating diabetic glomerulopathy. Additionally, diabetic kidney disease has been related to increased levels of adenosine. Therefore, we tested a link between insulin deficiency and dysregulated activity of the equilibrative nucleoside transporters (ENTs) responsible for controlling extracellular levels of adenosine. In ex vivo glomeruli, high D-glucose decreased nucleoside uptake mediated by ENT1 and ENT2 transporters, resulting in augmented extracellular levels of adenosine. This condition was reversed by exposure to insulin. Particularly, insulin through insulin receptor/PI3K pathway markedly upregulated ENT2 uptake activity to restores the extracellular basal level of adenosine. Using primary cultured rat podocytes as a cellular model, we found insulin was able to increase ENT2 maximal velocity of transport. Also, PI3K activity was necessary to maintain ENT2 protein levels in the long term. In glomeruli of streptozotocin-induced diabetic rats, insulin deficiency leads to decreased activity of ENT2 and chronically increased extracellular levels of adenosine. Treatment of diabetic rats with adenosine deaminase attenuated both the glomerular loss of nephrin and proteinuria. In conclusion, we evidenced ENT2 as a target of insulin signaling and sensitive to dysregulation in diabetes, leading to chronically increased extracellular adenosine levels and thereby setting conditions conducive to kidney injury.

Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal damage and produces phenotypic changes in resident cells. This review discusses the physiological and pathophysiological roles of adenosine and pays special attention to the mechanisms associated with switching homeostatic nucleoside levels to increased adenosine production in kidneys affected by CKD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Glioblastoma multiforme (GBM) is considered the most common and aggressive tumour of the central nervous system and is characterized for being highly chemoresistant. This property is mainly due to the activation of Multiple Drug Resistance (MDR) mechanisms that protect cancer cells from structurally and morphologically different drugs. Overexpression and increased ABC transporters activity is one of the most important MDR mechanisms at the clinical level, and both its expression and activity are elevated in GBM cells. Within the tumour, there is a subpopulation called glioblastoma stem-like cells (GSCs), which due to its high tumourigenic capacity and chemoresistance, have been postulated as the main responsible for tumour recurrence. The GSCs inhabit hypoxic tumour zones, niches that apart from maintaining and promoting stem phenotype have also been correlated with high chemoresistance. Of the signalling pathways activated during hypoxia, purinergic signalling has been highly associated to the induction of MDR mechanisms. Through its receptors, the nucleoside adenosine has been shown to promotes the chemoresistance mediated by ABC transporters. Therefore, targeting its components is a promising alternative for GBM treatment. In this review, we will discuss chemoresistance in GSCs and the effect of the hypoxic microenvironment and adenosine on MDR mechanisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

The most aggressive type of brain tumor is glioblastoma multiforme, which to date remains incurable. Thuja occidentalis is used in homeopathy for the treatment of cancer, however, its mechanism of action remains unknown. We set out to study the effects of thujone fractions of Thuja on glioblastoma using in vitro and in vivo models. We found that the α/ β-thujone fraction decrease the cell viability and exhibit a potent anti-proliferative, pro-apoptotic and anti-angiogenic effects in vitro. In vivo assays showed that α /β-thujone promotes the regression of neoplasia and inhibits the angiogenic markers VEGF, Ang-4 and CD31 into the tumor.

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.

The pathogenesis of diabetic nephropathy (DN) has not been clearly established, making diagnosis and patient management difficult. Recent studies using experimental diabetic models have implicated adenosine signaling with renal cells dysfunction. Therefore, the study of the biochemical mechanisms that regulate extracellular adenosine availability during DN is of emerging interest. Using streptozotocin-induced diabetic rats we demonstrated that urinary levels of adenosine were early increased. Further analyses showed an increased expression of the ecto 5'-nucleotidase (CD73), which hydrolyzes AMP to adenosine, at the renal proximal tubules and a higher enzymatic activity in tubule extracts. These changes precede the signs of diabetic kidney injury recognized by significant proteinuria, morphological alterations and the presence of the renal fibrosis markers alpha smooth muscle actin and fibronectin, collagen deposits and thickening of the glomerular basement membrane. In the proximal tubule cell line HK2 we identified TGF-β as a key modulator of CD73 activity. Importantly, the increased activity of CD73 could be screened in urinary sediments from diabetic rats. In conclusion, the increase of CD73 activity is a key component in the production of high levels of adenosine and emerges as a new tool for the early diagnosis of tubular injury in diabetic kidney disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Diabetic nephropathy (DN) continues being the primary cause of chronic hemodialysis and terminal renal disease worldwide. At tissue levels the DN occurs with glomerulopathy affecting the integrity of the filtration barrier and with an extensive glomerular and tubule-interstitial fibrosis. Current available therapeutic approaches have only demonstrated a modest effect on progression of kidney injury. Therefore, more research concerning the pathomechanisms and possible interventions are needed. Interestingly, in the last years it has been documented that DN progresses with growing levels of the nucleoside adenosine. This finding increased the interest in the events controlling the extracellular levels of the nucleoside. While the metabolism of extracellular ATP and cyclic AMP are well recognized sources, evidences regarding the role of the equilibrative nucleoside transporters in controlling adenosine availability and promoting diabetic glomerulopathy have recently acquired a pivotal role. The physiological effects of nucleoside are mediated by the P1 family of adenosine receptors. It has been shown in vivo that the use of an antagonist of the A2B receptor subtype can block the most remarkable early alterations seen in diabetic glomerulopathy. Furthermore, using models of chronic kidney injury it was demonstrated that fibrosis can also be blocked using treatment with the antagonist of A2B receptor subtype. This review highlights these findings that correlate the activity of a low affinity adenosine receptor with an increase in the ligand availability in the pathological state. In addition, we discuss the possible therapeutic interventions of adenosine signaling with regards to DN treatment.

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple-drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto-5'-nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A(3) receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells., (Copyright © 2012 Wiley Periodicals, Inc.)

Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A(2B) receptor (A(2B)AR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A(2B)AR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A(2B)AR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Old-growth forests of southern Chile represent an important reserve of temperate (rain) forests in the world. Wetter and colder forest ecosystems appear to be more efficient in conserving and recycling N such that mostly non-plant available N species are lost, which could be indicated by more depleted delta15N values of the soil and plants. Hydrological N loss from the old-growth forests in southern Chile occurs mainly via dissolved organic nitrogen and not via dissolved inorganic N. Forest disturbances (e.g. fire, clear-cutting or enhanced N deposition) cause (abrupt) changes in ecosystem N-cycling processes. In this study, we hypothesized that delta15N signatures of soil profiles under old-growth forests could be used as an integrator for ecosystem N-cycling, and changes of these delta15N profiles could be valuable to assess ecosystem resilience towards disturbances. Six old-growth forests were selected in the phytogeographical region of the Valdivian rain forest in southern Chile. One of the sites has been partly burned in February 2002. First, we observed that ecosystems with higher mean annual precipitation and lower mean annual temperature were relatively more depleted in 15N. Secondly, we found that a forest fire caused a 100-fold increase of the nitrate export and induced an enrichment of the soil delta15N signal in the upper 20 cm.

Antimicrobial proteins and peptides play an important role in the primary defence of epithelial barriers in vertebrates and invertebrates. Here we report the detection of the apolipoproteins A-I and A-II in the epidermis and epidermal mucus of the carp (Cyprinus carpio L.) by immunohistochemistry and Western blot analysis. Both apolipoproteins are major constituents of high density lipoprotein and have been shown to display antiviral and antimicrobial activity in mammals. Therefore the aim of this study was to evaluate if they could be part of the innate immune system of teleost fish. A cDNA clone containing most of the coding region for carp apoA-I was isolated and used as a probe to demonstrate the expression of apoA-I gene in the skin. In addition, mucus apoA-I was shown to be associated to small particles that could correspond to nascent HDL. Finally, affinity purified plasma HDL displayed bactericidal activity in vitro against a non-pathogenic Escherichia coli strain, suggesting a defensive role for HDL and its associated proteins in the carp epidermis and mucus.