Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR 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 A2BAR 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 A2BAR 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 A2BAR 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.

The TGF-ß profibrotic cascade targets ecto-5'-nucleotidase gene in proximal tubule epithelial cells and is a traceable marker of progressive diabetic kidney disease.

Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-ß and adenosinergic signaling contributes to the phenotypic transition of cells and to renal fibrosis in DN models. We evaluated the role of TGF-ß 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-ß results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-ß 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-ß was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-ß 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-ß cascade and is a traceable marker of progressive DN.

Intraglomerular Monocyte/Macrophage Infiltration and Macrophage-Myofibroblast Transition during Diabetic Nephropathy Is Regulated by the A2B Adenosine Receptor.

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 A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR 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-ß induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Author Correction: Deficient Insulin-mediated Upregulation of the Equilibrative Nucleoside Transporter 2 Contributes to Chronically Increased Adenosine in Diabetic Glomerulopathy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blockade of the Adenosine A3 Receptor Attenuates Caspase 1 Activation in Renal Tubule Epithelial Cells and Decreases Interleukins IL-1ß and IL-18 in Diabetic 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-1ß and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1ß, 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-1ß 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.

FK506 Attenuates the MRP1-Mediated Chemoresistant Phenotype in Glioblastoma Stem-Like Cells.

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-mediated Upregulation of the Equilibrative Nucleoside Transporter 2 Contributes to Chronically Increased Adenosine in Diabetic Glomerulopathy.

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.

High plasma adenosine levels in overweight/obese pregnant women.

We aim to investigate whether overweight/obese pregnant women have elevated plasma levels of adenosine associated with increased consumption of high-calorie food. Sixty women were included. They were divided into lean (n = 23 and n = 12) or overweight/obese (n = 7 and n = 18) non-pregnant and pregnant women, respectively. Clinical records and maternal blood samples were collected after informed consent. A self-reported dietary questionnaire was also completed. Plasma adenosine levels were determined with high-performance liquid chromatography. Biochemical parameters, including glucose, total protein, and lipid profile, were determined using standard colorimetric assays. Adenosine levels were higher in pregnant women than in non-pregnant women (18.7 ± 1.6 vs 10.8 ± 1.3 nM/µg protein, respectively, p < 0.0001). Overweight/obese pregnant women (21.9 ± 2.5 nM/µg protein) exhibited higher adenosine levels than lean pregnant (14.5 ± 1.0 nM/µg protein, p = 0.04) or non-pregnant women (11.7 ± 1.5 nM/µg protein, p = 0.0005). Also, pregnant women with elevated weight gain exhibited higher (26.2 ± 3.7 nM/µg protein) adenosine levels than those with adequate weight gain (14.9 ± 1.4 nM/µg protein, p = 0.03). These differences were not statistically significant compared with those of pregnant women with reduced weight gain (17.4 ± 2.1 nM/µg protein, p = 0.053). Body mass index and adenosine only in pregnant women were positively correlated (r = 0.39, p = 0.02). While, polyunsaturated fatty acid (PUFA) consumption was negatively correlated with plasma adenosine levels only in non-pregnant women (r = -0.33, p = 0.03). Pregnancy is associated with high plasma adenosine levels, which are further elevated in pregnant women who are overweight/obese. High PUFA intake might reduce plasma adenosine levels in non-pregnant women.

Multidrug resistance in glioblastoma stem-like cells: Role of the hypoxic microenvironment and adenosine signaling.

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.

Adenosine contribution to normal renal physiology and chronic kidney disease.

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.

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

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.

Pro-apoptotic and anti-angiogenic properties of the α /ß-thujone fraction from Thuja occidentalis on glioblastoma cells.

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/ ß-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 /ß-thujone promotes the regression of neoplasia and inhibits the angiogenic markers VEGF, Ang-4 and CD31 into the tumor.

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-ß 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.

Chemoresistance in high-grade gliomas: relevance of adenosine signalling in stem-like cells of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common glial cell-derived brain tumour, with one of the worst prognoses among all cancers. GBM cells are infiltrative and extremely resistant to radio- and chemotherapy, which inevitably leads to recurrence after surgical resection. These inherent GBM properties are the reasons that patient treatment has not seen major improvements in decades. Studies have consistently shown that glioblastoma stem-like cells (GSCs) are responsible for the tumourigenic properties in the GBM population. In fact, their self-renewal and proliferative potential are required for tumour growth, and their extreme chemoresistance leads to early recurrence of this tumour. Among those mechanisms associated with chemoresistance and having the greatest clinical impact in cancer treatment, are the activities of plasma membrane transporters that extrude antitumour drugs from the cell, thus notably decreasing the pharmacological efficiency of these drugs. The multiple drug resistance associated protein-1 (Mrp1) transporter has been shown to be particularly important in GBM, as inhibition of Mrp1 activity notably chemosensitises cells to antiproliferative drugs. As current therapeutic options for GBM offer only a poor improvement in overall survival rate, alternative strategies for overcoming tumour resistance are urgently sought after. To this end, it is of major clinical relevance to know more about the endogenous modulators that control Mrp1 expression within the pathological environment of the tumour. This review describes the particular properties of glioblastoma cells that overcome multimodal therapy and relapse, with an emphasis on the microenvironmental tumour properties that influence the chemoresistance phenotype to antiproliferative drugs. We also discuss alternative methods of reversal of Mrp1-mediated chemoresistance in these cells by targeting extracellular adenosine production or signalling through particular plasma membrane receptors.

Targeting adenosine signaling to treatment of diabetic nephropathy.

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.

5'-ectonucleotidase mediates multiple-drug resistance in glioblastoma multiforme cells.

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.

Adenosine A(2B) receptor-mediated VEGF induction promotes diabetic glomerulopathy.

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.

Radiation dose and image quality for paediatric interventional cardiology systems. A national survey in Chile.

Radiation dose and image quality for paediatric protocols in all five X-ray fluoroscopy systems used for interventional cardiology procedures existing in Chile have been evaluated. Entrance surface air kerma (ESAK) and image quality using a test object (TO) and polymethyl methacrylate (PMMA) phantoms have been measured for the typical paediatric patient thicknesses (4-16 cm of PMMA). Images from fluoroscopy (low (FL), medium and high) and cine (CI) modes have been archived in DICOM format. Signal-to-noise ratio (SNR), figure of merit (FOM) and high-contrast spatial resolution (HCSR) have been computed from the images. The ratio between the maximum and the minimum value of ESAK per frame for a given fluoroscopy mode between the five systems ranges from 2 to 5 and from 14 to 38 for CI mode. SNR, FOM and HCSR showed a great variability for the different acquisition modes (AMs) and PMMA thickness. In the near future, it is urgent to upgrade Chilean legislation on radiation protection to incorporate quality assurance programmes that will allow us to evaluate and optimise the X-ray systems used in medical applications. Increments in doses per frame when increasing phantom thickness and when used CI runs instead of FL runs can be considered by the cardiologist in the good management of patient dose and allow them to select the best imaging AM during clinical procedures.

Scatter and staff dose levels in paediatric interventional cardiology: a multicentre study.

Interventional cardiology procedures usually imply high doses to the staff, as paediatric cardiologists need to stay closer to the patient than during adult procedures. Also, biplane systems are used that imply an additional source of staff doses. The objective of this paper is to measure scatter doses in four X-ray systems, using polymethyl methacrylate phantoms with thicknesses ranging from 4 to 16 cm to simulate paediatric patients, for the different acquisition modes. Scatter dose rates measured at the position of cardiologist's eyes ranged from 0.8 to 12 mSv h(-1), and about twice the above values at lower extremities, as a linear function of the surface air kerma at the phantom, keeping the irradiated area constant. Therefore, the respective personal dose equivalent for the lens of the eyes may be around 0.5 and 1 mSv throughout the procedure, if additional protection is not used. Simultaneous cine acquisition in biplane systems yielded scatter doses to cardiologists, increased by factors from 5 to 21, compared with a single C-arm acquisition case and depending on geometry. Knowledge of scatter doses for different operation modes, patient thicknesses and the biplane operation should help paediatric cardiologists to adopt conservative attitudes in respect of their occupational radiation risks.

Soil delta15N patterns in old-growth forests of southern Chile as integrator for N-cycling.

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.