From Inflammation to the Onset of Fibrosis through A2A Receptors in Kidneys from Deceased Donors.

Pretransplant graft inflammation could be involved in the worse prognosis of deceased donor (DD) kidney transplants. A2A adenosine receptor (A2AR) can stimulate anti-inflammatory M2 macrophages, leading to fibrosis if injury and inflammation persist. Pre-implantation biopsies of kidney donors (47 DD and 21 living donors (LD)) were used to analyze expression levels and activated intracellular pathways related to inflammatory and pro-fibrotic processes. A2AR expression and PKA pathway were enhanced in DD kidneys. A2AR gene expression correlated with TGF-ß1 and other profibrotic markers, as well as CD163, C/EBPß, and Col1A1, which are highly expressed in DD kidneys. TNF-α mRNA levels correlated with profibrotic and anti-inflammatory factors such as TGF-ß1 and A2AR. Experiments with THP-1 cells point to the involvement of the TNF-α/NF-κB pathway in the up-regulation of A2AR, which induces the M2 phenotype increasing CD163 and TGF-ß1 expression. In DD kidneys, the TNF-α/NF-κB pathway could be involved in the increase of A2AR expression, which would activate the PKA-CREB axis, inducing the macrophage M2 phenotype, TGF-ß1 production, and ultimately, fibrosis. Thus, in inflamed DD kidneys, an increase in A2AR expression is associated with the onset of fibrosis, which may contribute to graft dysfunction and prognostic differences between DD and LD transplants.

Renal sodium transporters are increased in urinary exosomes of cyclosporine-treated kidney transplant patients.

BACKGROUND/AIMS: Cyclosporine (CsA) is a calcineurin inhibitor widely used as an immunosuppressant in organ transplantation. Previous studies demonstrated the relationship between CsA and renal sodium transporters such as the Na-K-2Cl cotransporter in the loop of Henle (NKCC2). Experimental models of CsA-induced hypertension have shown an increase in renal NKCC2. METHODS: Using immunoblotting of urinary exosomes, we investigated in CsA-treated kidney transplant patients (n = 39) the excretion of NKCC2 and Na-Cl cotransporter (NCC) and its association with blood pressure (BP) level. We included 8 non-CsA-treated kidney transplant patients as a control group. Clinical data, immunosuppression and hypertension treatments, blood and 24-hour urine tests, and 24-hour ambulatory BP monitoring were recorded. RESULTS: CsA-treated patients tended to excrete a higher amount of NKCC2 than non-CsA-treated patients (mean ± SD, 175 ± 98 DU and 90 ± 70.3 DU, respectively; p = 0.05) and showed higher BP values (24-hour systolic BP 138 ± 17 mm Hg and 112 ± 12 mm Hg, p = 0.003; 24-hour diastolic BP, 83.8 ± 9.8 mm Hg and 72.4 ± 5.2 mm Hg, p = 0.015, respectively). Within the CsA-treated group, there was no correlation between either NKCC2 or NCC excretion and BP levels. This was confirmed by a further analysis including potential confounding factors. On the other hand, a significant positive correlation was observed between CsA blood levels and the excretion of NKCC2 and NCC. CONCLUSION: Overall, these results support the hypothesis that CsA induces an increase in NKCC2 and NCC in urinary exosomes of renal transplant patients. The fact that the increase in sodium transporters in urine did not correlate with the BP level suggests that in kidney transplant patients, other mechanisms could be implicated in CsA-induced hypertension.

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.

New role of the human equilibrative nucleoside transporter 1 (hENT1) in epithelial-to-mesenchymal transition in renal tubular cells.

Epithelial-to-mesenchymal transition (EMT) is an important pro-fibrotic event in which tubular epithelial cells are transformed into myofibroblasts. Nucleoside transporters (NT) are regulated by many factors and processes, some of which are involved in fibrosis, such as cytokines, inflammation, and proliferation. Equilibrative nucleoside transporter 1 (ENT1) has been proved to be the most widely expressed adenosine transporter. In that sense, ENT1 may be a key player in cell damage signaling. Here we analyze the role of human ENT1 (hENT1) in the EMT process in proximal tubular cells. Addition of the main inducer of EMT, the transforming growth factor-ß1, to HK-2 cells increased hENT1 mRNA and protein level expression. ENT1-mediated adenosine uptake was also enhanced. When cells were incubated with dipyridamole to evaluate the potential contribution of ENT1 to EMT by blocking its transport activity, EMT was induced. Moreover, the knock down of hENT1 with siRNA induced EMT and collagen production in HK-2 cells. Kidneys isolated from ENT1 knockout mice showed higher levels of interstitial collagen and α-SMA positive cells than wild-type mice. Our results point to a new potential role of hENT1 as a modulator of EMT in proximal tubular cells. In this sense, hENT1 could be involved in renal protection processes, and the loss or reduced expression of hENT1 would lead to an increased vulnerability of cells to the onset and/or progression of renal fibrosis.

Are sodium transporters in urinary exosomes reliable markers of tubular sodium reabsorption in hypertensive patients?

BACKGROUND: Altered renal sodium handling has a major pathogenic role in salt-sensitive hypertension. Renal sodium transporters are present in urinary exosomes. We hypothesized that sodium transporters would be excreted into the urine in different amounts in response to sodium intake in salt-sensitive versus salt-resistant patients. METHODS: Urinary exosomes were isolated by ultracentrifugation, and their content of Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC) was analyzed by immunoblotting. Animal studies: NKCC2 and NCC excretion was measured in 2 rat models to test whether changes in sodium transporter excretion are indicative of regulated changes in the kidney tissue. Human studies: in hypertensive patients (n = 41), we investigated: (1) a possible correlation between sodium reabsorption and urinary exosomal excretion of sodium transporters, and (2) the profile of sodium transporter excretion related to blood pressure (BP) changes with salt intake. A 24-hour ambulatory BP monitoring and a 24-hour urine collection were performed after 1 week on a low- and 1 week on a high-salt diet. RESULTS: Animal studies: urinary NKCC2 and NCC excretion rates correlated well with their abundance in the kidney. Human studies: 6 patients (15%) were classified as salt sensitive. The NKCC2 and NCC abundance did not decrease after the high-salt period, when the urinary sodium reabsorption decreased from 99.7 to 99.0%. In addition, the changes in BP with salt intake were not associated with a specific profile of exosomal excretion. CONCLUSIONS: Our results do not support the idea that excretion levels of NKCC2 and NCC via urinary exosomes are markers of tubular sodium reabsorption in hypertensive patients.

Urinary Proteome Analysis Identified Neprilysin and VCAM as Proteins Involved in Diabetic Nephropathy.

Urinary proteome was analyzed and quantified by tandem mass tag (TMT) labeling followed by bioinformatics analysis to study diabetic nephropathy (DN) pathophysiology and to identify biomarkers of a clinical outcome. We included type 2 diabetic normotensive non-obese males with (n = 9) and without (n = 11) incipient DN (microalbuminuria). Sample collection included blood and urine at baseline (control and DN basal) and, in DN patients, after 3 months of losartan treatment (DN treated). Urinary proteome analysis identified 166 differentially abundant proteins between controls and DN patients, 27 comparing DN-treated and DN-basal patients, and 182 between DN-treated patients and controls. The mathematical modeling analysis predicted 80 key proteins involved in DN pathophysiology and 15 in losartan effect, a total of 95 proteins. Out of these 95, 7 are involved in both processes. VCAM-1 and neprilysin stand out of these 7 for being differentially expressed in the urinary proteome. We observed an increase of VCAM-1 urine levels in DN-basal patients compared to diabetic controls and an increase of urinary neprilysin in DN-treated patients with persistent albuminuria; the latter was confirmed by ELISA. Our results point to neprilysin and VCAM-1 as potential candidates in DN pathology and treatment.

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages.

Ciclosporin-induced hypertension is associated with increased sodium transporter of the loop of Henle (NKCC2).

BACKGROUND: Hypertension induced by cyclosporine is associated with renal sodium and water retention. Using immunoblotting of kidney homogenates, we investigated the regulation of sodium and water transport proteins in a rat model of cyclosporine-induced hypertension. METHODS: Rats were treated with cyclosporine (25 mg/kg/day intraperitoneally) during 7 days. Control rats received vehicle. RESULTS: Cyclosporine-treated rats had an increase in blood pressure with a decrease in renal sodium excretion compared with control rats. There were no differences either in sodium intake or in plasma creatinine levels between the two groups of rats. These data suggest that the decrease in sodium excretion in the cyclosporine-treated rats was due to an increase in renal sodium absorption. The densitometric analysis of the renal immunoblot showed an increase in the Na-K-2Cl cotransporter of the loop of Henle (NKCC2) in cyclosporine-treated rats (178% +/- 36) compared with control rats (100% +/- 18; P < 0.05*). This protein rise was associated with an increase in the NKCC2 mRNA pointing to a transcriptional regulation of this sodium transporter. There were no statistically significant changes in the sodium proton exchange (NHE-3) of the proximal tubule although in this renal segment, aquaporin-1 was increased in cyclosporine-treated rats compared with control rats (control 100% +/- 6 vs cyclosporine 119% +/- 6; P < 0.05*). CONCLUSIONS: Our results pointed to the thick ascending limb of the loop of Henle as an important site of sodium retention in cyclosporine-induced hypertension. This data may have potential clinical implications for the treatment of hypertension induced by cyclosporine.

Expression of the high-affinity fluoropyrimidine-preferring nucleoside transporter hCNT1 correlates with decreased disease-free survival in breast cancer.

PURPOSE: Nucleoside and nucleobase derivatives are currently used in the treatment of a variety of solid tumors; however, the role of plasma membrane transporters as biomarkers of drug metabolism has not been fully addressed. Thus, the purpose of this study was to determine whether the concentrative nucleoside transporter hCNT1 is a predictive marker of therapeutic response. METHODS: We studied a cohort of 90 breast cancer patients who were treated with cyclophosphamide-methotrexate-5-fluorouracil after surgery and then monitored for up to 108 months. hCNT1 and enzymes associated with nucleotide metabolism (thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase) were assessed immunohistochemically in tissue samples. RESULTS: Human CNT1 presence was mostly cytoplasmic, with some nuclear staining. The percentage of hCNT1-positive cells correlated positively with the expression of thymidine phosphorylase and dihydropyrimidine dehydrogenase. Nuclear staining correlated negatively with decreased disease-free survival, whereas the percentage of hCNT1-positive cells correlated positively with reduced long-term survival, with the hCNT1-positive index (>80%) being indicative of poor prognosis. A relative risk of relapse was associated with high hCNT1-positive indexes, whereas when this parameter was combined with the nodal status (positive), a high risk of relapse was found, suggesting that both parameters may reflect a poor prognosis. CONCLUSIONS: These results indicate that the expression of the high-affinity concentrative nucleoside transporter hCNT1 has a prognostic value in determining disease-free survival and risk of relapse in breast cancer patients undergoing surgery followed by cyclophosphamide-methotrexate-5-fluorouracil chemotherapy.

Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats.

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

Expression of the nucleoside-derived drug transporters hCNT1, hENT1 and hENT2 in gynecologic tumors.

Deoxynucleoside analogs are used in the treatment of a variety of solid tumors. Their transport across the plasma membrane may determine their cytotoxicity and thus nucleoside transporter (NT) expression patterns may be of clinical relevance. Lack of appropriate antibodies for use in paraffin-embedded biopsies has been a bottleneck to undertake high-throughput analysis of NT expression in solid tumors. Here we report the characterization of 2 new antibodies raised against the low-affinity equilibrative NTs, hENT1 and hENT2, suitable for that purpose. These 2 antisera, along with a previously characterized antibody that specifically recognizes the high-affinity Na-dependent concentrative NT, hCNT1, have been used to analyze, using a tissue array approach, NT expression in gynecologic cancers (90 ovarian, 80 endometrial and 118 uterine cervix carcinomas). Human CNT1 was not detected in 33% and 39% of the ovarian and uterine cervix carcinomas, respectively, whereas hENT1 and hENT2 expression was significantly retained in a high percentage of tumors (91% and 96% for hENT1, 84% and 98% for hENT2, in ovarian and cervix carcinomas, respectively). Only a few endometrial carcinomas (15%) were found to be negative for hCNT1, but they all retained hENT1 and hENT2 expression. In ovarian cancer, the loss of all 3 NT proteins was a more common event in the clear cell histologic subtype than in the serous, mucinous and endometrioid histotypes. In uterine cervix tumors, the loss of expression of hCNT1 was significantly associated with the adenocarcinoma subtype. In summary, hCNT1 was by far the isoform whose expression was most frequently reduced or lost in the 3 types of gynecologic tumors analyzed. Moreover, NT expression is related to the type of gynecologic tumor and its specific subtype, hCNT1 protein loss being highly correlated with poor prognosis histotypes. Since hCNT1, hENT1 and hENT2 recognize fluoropyrimidines as substrates, but with different affinities, this study anticipates high variability in drug uptake efficiency in solid tumors.