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 paraffinembedded biopsies has been a bottleneck to undertake highthroughput 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.

We attempt to identify the plasma membrane transporter involved in the uptake of 5Ј-deoxy-5-fluorouridine (5Ј-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5Ј-fluorouracil. We also examine the role of the transporter in 5Ј-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5Ј-DFUR, but not capecitabine or 5Ј-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5Ј-DFUR than vector-transfected or wild-type cells.

absorption 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.Copyright © 2010 S. Karger AG, Basel Key WordsExosomes ؒ Na-Cl cotransporter ؒ Na-K-2Cl cotransporter ؒ Renal sodium transporters ؒ Salt sensibility ؒ Urine biomarkers Abstract 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 re-

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 PKCa 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.