Nucleoside transport activity in renal epithelial tubule cells.

<p>A. Total and particular [³H]adenosine transport activities were assayed in HK2 cells. The effect of D-glucose concentration on transport activities was assayed in HK2 cells exposed to 5mM and 25mM for 24h. Total uptake activity mediated by concentrative (CNTs) and equilibrative (ENTs) systems was obtained in transport buffer containing Na⁺; while ENTs-mediated uptake was determined by using a Na⁺ free buffer. CNTs component was derived from the difference between total transport activity in Na⁺ containing buffer minus the transport activity in Na⁺ free buffer. The graphs depict particular ENT1 mediated nucleoside uptake as the fraction of the transport in Na⁺ free buffer inhibited by 1μM NBTI, while the ENT2 fraction that was inhibited by 2mM hypoxanthine. Data is expressed as mean ± SD of triplicate measurements from 20 independent assays. * <i>P</i> < 0.05 versus 5mM D-glucose. B. Renal proximal tubules were isolated from vehicle- and STZ-treated rats following 1 and 4 months. The effect of diabetes on sodium-independent transport uptake activities mediated by ENT1 and ENT2, was quantified. Data is expressed as mean ± SD of triplicate measurements for assays using extracts from 5 animals in each group. * <i>P</i> < 0.05 versus control. C. The protein level of ENT1 in tubule extracts was evaluated by western blot. Representative images of western blots using diabetic (db) and control (ctr) rat extracts are shown. The graph depicts the mean ± SD of ratio between immune signals of ENT1 vs tubulin. The ratio in control extracts were normalized to 1. A statistically significant difference was not found. n = 6. D. The extracellular adenosine levels were quantified in the medium of HK2 cells exposed to NBTI, an inhibitor of ENT1 activity. Data is mean ± SD from 5 experiments. * <i>P</i> < 0.05 versus control.</p

Effect of high D-glucose and experimental diabetes on the adenosine receptors content.

<p>A. The effect of D-glucose exposure (24 h) on the protein levels of adenosine receptor subtypes A₁, A_2A, A_2B and A₃ in HK2 cells was evaluated by western blot. Representative images of western blots of total protein extracts from cells exposed to 5 and 25 mM D-glucose are shown. B. The effect of STZ-induced diabetes on adenosine A₃ receptor content was evaluated by western blot of total protein extracts from tubules isolated from control (ctr) and diabetic (db) rats following 1 or 4 months post-induction. The graphs show quantitative analysis of immune signals. The means in controls were normalized to 1. A: n = 6, B: n = 3.</p

Evidences of renal fibrosis in diabetic rats and <i>ent1</i> knockout mice.

<p>A. Representative immunohistochemical detection of α-SMA in kidney sections from vehicle- and STZ-treated diabetic rats. The arrows indicate interstitial and periglomerular staining. B. The graph depicts plasmatic adenosine levels in control and diabetic rat groups. * <i>P</i> < 0.05 vs control, n = 6. C. Representative immunohistochemical detection of α-SMA in kidney sections from 8 weeks old <i>ent1-/-</i> and wild type mice. The graphs (A and C) show quantitative results derived from staining analyses of consecutive field from three animals per group using the UN-SCANIT 2.0 software. Values of control or wild type samples were normalized to 1. * <i>P</i> < 0.05 vs control or wild type. Original magnifications 200X.</p

An antagonist of adenosine A₃ receptor blocks profibrotic activation induced by TGF-β.

<p>A. The induction of the marker fibronectin was evaluated by western blots in HK2 cells upon exposure to TGF-β. The contribution of a particular adenosine receptor subtype in TGF-β-induced cell activation, was recognized by using selective pharmacological antagonists. Selective antagonists were DPCPX (30nM) for A₁, ZM241385 (10nM) for A_2A, MRS1754 (50nM) for A_2B and MRS1220 for A₃ (10nM) receptor subtypes. B. The particular contribution of the adenosine A₃ receptor was evidenced by knocking down the expression of the receptor using siRNA (siRNA A₃AR). The upper images show representative western blot detections of fibronectin content in total protein extracts (50 μg) from treated HK2 cells. The blocking effect was assayed in HK2 cells cultures 5mM (left white bars graphs) or 25mM (right black bars graphs) D-glucose. The graphs represent the mean ± SD of the ratio between immune signals of fibronectin vs β-actin. The ratio in HK2 cells without TGF-β treatment was normalized to 1. * <i>P</i> < 0.05 versus untreated cells, # <i>P</i> < 0.05 versus TGF-β, n = 5.</p

In vivo effects of pharmacological inhibition of A₃AR.

<p>Diabetic rats were treated with the adenosine A₃ receptor antagonist MRS1220 from days 31 to 60 post diabetes induction, A. Representative inmunohistochemical detection of α-SMA in cortical areas of kidney sections from untreated (control), diabetic (vehicle-treated rats) and MRS1220-treated diabetic rats. Original magnifications 400X. B. The graph shows quantitative results derived from staining analyses of consecutive field from three animals per group using the UN-SCANIT 2.0 software. Values of control samples were normalized to 1. * <i>P</i> < 0.05 vs control.</p

An antagonist of adenosine A₃ receptor blocks NECA-induced profibrotic markers.

<p>The particular adenosine receptor subtype involved in profibrotic activation was recognized by using selective pharmacological antagonists for each receptor subtype, in conjunction with the general agonist NECA (5 μM). The induction of the markers fibronectin (A) and α-SMA (B) in HK2 cells, was evaluated by western blots. Selective antagonists were DPCPX (30nM) for A₁, ZM241385 (10nM) for A_2A, MRS1754 (50nM) for A_2B and MRS1220 for A₃ (10nM) receptor subtypes. The upper images show representative western blot detections of marker content in total protein extracts (50 μg) from treated HK2 cells. The blocking effect was assayed in HK2 cell cultures in 5mM (left white bars graphs) or 25mM (right black bars graphs) D-glucose. The graphs represent the mean ± SD of the ratio between immune signals of fibronectin or α-SMA vs β-actin. The ratio in HK2 cells without any pharmacological modulator was normalized to 1. * <i>P</i> < 0.05 versus untreated cells, # <i>P</i> < 0.05 versus NECA, n = 6.</p

Expression of ENT₁ and A₃AR in human kidney.

<p>A. Immunohistochemical detection of ENT1 and A₃ receptor proteins were carried out in human kidney sections from non-diabetic normal tissue and biopsies from diabetic nephropathy patients. Selected images denote representative progressive stages of renal injury probed by the content of α-smooth muscle actin (α-SMA) and pathological analysis. Arrows indicate interstitial distribution of the immune signal. Original magnification 200x. Scale bars 50μm. B. Representative negative control without primary antibody. Quantitative image analyses of immune staining for ENT1 (C) A₃AR (D) and α-SMA (E) in human biopsies. The graphs show quantitative results derived from staining analyses in defined areas (at least 5 glomeruli, 10 tubules and 10 interstitium per sample) using the UN-SCANIT 2.0 software. Human biopsies from 9 diabetic nephropathy patient and 3 controls are individualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147430#pone.0147430.s002" target="_blank">S1 Table</a>. Values in normal tissue samples were normalized to 1. * <i>P</i> < 0.05 vs normal.</p

Phenotypic transition of HK2 cells.

<p>The induction of profibrotic markers α-SMA, fibronectin and pro-collagen were evaluated by western blot in HK2 cells treated for 24 h with the general agonist of adenosine receptors NECA (5 μM), TGF-β (2 ng/ml) and high D-glucose (25mM). Representative western blots are shown.</p