Journal: PLoS ONE

Article Title: Reduced Adenosine Uptake and Its Contribution to Signaling that Mediates Profibrotic Activation in Renal Tubular Epithelial Cells: Implication in Diabetic Nephropathy

doi: 10.1371/journal.pone.0147430

Figure Lengend Snippet: A. Total and particular [ 3 H]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. * P < 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. * P < 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. * P < 0.05 versus control.

Article Snippet: Immunodetections were performed as described previously [ ] using the primary polyclonal anti-ENT1 antibody from Protein Tech (II 337-1-A7) and the monoclonal anti-αSMA (sc130617) and anti-A 3 AR (sc-13938) from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Activity Assay, Derivative Assay, Isolation, Control, Western Blot

Journal: PLoS ONE

Article Title: Reduced Adenosine Uptake and Its Contribution to Signaling that Mediates Profibrotic Activation in Renal Tubular Epithelial Cells: Implication in Diabetic Nephropathy

doi: 10.1371/journal.pone.0147430

Figure Lengend Snippet: 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. * P < 0.05 vs control, n = 6. C. Representative immunohistochemical detection of α-SMA in kidney sections from 8 weeks old ent1-/- 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. * P < 0.05 vs control or wild type. Original magnifications 200X.

Article Snippet: Immunodetections were performed as described previously [ ] using the primary polyclonal anti-ENT1 antibody from Protein Tech (II 337-1-A7) and the monoclonal anti-αSMA (sc130617) and anti-A 3 AR (sc-13938) from Santa Cruz Biotechnology.

Techniques: Immunohistochemical staining, Staining, Control, Derivative Assay, Software

Journal: PLoS ONE

Article Title: Reduced Adenosine Uptake and Its Contribution to Signaling that Mediates Profibrotic Activation in Renal Tubular Epithelial Cells: Implication in Diabetic Nephropathy

doi: 10.1371/journal.pone.0147430

Figure Lengend Snippet: A. Immunohistochemical detection of ENT1 and A 3 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) A 3 AR (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 . Values in normal tissue samples were normalized to 1. * P < 0.05 vs normal.

Article Snippet: Immunodetections were performed as described previously [ ] using the primary polyclonal anti-ENT1 antibody from Protein Tech (II 337-1-A7) and the monoclonal anti-αSMA (sc130617) and anti-A 3 AR (sc-13938) from Santa Cruz Biotechnology.

Techniques: Immunohistochemical staining, Negative Control, Staining, Derivative Assay, Software