UA decreases cell viability in proximal tubular epithelial cells.

<p>Effects of increasing doses of UA on HK-2 viability at 24 and 48 hours (MTT test). A decrease in cell viability was observed with higher concentrations (9–12 mg/dl). For each treatment group the number of cells at t = 0 served as baseline value 100% and was used to express the percentage of living cells. Data shown as mean ± SEM; *p<0.05 vs. Ctrl. UA, uric acid; Ctrl, control untreated cells.</p

ROS production in UA treated cells.

<p>(A, B) The panels show representative images of DCFH-DA and HE accumulation in cells after UA treatment. Pictures show the same fields in bright and fluorescence light (magnification x 400). (C, D) The graphics show a quantitative analysis of ROS production. The results are reported as percentage of DCFH-DA and HE positive cells. For each condition ∼350 cells were counted; (D) 10 mMol NAC did not alter cell viability, assessed by MTT test (E) NAC decrease apoptosis of HK-2 cells treated with 12 mg/dl UA. Cells were treated with NAC (10 mM) and UA for 48 hours. Cells were stained by anti-cleaved caspase 3 antibody and examined under microscope. Apoptotic cells were expressed as percent of total cells counted (∼350 cells). Data shown as mean ± SEM, * = p<0.001 vs. Ctrl. DCFH-DA, 2′–7′ dichlorofluorescein-diacetate, HE Hydroethidine; Ctrl, control untreated cells; UA, uric acid; NAC = N-acetyl-cysteine.</p

Effects of UA on NOX 4 expression.

<p>(A) NOX 4 expression was evaluated by real time-PCR at time 0 and 5 hours after 12 mg/dl UA incubation. The results are reported as fold increase to T0; (B) NOX 4 expression was evaluated by western blot at 24 hours. The results are reported as fold increase to Ctrl. (C) 10 10 µM DPI had no cytotoxic effects on HK-2 as assessed by MTT test. (D) DPI inhibits ROS production in UA treated cells. The results are reported as percentage of DCFH-DA positive cells. For each condition ∼350 cells were counted; (E) Effects of NOX 4 inhibition on apoptosis. HK-2 cells were treated with DPI (10 µM) and 12 mg/dl UA. Cells were immunostained by anti-cleaved caspase 3 antibody and examined under microscope. For each condition ∼350 cells were counted. Apoptotic cells were expressed as percent of total cells counted. (F) HK-2 were transfected with 60 nM nonspecific negative control siRNA (NC siRNA) or Nox 4 specific siRNA. Gene expression was evaluated by real time PCR after 24 hours. (G) Down-regulation of Nox 4 by RNA interference decreased UA induced apoptosis in Nox4 siRNA respect to NC siRNA. Pictures show the effects of Nox 4 silencing on cleaved caspase 3 expression (magnification x 1000). Data shown as mean ± SEM. *p<0.05 vs. time 0, § = p<0.005 vs. cells treated with 12 mg/dl UA, **p<0.001 vs. NC siRNA+UA, #p<0.01 vs. NC<b>.</b> DPI, diphenylene iodonium; UA, uric acid; T0, time 0; Ctrl, control untreated cells. NC, Negative Control.</p

Efficacy of URAT1 inhibition.

<p>(A) Effects of Losartan and Probenecid on UA-induced apoptosis. HK-2 were treated with 1–10 µM Losartan or 20 µM Probenecid and 12 mg/dl UA for 48 hours. Cells were stained by anti-cleaved caspase 3 antibody and examined under microscope. Apoptotic cells were expressed as percent of total cells counted (∼350 cells). Data shown as mean ± SEM, * = p<0.0001 vs. Cells incubated with 12 mg/dl UA. (B) Pictures show the effects of different treatments on cleaved caspase 3 expression (magnification x 400). (C) HK-2 were transfected with 60 nM nonspecific negative control siRNA (NC siRNA) or URAT 1 specific siRNA. Gene expression was evaluated by real time PCR after 24 hours. (D) Down-regulation of URAT 1 by RNA interference decreased UA induced apoptosis in URAT1 siRNA respect to NC siRNA. (E) Pictures show the effects of URAT1 silencing on cleaved caspase 3 expression (magnification x 1000). Data shown as mean ± SEM. §p<0.001 vs. NC siRNA, §§p<0.0001 vs NC siRNA exposed to UA. Abbreviations: UA, Uric acid; NC, Negative Control.</p

Sequences of gene-specific primers used in qRT-PCR.

<p>Sequences of gene-specific primers used in qRT-PCR.</p

Effects of caspase inhibitor 8 (Z-IEDT-FMK) and caspase inhibitor 9 (Z-LEHD-FMK) (50 µM) on cell viability and 12 mg/dl UA-induced apoptosis, at 48 hours.

<p>No significant effect of Caspases inhibitors on cell viability was observed by MTT (A). Apoptosis was evaluated by anti-cleaved caspase 3 antibody (B) and by annexin V/propidium iodide (C) and examined under microscope. Positive cells were expressed as percentage of total cells counted (∼350 cells for each condition). Data shown as mean ± SEM of three different experiments. *p<0.01, **p<0.0001 vs. Ctrl. UA, uric acid; Ctrl, control untreated cells.</p

Time course of mitogen-activated protein kinase (MAPK) activity by UA.

<p>(A) Effects of MAPKs inhibitors on HK-2 viability evaluated by MTT. HK-2 were treated for different time intervals (0–240 minutes) with 12 mg/dl UA. Then, phosphorylated p44/42 (B), p38 (C), SAPK/JNK (D) MAPKs were detected by Western blot. The pictures shown are representative of 3 experiments. The graphs represent relative phospho-MAPKs protein abundance normalized to MAPKs and data are expressed as fold increase respect to basal value (T0) and as means ± SEM of 3 independent experiments. (E) Effects of MAPKs inhibitors on UA-induced apoptosis. HK2 were treated with PD 98059 (a p44/42 MAPK inhibitor), SB 203580 (a p38 MAPK inhibitor), SP 600125 (a SAPK/JNK inhibitor), for 60 min before treatment with 12 mg/dl UA. Values are means ± SEM of 3 independent experiments with duplicate wells. *p<0.05, **p<0.01, §p<0.0001 vs. Time 0. #p<0.0001 vs. UA treated cells. UA, uric acid; min, minutes; T0, time 0.</p

UA triggers apoptosis in proximal tubular epithelial cells.

<p>HK-2 were exposed for 24–48 hours to UA (7.5–12 mg/dl). Apoptosis was evaluated by anti-cleaved caspase 3 (A) antibody and by annexin V/propidium iodide (C) and examined under microscope. Apoptotic cells were expressed as percent of total cells counted (>400 cells for each condition). Photos are representative of cleaved caspase 3 immunostaining (B) and of Annexin V/propidium iodide staining (D) (magnification x400). Data shown as mean ± SEM; *p<0.05, **p<0.001, vs. Ctrl. UA, uric acid; Ctrl, control untreated cells.</p

MOESM2 of Normoalbuminuric kidney impairment in patients with T1DM: insights from annals initiative

Additional file 2: Table S1. Baseline clinical characteristics of 676 patients with T1DM with low eGFR on the basis of micro- and macro-albuminuria. Table S2. Baseline clinical characteristics of 277 patients with DMT1 with low eGFR on the basis of micro- and macro-albuminuria

Clinical characteristics of study patients on the basis of the presence/ absence of metabolic syndrome and hyperuricemia (top gender-specific quintile).

<p>Clinical characteristics of study patients on the basis of the presence/ absence of metabolic syndrome and hyperuricemia (top gender-specific quintile).</p