Uric acid promotes apoptosis in human proximal tubule cells by oxidative stress and the activation of NADPH oxidase NOX 4

Abstract

Mild hyperuricemia has been linked to the development and progression of tubulointerstitial renal damage. However the mechanisms by which uric acid may cause these effects are poorly explored. We investigated the effect of uric acid on apoptosis and the underlying mechanisms in a human proximal tubule cell line (HK-2). Increased uric acid concentration decreased tubule cell viability and increased apoptotic cells in a dose dependent manner (up to a 7-fold increase, p<0.0001). Uric acid up-regulated Bax (+60% with respect to Ctrl; p<0.05) and down regulated X-linked inhibitor of apoptosis protein. Apoptosis was blunted by Caspase-9 but not Caspase-8 inhibition. Uric acid induced changes in the mitochondrial membrane, elevations in reactive oxygen species and a pronounced up-regulation of NOX 4 mRNA and protein (p<0.05). In addition, both reactive oxygen species production and apoptosis was prevented by the NADPH oxidase inhibitor DPI as well as by Nox 4 knockdown. URAT 1 transport inhibition by probenecid and losartan and its knock down by specific siRNA, blunted apoptosis, suggesting a URAT 1 dependent cell death. In summary, our data show that uric acid increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signalling and URAT 1 transport. These results might explain the chronic tubulointerstitial damage observed in hyperuricaemic states and suggest that uric acid transport in tubular cells is necessary for urate-induced effects.

Figure 1. UA decreases cell viability in proximal tubular epithelial cells.

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.

Figure 2. UA triggers apoptosis in proximal tubular epithelial cells.

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.

Figure 3. 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.

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.

Figure 4. Effect of UA on the disruption of the mitochondrial transmembrane potential and apoptosis proteins.

Upper panel: UA causes an alteration in the mitochondrial membrane potential, detected by the MitoCapture technique. (A) In control cells, the cathionic dye accumulates in the mitochondria and it fluoresces red. (B) In UA treated cells the mitochondrial membrane potential is dissipated and the Mitocapture dye is dispersed into the cell as green fluorescent monomers. (C) UA induces a significant increase in green cells (magnification x 400). Data shown as mean ± SEM, *p<0.05. Lower panel: Effect of UA on proteins implicated in apoptosis. HK-2 cells were exposed for 48 hours to 7.5–12 mg/dl UA and cell lysates were separated by SDS-PAGE and immunoblotted with antibodies to Bax and XIAP. (D) The graph shows Bax and XIAP changes over Ctrl. (E) Representative immunoblots from 3 different experiments are shown. Data shown as mean ± SEM, *p<0.05. Ctrl, Control untreated cells; UA, uric acid.

Figure 5. ROS production in UA treated cells.

(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.

Figure 6. Effects of UA on NOX 4 expression.

(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. DPI, diphenylene iodonium; UA, uric acid; T0, time 0; Ctrl, control untreated cells. NC, Negative Control.

Figure 7. Time course of mitogen-activated protein kinase (MAPK) activity by UA.

(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.

Figure 8. Efficacy of URAT1 inhibition.

(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.