Abstract
Background/Aims: Endothelial dysfunction is associated with mitochondrial alterations. We hypothesized that uric acid (UA), which can induce endothelial dysfunction in vitro and in vivo, might also alter mitochondrial function. Methods: Human aortic endothelial cells were exposed to soluble UA and measurements of oxidative stress, nitric oxide, mitochondrial density, ATP production, aconitase-2 and enoyl Co-A hydratase-1 expressions, and aconitase-2 activity in isolated mitochondria were determined. The effect of hyperuricemia induced by uricase inhibition in rats on renal mitochondrial integrity was also assessed. Results: UA-induced endothelial dysfunction was associated with reduced mitochondrial mass and ATP production. UA also decreased aconitase-2 activity and lowered enoyl CoA hydratase-1 expression. Hyperuricemic rats showed increased mitDNA damage in association with higher levels of intrarenal UA and oxidative stress. Conclusions: UA-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP. These studies provide additional evidence for a deleterious effect of UA on vascular function that could be important in the pathogenesis of hypertension and vascular disease.
Introduction
Mitochondrial disturbances characterized by the altered expression of the enzymes aconitase-2 (ACO-2) and enoyl CoA hydratase-1 (ECoH-1), as well as a significant reduction of mitochondrial mass, could be a key feature of endothelial dysfunction [1].
Rats made hyperuricemic by the inhibition of uricase with oxonic acid (OA) develop endothelial dysfunction, hypertension and renal damage [2, 3]; l-arginine supplementation, antioxidants and treatment with the xanthine oxidase inhibitor allopurinol prevented these alterations [2, 3, 4]. Uric acid (UA) induces endothelial dysfunction by increasing oxidative stress and reducing nitric oxide (NO) bioavailability [4, 5, 6, 7]. Moreover, a large number of studies links UA with endothelial dysfunction in humans, and others report that lowering UA with allopurinol can improve endothelial dysfunction [8, 9, 10, 11, 12, 13, 14, 15].
No studies have investigated whether endothelial dysfunction induced by UA may be associated with mitochondrial alterations such as observed by Addabbo et al. [1]. We therefore tested whether UA might alter mitochondrial mass, as well as ACO-2 and ECoAH-1 levels in cultured human aortic endothelial cells (HAEC). We also investigated whether experimental hyperuricemia in rats might reduce mitochondrial DNA or induce mitochondrial DNA damage in kidney tissue, a target organ of hyperuricemia.
Methods
Cell Culture. HAEC (Lonza, Walkersville, Md., USA) were grown in EGM-2 plus bullet kit media (Clonetics, Walkersville, Md., USA). Cells between passages 4 and 7 were used in the experiments.
Experimental Conditions and Viability Assay. UA solutions were prepared as previously described [16]. In order to simulate human conditions, low normal (208 µm, 3.5 mg/dl), high normal (416 µm, 7 mg/dl) and hyperuricemic (714 µm, 12 mg/dl) concentrations of UA were used in these experiments. After incubation with or without UA, viability analysis was done by trypan blue dye exclusion.
Measurement of Reactive Oxygen Species (ROS) Generation and the Effect of Apocynin. This was determined using the 2′,7′-dichlorofluorescein diacetate dye (DCF-DA; Molecular Probes, Eugene, Oreg., USA). Preparations were imaged using a laser scanning confocal microscope (LSM510; Zeiss) and data analyzed by using the LSM Image Analyzer post-acquisition software (Zeiss). The effect of the NADPH oxidase inhibitor apocynin (100 µm) was also determined.
Determination of NO Production. Was assessed using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA; Cayman Chemical, Ann Arbor, Mich., USA) and examined using a laser scanning confocal microscope. The total amount of NOx (nitrate and nitrite) was also determined using a commercial Kit (Active Motif, Carlsbad, Calif., USA).
Quantification of Mitochondrial DNA. This was performed by real-time quantitative PCR. The primer sequences for mtDNA, and those for nDNA for loading normalization, 18S rRNA gene were reported previously [17]. Quantitative PCR was performed using a SYBR Green Master Mix (JumpStart® TaqReadymix®; Sigma) on a Bio-Rad iCycler. Nuclear internal control DNA and the amount of mitochondrial DNA were calculated by the comparative CT method.
Mitochondrial Labeling in Live Cells. This was assessed by using the specific mitochondrial dye MitoTracker Orange™ CMTMRos (Molecular Probes). A staining protocol was done following the manufacturer’s instructions. Cells were examined using a laser scanning confocal microscope with a peak excitation wavelength of 554 nm and a peak emission wavelength of 576 nm.
Immunoblotting. The following primary antibodies were assessed: anti-ACO-2 (Sigma-Prestige, St. Louis, Mo., USA), anti-ECoH-1 (Protein Tech Group, Chicago, Ill., USA), and anti-β-actin (Cell Signaling Technology, Beverly, Mass., USA). Blots were visualized using a horseradish peroxidase secondary antibody. Chemiluminescence was recorded and results analyzed with the 1D Image Software (Kodak Digital Science, Rochester, N.Y., USA).
Aconitase Activity Assay. Pure mitochondrial fractions were isolated using a reagent-based method that enables the isolation of intact mitochondria by differential centrifugation (No. 89874; Thermo Scientific, Rockford, Ill., USA) and assessed for ACO-2 activity using a commercial kit (BioVision, Milpitas, Calif., USA).
Measurement of Intracellular ATP Content. This was measured by using an ATP bioluminescence assay kit (CLS II; Roche, Mannheim, Germany).
Animal Model. Ten male Sprague-Dawley rats were studied, 5 received OA (750 mg/kg b.w. by gavage) during 8 weeks, 5 animals received vehicle and served as controls. Awake systolic blood pressure (XBP1000; Kent Scientific, Torrington, Conn., USA), proteinuria (Bradford method) and plasma UA (Amplex-Red Kit Molecular Probes) were measured at the end of 8 weeks. At sacrifice the right kidney was extirpated and the renal cortex separated from the medulla, frozen in liquid nitrogen and stored at –80°C until processed. Left kidney was fixed in 4% paraformaldehyde for further processing and histological analysis. All animal experiments and procedures were approved by the INC Ignacio Chávez Ethics Committee.
Renal Cortex UA Measurement. UA was extracted as previously described [18] and measured using Amplex-Red Kit (Molecular Probes).
Renal Cortex Protein Carbonyl Measurement. Tissue was homogenized in phosphate buffer containing a cocktail of protease inhibitors. Protein carbonyls reacted with dinitrophenylhydrazine to form protein hydrazones which was measured at 370 nm [26].
Evaluation of Glomerulosclerosis and Tubulointerstitial Fibrosis. Two-micrometer sections were stained with Masson’s trichrome. Ten non-crossed fields of cortex (640 × 477 mm, 10×) per biopsy were analyzed by light microscopy (Olympus BX51; Olympus American, Melville, N.Y., USA) and captured with a digital camera (VF Evolution; Media Cybernetics, Silver Spring, Md., USA). Positive blue color areas (excluding glomeruli and vessels) were analyzed in Image Pro Plus (Media Cybernetics). Glomerulosclerosis was also evaluated in Masson’s trichrome-stained slides divided in four quadrants. Segmental and global sclerosed glomeruli were reported as percent of the total number of glomeruli counted in one quadrant.
Evaluation of Renal Inflammatory Infiltration. This was assessed by indirect peroxidase immunostaining using an antibody against the common leukocyte antigen (CD45) counterstained with hematoxylin. Negative control consisted of slides incubated with non-relevant antisera. Inflammation was evaluated in 30 non-overlapping ×400 cortical fields, excluding glomeruli, per biopsy. Results were expressed as positive cells per 0.074 mm2 using the mean of the 30 fields.
Assay for Relative mitDNA Copy Number and the Common mitDNA Deletion. Total renal cortex DNA was isolated using Quick-gDNA MiniPrep (Zymo Research, Irvine, Calif., USA). Primers and probes for the rat D-loop (mitDNA copy number) and the rat mitDNA deletion from the rat mitochondrial genome were previously reported [19]. mitDNA was compared against the nuclear gene for 18S rRNA. Fluoresce spectra was continuously monitored by the ABI-Prism 7300 Sequence Detection System (Applied Biosystems, Carlsbad, Calif., USA) with sequence detection software version 1.3.1. Data analysis was based on measurement of the CT and relative copy numbers were calculated as 2-ΔΔCT[19].
Statistical Analysis. All data are presented as the mean ± SEM. Data graphics and statistical analysis were performed using Prism 5 (GraphPad Software, San Diego, Calif., USA). Multiple group analysis was done by ANOVA and post hoc comparisons were done by a Bonferroni test. Analysis of two groups was done by a t test. In most cases, experiments were performed three times with independent replicates. p values <0.05 were considered statistically significant.
Results
Cell Culture
UA Did Not Affect HAEC Viability. Exposure of HAEC to varying concentrations of UA or control for 48 h had no effect on cell viability, with 90–95% of cells remaining viable for this time period.
UA Exposure Increased Intracellular Oxidative Stress. Incubation with UA for 48 h produced a 200% increment in intracellular oxidative stress. Co-incubation with the antioxidant apocynin almost completely prevented this effect (fig. 1a). Thus, we confirmed the pro-oxidant effect of UA inside the cell, as previously reported in other lines or primary cultures of human and rodent endothelial cells [7].
UA Incubation Decreased eNOS-Stimulated NO Synthesis. As shown in figure 1b, an incubation with 714 µm of UA for 48 h significantly decreased DAF-2-labeled NO level in response to the combination of insulin and A23187. Likewise, the accumulation of NOx products (nitrates plus nitrites) in culture media after activation of eNOS with insulin and A23187 showed the same trend (fig. 1c). These findings are in agreement with previous reports [4, 7].
UA-Induced Mitochondrial Alterations in HAEC (fig. 2). Since it was recently reported that specific mitochondrial alterations are an early marker of endothelial dysfunction [1], we determined whether UA exposure might induce the same effect in HAEC. Endothelial cells incubated with UA for 24 and 48 h showed a 50% reduction in mitochondrial DNA levels (fig. 2a). Quantification of mitochondrial labeling intensity with MitoTracker Orange showed a significant lower fluorescence in cells treated with UA (714 µm) for 48 h compared to control cultures (fig. 2b) indicating a decrease in mitochondrial mass. In addition, we found a significant reduction in the protein expression of the mitochondrial enzyme ECoAH-1 at 24 and 48 h (fig. 2c), while ACO-2 protein expression was modestly and transiently reduced at 24 h but not at 48 h (data not shown). Nevertheless, incubation of HAEC with UA for 48 h resulted in a significant reduction in ACO-2 (fig. 2d).
UA Treatment Induced a Significant Reduction in the Basal Concentration of ATP. HAEC exposed to UA for 48 h showed a reduced basal intracellular ATP concentration of 30, 39 and 43% for concentrations of 208, 416 and 714 µm of UA, respectively (fig. 2e).
Animal Model
As previously shown, we confirmed that treatment with the uricase inhibitor OA could induce mild hyperuricemia and endothelial dysfunction characterized by systemic hypertension, glomerulosclerosis and renal inflammatory infiltration (table 1; fig. 3a). At this time point, tubulointerstitial fibrosis was not evident. In the present study we also report that OA-treated rats had significantly higher intrarenal levels of UA associated with greater oxidative stress (fig. 3b). The increase in plasma and intrarenal UA was associated with lower relative mitDNA copy number and significantly higher relative mitDNA common deletion copy number in the renal cortex, resulting in a high mitDNA deletion/mitDNA ratio in OA-treated rats (fig. 3c).
Discussion
We report that UA exposure reduced mitochondrial mass, decreased the expression of ECoAH1 and reduced the activity of ACO-2 in isolated mitochondria from HAEC. Collectively these alterations are considered the mitochondrial manifestations of early endothelial dysfunction [1]. In addition, UA decreased the basal concentration of ATP in these cells. Importantly, we reproduced mitDNA alterations in renal tissue of hyperuricemic rats induced by the uricase inhibitor, OA, providing additional evidence for a general deleterious effect of UA on mitDNA.
In the present studies, we first corroborated the most distinctive effects of UA on endothelial cells, those consisting of increased oxidative stress partially mediated by NADPH activation (suggested by the protective effect of apocynin), and reduced bioavailability of NO (fig. 1) [4, 7].
Mitochondrial dysfunction has recently been described as a characteristic feature associated with endothelial dysfunction and was characterized by a reduction of mitochondrial mass and selective depletion of ACO-2 and ECoAH-1 [1]. In the present study, we observed similar effects induced by UA exposure: a significant reduction of mitochondrial mass coupled with reduced expression of ECoH-1 and ACO-2 activity, although ACO-2 expression remained without changes. Contrary to ACO-2, for ECoH-1, there is no evidence for the participation of metal ions or cofactors in its catalytic mechanism that can be inactivated by oxidative stress [20]. Moreover, ECoH-1 is one of the most proficient catalysts known; therefore, in order to accomplish a decrement in activity, the enzyme levels need to be downregulated [21]. On the other hand, the structural predilection of iron-sulfur center in ACO-2 to ROS and peroxynitrite may in part explain its selective vulnerability [22]. In the present study, we report a significantly reduced activity of the enzyme in mitochondria isolated from cells treated with UA with evidence for increased oxidative stress in the same conditions. Thus we speculate that UA-induced oxidative stress acts to inactivate ACO-2 in this setting.
We also documented a lower basal concentration of ATP in cells exposed to UA. We can provide three mechanisms that might contribute to this effect: (1) by blocking ACO-2 in the Krebs cycle, and by reducing β-fatty acid oxidation via reduction in ECoAH it is possible that both effects participate in decreasing ATP generation; (2) UA treatment was also associated with a slight but significant reduction in mitochondrial mass as noted by the reduction in mitochondrial DNA/nuclear DNA ratio and as determined by the MitoTracker assay, and (3) it was shown that acute high concentrations of UA produced mitochondrial calcium overload in HUVEC, resulting in a significant increment of mitochondrial membrane potential that could lead to excess generation of ROS [23]. These effects are suggestive of mitochondrial uncoupling, which can be associated with a decreased synthesis of ATP through oxidative phosphorylation. Nevertheless, we cannot rule out a detrimental effect of UA on the ATP produced by glycolysis, since endothelial cells tend to have a strong glycolytic pathway.
Since we previously found increased oxidative stress and endothelial dysfunction in the kidney of hyperuricemic rats, we also examined whether similar mitochondrial changes might occur in this model. To assess mitochondrial injury we evaluated both the relative mitDNA copy number and the relative mitDNA common deletion copy number. The increase of deleted mitDNA is considered a marker of increased mitDNA damage induced by oxidative stress; as such it is augmented in a number of pathological conditions and during aging [24, 25]. We found that increased levels of plasma and renal UA and augmented renal oxidative stress were associated with a decrement in the relative mitDNA copy number and an increased proportion of mitDNA common deletion. These findings are in agreement with the results in HAEC exposed to UA. Since renal tissue includes more cell types than endothelial cells, we speculate that the effect of UA on mitochondrial damage is a more general process that may affect diverse cell types. Figure 4 summarizes the mechanisms potentially involved in the endothelial dysfunction induced by UA.
In summary, UA-induced endothelial dysfunction is associated with mitochondrial dysfunction and reduced ATP generation. These studies provide additional evidence for a deleterious effect of UA on vascular function that could be important in the pathogenesis of hypertension and vascular disease.
Acknowledgements
Support for this paper was provided by NIH grants HL-68607 and DK-90859, Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea grant A101742, and National Council of Science and Technology (CONACyT) Mexico grants 081054 and 0133232. We thank Benito Chavez-Renteria for processing the renal sections for histological evaluation.
Disclosure Statement
Dr. Johnson has a patent with the University of Washington for allopurinol in the treatment of hypertension (7,799,794) and has patent applications with the University of Florida and with the University of Colorado for UA-lowering therapy and/or treatments to block fructose metabolism in the treatment of metabolic syndrome or diabetic nephropathy.