Protein carbamylation exacerbates vascular calcification

Protein carbamylation is a posttranslational modification that can occur non-enzymatically in the presence of high concentrations of urea. Although carbamylation is recognized as a prognostic biomarker, the contribution of protein carbamylation to organ dysfunction remains uncertain. Because vascular calcification is common under carbamylation-prone situations, we investigated the effects of carbamylation on this pathologic condition. Protein carbamylation exacerbated the calcification of human vascular smooth muscle cells (hVSMCs) by suppressing the expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1), a key enzyme in the generation of pyrophosphate, which is a potent inhibitor of ectopic calcification. Several mitochondrial proteins were carbamylated, although ENPP1 itself was not identified as a carbamylated protein. Rather, protein carbamylation reduced mitochondrial membrane potential and exaggerated mitochondria-derived oxidative stress, which down-regulated ENPP1. The effects of carbamylation on ectopic calcification were abolished in hVSMCs by ENPP1 knockdown, in mitochondrial-DNA-depleted hVSMCs, and in hVSMCs treated with a mitochondria-targeted superoxide scavenger. We also evaluated the carbamylation effects using ex vivo and in vivo models. The tunica media of a patient with end-stage renal disease was carbamylated. Thus, our findings have uncovered a previously unrecognized aspect of uremia-related vascular pathology. Protein carbamylation is a posttranslational modification that can occur non-enzymatically in the presence of high concentrations of urea. Although carbamylation is recognized as a prognostic biomarker, the contribution of protein carbamylation to organ dysfunction remains uncertain. Because vascular calcification is common under carbamylation-prone situations, we investigated the effects of carbamylation on this pathologic condition. Protein carbamylation exacerbated the calcification of human vascular smooth muscle cells (hVSMCs) by suppressing the expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1), a key enzyme in the generation of pyrophosphate, which is a potent inhibitor of ectopic calcification. Several mitochondrial proteins were carbamylated, although ENPP1 itself was not identified as a carbamylated protein. Rather, protein carbamylation reduced mitochondrial membrane potential and exaggerated mitochondria-derived oxidative stress, which down-regulated ENPP1. The effects of carbamylation on ectopic calcification were abolished in hVSMCs by ENPP1 knockdown, in mitochondrial-DNA-depleted hVSMCs, and in hVSMCs treated with a mitochondria-targeted superoxide scavenger. We also evaluated the carbamylation effects using ex vivo and in vivo models. The tunica media of a patient with end-stage renal disease was carbamylated. Thus, our findings have uncovered a previously unrecognized aspect of uremia-related vascular pathology. Vascular calcification, a pathologic condition that erodes the compliance and elastance of the aorta, is common in aging and in chronic kidney disease (CKD).1Persy V. D’Haese P. Vascular calcification and bone disease: the calcification paradox.Trends Mol Med. 2009; 15: 405-416Google Scholar Because vascular calcification is an independent predictor of mortality,2Wilson P.W.F. Kauppila L.I. O’Donnell C.J. et al.Abdominal aortic calcific deposits are an important predictor of vascular morbidity and mortality.Circulation. 2001; 103: 1529-1534Google Scholar, 3Hernández D. Rufino M. Bartolomei S. et al.Clinical impact of preexisting vascular calcifications on mortality after renal transplantation.Kidney Int. 2005; 67: 2015-2020Google Scholar numerous studies in both animals and humans have investigated its underlying pathologic mechanisms.4Walsh C.R. Cupples L.A. Levy D. et al.Abdominal aortic calcific deposits are associated with increased risk for congestive heart failure: the Framingham heart study.Am Heart J. 2002; 144: 733-739Google Scholar, 5Cannata-Andia J.B. Roman-Garcia P. Hruska K. The connections between vascular calcification and bone health.Nephrol Dial Transplant. 2011; 26: 3429-3436Google Scholar, 6Leening M.J.G. Elias-Smale S.E. Kavousi M. et al.Coronary calcification and the risk of heart failure in the elderly: the Rotterdam study.JACC Cardiovasc Imaging. 2012; 5: 874-880Google Scholar However, several key aspects of vascular calcification remain unresolved. Posttranslational protein modifications are involved in the mechanisms of various human diseases. Carbamylation comprises an irreversible posttranslational protein modification that can occur nonenzymatically in the presence of urea.7Stark G.R. Stein W.H. Moore S. Reactions of the cyanate present in aqueous urea with amino acids and proteins.J Biol Chem. 1960; 235: 3177-3181Abstract Full Text PDF Google Scholar, 8Kalim S. Karumanchi S.A. Thadhani R.I. et al.Protein carbamylation in kidney disease: pathogenesis and clinical implications.Am J Kidney Dis. 2014; 64: 793-803Google Scholar In the human body, urea exists in equilibrium with cyanate and its reactive form isocyanate, which can add the carbamoyl moiety [-CONH2] to the amino groups of free amino acids and the Nε-amino group of protein lysine residues (homocitrulline).9Jaisson S. Pietrement C. Gillery P. Carbamylation-derived products: bioactive compounds and potential biomarkers in chronic renal failure and atherosclerosis.Clin Chem. 2011; 57: 1499-1505Google Scholar Myeloperoxidase-dependent oxidation of thiocyanate has been recently revealed as an alternative pathway that induces cyanate in the body.10Wang Z. Nicholls S.J. Rodriguez E.R. et al.Protein carbamylation links inflammation, smoking, uremia and atherogenesis.Nat Med. 2007; 13: 1176-1184Google Scholar Notably, carbamylation is recognized as a prognostic biomarker in humans. Positive association with mortality has been reported for the serum level of protein-bound homocitrulline in patients undergoing maintenance hemodialysis, the plasma level of carbamylated low-density lipoprotein in predialysis patients with CKD, and the plasma level of protein-bound homocitrulline in non-CKD study participants.10Wang Z. Nicholls S.J. Rodriguez E.R. et al.Protein carbamylation links inflammation, smoking, uremia and atherogenesis.Nat Med. 2007; 13: 1176-1184Google Scholar, 11Koeth R.A. Kalantar-Zadeh K. Wang Z. et al.Protein carbamylation predicts mortality in ESRD.J Am Soc Nephrol. 2013; 24: 853-861Google Scholar, 12Speer T. Owala F.O. Holy E.W. et al.Carbamylated low-density lipoprotein induces endothelial dysfunction.Eur Heart J. 2014; 35: 3021-3032Google Scholar These reports suggested that protein carbamylation is associated with mortality regardless of CKD stage. However, the mechanism by which protein carbamylation affects mortality remains uncertain. Here, we investigated whether protein carbamylation exacerbates medial vascular calcification (VC). We hypothesized that protein carbamylation would exaggerate medial VC based on several points. First, medial calcification is common in patients with CKD whose protein carbamylation ratios are elevated.13Berg A.H. Drechsler C. Wenger J. et al.Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure.Sci Transl Med. 2013; 5: 175ra29Google Scholar Second, medial calcification is also common in elderly individuals,1Persy V. D’Haese P. Vascular calcification and bone disease: the calcification paradox.Trends Mol Med. 2009; 15: 405-416Google Scholar and Gorisse et al.14Gorisse L. Pietrement C. Vuiblet V. et al.Protein carbamylation is a hallmark of aging.Proc Natl Acad Sci. 2016; 113: 1191-1196Google Scholar recently reported that the protein carbamylation ratio is elevated in the elderly. Third, a low-protein diet exacerbates medial VC in uremic rats through unknown mechanisms,15Price P.A. Roublick A.M. Williamson M.K. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate.Kidney Int. 2006; 70: 1577-1583Google Scholar and Berg et al.13Berg A.H. Drechsler C. Wenger J. et al.Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure.Sci Transl Med. 2013; 5: 175ra29Google Scholar have reported that mice with low-protein-diet–induced amino acid deficiencies showed increased susceptibility to carbamylation. Accordingly, we performed various assays to examine carbamylation effects using in vitro, ex vivo, and in vivo models of medial calcification. Our results provide novel insights into the biology of protein carbamylation. We used cultured human vascular smooth muscle cells (hVSMCs) to assess the effects of protein carbamylation on medial VC. hVSMCs were first treated with urea at 250 mmol/l for 3 days and then cultured in normal or calcifying medium for 20 days without urea. Urea was not included during the 20-day culture because we aimed to examine effects of carbamylation, not the direct effects of urea. Equimolar mannitol-pretreated cells served as control cells. Both quantification of cellular minerals and alizarin red staining revealed that urea pretreatment exaggerated calcification under calcifying conditions (Figure 1a and b). To more directly evaluate the effects of carbamylation, we performed similar experiments using 10 mmol/l of cyanate as a carbamylation inducer. Mineral quantification revealed that cyanate pretreatment also exaggerated hVSMC calcification in the calcifying medium (Figure 1a and b). To investigate the molecular mechanisms underlying the aforementioned process, we performed real-time polymerase chain reaction (PCR) analyses, which revealed that culturing hVSMCs in the calcifying medium resulted in up-regulated mRNA expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1) (Figure 1c). ENPP1 constitutes the key enzyme that hydrolyzes extracellular adenosine triphosphate (ATP) into adenosine monophosphate and pyrophosphate (PPi). Because PPi is a potent inhibitor of ectopic mineralization, the up-regulation of ENPP1 in the calcifying medium serves as a counterregulatory response that suppresses calcification.16Fleisch H. Russell R.G. Straumann F. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis.Nature. 1966; 212: 901-903Google Scholar, 17Meyer J.L. Can biological calcification occur in the presence of pyrophosphate?.Arch Biochem Biophys. 1984; 231: 1-8Google Scholar, 18Addison W.N. Azari F. Sørensen E.S. et al.Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity.J Biol Chem. 2007; 282: 15872-15883Google Scholar Here, both urea pretreatment and cyanate pretreatment suppressed the counterregulatory elevation of ENPP1 under calcifying conditions (Figure 1c). Pretreatment with cyanate but not urea suppressed runt-related transcription factor 2 (RUNX2) expression under calcifying conditions. Because RUNX2 is an inducer of VC, down-regulated RUNX2 did not contribute to the pathogenesis in this study.19Ducy P. Zhang R. Geoffroy V. et al.Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.Cell. 1997; 89: 747-754Google Scholar, 20Inada M. Yasui T. Nomura S. et al.Maturational disturbance of chondrocytes in Cbfa1-deficient mice.Dev Dyn. 1999; 214: 279-290Google Scholar Moreover, protein carbamylation did not affect the levels of other calcification-related molecules, including bone morphogenic protein 2, tissue-nonspecific alkaline phosphatase (TNAP), osteoprotegerin, and human homolog of progressive ankyloses (Figure 1c). Western blotting analyses further confirmed that protein carbamylation suppressed the counterregulatory elevation of ENPP1 under calcifying conditions (Figure 1d). Consequently, PPi levels in the calcifying culture medium decreased when cells were pretreated with either urea or cyanate (Figure 1e). Cyanate pretreatment also suppressed PPi levels in the normal culture medium (Figure 1e). Exogenous PPi added into the culture media suppressed calcification of urea- or cyanate-pretreated hVSMCs in a dose-dependent manner (Figure 2a). Calcification was exacerbated by protein carbamylation in nontargeted short hairpin RNA (shRNA)-transfected control hVSMCs, but not in ENPP1-knockdown cells (Figure 2b and c). All these data indicated that the suppression of ENPP1 played essential roles in the effects of carbamylation on ectopic calcification. We also examined whether inflammation, a key factor that exacerbates VC, contributed to the pathogenesis of protein carbamylation. Levels of interleukin-6 and -1β were not elevated by carbamylation (Supplementary Figure S1). Western blotting analyses of proteins isolated from urea- or cyanate-treated hVSMCs revealed that multiple proteins were carbamylated (Figure 3a and b). We also analyzed protein carbamylation levels in rat aortic tissues. Samples were obtained from 6-week-old male Sprague Dawley (SD) rats that had been randomly divided into 2 groups and injected for 3 days with either isotonic saline (“vehicle” group) or cyanate (“cyanate” group). In agreement with the in vitro results, multiple aortic proteins were carbamylated in the tissue from the cyanate group (Figure 3c). To analyze effects of carbamylation on ectopic calcification more precisely, we inhibited carbamylation by treating hVSMCs with glycylglycine, a compound that has been reported to inhibit carbamylation.13Berg A.H. Drechsler C. Wenger J. et al.Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure.Sci Transl Med. 2013; 5: 175ra29Google Scholar Western blotting analysis showed that glycylglycine inhibited protein carbamylation (Figure 3d). The extent of calcification was attenuated in glycylglycine-pretreated hVSMCs (Figure 3e). We confirmed the specificity of the anti-carbamylated-lysine antibody used in this study, which detected carbamylated bovine serum albumin (BSA) but not unmodified or glycated BSA in Western blots (Supplementary Figure S2). We used immunoprecipitation in combination with mass spectrometry (MS) and identified carbamylated proteins in hVSMCs and rat aortae (Tables 1 and 2). The results showed that several of the same mitochondrial and cytoskeletal proteins were carbamylated both in vitro and in vivo (underlined molecules in Tables 1 and 2). We confirmed the carbamylation of vimentin, a molecule listed in Tables 1 and 2, by an immunoprecipitation assay (Supplementary Figure S3). Notably, ENPP1 was not detected as a carbamylated protein (Tables 1 and 2). In addition, we confirmed that ENPP1 was not carbamylated through an immunoprecipitation assay (Supplementary Figure S3). Because ENPP1 expression was inhibited at the transcriptional level (Figure 1c), our results suggested that the carbamylation of proteins other than ENPP1 caused the suppression of ENPP1 expression.Table 1Carbamylated proteins in urea-treated hVSMCsGroupProteinMW (kDa)Percentage of sequence homology/coverageMascot scoreMitochondrialATP synthase subunit α, mitochondrial59.752100/32644.32proteinsATP synthase subunit β, mitochondrial56.561100/20392.71ADP/ATP translocase 133.065100/11220.36ADP/ATP translocase 332.86799/10147.97MICOS complex subunit MIC6078.975100/20447.58CytoskeletalFilamin-A280.729100/6440.89proteinsFilamin-C291.015100/7542.21α-internexin55.392100/33512.08Nestin177.437100/3123.18Vimentin53.653100/581990.07Tubulin α-1C chain57.731100/35846.43Tubulin β chain47.767100/25382.41Tubulin β-2A chain49.907100/18332.63Plectin531.784100/244162.83Cell adhesionCollagen α-3(VI) chain343.667100/171546.16proteinsFibronectin262.617100/231898.8Integrin α-2129.298100/8258.17Vitronectin54.306100/4105.61Nuclear proteinsHistone H411.368100/1264.85IgsIg γ-1 chain C region52.425100/10165.21EnzymesATP-dependent 6-phosphofructokinase85.019100/22375.53ATP-dependent RNA helicase DDX3X73.246100/7182.07β-galactosidase76.078100/15389.75Glutamine-fructose-6-phosphate aminotransferase [isomerizing] 178.808100/5110.99Peroxisomal multifunctional enzyme type 279.689100/6156.99Probable ATP-dependent RNA helicase DDX1780.274100/4101.05Pyruvate kinase57.938100/22434.72Vesicle-fusing ATPase82.095100/15248.32OthersCoagulation factor XIII A chain83.267100/3111.27Coatomer subunit α138.349100/23942.01Heat shock protein β-122.783100/1672.32Interferon-induced GTP-binding protein Mx175.522100/10181.48Myosin, heavy polypeptide 9226.538100/3107.83Prothrombin65.408100/10403.22Ras GTPase-activating-like protein189.259100/9511.26Ribonuclease inhibitor49.974100/568.87Sequestosome-147.687100/12267.36Signal transducer and activator of transcription83.363100/9174.09Spermatogenesis-associated protein 5-like protein 180.712100/3105.54T-complex protein 1 subunit ε59.672100/9184.260 kDa heat shock protein, mitochondrial61.056100/24453.5978 kDa glucose-regulated protein72.335100/421116.7ADP, adenosine diphosphate; ATP, adenosine triphosphate; DDX3X, DEAD box helicase 3, x-linked; DDX17, DEAD box protein 17; GTP, guanosine triphosphate; hVSMCs, human vascular smooth muscle cells; MICOS, mitochondrial contact site; MW, molecular weight; Mx1, myxovirus resistance protein 1. Carbamylated proteins in urea-treated hVSMCs (Figure 3a) were identified by performing immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Underlined molecules are also identified in Table 2. Open table in a new tab Table 2Carbamylated proteins in cyanate-treated rat aortaGroupProteinMW (kDa)Percentage of sequence homology/coverageMascot scoreMitochondrialATP synthase subunit α, mitochondrial59.755100/38336.48proteinsATP synthase subunit β, mitochondrial56.345100/1270.26Trifunctional enzyme subunit α, mitochondrial82.667100/7108.32CytoskeletalActin, cytoplasmic 142.087100/33150.45proteinsFilamin-A280.485100/4103.88Desmin53.458100/41162.85Vimentin53.702100/3488.11OthersDesmoplakin332.401100/1136.68Myosin, heavy polypeptide 9226.419100/28847.78Myosin, heavy polypeptide 10229.004100/22441.65Myosin, heavy polypeptide 11227.302100/331174.71Myosin regulatory light chain 12B19.780100/1750.17PDZ and LIM domain protein 749.913100/20126.62Polymerase I and transcript release factor43.909100/15186.04ATP, adenosine triphosphate; LIM, lin11, isl-1, and mec-3; MW, molecular weight; PDZ, PSD-95, Dlg, and ZO-1. Carbamylated proteins in cyanate-treated rat aorta (Figure 3c) were identified by performing immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Underlined molecules are also identified in Table 1. Open table in a new tab ADP, adenosine diphosphate; ATP, adenosine triphosphate; DDX3X, DEAD box helicase 3, x-linked; DDX17, DEAD box protein 17; GTP, guanosine triphosphate; hVSMCs, human vascular smooth muscle cells; MICOS, mitochondrial contact site; MW, molecular weight; Mx1, myxovirus resistance protein 1. Carbamylated proteins in urea-treated hVSMCs (Figure 3a) were identified by performing immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Underlined molecules are also identified in Table 2. ATP, adenosine triphosphate; LIM, lin11, isl-1, and mec-3; MW, molecular weight; PDZ, PSD-95, Dlg, and ZO-1. Carbamylated proteins in cyanate-treated rat aorta (Figure 3c) were identified by performing immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Underlined molecules are also identified in Table 1. To investigate whether the carbamylation of mitochondrial proteins, such as ATP synthase subunits α and β (Tables 1 and 2), plays essential roles in exacerbating calcification, we used hVSMCs in which the mitochondrial DNA had been depleted but the nuclear DNA had been retained: ρ0-hVSMCs. Although ATP synthase subunits α and β are encoded by nuclear DNA, these subunits cannot fully function as ATP synthase (complex V of the oxidative phosphorylation pathway) without 2 subunits encoded by mitochondrial DNA, ATPase 6 and ATPase 8.21García J.J. Ogilvie I. Robinson B.H. et al.Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation: comparison with the enzyme in Rho(0) cells completely lacking mtDNA.J Biol Chem. 2000; 275: 11075-11081Google Scholar, 22Baracca A. Barogi S. Carelli V. et al.Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a.J Biol Chem. 2000; 275: 4177-4182Google Scholar, 23Sgarbi G. Baracca A. Lenaz G. et al.Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA.Biochem J. 2006; 500: 493-500Google Scholar Depletion of mitochondrial DNA in ρ0-hVSMCs was confirmed using PCR (Figure 4a). We used ρ0-hVSMCs in experiments similar to those shown in Figure 1 and found that the exacerbating effect of urea on calcification was not observed in ρ0-hVSMCs (Figure 4b). Furthermore, mitochondrial DNA depletion also abolished the effects of cyanate on hVSMC calcification (Figure 4c). The absolute values of Figure 4b and c were higher than those of Figure 1a, indicating that proper mitochondrial function is required for the protection of hVSMCs from calcification stress. We analyzed how carbamylation of ATP synthase subunits α and β exacerbated ectopic calcification. Immunohistochemical analyses of urea- or cyanate-treated hVSMCs showed that ATP synthase subunit α and carbamylated lysine were colocalized (Figure 5a). The carbamylation of ATP synthase subunit α in cyanate-treated hVSMCs was also confirmed by an immunoprecipitation assay (Figure 5b). Using liquid chromatography and tandem MS (LC-MS/MS) analysis, we found that at least 4 lysine residues in ATP synthase subunit α were carbamylated (Figure 5c). Furthermore, we tested whether carbamylation induces mitochondrial dysfunction and resultant oxidative stress. Mitochondrial membrane potential, assessed by tetramethylrhodamine ethyl ester (TMRE) staining, was reduced after urea or cyanate treatment (Figure 6a). All parameters of mitochondrial oxygen consumption rate (OCR)—basal respiration, maximal respiration, and ATP production—were suppressed in cyanate-treated hVSMCs (Figure 6b). Mitochondrial superoxide measurement by MitoSOX Red (Thermo Fisher Scientific, Waltham, MA) indicated that carbamylated hVSMCs were exposed to mitochondria-derived oxidative stress (Figure 6c). Mitochondrial-specific scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-TEMPO; ENZO Life Sciences International, Inc., Plymouth Meeting, PA) suppressed oxidative stress induced by protein carbamylation (Figure 6c). To investigate whether mitochondria-derived oxidative stress affects the extent of calcification, we performed experiments similar to those in Figure 1a under the presence of Mito-TEMPO (Figure 6d). The effects of protein carbamylation on hVSMC calcification was abolished by Mito-TEMPO (Figure 6d). Because carbamylated hVSMCs were exposed to oxidative stress, we examined whether oxidative stress affected ENPP1 expression in these cells. The induction of ENPP1 expression under calcifying conditions was suppressed by H2O2 in a dose-dependent manner at both mRNA and protein levels without affecting cell viability (Figure 6e and f). Antimycin A, a mitochondrial reactive oxygen species generator, also suppressed the expression of ENPP1 in hVSMCs under calcifying conditions (Figure 6g). These findings indicated that oxidative stress derived from mitochondria suppressed the counterregulatory up-regulation of ENPP1 in hVSMCs.Figure 6Protein carbamylation in vitro causes mitochondrial dysfunction and a resultant potentiation of oxidative stress, which suppresses ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1) expression. (a) Protein carbamylation reduced mitochondrial membrane potential in human vascular smooth muscle cells (hVSMCs). hVSMCs treated with urea (250 mmol/l) for 3 days (Ur) or cyanate (10 mmol/l) for 4 hours (Cyn) were analyzed, with mannitol (Man)- or distilled water (Veh)-treated cells serving as their respective control cells. Mitochondrial membrane potential was assessed through tetramethylrhodamine ethyl ester (TMRE) staining. Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation in mitochondria, served as a positive control (20 μmol/l for 10 minutes). (n = 9–10 in each group; ***P < 0.001. Dunnett test). (b) To investigate effects of carbamylation on mitochondrial function, oxygen consumption rates (OCR) were recorded in cyanate-treated hVSMCs. Measurements were performed under basal status and after injection of 1.0 μg/ml oligomycin (point a), 1.0 μmol/l FCCP (point b), and 0.1 μmol/l rotenone and antimycin A (point c). Parameters were calculated as follows: basal respiration; (last rate measurement before first injection) – (nonmitochondrial respiration rate), maximal respiration; (maximum rate measurement after FCCP injection) – (nonmitochondrial respiration), and adenosine triphosphate (ATP) production; (last rate measurement before oligomycin injection) – (minimum rate measurement after oligomycin injection). The OCR was normalized to the total protein level after the assay (n = 5–9 in each group; *P < 0.05, **P < 0.01. Mann-Whitney U test). (c) MitoSOX Red staining of urea- or cyanate-treated hVSMCs showed that carbamylated hVSMCs were exposed to mitochondria-derived oxidative stress. hVSMCs exposed to antimycin A, an inhibitor of mitochondrial electron transport chain, at 5.0 μmol/l for 10 minutes served as the positive control. Mitochondrial reactive oxygen species production induced by urea or cyanate was suppressed in the presence of mito-TEMPO (MT), an inhibitor of mitochondria-derived oxidative stress. (n = 10–15 in each group; *P < 0.05, **P < 0.01, ***P < 0.001. Differences were tested by analysis of variance with post hoc Tukey-Kramer honest significant difference test.) (d) Effects of protein carbamylation on calcification of hVSMCs under mito-TEMPO at day 20 were analyzed (n = 3 in each group under normal conditions; n = 6 in each group under calcifying conditions. Mann-Whitney U test). (e) Effects of hydrogen peroxide on counterregulatory elevation of ENPP1 expression in calcifying medium at both mRNA and protein levels were analyzed (for real-time polymerase chain reaction, n = 6 in each group; *P < 0.05, ***P < 0.001, Dunnett test; for Western blotting, n = 3 in each group; ***P < 0.001, Dunnett test). (f) Cell viability was assessed by a lactate dehydrogenase (LDH) assay. The levels of LDH released into the culture media were not elevated by H2O2 under calcifying conditions (n = 3 in each group. Dunnett test). (g) Effects of antimycin A on ENPP1 expression under calcifying conditions were analyzed by Western blotting. Bars = 10 μm. AMA, antimycin A-treated hVSMCs; NS, not significant; ref, reference. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Male SD rats were divided randomly into cellulose and urea groups to evaluate the effects of protein carbamylation on mitochondrial functions in vivo. Rats were heminephrectomized on day 1 and fed a CE-2 diet containing 20% urea or 20% cellulose from days 4 to 10. Aortic samples were obtained on day 11. In accordance with the in vitro results, immunohistochemical staining of the aorta showed that ATP synthase subunit α of rats in the urea group was carbamylated (Figure 7a). Similar results were obtained from aortic tissues of rats in the vehicle and cyanate groups (Figure 7b). The carbamylation of ATP synthase subunit α in cyanate-treated rat aorta was also confirmed by an immunoprecipitation assay (Figure 7c). Western blotting analysis of thoracic aorta demonstrated that protein carbamylation induced oxidative stress also in vivo. (Figure 7d). Moreover, enzymatic histochemical staining for cytochrome C oxidase and succinate dehydrogenase was diminished in the aortic tissues of rats in the urea and cyanate groups, indicating that protein carbamylation in vivo resulted in mitochondrial dysfunction (Figure 7e and f). We further analyzed the effect of carbamylation on VC using the aortic ring culture system. Aortic rings obtained from urea and cellulose groups and cyanate and vehicle groups shown in Figure 7 were cultured in normal or calcifying medium for 8 days (Figure 8). Serum parameters including creatinine, calcium, and phosphate levels were at comparable levels between the urea and cellulose groups (Table 3). These parameters were also at comparable levels between the cyanate and vehicle groups (Table 3). However, urea nitrogen was elevated in the urea and cyanate groups (Table 3). Protein carbamylation suppressed the counterregulatory elevation of ENPP1 expression in the calcifying medium (Figure 8a and b). Mineral quantification showed that protein carbamylation exacerbated calcification of the cultured aortic rings under the calcifying conditions (Figure 8c and d).Table 3Serum parameters of the ratsParameterGroupCelluloseUreaVehicleCyanateCreatinine (μmol/l)54.25 ± 14.35ref49.22 ± 12.51NS51.23 ± 8.64ref47.72 ± 8.98NSUrea nitrogen (mmol/l)11.46 ± 0.87ref31.52 ± 4.25a6.89±0.53ref8.67 ± 0.98aCalcium (mmol/l)2.51 ± 0.074ref2.52 ± 0.049NS2.66 ± 0.048ref2.61 ± 0.11NSPhosphate (mmol/l)3.47 ± 0.40ref3.11 ± 0.10NS2.46 ± 0.11ref2.31 ± 0.44NSNS, not significant; ref, reference. Serum parameters of the rats used in the experiment shown in Figure 7 a