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see basic research on page 923

Role of cyanate in the induction of vascular dysfunction during uremia: more than protein carbamylation? Clare L. Hawkins1,2 Cyanate is a uremic toxin responsible for the carbamylation of proteins, which has been implicated as playing a key role in accelerating the progression of atherosclerosis in patients with chronic kidney disease. El-Gamal et al. report that while cyanate promotes protein carbamylation in vivo, the resulting endothelial dysfunction observed is consistent with reactions mediated by cyanate itself, rather than by carbamylated proteins. This provides new insight into the relationship between uremia and cardiovascular disease. Kidney International (2014) 86, 875–877. doi:10.1038/ki.2014.256

It is well established that chronic kidney disease (CKD) is strongly associated with the development of atherosclerosis, which results in an elevated risk of developing cardiovascular disease. Uremic toxins, including cyanate, have been implicated as playing a key role in accelerating the progression of atherosclerosis by the induction of vascular dysfunction.1 Cyanate is formed via the decomposition of urea, and exists in plasma in equilibrium with its reactive form, isocyanic acid (Figure 1). Plasma levels of cyanate and isocyanic acid have been reported to reach 150 nM in patients with CKD, though urea levels can exceed 100 mM in patients with chronic renal failure.2 The discrepancy in plasma cyanate levels compared with urea concentrations may reflect its reactivity, particularly with abundant proteins, such

1 Heart Research Institute, Newtown, New South Wales, Australia and 2Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia Correspondence: Clare L. Hawkins, Heart Research Institute, 7 Eliza Street, Newtown, New South Wales 2042, Australia. E-mail: [email protected]

Kidney International (2014) 86

as albumin and lipoproteins, which are present in the circulation.1 Exposure of proteins to cyanate results in carbamylation, which is a post-translational modification resulting from the non-enzymatic reaction of isocyanic acid with amino (-NH2) and sulfhydryl (-SH) functional groups (Figure 1). The reaction of isocyanic acid with the e-amino group of lysine residues results in the formation of homocitrulline, which is often used as a marker to assess the extent of carbamylation in biological systems.3 Protein carbamylation is prevalent in patients with CKD4 and is elevated in human atherosclerotic lesions.3 Importantly, carbamylation is also recognized as a prognostic marker of patient outcome, with serum levels of carbamylated proteins predicting mortality in patients with CKD,5 and homocitrulline shown to be an independent risk factor for the development of coronary artery disease, future myocardial infarction, stroke, and death.3 Myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate has been recently demonstrated to be an alternative source of cyanate (Figure 1),

particularly at inflammatory sites and atherosclerotic lesions, where MPO levels are elevated.3 Given the compelling evidence linking MPO with multiple stages of atherogenesis, it has been suggested that MPO may be the dominant pathway to protein carbamylation associated with atherosclerosis, particularly in smokers, who have elevated plasma levels of thiocyanate.3 The data from El-Gamal et al.6 (this issue) provide further support for a role of MPO in protein carbamylation in atherosclerosis, as elevated proteinbound homocitrulline was observed in human atheroma from non-uremic patients with normal plasma urea levels. In intermediate atherosclerotic lesions, El-Gamal and colleagues show an association between carbamylated epitopes and endothelial cells, consistent with exposure of the endothelium to high localized concentrations of cyanate. This may be rationalized in non-uremic patients by the presence of elevated MPO resulting from the infiltration of neutrophils and other phagocytes at sites of inflammation. Protein carbamylation alters protein charge and structure and is associated with alterations to functionality and cellular interactions, which have been linked to disease development. For example, carbamylation of albumin during uremia is reported to exacerbate renal fibrosis in patients with CKD, and a number of studies provide compelling evidence to link lipoprotein carbamylation with atherosclerosis.1,7 Carbamylated low-density lipoprotein is poorly cleared from the plasma and is recognized by scavenger receptors, which promotes vascular-cell dysfunction by multiple pathways, including increased foam-cell formation, monocyte adhesion, endothelial-cell death, and smooth muscle cell proliferation.3,7 El-Gamal et al.6 propose an alternative hypothesis and demonstrate that while cyanate inhalation promotes plasma homocitrulline formation, and the generation of carbamylated epitopes within the aortas of mice, the resulting endothelial dysfunction observed is consistent with effects 875

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Uremia H2N

NH2

Inflammation O

N C S– Thiocyanate

Atherosclerosis

Urea

MPO

H2O2 C O– N Cyanate

HN C O Isocyanic acid R1 C HC

Endothelial dysfunction

R1 C

O (CH2)4

NH2

HC

NH

NH

R2

R2

O

O (CH2)4

NH

C NH2

Protein-bound ε-carbamyl-lysine (homocitrulline)

Figure 1 | Pathways responsible for cyanate formation and protein carbamylation during uremia and inflammation.

induced by cyanate rather than by carbamylated proteins. Treatment of mice with cyanate resulted in an increase in plasma homocitrulline levels, which compared well with the levels of this marker reported in patients with renal and cardiovascular disease. Carbamylated epitopes were also detected within the aortas of the cyanate-exposed mice, similar to the endothelium-associated homocitrulline staining seen in human atheroma. Importantly, cyanate treatment attenuated aortic vasorelaxation induced by acetylcholine, but not the nitric oxide donor sodium nitroprusside. This is consistent with a decrease in the production of an endothelium-derived vasodilator, such as nitric oxide, particularly as the contractile response of the aortic smooth muscle cells also remained unaltered on cyanate treatment. This led El-Gamal and colleagues6 to assess the levels of nitrite released from aortic segments of cyanate-exposed mice as a surrogate marker for nitric oxide production, and determine the expression of endothelial nitric oxide synthase (eNOS), which is a key regulator of nitric oxide production. As expected, nitrite levels and the expression of eNOS were significantly reduced in the aortas of cyanate-treated mice. Cyanate also increased the expression of tissue factor and plasminogen activator inhibitor-1 (PAI-1), suggesting a switch to a prothrombogenic 876

endothelial phenotype in addition to endothelial dysfunction. The mechanism involved in the reduction in eNOS expression and increased tissue factor and PAI-1 expression was examined further with the use of human coronary artery endothelial cells (HCAECs). Exposure of HCAECs to cyanate also resulted in a decrease in eNOS expression (both protein and mRNA) and an increase in tissue factor and PAI-1 expression, which occur without a significant loss in cellular viability. The upregulation of tissue factor, which is a key initiator of the coagulation cascade and hence critical in the development of atherosclerosis, appears to involve activation of the mitogen-activated protein kinase (MAPK) cascade, as inhibitors of p38, JNK, ERK, and nuclear factor-kB attenuate the effects of cyanate in this case. MAPKs are involved in numerous stress-related signaling pathways and play a role in the regulation of cell survival and apoptosis as well as proliferation, gene expression, and differentiation. Thus, although further work will be required to delineate the specific pathway involved in cyanate-induced changes in protein expression, the fact that inhibitors of p38, JNK, ERK, and nuclear factor-kB all influenced the expression of tissue factor suggests that cyanate is likely to be able to perturb other stress-related signaling pathways

of relevance to disease development. Indeed, previous work by El-Gamal et al.8 showed that cyanate triggered intercellular cell adhesion molecule-1 (ICAM-1) expression in HCAECs via MAPK-related pathways. Given that the HCAECs were incubated for 24–48 h with relatively high concentrations of cyanate (1–2 mM), it was important for El-Gamal et al.6 to demonstrate that the cellular effects observed are directly attributable to cyanate rather than via the action of carbamylated proteins, which are formed in the serum-containing medium during the incubation. Treatment of serum-containing culture medium with cyanate resulted in a marked increase in the formation of protein-bound homocitrulline, which saturated after 48 h of incubation. However, no change in either eNOS or tissue factor expression was observed on exposure of HCAECs to dialyzed cyanate-treated medium, where residual cyanate had been removed before addition to the cells. Supplementing this preconditioned medium with cyanate again resulted in alterations to eNOS and tissue factor expression. In contrast, the expression of PAI-1 was influenced by both cyanate and carbamylated serum proteins. These data support earlier work by El-Gamal et al.,8 where increased ICAM-1 expression was observed in HCAECs exposed to cyanate, but not dialyzed cyanateconditioned media or carbamylated lowdensity lipoprotein (cLDL), though it is notable that cLDL (in the absence of serum) has been reported by others to increase both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) expression in the same cell type.9 Taken together, the results reported by El-Gamal et al.6,8 support a novel role for cyanate, in addition to carbamylated proteins, in the induction of endothelial dysfunction and a prothrombogenic endothelial phenotype. This may be particularly significant in light of a recent study showing that impaired endogenous thrombolysis is a risk factor in patients with end-stage renal disease, which correlates with cardiovascular events.10 However, Kidney International (2014) 86

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further work will be needed to clarify the relevance of this pathway in vivo, particularly given the high (millimolar) cyanate concentrations required for effects in the cell-culture studies. Moreover, it is difficult in the in vivo cyanate supplementation studies to delineate whether carbamylation is contributing to endothelial dysfunction or is simply a by-product. Regardless of mechanism, the studies by El-Gamal et al.6,8 do provide new insight into the association between uremia and atherosclerosis, and highlight that therapeutic interventions to reduce cyanate, which would also decrease carbamylation, may have utility in reducing cardiovascular risk in patients with CKD.

see clinical investigation on page 991

Patient survival on dialysis in Korea: a different story? Marlies Noordzij1 and Kitty J. Jager1 In a propensity score–matched analysis of their cohort study including more than 32,000 hemodialysis and peritoneal dialysis patients in South Korea, Kim and colleagues found that mortality risk was higher in peritoneal dialysis than in hemodialysis patients aged 55 years or older. Among younger patients, survival was similar for both treatment modalities. These findings are in contrast to most results from studies in Western countries. We discuss a number of factors that may explain these differences. Kidney International (2014) 86, 877–880. doi:10.1038/ki.2014.194

DISCLOSURE

The author declared no competing interests. ACKNOWLEDGMENTS

The author is supported by the Australian Research Council (FT120100682). REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Jaisson S, Pietrement C, Gillery P. Carbamylation-derived products: bioactive compounds and potential biomarkers in chronic renal failure and atherosclerosis. Clin Chem 2011; 57: 1499–1505. Nilsson L, Lundquist P, Kagedal B et al. Plasma cyanate concentrations in chronic renal failure. Clin Chem 1996; 42: 482–483. Wang Z, Nicholls SJ, Rodriguez ER et al. Protein carbamylation links inflammation, smoking, uremia and atherogenesis. Nat Med 2007; 13: 1176–1184. Apostolov EO, Shah SV, Ok E et al. Quantification of carbamylated LDL in human sera by a new sandwich ELISA. Clin Chem 2005; 51: 719–728. Koeth RA, Kalantar-Zadeh K, Wang Z et al. Protein carbamylation predicts mortality in ESRD. J Am Soc Nephrol 2013; 24: 853–861. El-Gamal D, Rao SP, Holzer M et al. The urea decomposition product cyanate promotes endothelial dysfunction. Kidney Int 2014; 86: 923–931. Basnakian AG, Shah SV, Ok E et al. Carbamylated LDL. Adv Clin Chem 2010; 51: 25–52. El-Gamal D, Holzer M, Gauster M et al. Cyanate is a novel inducer of endothelial ICAM-1 expression. Antioxid Redox Signal 2012; 16: 129–137. Apostolov EO, Shah SV, Ok E et al. Carbamylated low-density lipoprotein induces monocyte adhesion to endothelial cells through intercellular adhesion molecule1 and vascular cell adhesion molecule-1. Arterioscler Thromb Vasc Biol 2007; 27: 826–832. Sharma S, Farrington K, Kozarski R et al. Impaired thrombolysis: a novel cardiovascular risk factor in end-stage renal disease. Eur Heart J 2013; 34: 354–363.

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The utilization of peritoneal dialysis varies strongly across the world. In a recent study, Jain and colleagues gave an overview of the use of peritoneal dialysis treatment in 130 countries worldwide between 1997 and 2008. Focusing on developed countries, the top five of countries with the highest prevalence of peritoneal dialysis per million population (pmp) consisted, in addition to New Zealand (182.6 pmp), of four Asian countries: Hong Kong (488.5 pmp), Taiwan (216.0 pmp), South Korea (162.5 pmp), and Singapore (158.3 pmp). There was, however, a huge variation in the proportion of patients who received peritoneal dialysis as opposed to hemodialysis; in Hong Kong this proportion was as high as 79.4%, while it was 19.0% in South Korea.1 Numerous studies have compared patient survival between peritoneal dialysis and hemodialysis patients, but 1 European Renal Association–European Dialysis and Transplant Association (ERA-EDTA) Registry, Department of Medical Informatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Correspondence: Marlies Noordzij, ERA-EDTA Registry, Department of Medical Informatics, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: [email protected]

few large ones were performed in Asian populations.2,3 In general, subgroups of Asian patients in studies from other countries comparing patient survival on hemodialysis and peritoneal dialysis are too small to obtain meaningful results. For example, a recent study based on the DaVita database in the United States did not present any separate analyses for Asian patients because of the low sample size of this subgroup.4 Also in Europe survival data on non-Caucasian patients, including Asians, are scarce.5 It is therefore noteworthy that Kim and colleagues6 (this issue) report on more than 32,000 incident dialysis patients from South Korea using data from the Korean Health Insurance Review and Assessment Service database. To compare the survival of peritoneal dialysis and hemodialysis patients the authors applied different statistical methods, including the Kaplan–Meier method, standard Cox regression, and Cox regression after propensity score matching. Using the latter method they found that overall the mortality risk was 20% higher in peritoneal dialysis patients than in hemodialysis patients. When the patients were categorized according to their age, this difference was present only in patients of 55 years or older, while patient survival was 877