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Urea and protein carbamylationin ESRD: surrogate markers orpartners in crime?Manuel T. Velasquez1, Ali Ramezani1 and Dominic S. Raj1

Protein carbamylation may result from chronic exposure to elevatedlevels of urea in patients with chronic kidney disease. Carbamylationcould cause conformational changes in proteins resulting in alterationsin binding sites and disturbances in cellular functions. Elevatedlevels of carbamylated protein have been shown to be associatedwith increased risk of death from cardiac causes in patients withend-stage renal disease. The precise mechanism by whichcarbamylated proteins mediate toxicity in uremia needs furtherinvestigation.Kidney International (2015) 87, 10921094. doi:10.1038/ki.2015.78

Since the identification of urea byFourcroy and Vauquelin in the eight-eenth century, it has been shroudedwith controversy. In Richard Brightsera, morbid anatomy was the doctrine,and the idea that urea could be a toxinwas almost heretical. During subsequentyears, the term uremia came intogeneral usage, and urea became acceptedas a marker for kidney failure, but stillnot accepted as a uremic toxin. Thiswas supported by the observation thatmaintaining a high concentration ofblood urea in end-stage renal disease(ESRD) patients by adding urea to thedialysate did not present any acute toxicconsequences. Also, data from theHemodialysis (HEMO) and Adequacyof PD in Mexico (ADEMEX) studiesshowing no survival benefit from higherdialysis dose argue against urea being theuremic toxin. However, a growing bodyof evidence indicates that urea may

mediate at least part of its toxic effectsthrough carbamylation of serum ortissue proteins (Figure 1).

UREA AND CARBAMYLATION: OLDWINE IN A NEW BOTTLE?Urea is in equilibrium with electrophilicspecies, cyanate and isocyanate, whichcan react with amino groups of freeamino acids and with lysine residuesof proteins, resulting in -amino-carbamoyl-lysine (homocitrulline).Carbamylation is an irreversible post-translational modification and occurs atmultiple lysine sites within a protein,with accumulation over the lifespan ofthe protein. Recent evidence suggeststhat oxidation of thiocyanate catalyzedby myeloperoxidase is an importantmechanism of cyanate formation andprotein carbamylation, especially atsites of inflammation.1 Carbamylationreaction could cause conformationalchanges in proteins resulting in inhibi-tion of enzyme activity, alterations inbinding sites, and disturbances in othercellular functions. A number of studieshave shown increased carbamylation ofproteins in patients with chronic kidneydisease.2 Protein carbamylation hasbeen shown to be associated withincreased mortality in subjects with

and without impaired kidney func-tion.1,3,4 Koeth et al.3 quantified theplasma level of protein-bound homo-citrulline (PBHCit), a marker of carba-mylation, in a cohort of 347 patientsundergoing maintenance hemodialysiswith 5 years of follow-up and foundthat the risk for death among patientswith PBHCit values in the highest tertilewas more than two times the riskamong those in lower tertiles.

CARBAMYLATION: A NOVELBIOMARKER OR AGENTPROVOCATEUR?It is well recognized that patients withESRD have a high risk of cardiovascular(CV) events. This excess mortality doesnot appear to be explained entirely bytraditional CV risk factors. Increasedattention has, therefore, been directedtoward investigation of nontraditionalrisk factors, including, among others,uremic toxins, oxidative stress, andsystemic inflammation, as potentialmechanisms to explain the excess CVdisease burden associated with chronickidney disease. Berg and associates5

(this issue) report an associationbetween elevated levels of carbamylatedalbumin (C-Alb) and mortality in asecondary analysis of the data from DieDeutsche Diabetes Dialyse Studie (4D),which investigated the CV benefits ofatorvastatin in maintenance hemodia-lysis patients with diabetes. The presentstudy is a continuation of their previouswork in which they showed that theproportion of albumin carbamylated onLys-549 was associated with increasedrisk of death at 12 months in 187Accelerated Mortality on Renal Replace-ment (ArMORR) study participantsand 1161 4D study participants.6 Theexciting new findings in this study arethat elevated C-Alb levels were asso-ciated with risk for CV mortality,especially with short-term risk of sud-den death and long-term risk of deathfrom congestive heart failure (CHF). Itis possible that those with more severeheart disease died from sudden cardiacdeath early and others with less severedisease developed and died from CHFlater. The mechanisms for the associa-tion between elevated serum protein

1Division of Renal Diseases and Hypertension, TheGeorge Washington University, Washington, DC,USACorrespondence: Dominic S. Raj, Division ofRenal Diseases and Hypertension, Medical FacultyAssociates, School of Medicine, The GeorgeWashington University, 2150 PennsylvaniaAvenue NW, Washington, DC 20037, USA.E-mail: draj@mfa.gwu.edu

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carbamylation and makers of cardiacdamage, sudden cardiac death, and riskof CHF are unknown. The authorsspeculate that the risk of sudden cardiacdeath is consistent with the hypothesisthat cardiac protein hypercarbamyla-tion represents an acute insult to thecardiac conduction system, whereasthe long-term risk of CHF may be aconsequence of the chronic effects ofcarbamylation resulting in myocardialprotein dysfunction due to uremiccardiomyopathy and fibrosis.

LACK OF ASSOCIATION WITH ISCHEMICHEART DISEASE: LOST IN TRANSLATION?Epidemiological studies have shown thatabout 64% of all cardiac deaths amonghemodialysis patients are sudden cardiacdeaths. Many triggering events for fatalarrhythmias and sudden cardiac deathhave been identified, but acute myocar-dial ischemia is believed to be the mostcommon precipitating event. Substantialevidence from clinical and laboratoryscience suggests that carbamylation of

proteins may promote atherosclerosis.Animal studies in a mouse model ofchronic kidney disease have shown thaturemia increases carbamylation of plas-ma low-density lipoproteins (cLDLs)and high-density lipoproteins (cHDLs),and this modification inhibits receptor-mediated uptake and instead promotesfoam-cell formation and accumulationin atherosclerotic tissues. In addition,cLDL exhibits distinct cytotoxic effectswhen tested in vitro on endothelial cells,induces the expression of adhesionmolecules, and aggravates the monocyteadhesion to endothelial cells. LDLsisolated from uremic patients and ure-mic aortic plaques exhibit a higherdegree of carbamylation than those fromcontrols. cLDL binds to macrophagescavenger receptors, facilitating foam-cell formation, inflammatory signaling,endothelial-cell apoptosis, and vascularsmooth muscle proliferation. In patientsundergoing cardiac catheterization, thosewith elevated serum carbamylated pro-tein were at increased risk of coronary

artery disease, future myocardial infarc-tion, stroke, and death.1 However, inthe study by Berg and associates,5 nospecific association between C-Alb andrisk for myocardial infarction was evi-dent. Diabetics with advanced kidneydisease have a large burden of traditionaland novel CV risk factors. Thus, it ispossible that C-Alb may not be themost important mechanism for acce-lerated atherosclerosis in this patientpopulation.

DOES CARBAMYLATION MEDIATE CHFTHROUGH ABERRANT CARDIACREMODELING?Elevated C-Alb was associated with long-term risk for death from CHF in thestudy by Berg and associates.5 Althoughleft ventricular hypertrophy begins as anadaptive response to pressure or volumeoverload, it often results in diastolicdysfunction, eventually leading to heartfailure. In a small study involving 115patients with chronic systolic heart fail-ure, Tang et al.4 showed that PBHCitlevels were not related to indices ofcardiac structure or function, but itpredicted poor long-term survival inthese patients. In a recent study, plasmalevels of carbamylated proteins increasedabout twofold in nephrectomized versuscontrol mice over the 20 weeks of theexperiment.7 The progressive accumula-tion of carbamylated proteins was evi-dent in extracellular matrix proteins suchas collagen in multiple organs includingthe heart and the kidney.7 Carbamylationcould potentially alter interaction withinflammatory cells and susceptibility toproteolysis, leading to aberrant tissueremodeling in target organs.8 Highresistance of electrical conduction path-ways in the fibrotic tissue could con-tribute to intraventricular conductiondisturbance and arrhythmia leading tosudden cardiac death. Future studiesshould examine the role of carbamyla-tion in abnormal cardiac remodeling andcontractile function in ESRD.

CARBAMYLATION AND CHOLESTEROL:ONE MORE TWIST IN THE PARADOX?Serum cholesterol and CV diseaseexhibit a U-shaped association in ESRD,with increased CV event rates noted at

Inflammation

Diet Smoking

Endothelialdysfunction

Atherosclerosis

Cardiomyopathy

PEW

Amino aciddepletion

Kidney disease uremia/amino acid deficiency

NThiocyanate

NCyanate

HN

C OR1

R2Protein

HCNH

(CH2)4NH2C OR1

R2Carbamyl-protein(homocitrulline)

HCNH

(CH2)4NHCNH2

O

Isocyanate

MPOH2N N2H

NH4+

OUrea

Carbamylation

H2O2

CS

COHC O

Figure 1 |Carbamylation of protein and potential toxicity. MPO, myeloperoxidase;PEW, protein energy wasting.

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both ends of the spectrum. The 4D studydemonstrated that lowering LDL choles-terol with atorvastatin had no statisticallysignificant effect on mortality or CVevents. An interesting finding of Bergand associates5 is that patients with lowC-Alb did benefit from atorvastatintreatment. It is now well recognized thatcarbamylation alters the LDL and HDLfunction, and the authors speculate thatthe beneficial effect of lipid lowering waslost because of excessive carbamylation.Given the complexity of the uremic stateand cholesterol metabolism in ESRD,further clinical and experimental studiesare warranted exploring this possibility.

DOES CARBAMYLATION CAUSEFUNCTIONAL AMINO ACIDDEFICIENCY?Protein carbamylation could be thepotential link between inflammation,protein energy wasting, and CV diseasein ESRD patients. Carbamylation reactioncan also occur on amino acids andconvert free to carbamoyl-amino acids.Carbamoyl-amino acids cannot formpeptide bonds with the carboxyl groupof other amino acids in protein synthesis,thus contributing to protein energy wast-ing (Figure 1). Taking advantage of thefact that free amino acids are potentialscavengers for reactive isocyanate, thisgroup of investigators is examining theeffect of supplementation with specificamino acids on carbamylation in ESRDpatients undergoing maintenance hemo-dialysis (NCT no. NCT01612429).

Advances in analytical techniquesand the emergence of omics haveidentified a myriad of retention solutesin patients with ESRD, which is evergrowing. The journey, however, startedwith urea, used as a surrogate markerprimarily because of its abundance andnot necessarily because of its toxicity,which has re-emerged as a strongcontender as a uremic toxin in recentyears. This study by Berg and associates5

fortifies the notion that urea could be apotential uremic toxin that mediates itstoxicity through carbamylation. How-ever, given the complexity of uremicsyndrome and the enormous number ofsolutes retained, generated, and mod-ified in uremia, protein carbamylation

may be one of the toxins but certainly isnot the only uremic toxin. This shouldbe considered as we move forward withtargeted intervention to reduce proteincarbamylation.

DISCLOSUREAll the authors declared no competinginterests.

ACKNOWLEDGMENTSDominic S. Raj is supported by NationalInstitutes of Health grants 1R01-DK073665-01A1, 1U01-DK099924-01, and 1U01-DK099914-01.

REFERENCES1. Wang Z, Nicholls SJ, Rodriguez ER et al. Protein

carbamylation links inflammation, smoking,uremia and atherogenesis. Nat Med 2007; 13:11761184.

2. Kalim S, Karumanchi SA, Thadhani RI et al.Protein carbamylation in kidney disease:

pathogenesis and clinical implications. Am JKidney Dis 2014; 64: 793803.

3. Koeth RA, Kalantar-Zadeh K, Wang Z et al.Protein carbamylation predicts mortalityin ESRD. J Am Soc Nephrol 2013; 24: 853861.

4. Tang WH, Shrestha K, Wang Z et al. Proteincarbamylation in chronic systolic heart failure:relationship with renal impairment andadverse long-term outcomes. J Card Fail 2013;19: 219224.

5. Drechsler C, Kalim S, Wenger JB et al. Proteincarbamylation is associated with heart failureand mortality in diabetic patients with end-stagerenal disease. Kidney Int 2015; 87: 12011208.

6. Berg AH, Drechsler C, Wenger J et al.Carbamylation of serum albumin as a riskfactor for mortality in patients with kidneyfailure. Sci Transl Med 2013; 5: 175ra29.

7. Pietrement C, Gorisse L, Jaisson S et al. Chronicincrease of urea leads to carbamylatedproteins accumulation in tissues in a mousemodel of CKD. PLoS One [online] 2013; 8:e82506.

8. Jaisson S, Lorimier S, Ricard-Blum S et al.Impact of carbamylation on type I collagenconformational structure and its ability toactivate human polymorphonuclearneutrophils. Chem Biol 2006; 13: 149159.

see clinical investigation on page 1209

Comparative health servicesresearch across populations: theunused opportunities in big dataBenjamin A. Goldstein1 and Wolfgang C. Winkelmayer2

Large population registries present opportunities to understand theepidemiology of disease, study patters of care, and compare theeffectiveness of different care practices. While these big data temptinvestigators to examine all available individuals, a targeted approachthat restricts the study to an optimal sub-population may yield morerelevant insights. We discuss how researchers might adapt their studiesexisting literature to generate additional insights.Kidney International (2015) 87, 10941096. doi:10.1038/ki.2015.91

Historically, conducting large-scale po-pulation-based research using insuranceor electronic health records databases

used to be the privilege of a fewinstitutions and investigators that hadexclusive access to highly guarded dataas well as rather unique data storage,manipulation, and analytical capabil-ities. The use of Medicare or stateMedicaid claims in the United States,universal health insurance claims datafrom some Canadian provinces, or whatused to be called the General PracticeResearch Database in the UnitedKingdom comes to mind. The uses of

1Department of Biostatistics and Bioinformatics,Duke University School of Medicine, Durham,North Carolina, USA and 2Section of Nephrology,Department of Medicine, Baylor College ofMedicine, Houston, Texas, USACorrespondence: Wolfgang C. Winkelmayer,Section of Nephrology, Baylor College of Medicine,One Baylor Plaza, ABBR R705, Houston, Texas77030, USA. E-mail: winkelma@bcm.edu

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