Protein Carbamylation in Chronic Kidney Disease and Dialysis

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Protein Carbamylation in Chronic Kidney Disease and Dialysis Joshua Long, Xavier Vela Parada and Sahir Kalim1 Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States 1 Corresponding author: E-mail: [email protected]

Contents 1. Introduction and Background 1.1 Introduction 1.2 A Brief Overview of the Chemistry of Carbamylation and Human Causes 1.3 What’s in a Name? 2. The Impact of the Carbamylation Reaction on Human Proteins 2.1 Altered Protein Structure Begets Altered Protein Function 2.2 Carbamylation Appears Diffuse 3. Mechanistic Underpinnings of Carbamylation and Clinical Outcomes Research in Kidney Disease 3.1 Cardiovascular Disease 3.2 Kidney Fibrosis 3.3 Erythropoietin Resistance 3.4 Autoimmunity 4. Biomarkers of Carbamylation Burden: How Carbamylation has Been Assessed in Kidney Disease Studies 5. Clinical Association Studies in Kidney Disease 5.1 Carbamylation and Cardiovascular Outcomes 5.2 Carbamylation in End Stage Kidney Disease 6. Possible Targeted Therapies to Mitigate Carbamylation in Kidney Disease 7. Future Directions for Research 8. Conclusion References

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Abstract Protein carbamylation is a nonenzymatic posttranslational protein modification that can be driven, in part, by exposure to urea’s dissociation product, cyanate. In humans, when kidney function is impaired and urea accumulates, systemic protein carbamylation levels increase. Additional mediators of protein carbamylation have been identified Advances in Clinical Chemistry, Volume 87 ISSN 0065-2423 https://doi.org/10.1016/bs.acc.2018.07.002

© 2018 Elsevier Inc. All rights reserved.

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including inflammation, diet, smoking, circulating free amino acid levels, and environmental exposures. Carbamylation reactions on proteins are capable of irreversibly changing protein charge, structure, and function, resulting in pathologic molecular and cellular responses. Carbamylation has been mechanistically linked to the biochemical pathways implicated in atherosclerosis, dysfunctional erythropoiesis, kidney fibrosis, autoimmunity, and other pathological domains highly relevant to patients with chronic kidney disease. In this review, we describe the biochemical impact of carbamylation on human proteins, the mechanistic role carbamylation can have on clinical outcomes in kidney disease, the clinical association studies of carbamylation in chronic kidney disease, including patients on dialysis, and the promise of therapies aimed at reducing carbamylation burden in this vulnerable patient population.

1. INTRODUCTION AND BACKGROUND 1.1 Introduction Proteins within the human body, during both health and disease, are exposed to a multitude of chemical reactions capable of altering their structural and functional properties. Spontaneous post-translational protein modifications can occur through the non-enzymatic binding of reactive molecules to protein functional groups, as seen, for example, in glycation and oxidation reactions. Because post-translational modifications are capable of changing original protein structure and function, they can create a mechanistic chemical link to the adverse pathophysiology underlying certain diseases with metabolic consequences. Protein carbamylation describes a post translational protein modification that is driven by cyanate, which notably, is the reactive dissociation product of urea (Fig. 1A). Irreversible carbamylation reactions with amine groups can change the charge, structure, and function of proteins, resulting in molecular and cellular dysfunction. While generally found in low concentrations in vivo, urea (the chief end-product of nitrogen metabolism), and thus cyanate and carbamylation, all naturally increase when kidney function is impaired. Protein carbamylation, however, is not solely related to urea; it has also been shown that free amino acids compete with proteins for reaction with cyanate, in essence shielding proteins from carbamylation, and amino acid deficiencies from a variety of causes can exacerbate carbamylation burden [1]. Additionally, the heme protein myeloperoxidase (MPO), which is secreted in high concentrations at inflammatory sites from stimulated neutrophils and monocytes, can catalyze production of hypochlorous acid and chloramines which oxidize thiocyanate or cyanide (derived from diet, smoking, and even air

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Figure 1 (A) Pathways that promote the protein carbamylation reaction. Cyanate and its tautomer isocyanate can be derived from sources such as diet or smoking, and from urea which accumulates with impaired kidney function. The reactive species can carbamylate the N-terminal of a protein or an amino acid side chain. This reaction can occur on free individual amino acids as well in a similar fashion. Carbamylated proteins may undergo change in charge, structure, and function. (B) Carbamylation per the official IUPAC nomenclature. Binding of CO2 leads to the addition of a carbamate group as shown and this officially represents “carbamylation.” However, the biomedical literature has largely used the term “carbamylation” to represent the reaction in Fig. 1A in which isocyanate binding results in the addition of a carbamoyl group. While official IUPAC nomenclature would deem the reaction in Fig. 1A “carbamoylation,” in current biomedical literature term carbamylation designates carbamoylation.

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pollution) into cyanate, contributing to carbamylation at sites of local inflammation (Fig. 1A) [2,3]. Protein carbamylation can therefore stem from such divergent exposures as kidney disease, smoking, air pollution, and certain foods, ultimately leading to common pathways of molecular and cellular dysfunction. Because carbamylation is a protein modification that can result from constant exposure to urea and its byproduct, cyanate, which both increase as kidney function declines, it has been an increasing focus of nephrology research through the years [4]. In this report, we will review the direct impact of the carbamylation reaction on protein structure and function, the mechanistic rationale for carbamylation driving clinical outcomes in kidney disease along with methods to assess carbamylation burden in vivo, and, finally, clinical studies of carbamylation in kidney disease with attention to possible therapeutic approaches.

1.2 A Brief Overview of the Chemistry of Carbamylation and Human Causes Under physiologic conditions, urea can dissociate into ammonia and cyanate, the latter being in equilibrium with its tautomer, isocyanate, a highly reactive electrophile that can react with the amine and sulfhydryl groups on proteins and free amino acids [5,6]. Classic early experiments showed that when certain proteins are incubated with urea, a modified form of the protein accumulates that is less positively charged, and urea exposure results in lysine within the protein becoming irreversibly modified into N (6)-carbamoyl-L-lysine (homocitrulline). It is now established that cyanate/isocyanate can react with amino, sulfhydryl, carboxyl, hydroxyl, and imidazole groups in what is now known as the carbamylation reaction [7e10]. While urea-derived cyanate is a central source for carbamylation in humans, particularly those with kidney disease, the enzymatic action of MPO on thiocynate in the presence of hydrogen peroxide can also generate cyanate (Fig. 1A) [2]. MPO is an abundant enzyme contained in inflammatory cells such as polymorphonuclear neutrophils and monocytes/ macrophages, which transforms thiocyanate into hypothiocyanic acid and cyanate [11]. The thiocyanate necessary for this reaction can be derived from eating various fruits and vegetables, milk by-products, and from cigarette smoking [2,4,12]. Similarly, an additional source of protein carbamylation that is largely unstudied is the consumption of improperly prepared cassava root, a popular staple food in parts of Africa. Cassava is a rich source

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of cyanogens that are metabolized to cyanate, which may contribute significantly to carbamylation in populations consuming large quantities of raw cassava [13]. More recently, it was shown that MPO is also able to catalyze production of hypochlorous acid and the two-electron oxidation of cyanide (a known hazardous environmental exposure) into cyanate, thus marking another potential source of carbamylation [3]. Moreover, direct environmental exposure to isocyanate in the atmosphere, generated by the combustion of biomass, has been reported as a carbamylation source [14].

1.3 What’s in a Name? The net result of these reactions, which we will call “carbamylation,” is the addition of a “carbamoyl” moiety (-CONH2) to a functional group (Fig. 1A). It must be noted that several authorities accurately state that the reaction in question is more correctly deemed “carbamoylation” and, in fact, “carbamylation” denotes a different chemical reaction (see Fig. 1B) [15]. However, over the past decades, the biomedical literature has skewed towards calling the reaction involving a carbamoyl moiety, “carbamylation,” and we will continue in this manner for convention.

2. THE IMPACT OF THE CARBAMYLATION REACTION ON HUMAN PROTEINS N-carbamylation yields a relatively stable moiety which does not readily dissociate or oxidize and can remain stable on the carbamylated protein. Consequently, proteins can accumulate these modifications on their N-terminal aeamino groups or the ε-amino groups of lysine side chains throughout their lifespan. Similarly, free amino acids may also be carbamylated on their a-amino group or on nucleophilic amino and sulfhydryl groups on their side chains [8,16]. Because of differences in pKa, a-amino groups of free amino acids react about 100 times faster than lysine side chain ε-amino groups on proteins at physiologic pH [8,17]. When an ε-lysine is carbamylated, it is called “homocitrulline,” a key marker used to assay carbamylation load in vivo (see Biomarkers of carbamylation burden below).

2.1 Altered Protein Structure Begets Altered Protein Function A major chemical effect of the binding of a carbamoyl group on protein amino groups is the neutralization of a positive charge which in turn alters

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ionic protein-water interactions on the protein surface [11,18,19]. This disturbance can destabilize secondary and tertiary protein structures resulting in subsequent conformational changes. With the conformational change of a protein, it is easy to see how functional changes can ensue. Dozens of studies through the years have shown results implicating carbamylation in changes in protein charge [20,21], conformation [19,22], and stability [23], with consequent alterations in enzyme and hormone activity [24e28], binding properties [29e31], receptor-drug interaction [10,32], and cellular expression and responses [33e38]. Descriptions of the numerous demonstrations of these phenomena are beyond the scope of this report and the reader is referred to other excellent broad reviews of carbamylation recently published [4,11].

2.2 Carbamylation Appears Diffuse The broad impact carbamylation can have on proteins is important when considering a leading pathway to cyanate formation and protein carbamylation in the human body is the spontaneous dissociation of urea [39]. Given the ubiquity of urea in all tissue compartments, the potential resulting effects of carbamylation are far reaching. Pietrement et al. demonstrated this in a chronic kidney disease animal model where the authors were able to quantitatively demonstrate, using high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS), that impaired kidney function accelerated carbamylation across a diverse set of tissues including aorta, kidney, bone, skin, liver, and heart, with greater accumulations in the long-lived extracellular matrix proteins [40]. While there appeared to be a basal level of carbamylation occurring in control mice without renal disease, there was a twofold increase in carbamylation burden in 75% nephrectomized mice at 20 weeks across these tissues. The notion that the reaction also occurs in physiological conditions at a basal level and can naturally accumulate on proteins with long half-lives, was the basis for investigating the age-related kinetics of carbamylation accumulation [41]. Interestingly, the accumulation rate of carbamylation over time in the skin of different mammalian species with different life expectancies is inversely correlated with longevity. This suggests that some animals may harbor potential protective mechanisms against carbamylation that are yet to be identified [41]. Similar speculation of some intrinsic guard against carbamylation’s harmful effects has also been postulated when considering the biochemistry of elasmobranchs (sharks, rays, and skates), the physiology of which is characterized by urea retention resulting in body fluids that

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contain extremely elevated urea concentrations (350e500 mM) [4,42]. How these animals handle high urea levels without enduring adverse effects from carbamylated proteins is not known, but could offer insights to possible treatment strategies in humans. Understanding protective strategies is appealing as reports across numerous disciplines implicate protein carbamylation in specific human disease processes such as cataract formation [19,43e 50], arthritis and autoimmune disease [51e56], and even dementia (Fig. 2) [57]. However, given the relationship between urea, cyanate, and decreased kidney function, the disease entity with perhaps the greatest clinical relevance is kidney disease.

Figure 2 The systemic effects of protein carbamylation. The carbamylation reaction, which can be driven by a variety of exposures including smoking, inflammation, and kidney disease, can result in diffuse and diverse pathophysiology.

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3. MECHANISTIC UNDERPINNINGS OF CARBAMYLATION AND CLINICAL OUTCOMES RESEARCH IN KIDNEY DISEASE Chronic kidney disease (CKD) is a significant public health problem in the United States and an even greater issue worldwide [58,59]. The presence of CKD can increase the risk of death up to 26% per year when compared to the general population [58], with cardiovascular diseases (CVDs) representing the major cause of mortality in this population [60,61]. While traditional risk factors, such as hypertension, atherosclerosis, and left ventricular hypertrophy are highly prevalent in CKD and contribute to CV risk, studies employing conventional treatments (e.g., statins) have failed to change key outcomes in the CKD population [62,63] suggesting novel pathophysiology. Considerable efforts have been directed towards understanding the unique aspects of kidney disease that contribute to adverse outcomes and, in this framework, protein carbamylation has garnered significant attention.

3.1 Cardiovascular Disease Because patients with CKD suffer disproportionately from CVD, novel risk factors in CKD would presumably have a mechanistic link to cardiovascular outcomes. Multiple studies have suggested that carbamylation can contribute to atherosclerosis and cardiovascular risk via its effects on lipoproteins, collagen, fibrin, proteoglycans, and fibronectin (Fig. 2 and Table 1) [64]. Carbamylation of low density lipoprotein (LDL) may enhance its atherogenic properties partly by decreasing its binding to the LDL-receptor [2] and by preventing its clearance from circulation (a phenomenon seen in both animal models of chronic renal failure [65,66], as well as in uremic patients [67e70]). Carbamylated LDL has also been shown to readily bind macrophage scavenger receptors, facilitating foam cell formation and inflammatory signaling [2,71,72]. Moreover, carbamylated LDL can induce endothelial cell apoptosis [73,74] and stimulate vascular smooth muscle proliferation [75] while carbamylated collagen increases release of matrix metalloproteinases by monocytes, possibly stimulating remodeling of the extracellular matrix [38,76]. Carbamylation of high density lipoprotein (HDL) has similar atherogenic properties by impacting migration, angiogenesis, and proliferation in endothelial cells [77,78], while other studies suggest cyanate itself has direct pathologic effects on endothelial cells [79,80]. Similar mechanistic links to endothelial dysfunction can be seen through carbamylation’s contribution to oxidative stress. For example, S-carbamylation of

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Table 1 Mechanisms of Atherosclerosis Accelerated by Carbamylation Substrate Effect References

LDL

HDL

Collagen

Fibrin Proteoglycans, fibronectin

Reduces LDL-receptor recognition, clearance Increases macrophage scavenger receptor recognition and foam cell formation Promotes endothelial/monocyte adhesion Induces endothelial cell injury/death/ autophagy Stimulates proliferation of vascular smooth muscle cells Increases oxidative stress Reduces endothelial nitric oxide synthase, increases tissue factor and plasminogen activator inhibitor-1 expression Enhances platelet aggregation Accumulates in atherosclerotic lesions Promotes cholesterol accumulation in macrophages and foam cell formation Impairs HDL’s anti-inflammatory and antioxidative activity Inhibits endothelial cell repair function and promotes apoptosis Decreases ability to polymerize into normal fibrils, alters interactions with inflammatory cells and alters extracellular matrix remodeling Increased adhesion of monocytes Alters fibrin clot formation, clot structure, and resistance to fibrinolysis. Alters extracellular matrix organization via decreased collagen and basement membrane affinity

[2,29,66,67,141,142] [2,71,141,143] [71,72] [2,73,74,143e146] [2,73,75] [144] [147,148]

[149] [2,69,143] [77] [150] [2,78] [76,86]

[38] [151] [64]

free cysteine and glutathione sulfhydryl groups as well as of hydrogen sulfide interferes with their antioxidant functions (e.g., free radical scavenging) [81,82].

3.2 Kidney Fibrosis Beyond CVD, there are additional reasons to believe that carbamylation contributes to adverse outcomes in kidney disease. Carbamylated proteins at concentrations present in uremia have been shown to activate glomerular mesangial cells to a profibrogenic phenotype and stimulate collagen

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deposition [83], and cyanate exposure strongly inhibits the collagenase activity of purified human and rat mesangial cell MMP-2 [84]. Intraperitoneal injection of carbamylated albumin in the amphibian kidney results in tubular cell damage and peritubular fibrosis via stimulation of TGF-b, EGF, NF-kB and endothelin-1 [84]. In general, it is suggested that carbamylation of collagen or enzymes involved in extracellular-matrix remodeling can disrupt the balanced remodeling of extracellular proteins and enhance urea associated fibrosis [85,86]. Thus, accelerated carbamylation due to kidney failure may further accelerate the progression of kidney disease itself, leading to a detrimental positive feedback loop. Such processes may not be limited to the kidney and could occur in other susceptible organs including the heart.

3.3 Erythropoietin Resistance The kidney’s production of erythropoietin, the most important hormone of hemoglobin regulation, is impaired in CKD, and erythropoiesis-stimulating agents (ESAs) such as recombinant human erythropoietin (EPO) are commonly used to manage the anemia associated with CKD. While most patients experience a dose-dependent response to ESAs, a subset of patients are considered resistant, showing a limited or absent response despite high dose treatment [87]. The mechanisms of EPO resistance remain poorly understood and are likely multi-factorial, relating to iron stores, inflammation, and other causes [87]. While lower EPO responsiveness is associated with an increased risk of adverse cardiovascular events including death [88e90], it is unclear if these observed risks are caused by ESAs themselves or by underlying processes leading to higher EPO requirements. This dilemma became relevant to carbamylation research when investigators seeking to leverage the anti-inflammatory effects of EPO in various disease states (including stroke and acute kidney injury) wanted to create a form of EPO that would only be anti-inflammatory without the other undesired effects of the hormone in these settings (i.e., erythropoiesis). Erythropoietin receptors can be found in the brain, spinal cord, heart and skin and many tissues produce and locally release it in response to hypoxic, biochemical, and physical stress [91]. In cellular, animal, and clinical studies, EPO protects tissues from ischemia and reperfusion injury, has antiapoptotic effects, and improves regeneration after injury [91]. Surprisingly, it was found that carbamylated EPO lacks erythropoietic activity yet still engages tissueprotective receptors [92]. It is now well noted that carbamylation of erythropoietin abrogates its erythropoietic activity both in vitro and in vivo

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and thus, carbamylation of erythropoietin or proteins in the erythropoietic pathway may directly contribute to the pathophysiology of EPO resistance in CKD [92e95].

3.4 Autoimmunity An additional consequence of protein carbamylation appears to be the induction of autoantibody responses. Protein carbamylation creates potential neo-epitopes on proteins and this has been shown to result in multiple autoantibody responses in conditions such as rheumatoid arthritis (RA) [51e54]. For example, anti-homocitrulline antibodies were not only present and higher in individuals with RA and arthralgias compared to healthy subjects, they also showed positive associations with joint damage and could predict disease development in “pre-RA” patients independent of established biomarkers [53,55]. Similar findings have been reported across multiple groups and this remains a highly active area of investigation [96e98]. Whether similar stimuli for auto-immune activity could be relevant to the many autoimmune driven renal diseases (e.g., several form of glomerulonephritis), remains unknown.

4. BIOMARKERS OF CARBAMYLATION BURDEN: HOW CARBAMYLATION HAS BEEN ASSESSED IN KIDNEY DISEASE STUDIES Protein carbamylation has largely been assessed in studies of kidney disease using two methods-measuring homocitrulline or measuring specific carbamylated proteins, namely albumin, hemoglobin, and LDL (Table 2). Analogous to hemoglobin A1c’s relationship to glucose levels in diabetes mellitus, when urea-derived cyanate makes a stable attachment to a protein, it takes on the half-life of that protein. In this sense, carbamylated proteins can give a time averaged sense of total body “carbamylation burden.” In 1981 Fl€ uckiger et al. observed that a sub-fraction of hemoglobin A1 was carbamylated and that the proportion of carbamylated hemoglobin was related to patient’s blood urea concentrations [99]. Subsequent reports confirmed this finding with several clinical research studies in carbamylation following [100e103]. Thus carbamylated hemoglobin was one of the earlier markers of systemic carbamylation burden in patients with kidney disease. However, the levels of carbamylated hemoglobin may be confounded in CKD patients due to the significant variability in the age of erythrocytes in this patient population, in the same way that erythrocyte aging influences

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Table 2 Biomarkers of Protein Carbamylation Biomarker

Main Analytical Methods

Carbamylated albumin

HPLC- tandem mass spectrometry Capillary electrophoresis Gas Chromatography HPLC HPLC- tandem mass spectrometry HPLC Enzyme linked immunosorbent assay Enzyme linked immunosorbent assay Chemiluminescent immunoassays

Carbamylated hemoglobin Homocitrulline Carbamylated amino acids Carbamylated LDL Plasma antibodies to carbamylated proteins

Example Reference

[1,152] [99,123] [2] [116] [108] [53,54]

Abbreviations: HPLC, high performance liquid chromatography; LDL, low density lipoprotein.

hemoglobin A1c levels [104,105]. The age of erythrocytes in patients with end stage renal disease (ESRD) further depends upon their degree of ESRD-related anemia, dialysis related red cell loss, EPO prescription, and whether they have been recently transfused [104e107]. To overcome some of these limitations, our group recently devised an assay using HPLC-MS/MS for measuring the most abundant site of carbamylation on human serum albumin (Lysine-549) [1]. This assay has excellent linearity with a coefficient of variation of 4.2% and has subsequently been employed in several clinical studies (see below). Additional blood proteins such as carbamylated LDL can be determined by ELISA or by colorimetric measurement of protein carbamylation after isolation of the LDL fraction by ultracentrifugation [108]. While not standardized, antibodies to carbamylated LDL have allowed for tissue localization permitting better study of carbamylation’s roll in endothelial dysfunction and atherosclerotic vascular disease [2]. Despite much work targeting specific carbamylated proteins or their antibodies, the most wide-spread biomarker for carbamylation in the literature is protein-bound homocitrulline. Homocitrulline (or ε -aminocarbamoyl-lysine) is an established marker of carbamylation burden and the levels of protein-bound homocitrulline (w90% of total homocitrulline [109]) can be specifically quantified in plasma or tissue samples with high specificity and low detection threshold using HPLC-MS/MS after protein hydrolysis. One potential drawback to measurement of protein-bound homocitrulline, however, is that it represents the accumulation of carbamylation modifications on different proteins which are carbamylated at different rates and have differing circulating half-lives. Thus, when

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measuring plasma protein carbamylation in human subject studies, differences in the proportions of albumin versus immunoglobulins and other abundant proteins may significantly alter protein-bound homocitrulline values independent of the underlying causes of carbamylation.

5. CLINICAL ASSOCIATION STUDIES IN KIDNEY DISEASE Under physiologic conditions, the equilibrium between cyanate and urea favors urea, with the cyanate: urea ratio averaging less than 1:100 [39,110,111]. Nevertheless, because urea levels in the body are relatively high compared to many other biomolecules, significant amounts of cyanate can be generated, and as urea levels increase with declining kidney function, so does protein carbamylation [1]. The plasma concentration of cyanate in healthy individuals is about 45 nmol/L, and in patients with ESRD needing dialysis it reaches 140 nmol/L [112]. While this concentration may seem low compared to other biomolecules, recall that urea dissociation is constantly generating more cyanate, which rapidly binds to nearby proteins and amino acids, and thus the rate of protein and amino acid carbamylation may become quite significant. Furthermore, thiocyanate is also known to elevate in patients undergoing dialysis compared to healthy controls, introducing another pathway by which carbamylation can accelerate in this population [113e115]. Kraus et al. demonstrated that serum concentrations of many carbamylated free amino acids in patients with ESRD exceeded the concentrations of their unmodified precursors [116]. This finding could have further implications for the nutritional depletion, specifically protein energy malnutrition, well described in CKD and ESRD patients [117]. Similarly, carbamylated albumin levels were about twofold higher in CKD and ESRD patients compared to non-uremic subjects [1]. While it seems urea accumulation plays a dominant role in the carbamylation burden of patients with CKD, comparative assessments of the relative carbamylation impact of the MPO catalyzed pathway or by amino acid balance, have not been well studied. Given carbamylation’s impact on protein charge, structure, and function, effecting distinct pathways relative to kidney disease progression and outcomes (such as renal fibrosis and cardiovascular disease), investigators have pursed several human association studies to quantify carbamylation’s independent role in clinical outcomes in patients with kidney disease. These studies have largely employed homocitrulline or carbamylated proteins

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(albumin, hemoglobin) as markers of total body carbamylation burden (detailed above) to predict meaningful clinical outcomes.

5.1 Carbamylation and Cardiovascular Outcomes One of the earliest clinical outcome carbamylation studies linked protein bound homocitrulline levels in blood samples to cardiovascular outcomes in participants of the Genebank study, a large (n ¼ 10,000) and well characterized tissue repository with longitudinal data from subjects undergoing elective diagnostic left heart catheterization [2]. Notably, the individuals selected from this cohort for the carbamylation study had relatively preserved kidney function (average estimated glomerular filtration rate [GFR] > 80 mL/min/1.73 m2), highlighting that carbamylation’s relevance is not solely reliant upon grossly elevated urea levels as seen in advanced CKD. The investigators found, using a nested case-control study design of 150 patients with CVD and 300 age and gender matched controls, that protein bound homocitrulline levels significantly associated with coronary or peripheral artery disease as diagnosed on angiography. Subjects in the highest quartile of carbamylation had a 7e8 fold higher risk of CVD as compared to the lowest quartile. The same report went on to confirm these findings in a second case (n ¼ 275) - control (n ¼ 275) study stemming from the same cohort, with case definition including individuals experiencing a major adverse cardiac event (MACE) over a 3-year period including subjects undergoing a revascularization procedure (angioplasty, stent, or bypass grafting), or experiencing nonfatal myocardial infarction, stroke, or death. Subjects who experienced a MACE had significantly higher carbamylation levels than controls, and these findings remained significant following adjustment for traditional CVD risks factors, GFR, and inflammatory markers. Others have shown similar results in smaller studies, thus it seems that measures of systemic protein carbamylation burden can independently predict cardiovascular outcomes [118,119].

5.2 Carbamylation in End Stage Kidney Disease Early studies looking at carbamylation burden in patients with ESRD requiring dialysis were partially motivated by identifying a reliable marker of dialysis adequacy. It was shown in a small cohort of hemodialysis subjects (n ¼ 55) that carbamylated hemoglobin was higher in individuals with a Kt/V (the standard dialysis adequacy metric based on urea kinetic modelling) less than 1.1 compared to those with a Kt/V greater than this, and there was a negative correlation between carbamylation levels and both Kt/V

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and urea reduction ratios (URR, a measure of the pre vs. post urea reduction across a single dialysis treatment) [58]. Tarif and colleagues directly set out to determine if carbamylated hemoglobin could serve as a better predictor of adequacy of hemodialysis than the URR [103]: Studying 50 individuals for 14 months, they found while carbamylated hemoglobin levels were correlated to URR, the levels were more strongly correlated to pre-dialysis urea levels as well as urea changes over time. The results imply that carbamylated hemoglobin could be a more accurate indicator of time averaged urea, and thus overall dialysis adequacy, than the conventional indices of dialysis adequacy, Kt/V and URR. Others have found similar associations between carbamylated hemoglobin, time-averaged urea, and Kt/V [120]. In contrast, in a study limited by sample size (n ¼ 7), Balion et al. found significant variability over time in individuals’ carbamylated hemoglobin levels, with little association to markers of dialysis adequacy [36]. The aforementioned studies were limited in scope as they did not ultimately test if carbamylation measures could predict clinical outcomes such as mortality in dialysis patients better than the standard dialysis adequacy measures. However, we found that in the modern era of dialysis, adequacy measures typically fail to show a mortality association presumably because the clear majority of hemodialysis patients meet minimum recommendations for Kt/V and, informed by large scale landmark clinical trials, dialysis doses beyond the minimal requirements have not proven to add benefit [1,121]. In this context we have shown that carbamylation levels are indeed stronger predictors of mortality in ESRD patients on hemodialysis than the conventional urea kinetic based markers Kt/V or URR [1,122]. Such findings reiterate that carbamylation should not be considered a simple urea equivalent, rather its levels likely integrate multiple pathologic pathways including nutritional status, inflammation, and time averaged solute clearance and accumulation, making it a potent assessment of health risk. In addition to the application of carbamylated protein measurements as indicators of the adequacy of dialysis therapy, these biomarkers may also be useful in discriminating between acute and longer-term increases in urea levels [100,101,123e125]. When looking at individuals with acute (n ¼ 42), acute on chronic (n ¼ 22), or stable chronic (n ¼ 24) kidney disease, matched for degree of renal impairment, carbamylated hemoglobin could sufficiently discern between the groups, offering utility in identifying individuals with acute and possibly reversible kidney injury from those with chronic and likely stable disease [126]. Others have shown similar findings in slightly larger cohorts and using homocitrulline assays [127].

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The time-dependence and reversibility of carbamylation has also been investigated by measuring carbamylated hemoglobin in patients with acute kidney injury (n ¼ 37), CKD (n ¼ 53), post successful kidney transplant (n ¼ 6), and normal kidney function (n ¼ 31) [128]. As predicted, patients with CKD showed significantly higher carbamylation than those with acute kidney injury. Moreover, the carbamylation burden in individuals with acute injury was positively correlated with the number of days of illness (r ¼ 0.74; P < .01). This fact was despite similar blood urea nitrogen and serum creatinine values. In patients with a kidney transplant, carbamylated hemoglobin concentration declined by 19.7% within 2e3 weeks after receiving the graft but this occurred with an average hemoglobin increase of 25% making the true in vivo post-transplant kinetics less clear. More recently, nearly all clinical outcomes studies have focused on subjects with ESRD on dialysis [1,122,129e134]. In 2013, two groups independently and simultaneously reported on the all-cause mortality risk associated with elevated protein carbamylation levels in hemodialysis patients. It was noteworthy that the investigator groups were completely independent, used different measures of carbamylation burden (carbamylated albumin and protein bound homocitrulline) and studied three distinct hemodialysis cohorts in total, each with unique attributes (incident dialysis, prevalent dialysis, and prevalent dialysis patients with diabetes). Even after adjusting for relevant covariates, in each of these cohorts, baseline protein carbamylation was found to be an independent risk factor for death in follow up periods ranging from 1 to 5 years [1,134]. In the first of these studies, Koeth et al. looked at serum protein bound homocitruline levels in a group of 347 patients undergoing maintenance hemodialysis with 5 years of follow up [134]. After multivariable adjustments, carbamylation levels in the highest tertile conferred a more than double risk of death compared to patients with carbamylation levels in the middle or lowest tertiles (adjusted hazard ratio [HR] 2.4; 95% confidence interval [CI], 1.5e3.9; and adjusted HR, 2.3; 95%CI, 1.5e3.7; respectively). Nearly simultaneously, we reported similar findings using two distinct cohorts of hemodialysis patients [1]. The first cohort consisted of 187 participants from the Accelerated Mortality on Renal Replacement cohort which included only incident dialysis patients followed for up to 1 year. Like the Koeth et al. study, we found a risk ratio 3.23; 95% CI, 1.74 to 6.00 for the top tertile of carbamylation (measured using carbamylated albumin) compared to the lowest. In the largest hemodialysis cohort in which carbamylation has been studied, we employed data and blood samples

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from the randomized controlled “German Diabetes and Dialysis study” which investigated the benefits of the cholesterol lowering drug atorvastatin in diabetic dialysis patients (n ¼ 1161) [62]. In this group of prevalent diabetic hemodialysis subjects, we again found a significant risk of death among subjects with the highest tertile of carbamylated albumin (HR 2.25; 95%CI, 1.42e3.56) even after multivariable adjustments. The actual cause of death in these carbamylation-mortality association studies next became of interest to shed light onto any mechanistic underpinnings from the findings. Dreschler et al. addressed this by reanalyzing the aforementioned German Diabetes and Dialysis study data [131]. When looking at specific causes of death, carbamylation levels were most strongly associated with the 4-year risk of death from congestive heart failure (but not myocardial infarction [MI] or stroke). This has been the only report of cause of death in ESRD in association with carbamylation to date and further work is required. While it may seem at odds with the earlier Genebank studies strongly implicating carbamylation in pathways of atherosclerosis, ischemia, and need for revascularization, the differences in the patient populations (ESRD vs. largely preserved renal function) could account for this. It is reported that with declining kidney function, there is a progressive shift from ischemic to non-ischemic etiologies of cardiac death, particularly sudden death in the absence of evidence of an acute MI [135]. We interpreted the long-term risk of heart failure to be consistent with the hypothesis that carbamylation is contributing to uremic cardiomyopathy and fibrosis, which are irreversible but take time to progress before fulminant cardiac failure occurs. While the comparative rates of carbamylation-linked accelerated atherosclerosis versus cardiomyopathy are unknown and surely variable, the increase in heart failure with greater observation time may suggest a “late look bias.” Notably, carbamylation also associated with cardiac stress markers and adverse long-term events in a smaller cohort of non- ESRD patients with chronic systolic heart failure [136]. Another key observation that came of the post-hoc study of the German Diabetes and Dialysis statin trial, was that in subgroup analysis, subjects in the lowest carbamylation tertile appeared to derive a survival benefit from the cholesterol lowering drug whereas subjects in the second and top tertile of carbamylation showed no benefit from statin therapy [131]. As the original trial showed that statins, one of the most potent and efficacious CVD risk modifiers in the general population, conferred no benefit to dialysis patients, detecting this signal of benefit in an ESRD sub-group is significant. It is plausible that a statin benefit is only evident in subjects who are not

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burdened with the strong competing risk of excessive carbamylation, and at some degree of carbamylation the protein modification pathway then becomes the dominant effector. This hypothesis needs additional work to determine its merit. For example, it remains unclear what effect a patients’ carbamylated LDL levels have on the net efficacy of statin therapy. If hyper-carbamylation resulted in more carbamylated LDL, perhaps this would modify the typical beneficial effects of the statin therapy seen in non-ESRD patients. If true, this could potentially introduce a new therapeutic target (either the reduction of global carbamylation load or carbamylated LDL specifically). Morbidity in ESRD has also been interrogated with regards to carbamylation. We demonstrated, among 161 hemodialysis patients, that subjects in the highest quartile of carbamylated albumin had the lowest hemoglobin levels, yet used the greatest EPO doses. This so-called “hyporesponsiveness” to EPO that independently associates with carbamylation (independent of traditional EPO hyporesponsiveness risks such as iron stores, inflammatory markers, etc.) has become a promising intermediary clinical endpoint in interventional studies that aim to ameliorate carbamylation levels in chronic dialysis patients (e.g., NCT02472834, see next section) [130]. Changes in EPO responsiveness are likely to manifest on the order of months verses the years it takes to chart differences in mortality. Interest in reducing protein carbamylation burden further increased when we began to better understand the natural history of carbamylation in ESRD overtime. In another case-control study of 122 incident ESRD hemodialysis subjects who survived at least the first 90 days of dialysis but died within 1 year (cases) and 244 incident dialysis patients who survived beyond 1 year (controls), carbamylated albumin levels measured at the start of dialysis and every 90-day period until 1-year or death, showed that at the time of dialysis initiation, carbamylation levels were markedly higher than levels observed in prevalent maintenance dialysis [122]. This suggests that as CKD progresses and before dialysis restores some degree of solute clearance, patients are likely in a “peak” carbamylation state. Nevertheless, the high levels of carbamylation at dialysis initiation were similar between the cases and controls. Adjusted repeated measures analysis of carbamylation changes over time then showed that 1-year survivors experienced a significantly greater mean carbamylation decrease from baseline compared to cases. Interestingly, similar analysis of urea levels, serum albumin, or standard dialysis metrics such a Kt/V or normalized protein catabolic rate did not show such a difference between the groups. This implies that a greater reduction

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in carbamylation over time is associated with a lower risk of mortality and this is a phenomenon independent of traditional risk factors. Indeed, multiple studies aimed at understanding how carbamylation can be modulated seek to prove such a benefit.

6. POSSIBLE TARGETED THERAPIES TO MITIGATE CARBAMYLATION IN KIDNEY DISEASE As with all association studies, the correlations between protein carbamylation and adverse clinical outcomes, however, compelling, cannot definitively declare causation. For this determination, the gold standard burden of proof would be evidence that the direct modulation of protein carbamylation results in changes in clinical outcomes. No completed studies have attempted to look at this, though there are some ongoing (e.g., NCT02472834). Rather, to date the focus has been mainly on determining precisely how to modulate carbamylation. Nutritional interventions and dialysis modifications have been the two most promising avenues studied thus far (Table 3). The transition from late stage CKD not on dialysis to ESRD on maintenance dialysis has been shown to confer a substantial reduction in protein carbamylation [122]. This is presumably from the marked increase in urea clearance that dialysis initiation confers. It is notable, however, that even with ongoing hemodialysis, ESRD is clearly a state of excess carbamylation relative to healthy controls or individuals with largely preserved kidney function [1]. Thus, additional urea clearance by way of more intensive hemodialysis (i.e., extended duration), might reduce carbamylation levels. It was shown in a small study of 33 subjects receiving routine thrice weekly hemodialysis that conversion to extended duration (double treatment time), thrice weekly, inecenter hemodialysis resulted in a significant reduction in protein carbamylation (3.2 mmol/mol) compared to no significant changes in a small non-randomized control group (n ¼ 20) that did not change from their conventional thrice weekly dialysis prescription [129]. This study went on to show that individuals who experienced the greatest reductions in carbamylation in the extended dialysis group also demonstrated reductions in left ventricular mass as assessed by cardiac MRI. Another interesting finding from this small study was that subjects undergoing the intensive dialysis strategy also demonstrated an increase in serum amino acid levels. Given the background mechanisms of elevated urea driving increases in cyanate and carbamylation, and the demonstration

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Table 3 Possible Targeted Therapies for Attenuating Carbamylation Intervention Effect

Extended duration hemodialysis

Amino Acid Supplementation Nutritional Therapy

Ibuprofen

Aspirin

Bendazac

Triclosan

Associated with reduction in carbamylated albumin levels, increases in average essential amino acids, and reduction in urea compared to patients on conventional hemodialysis Amino acid infusion 3 times per week post-dialysis over 8 weeks reduced C-Alb levels in hemodialysis patients Mediterranean diet and a very low protein diet with essential amino acid and ketoanalogue supplementation reduced protein carbamylation and reduced urea levels in patients with CKD Ascorbic acid, alpha-tocopherol, and lycopene with antioxidant activity inhibited LDL carbamylation in vitro Flavonoids inhibit LDL carbamylation in vitro (likely by scavenging cyanate ions) Eicosapentaenoic acid supplementation in mice decreased mitochondrial protein carbamylation, improving mitochondrial protein quality and attenuating the age-related loss of mitochondrial function Inhibited in vitro cross-linking events from carbamylation that cause the loss of chaperone-like activity of alpha-crystallin Reduced the rate of cyanate driven carbamylation of most soluble lens proteins in vitro Decreased cyanate binding to lens proteins (and thus, carbamylation) in vitro; its major metabolite, 5-hydroxybendazac also inhibits the binding of cyanate to lens proteins Carbamylation was inhibited by triclosan in a dose dependent manner in vitro using doses well below those found in typical consumer products.

Reference

[129]

[130]

[140]

[153]

[154] [155]

[156]

[157,158]

[159]

[160]

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that serum free amino acid levels effectively compete with proteins for carbamylation, either mechanism could be attributed (or most likely both) to the significant reduction in carbamylation observed in the study. The reasons for an increase in amino acid levels might be related to nutritional improvements in the well dialyzed patients but, disappointingly, intensive dialysis has failed to show substantial clearance of several other uremic toxins possibly limiting the ultimate impact this strategy will have on clinical outcomes [137e139]. The notion of elevating circulating amino acid levels to effectively shield proteins from carbamylation has been more directly studied as well. Based on the in vitro and animal studies demonstrating that amino acid supplementation can reduce protein carbamylation load in the presence of urea or cyanate [1], and acknowledging that high carbamylation states may contribute to effective amino acid deficiency because carbamylated amino acids cannot be used for protein synthesis, in human studies of amino acid supplementation during hemodialysis are being pursued. We reported the first proof-of-concept investigation of amino acid therapy aimed at reducing carbamylation in a small pilot study of hemodialysis subjects [130]. Carbamylated albumin levels measured across an 8-week period (2 half-lives of normal serum albumin) fell by 15% in individuals receiving a commercially available amino acid infusion during routine thrice weekly dialysis treatments when compared to individuals receiving no treatment. Notably, there was not an appreciable change in blood urea levels in the treated group which would be one concern if giving additional nitrogen loads to patients. While such results are promising, it is important to note that no study has yet examined whether the modulation of carbamylation will result in actual changes in clinical outcomes. This point is being addressed through an ongoing randomized controlled clinical trial that is effectively an extension of the aforementioned pilot study. The difference in the new study is randomization of subjects to address the possibility of residual confounding and the addition of intermediary clinical end-points such as changes in erythropoietin responsiveness (NCT02472834). The data highlighting that urea reduction and amino acid increases might be effective strategies at mitigating protein carbamylation in hemodialysis patients, raise the question whether similar strategies are applicable to other

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populations. A recently conducted study in peritoneal dialysis subjects receiving intraperitoneal amino acid solutions offered some insights [133]. In a post-hoc analysis from the IMPENDIA trial which originally compared the metabolic effects of low-glucose peritoneal dialysis solutions (incorporating icodextrin and intraperitoneal amino acids) to a control group (dextrose-only solutions), researchers examined the impact the two treatment strategies had on carbamylation levels. First, this report noted that when carbamylated albumin levels of diabetic peritoneal dialysis patients were compared to matched hemodialysis subjects, peritoneal dialysis patients had significantly higher baseline urea levels and higher carbamylated albumin levels (mean  standard deviation [SD] urea 58.6  16.7 vs. 48.4  15.2 mg/dL; carbamylated albumin 11.4  4.0 vs. 10.1  4.1 mmol/mol). Among IMPENDIA participants, there was no difference in carbamylation change in either arm, but amino acid treated subjects showed a trend towards increased carbamylation. Notably, the treated subjects also demonstrated an increase in urea, possibly explaining the carbamylation trend. This data would suggest that strategies other intraperitoneal amino acid treatment might be preferred if aiming to reduce carbamylation in this population. As detailed above, among incident dialysis populations, carbamylation tends to be the highest at the time of dialysis initiation (reflecting the carbamylation burden of the later stages of CKD before dialysis) and progressive dialysis treatments reduce carbamylation over time as does intensive dialysis and intra-dialytic amino acid supplementation [122,129,130]. Because nondialysis CKD populations do not have such modifications of dialysis as a treatment option, studies are now looking at what can be accomplished through alternative means. In a well-designed and rigorous study of dietary modifications, Di Iorio and colleagues achieved significant carbamylation reductions through dietary interventions [140]. In a prospective, randomized, crossover controlled trial, 60 patients with CKD stage 3be4 were assigned to different nutritional treatment arms: 3 months of free diet (FD), 6 months of a very low protein diet supplemented with ketoanalogues (VLPD þ KA), and 6 months of a Mediterranean diet (MD). All subjects started with 3 months of FD, then half went to VLPD þ KA and half to MD, with another 3 month FD period, and then cross over into the remaining alternative dietary intervention group. At the study completion, the investigators noted lower blood pressure, decreased serum urea and

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phosphorous, and higher levels of bicarbonate and hemoglobin with MD and VLPD þ KA. When compared with FD, both MD and VLPD þ KA were associated with a decrease in homocitrulline levels. While urea reduction is presumably the primary effect these dietary interventions had on carbamylation, it is notable that the VLPD þ KA showed the most dramatic carbamylation reduction and the impact of KA supplementation on circulating AA’s is not entirely known. In many ways, this study corroborates the studies in dialysis that suggest urea reduction and AA supplementation (without increasing urea) are effective strategies to reduce carbamylation. To date there have not been association studies linking carbamylation to clinical outcomes in a CKD cohort not on dialysis and this is a key future direction for research.

7. FUTURE DIRECTIONS FOR RESEARCH While the number of studies of protein carbamylation in kidney disease continue to grow, several gaps in our knowledge exist. With additional attention, several key questions can be answered to guide this promising field of research. Carbamylation load increases with kidney disease and there is ample evidence how this could mechanistically confer increased risk for clinical outcomes. Carbamylation measures appear to carry an independent association to adverse outcomes in end stage kidney disease patients on hemodialysis. Presently, clinical outcome studies in peritoneal dialysis patients and in CKD patients not yet on dialysis are lacking. Such studies would add to the robustness of the risk association data and further justify interest in pursuing studies aimed at modifying carbamylation. As highlighted above, approaches targeting the lowering of serum urea, while not compromising nutritional balance, and enhancing amino acid balance (possibly through ketoanalogue supplementation) represent early successful attempts at modifying carbamylation that appear applicable to both ESRD and CKD populations. These findings are preliminary and need replication as well as assessment for clinical impact. Lastly, standardization of the assays used to measure carbamylation in human research could help validate results across different studies. For example, the characteristics of carbamylated proteins such as albumin and hemoglobin versus protein bound homocitrulline levels have not been directly compared and could be useful in interpreting future studies which utilized any given assay.

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8. CONCLUSION Decades of research have coalesced to demonstrate protein carbamylation is a post-translational protein modification that occurs ubiquitously in humans and can increase through a variety of pathways including kidney disease, smoking, air pollution, and diet. Carbamylated proteins can undergo changes in charge, structure, and function, resulting in molecular and cellular dysfunction. CKD is a state of hyper-carbamylation, in part due to excessive urea accumulation, and rises in carbamylation have been strongly and independently associated with adverse outcomes including death. Thus far, interventions that reduce urea levels and restore amino acid levels appear to be efficacious at reducing carbamylation but whether such interventions result in meaningful clinical outcomes is yet to be determined. Replication and validation of existing studies are needed as well as expansion to investigate the effects of protein carbamylation in CKD patients not yet on dialysis. Standardization of the methods to assess carbamylation load in patients will help to further advance this promising line of research.

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