A novel DNase assay reveals low DNase activity in severe asthma.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Antibody profiles in the mosaic of "seronegative" APS syndrome.

Clinical manifestations, as distinct from thrombotic and obstetric morbidity, were recently included in the update of classification criteria of the antiphospholipid syndrome (APS). However, the existence of several patients with clinical manifestations suggestive of APS, but negative for criteria antiphospholipid antibodies (aPLs) [anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant] may suggest an update of diagnostic criteria. In this study, we analyzed the prevalence of six non-criteria aPLs in a large monocentric cohort of patients with seronegative APS (SN-APS), to investigate their possible diagnostic role. aCL IgA, aß2-GPI IgA and aß2-GPI Domain 1 antibodies were detected by chemiluminescence, anti-phosphatidylserine/prothrombin (aPS/PT) IgG, anti-vimentin/cardiolipin (aVim/CL) IgG and anti-carbamylated-ß2-glycoprotein I (aCarb-ß2-GPI) IgG by ELISA in sera from 144 SN-APS patients. In SN-APS patients, aCL IgA were detected in 4/144 (2.77%), aß2-GPI IgA in 2/144 (1.39%), aß2-GPI-Domain 1 in 1/144 (0.69%), aPS/PT in 16/144 (11.11%), aVim/CL in 37/144 (25.69%) and aCarb-ß2-GPI in 43/144 patients (29.86%). Patients negative for all non-criteria aPL assays were 77/144 (53.47%). Notably, the Venn diagram showed that aCarb-ß2-GPI together with aVim/CL represented the prevalent combination of positive antibodies. In SN-APS patients, aCL IgA were associated with recurrent thrombosis (OR11.48; p=0.03); in obstetric SN-APS patients, aPS/PT were significantly associated with foetal deaths (OR4.84; p=0.01), aVim/CL with spontaneous abortions (OR2.71; p=0.016). This study indicates that aPS/PT, aVim/CL and aCarb-ß2-GPI antibodies may represent useful tools to identify "seronegative" APS patients, who are negative for criteria aPLs, supporting the need to make testing for non-criteria aPLs more accessible in patients with SN-APS.

Accumulation of Carbamylation-Derived Products in Aneurysmal Aorta.

INTRODUCTION: Carbamylation is a nonenzymatic post-translational modification of proteins characterized by the binding of isocyanic acid to amino groups of proteins, which leads to the alteration of their properties. An increase in serum carbamylation-derived products, including homocitrulline (HCit), has been shown to be associated with the development of cardiovascular diseases. METHODS: HCit was quantified by LC-MS/MS within extracts of aneurysmal and control human aortas. A mouse model of aortic aneurysm (ApoE-/- mice perfused with angiotensin II and fed with sodium cyanate) was used to evaluate the role of carbamylation in aneurysm development. RESULTS: HCit quantification showed a greater heterogeneity of values in aneurysmal aortas in comparison with control ones. At the maximum diameter of dilation, HCit values were significantly higher (+94%, p < 0.05) compared with less dilated areas. No differences were observed according to aneurysm size or when comparing ruptured and unruptured aneurysms. No significant effect of carbamylation on aneurysm development was observed using the animal model. CONCLUSIONS: These results evidenced the accumulation of HCit within aneurysmal aortas but do not allow concluding about the exact participation of protein carbamylation in the development of human abdominal aortic aneurysms.

Neutrophils in Inflammatory Bone Diseases.

PURPOSE OF REVIEW: In this review, we summarize the current evidence that suggests that neutrophils play a key role in facilitating damage to local bone structures. RECENT FINDINGS: Neutrophil infiltration is a hallmark of inflammatory bone diseases such as rheumatoid arthritis (RA) and periodontitis disease (PD). Both of these human diseases are marked by an imbalance in bone homeostasis, favoring the degradation of local bone which ultimately leads to erosions. Osteoclasts, a multinucleated resident bone cell, are responsible for facilitating the turnover of bone and the bone damage observed in these diseases. The involvement of neutrophils and neutrophil extracellular trap formation have recently been implicated in exacerbating osteoclast function through direct and indirect mechanisms. We highlight a recent finding that NET proteins such as histones and elastase can generate non-canonical, inflammatory osteoclasts, and this process is mediated by post-translational modifications such as citrullination and carbamylation, both of which act as autoantigens in RA. It appears that NETs, autoantibodies, modified proteins, cytokines, and osteoclasts all ultimately contribute to local and permanent bone damage in RA and PD. However, more studies are needed to fully understand the role of neutrophils in inflammatory bone diseases.

Comparative CKD risk prediction using homocitrulline and carbamylated albumin: two circulating markers of protein carbamylation.

BACKGROUND: Protein carbamylation, a post-translational protein modification primarily driven by urea, independently associates with adverse clinical outcomes in patients with CKD. Biomarkers used to quantify carbamylation burden have mainly included carbamylated albumin (C-Alb) and homocitrulline (HCit, carbamylated lysine). In this study, we aimed to compare the prognostic utility of these two markers in order to facilitate comparisons of existing studies employing either marker alone, and to inform future carbamylation studies. METHODS: Both serum C-Alb and free HCit levels were assayed from the same timepoint in 1632 individuals with CKD stages 2-4 enrolled in the prospective Chronic Renal Insufficiency Cohort (CRIC) study. Adjusted Cox proportional hazard models were used to assess risks for the outcomes of death (primary) and end stage kidney disease (ESKD) using each marker. C-statistics, net reclassification improvement, and integrated discrimination improvement were used to compare the prognostic value of each marker. RESULTS: Participant demographics included mean (SD) age 59 (11) years; 702 (43%) females; 700 (43%) white. C-Alb and HCit levels were positively correlated with one another (Pearson correlation coefficient 0.64). Higher C-Alb and HCit levels showed similar increased risk of death (e.g., the adjusted hazard ratio [HR] for death in the 4th carbamylation quartile compared to the 1st was 1.90 (95% confidence interval [CI] 1.35-2.66) for C-Alb, and 1.89 [1.27-2.81] for HCit; and on a continuous scale, the adjusted HR for death using C-Alb was 1.24 [1.11 to 1.39] per standard deviation increase, and 1.27 [1.10-1.46] using HCit). Both biomarkers also had similar HRs for ESKD. The C-statistics were similar when adding each carbamylation biomarker to base models (e.g., for mortality models, the C-statistic was 0.725 [0.707-0.743] with C-Alb and 0.725 [0.707-0.743] with HCit, both compared to a base model 0.723). Similarities were also observed for the net reclassification improvement and integrated discrimination improvement metrics. CONCLUSIONS: C-Alb and HCit had similar performance across multiple prognostic assessments. The markers appear readily comparable in CKD epidemiological studies.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta.

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Serum carboxymethyllysine concentration is associated with erosive hand osteoarthritis.

OBJECTIVE: Carboxymethyllysine (CML) and homocitrulline (HCit) are the products of two non-enzymatic post-translational modifications of protein, a process related to age. We investigated whether serum CML and HCit concentrations were associated with hand osteoarthritis (HOA), especially erosive HOA. DESIGN: Serum CML and HCit were measured by using liquid chromatography coupled with tandem mass spectrometry at inclusion in 386 patients included in the DIGItal Cohort Design (DIGICOD) cohort. We investigated whether serum CML and/or HCit concentrations were associated with erosive HOA or with HOA clinical and radiological features. Moreover, we compared the tissular concentrations of CML and HCit in OA and non-OA cartilage from proximal interphalangeal and metacarpo-phalangeal (MCP) joints from human cadaveric donors. RESULTS: Median (IQR) serum CML concentration was lower in patients with erosive HOA than those with non-erosive HOA (178.7 [157.1-208.8] vs 194.7 [168.9-217.1] µmol/mol Lys, P = 0.002), but median HCit concentration did not differ between the groups (193.9 [162.9-232.0] vs 193.9 [155.9-224.6] µmol/mol Lys). Cartilage HCit and CML concentrations were not correlated with clinical features. Serum CML concentration was higher in OA than non-OA MCPs (7.0 vs 4.0 mmol/mol Lys, P = 0.01). CONCLUSIONS: Serum CML concentration was lower in erosive HOA than non-erosive HOA, and cartilage CML concentration was higher in OA than non-OA cartilage. These results encourage further studies to test whether serum CML could be a new prognostic biomarker in HOA.

Comparison of homocitrulline and carbamylated albumin as biomarkers of carbamylation reactions in hemodialyzed patients.

To describe the association between levels of homocitrulline (HCit) and the degree of albumin carbamylation in a cohort of hemodialyzed patients. Plasma total and protein-bound HCit concentrations in samples from hemodialyzed patients included in NICOREN trial were determined by LC-MS/MS at baseline and after 24 weeks of treatment with either sevelamer or nicotinamide. HCit concentrations at all timepoints and in both groups were positively and significantly correlated with the degree of albumin carbamylation. Plasma concentrations of total HCit, protein-bound HCit and carbamylated albumin did not decrease after 24 weeks of treatment with either sevelamer or nicotinamide. The present results demonstrate that plasma total and protein-bound HCit concentrations were closely associated with albumin carbamylation in hemodialyzed patients. Therefore, total and protein-bound HCit concentrations might be valuable biomarkers of the overall intensity of protein carbamylation in this context. Given the less complex and time-consuming analytical methods required, these markers should be favored in future clinical studies of carbamylation reaction.

Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk.

BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. RESULTS: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. CONCLUSIONS: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. Video Abstract.

Potential interference of in vitro carbamylation on C-reactive protein laboratory measurement.

Carbamylation is a nonenzymatic post-translational modification observed during the reaction between cyanate and amino acids and/or proteins that may occur during some pathologies such as chronic kidney disease. Evidence suggests that carbamylation may interfere with the quantification of some analytes measured using immunoturbidimetric assays. C-reactive protein (CRP) is an inflammatory response protein that is commonly quantified through immunoturbidimetry in clinical laboratories. Because the presence of modified proteins in serum can lead to impaired quantification, this study aimed to verify the impact of in vitro carbamylation on the measurement of CRP in a CRP standard solution and serum pool. The samples were incubated with 150 nM, 150 µM, or 150 mM potassium cyanate (KOCN) or 20, 100, or 500 mg/dL urea at 37 °C for 24 h. CRP concentrations were measured using an immunoturbidimetric assay. The results showed a 61%-72% decrease in the CRP detection rate after incubation with KOCN. Incubation with urea resulted in a 0.7%-8% lower CRP detection rate. The results of this study indicate that high concentrations of cyanate can lead to falsely decreased CRP levels, as measured by immunoturbidimetry.

Carbamylation of Integrin α IIb ß 3: The Mechanistic Link to Platelet Dysfunction in ESKD.

BACKGROUND: Bleeding diatheses, common among patients with ESKD, can lead to serious complications, particularly during invasive procedures. Chronic urea overload significantly increases cyanate concentrations in patients with ESKD, leading to carbamylation, an irreversible modification of proteins and peptides. METHODS: To investigate carbamylation as a potential mechanistic link between uremia and platelet dysfunction in ESKD, we used liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to quantify total homocitrulline, and biotin-conjugated phenylglyoxal labeling and Western blot to detect carbamylated integrin α IIb ß 3 (a receptor required for platelet aggregation). Flow cytometry was used to study activation of isolated platelets and platelet-rich plasma. In a transient transfection system, we tested activity and fibrinogen binding of different mutated forms of the receptor. We assessed platelet adhesion and aggregation in microplate assays. RESULTS: Carbamylation inhibited platelet activation, adhesion, and aggregation. Patients on hemodialysis exhibited significantly reduced activation of α IIb ß 3 compared with healthy controls. We found significant carbamylation of both subunits of α IIb ß 3 on platelets from patients receiving hemodialysis versus only minor modification in controls. In the transient transfection system, modification of lysine 185 in the ß 3 subunit was associated with loss of receptor activity and fibrinogen binding. Supplementation of free amino acids, which was shown to protect plasma proteins from carbamylation-induced damage in patients on hemodialysis, prevented loss of α IIb ß 3 activity in vitro. CONCLUSIONS: Carbamylation of α IIb ß 3-specifically modification of the K185 residue-might represent a mechanistic link between uremia and dysfunctional primary hemostasis in patients on hemodialysis. The observation that free amino acids prevented the carbamylation-induced loss of α IIb ß 3 activity suggests amino acid administration during dialysis may help to normalize platelet function.

Carbamylated sortilin associates with cardiovascular calcification in patients with chronic kidney disease.

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD.

Carbamylation of ß2-glycoprotein I generates new autoantigens for antiphospholipid syndrome: a new tool for diagnosis of 'seronegative' patients.

OBJECTIVES: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-ß2-glycoprotein I (ß2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ('seronegative APS', SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-ß2-GPI (Carb-ß2-GPI) as a new autoantigen of APS. METHODS: ß2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived dendritic cell (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with RA, 20 non-APS thrombosis and 50 healthy donors were analysed for anti-Carb-ß2-GPI by ELISA. RESULTS: Carb-ß2-GPI is able to activate moDCs, inducing upregulation of CD80, CD86 and CD40, activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB, and IL-12p70 release. Serological results showed that both 37/114 SN-APS (32.46%) and 23/60 APS (38.33%) patients resulted positive for anti-Carb-ß2-GPI. Interestingly, SN-APS patients who tested positive for anti-Carb-ß2-GPI showed a higher prevalence of thrombocytopenia (P = 0.04, likelihood positive ratio of 3.9). CONCLUSION: Data obtained from both functional tests on moDCs and immunological approaches prompted identification of Carb-ß2-GPI as a 'new' antigenic target in APS. In particular, anti-Carb-ß2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients who tested positive for anti-Carb-ß2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

HDL functionality in type 1 diabetes: enhancement of cholesterol efflux capacity in relationship with decreased HDL carbamylation after improvement of glycemic control.

BACKGROUND: Reduced cholesterol efflux capacity (CEC) of HDLs is likely to increase cardiovascular risk in type 1 diabetes (T1D). We aimed to assess whether improvement of glycemic control in T1D patients is associated with changes in CEC in relation with changes in carbamylation of HDLs. METHODS: In this open-label trial, 27 uncontrolled T1D patients were given a three-month standard medical intervention to improve glycemic control. HDL fraction was isolated from plasma, and CEC was measured on THP-1 macrophages. Carbamylation of HDLs was evaluated by an immunoassay. Control HDLs from healthy subjects were carbamylated in vitro with potassium cyanate. RESULTS: HbA1c decreased from 11.4% [10.2-12.9] (median [1st-3rd quartiles]) at baseline to 8.1% [6.6-9.0] after the three-month intervention (P < 0.00001). The CEC of HDLs increased after intervention in 19 (70%) patients (P = 0.038). At the same time, the carbamylation of HDLs decreased in 22 (82%) patients after intervention (P = 0.014). The increase in CEC significantly correlated with the decrease in carbamylated HDLs (r = -0.411, P = 0.034), even after adjustment for the change in HbA1c (ß = -0.527, P = 0.003). In vitro carbamylation of control HDLs decreased CEC by 13% (P = 0.041) and 23% (P = 0.021) using 1 and 10 mmol/L of potassium cyanate, respectively. CONCLUSIONS: The improvement of CEC in relation to a decrease in the carbamylation of HDLs may likely contribute to the beneficial cardiovascular effect of glycemic control in T1D patients. TRIAL REGISTRATION: NCT02816099 ClinicalTrials.gov.

High-resolution capillary electrophoresis for the determination of carbamylated albumin.

OBJECTIVES: Carbamylation is a non-enzymatic post-translational reaction of a primary amino group of a protein with isocyanate. The albumin carbamylation is a negative prognostic factor in chronic kidney disease (CKD) patients and induce charge difference implying an observed shift in electrophoretic mobility that can be measured through a symmetry factor (SF). METHODS: The Helena V8 and the Sebia Capillarys 2 systems were used for all experiments. The effect of in vitro carbamylation on the SF by spiking increasing concentrations of potassium isocyanate (KCNO) in serum of three healthy volunteers was investigated. Theoretical plate numbers (N) as a surrogate of separation efficiency were also calculated and correlations between SF and renal function biomarkers were performed on 284 patients. RESULTS: A dose-dependent impact of KCNO on the SF was observed for both methods with the Helena V8 being more sensitive. The mean N was significantly higher on the Helena V8 as compared to the Sebia Capillarys 2 (2,972 vs. 444.1, p<0.0001). The SF correlated significantly with eGFR (r=0.50, p<0.0001), creatinine (r=-0.31, p<0.0001) and urea (r=-0.34, p<0.0001) on the Helena V8. On the Sebia Capillarys 2, a significant correlation was only observed with eGFR (r=0.17, p=0.004). A better discrimination between CKD stages was also observed using the Helena V8. CONCLUSIONS: Thanks to a higher mean N, the Helena V8 might offer new possibilities, including detection of carbamylated albumin through SF calculation. Further studies are still needed to confirm the interest of using this type of assays in clinical routine.

Carbamylated Proteins in Renal Disease: Aggravating Factors or Just Biomarkers?

Carbamylation is a nonenzymatic post-translational modification resulting from the reaction between cyanate, a urea by-product, and proteins. In vivo and in vitro studies have demonstrated that carbamylation modifies protein structures and functions, triggering unfavourable molecular and cellular responses. An enhanced formation of carbamylation-derived products (CDPs) is observed in pathological contexts, especially during chronic kidney disease (CKD), because of increased blood urea. Significantly, studies have reported a positive correlation between serum CDPs and the evolutive state of renal failure. Further, serum concentrations of carbamylated proteins are characterized as strong predictors of mortality in end-stage renal disease patients. Over time, it is likely that these modified compounds become aggravating factors and promote long-term complications, including cardiovascular disorders and inflammation or immune system dysfunctions. These poor clinical outcomes have led researchers to consider strategies to prevent or slow down CDP formation. Even if growing evidence suggests the involvement of carbamylation in the pathophysiology of CKD, the real relevance of carbamylation is still unclear: is it a causal phenomenon, a metabolic consequence or just a biological feature? In this review, we discuss how carbamylation, a consequence of renal function decline, may become a causal phenomenon of kidney disease progression and how CDPs may be used as biomarkers.

Anti-carbamylated protein antibodies positivity and disease activity in Hispanic patients with established rheumatoid arthritis: An observational study.

OBJECTIVES: We aimed to determine the prevalence of anti-carbamylated protein (anti-CarP) antibodies in Mexican Hispanics with established rheumatoid arthritis (RA) and to assess their relationship with disease activity. METHODS: A cohort study was conducted in 278 patients with established RA during an 18-month follow-up. We measured IgG/IgM/IgA rheumatoid factor (RF), IgG anticitrullinated protein antibodies (ACPA) and IgG/IgM/IgA anti-CarP antibodies using enzyme-linked immunosorbent assay (ELISA). For disease activity, we performed the 28-joint disease activity score with erythrocyte sedimentation rate (DAS28-ESR). Repeated measures one-way ANOVA was used to test the association between anti-CarP IgG antibody status and longitudinal DAS28-ESR scores. Patients were evaluated at baseline and at 6, 12, and 18 months during follow-up. RESULTS: Anti-CarP IgG antibodies were positive in 47.8% of patients and, accounting for all isotypes, in 9.5% of patients with negative RF and ACPA. Triple antibody positivity was present in 42.6% of patients in our sample. Anti-CarP IgG antibody positivity did not show statistically significant differences in mean DAS28-ESR when compared to anti-CarP IgG antibody negative patients at baseline, 6, 12 or 18 months. CONCLUSION: Anti-CarP IgG antibodies are not associated to a higher disease activity in Hispanic patients with established RA. Our findings suggest that the clinical value of measuring anti-CarP antibodies in RA diminishes over time.

Confident Identification of Citrullination and Carbamylation Assisted by Peptide Retention Time Prediction.

The chromatographic behavior of peptides carrying citrulline and homocitrulline residues in proteomic two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS) experiments has been investigated. The primary goal of this study was to determine the chromatographic conditions that allow differentiating between arginine citrullination and deamidation of asparagine based on retention data, improving the confidence of MS-based identifications. Carbamylation was used as a reference point due to a high degree of similarity between modification products and anticipated changes in chromatographic behavior. We applied 2D LC-MS/MS (a high-pH-low-pH reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC)-low-pH RP, and strong cation exchange (SCX)-low-pH RP) to acquire retention data for modified-nonmodified peptide pairs in the four separation modes. Modifications of a standard protein mixture were induced enzymatically (PAD-2) or chemically (urea) for citrullination and carbamylation, respectively. Deamidation occurs spontaneously. Similar retention shifts were observed for all three modifications in a high-pH RP (decrease) and a low-pH RP (increase), thus limiting the applicability of this 2D LC combination. HILIC on bare silica and strong cation exchange separations have been probed to amplify the effect of charge loss upon citrullination, with SCX demonstrating the most differentiating power: the elimination of basic residues upon citrullination/carbamylation results in an ∼58 mM KCl retention decrease, while retention of deamidated products decreases slightly.

Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Impact of albumin carbamylation on immunoturbidimetric measurement of urinary albumin.

Urinary albumin is one of the main markers used in clinical practice to assess kidney damage. It is usually measured in laboratories through immunological assays, but these assays may not detect molecules with conformational changes, such as carbamylated albumin/proteins. Therefore, this study aimed to investigate the impact of albumin carbamylation on the measurement of albuminuria by an immunoturbidimetric assay. The addition of the carbamylating agent to PBS buffer and urine pool promoted a lower quantification of albumin measured by the immunoturbidimetric method, indicating that this process may be responsible for an underestimation of the results in clinical practice.