Carbamylation of serum albumin as a risk factor for mortality in patients with kidney failure

Abstract

Urea, the toxic end product of protein catabolism, is elevated in end-stage renal disease (ESRD), although it is unclear whether or how it contributes to disease. Urea can promote the carbamylation of proteins on multiple lysine side chains, including human albumin, which has a predominant carbamylation site on Lys(549). The proportion of serum albumin carbamylated on Lys(549) (%C-Alb) correlated with time-averaged blood urea concentrations and was twice as high in ESRD patients than in non-uremic subjects (0.90% versus 0.42%). Baseline %C-Alb was higher in ESRD subjects who died within 1 year than in those who survived longer than 1 year (1.01% versus 0.77%) and was associated with an increased risk of death within 1 year (hazard ratio, 3.76). These findings were validated in an independent cohort of diabetic ESRD subjects (hazard ratio, 3.73). Decreased concentrations of serum amino acids correlated with higher %C-Alb in ESRD patients, and mice with diet-induced amino acid deficiencies exhibited greater susceptibility to albumin carbamylation than did chow-fed mice. In vitro studies showed that amino acids such as cysteine, histidine, arginine, and lysine, as well as other nucleophiles such as taurine, inhibited cyanate-induced C-Alb formation at physiologic pH and temperature. Together, these results suggest that chronically elevated urea promotes carbamylation of proteins in ESRD and that serum amino acid concentrations may modulate this protein modification. In summary, we have identified serum %C-Alb as a risk factor for mortality in patients with ESRD and propose that this risk factor may be modifiable with supplemental amino acid therapy.

Figures

Fig. 1

Identification of carbamylation on Lys-549 of cyanate-treated albumin. Cyanate-treated and untreated purified human albumin from a commercial source was digested with glutamyl endoproteinase and analyzed by LC-MS/MS. Peptides were identified by matching fragmentation spectra to sequences from the human SwissProt proteome with the AB Sciex Protein Pilot software programmed to search for both carbamylated and non-carbamylated peptide forms. (A) Summary reports for the carbamylated and non-carbamylated forms of the peptide encompassing lysine residue 549 (sequence RQIKXQTALVE where X = N-ε-carbamoyl-L-lysine). Contrib, contribution of identified peptide (in ProtScore units) to protein identification; Conf, percent confidence of peptide identification; z, peptide charge; Spectrum, within-run identifier for MS/MS spectra used for peptide identification. (B) MS/MS spectra for carbamylated and non-carbamlyated digested albumin peptides encompassing Lys-549; the modification state-revealing fragment ions are shown in red. (C) Predicted ion fragments of carbamylated and non-carbamylated peptide forms; detected fragment ions matching the predicted fragmentation spectra are highlighted in red. Both peptides were identified with 99+% confidence using the Paragon algorithm, which compares the confidence of the match between the observed spectra and the identified peptide sequence to the combined confidence for all other possible peptide matches.(54).

Fig. 2

Average carbamylated albumin values in uremic and non-uremic patients. (A) Average %C-Alb values in non-uremic subjects (n = 20) and in patients with stage 3 or 4 chronic kidney disease (CKD) (n = 122) and ArMORR HD subjects (n = 187). (B) Average %C-Alb in ArMORR survivors who lived longer than 12 months (n = 106) and in ArMORR cases who died during the 12-month study period (n = 81) . Individual %C-Alb values for each group are shown in Table S2. Data are expressed as average carbamylated albumin as a % of total; error bars, 95% confidence intervals of the mean; Student’s t-test P-values shown.

Fig. 3

Kaplan-Meier curve estimates of the incidence of all-cause mortality in ESRD patients. Subjects were categorized into upper, middle, and lower tertiles according to serum %C-Alb values measured at the outset of the study. (A) 12 month survival rates in ArMORR study subjects. (B) 12 month survival rates in 4D study subjects. Numbers of subjects at risk at different time points during each study shown in the tables at bottom.

Fig. 4

Correlation between %C-Alb and blood urea concentrations. (A) Correlation between blood urea and %C-Alb in ArMORR study subjects with ESRD (n = 187). (B) Correlation between blood urea and %C-Alb in 4D study subjects with ESRD (n = 1,161). (C) Correlation between %C-Alb and blood urea in non-hemodialysis CKD subjects (n = 122). Pearson correlation coefficients (r) and P-values are shown.

Fig. 5

Effects in mice of low protein diet on albumin carbamylation by urea or cyanate. (A) Serum amino acid concentrations after 15 days of low- or normal-protein diets. Values are normalized to average concentrations of amino acid in animals on a normal protein diet. *P < 0.05 using unpaired T test, comparing average amino acids in animals on low and normal protein diets. (B) %C-Alb values in low- or normal-protein fed animals before and 30 minutes after cyanate injection (100 mg/kg body weight). (C) %C-Alb values in animals fed low or normal protein diets supplemented with or without urea (67 mg per gram of feed). Bars indicate mean ± SD, n = 6 animals per group. P-values were calculated using unpaired T test.