Supplementary Materials
This appendix has been provided by the authors to give readers additional information about their work (Kalim et al, “Protein Carbamylation and the Risk of End Stage Kidney Disease in Patients with Chronic Kidney Disease”)
Contents
Calibration methods for the carbamylated albumin assay.
Supplemental Table 1: Baseline characteristics of the study population vs. the parent cohort.
Supplemental Table 2: Risk of a 50% eGFR decline according to carbamylated albumin quartiles
Supplemental Table 3: Estimated GFR slope per year based on baseline carbamylated albumin quartile
Supplemental Table 4: Risk of ESKD and eGFR decline according to carbamylated albumin quartile using different GFR estimation equations
Supplemental Table 5: P-interaction for all subgroups compared with carbamylated albumin Supplemental Table 6: Model comparisons to show impact of adding carbamylated albumin to fully adjusted ESKD models
Supplemental Figure 1: Adjusted hazard ratios for reaching ESKD according to baseline carbamylated albumin value
Calibration methods:
Carbamylated albumin (C-Alb) measurements were obtained using high performance liquid chromatography and tandem mass spectrometry. Measurements were calibrated using three pooled serum calibrators previously used in an initial large scale C-Alb study.
1Calibrators and assay values in this original study were reported as percent carbamylated albumin relative to non-carbamylated albumin; however, the assay has since then been adapted to conform to SI units (in mmol carbamylated albumin per mol non-carbamylated albumin) similar to guidelines for reporting of glycated hemoglobin.
2Low, medium, and high C-Alb calibrators were previously assayed to be 3.17, 8.75, and 18.18 mmol of carbamylated albumin per mol non-carbamylated albumin, respectively. Each of these calibrators were aliquoted when they were first produced and have been stored at -80°C since that time. During analysis of the CRIC study samples, on each day of analysis, fresh aliquots of each calibrator were thawed and included in the final 3 wells of each 96-well plate of samples for measurement. In this way, each 96-well plate of samples was independently calibrated with calibrators traceable to the original study.
The area under the curve (AUC) for carbamylated and non-carbamylated albumin and their
isotopically labeled peptide internal standards (IS) were integrated and the ratio of
carbamylated-to-non carbamylated albumin was calculated and normalized to their respective internal standards:
Normalized C-Alb ratio = (carbamylated albumin AUC/non-carbamylated albumin AUC) ÷ (carbamylated albumin IS AUC/non-carbamylated albumin IS AUC)
For each plate of samples, normalized C-Alb ratios for each calibrator were plotted against their assigned C-Alb concentrations. A linear standard curve was generated using ordinary least squares linear regression in Microsoft Excel, generating a plate-specific standard curve:
C-Alb value in mmol/mol = Slope x [Normalized C-Alb ratio] + Constant
Patient sample C-Alb concentrations were then calculated using their plate-specific standard
curves.
Supplemental Table 1: Baseline characteristics of the study population vs. the parent cohort
CRIC initial cohort at recruitment
CRIC cohort without ESKD at Year 1 visit
Year 1
patients without samples available for the study
Year 1 patients with samples included in the study
n 3939 3455 344 3111
Age, years 57.7 (11.0) 59.0 (10.7) 59.4 (10.1) 59.0 (10.8)
Female, No (%) 1778 (45.1) 1569 (45.4) 212 (61.4) 1357 (43.6)
Race, No (%)
Non-Hispanic White 1638 (41.6) 1504 (43.5) 171 (49.6) 1334 (42.9)
Non-Hispanic Black 1650 (41.9) 1436 (41.6) 115 (33.3) 1321 (42.5)
Hispanic 497 (12.6) 377 (10.9) 39 (11.3) 338 (10.9)
Other 154 (3.9) 138 (4.0) 20 (5.8) 118 (3.8)
Past Medical History, No (%)
Hypertension 3391 (86.1) 3072 (89.0) 270 (78.3) 2802 (90.2)
Diabetes 1908 (48.4) 1696 (49.1) 146 (42.3) 1550 (49.8)
CHF 382 (9.7) 357 (10.3) 31 (9.0) 326 (10.5)
Stroke 392 (10.0) 371 (10.7) 29 (8.4) 342 (11.0)
PVD 262 (6.7) 257 (7.4) 19 (5.5) 238 (7.7)
Current smoking 517 (13.1) 422 (12.2) 33 (9.6) 389 (12.5)
BMI 32.1 (7.8) 32.2 (7.7) 31.4 (8.5) 32.2 (7.6)
SBP, mmHg 128.5 (22.2) 126.9 (21.7) 124.1 (23.7) 127.2 (21.4)
Medication use, No (%)
Aspirin 1677 (42.9) 1606 (46.7) 138 (40.6) 1468 (47.4)
Beta blocker 1930 (49.3) 1740 (50.6) 151 (44.4) 1589 (51.3)
Statin 2153 (55.0) 2037 (59.3) 191 (56.2) 1846 (59.6)
ACE or ARB 2689 (68.8) 2398 (69.8) 206 (60.6) 2192 (70.8)
Laboratory data
Serum creatinine, mg/dL 1.8 (0.7) 2.0 (0.9) 1.7 (1.0) 2.0 (0.9)
eGFR ml/min/1.73 m2 44.9 (16.7) 42.5 (16.8) 49.1 (18.9) 41.8 (16.4)
Urinary protein, g/ 24h 0.2 [0.1, 0.9] 0.2 [0.1, 0.9] 0.1 [0.1, 0.3] 0.2 [0.1, 0.9]
Serum urea nitrogen, mg/dL 29.7 (13.6) 31.0 (16.0) 27.1 (14.9) 31.4 (16.1)
Serum albumin, g/dL 3.9 (0.5) 4.0 (0.4) 4.1 (0.5) 4.0 (0.4)
Hemoglobin, g/dL 12.6 (1.8) 12.8 (1.8) 13.0 (1.6) 12.8 (1.8)
Abbreviations: No, number; CHF, congestive heart failure; PVD, peripheral vascular disease; BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); SBP, systolic blood pressure; ACE, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; eGFR, estimated glomerular filtration rate; HS-CRP, high sensitivity C-reactive protein.
Values for continuous variables are presented as mean (SD) or median [interquartile range].
Supplemental Table 2: Risk of a 50% eGFR decline according to carbamylated albumin quartiles Carbamylated albumin, mmol/mol
(mean, range) Hazard Ratio Overall population per SD in C-Alb 1.21 (1.06, 1.36) Quartile 1: 4.71 (<= 5.80) ref
Quartile 2: 6.75 (5.80 - 7.80) 1.48 (1.13, 1.93) Quartile 3: 9.15 (7.80 - 10.71) 1.54 (1.15, 2.04) Quartile 4: 15.15 (> 10.71) 1.58 (1.14, 2.20)
Model is stratified by center and adjusts for age, sex, race/ ethnicity, systolic blood pressure, body mass index, smoking status, history of diabetes, cardiovascular disease, use of angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker medications, serum total albumin, natural log-transformed high sensitivity C- reactive protein levels, baseline estimated glomerular filtration rate (eGFR), natural log-transformed proteinuria, and cause of kidney disease.
Supplemental Table 3: Estimated eGFR slope per year based on baseline carbamylated albumin quartile
C-Alb quartile groups
eGFR CRIC equation 2021 CKD-EPI Creatinine equation eGFR slope per
year P value eGFR slope per
year P value
4.71 (<= 5.80) -0.85 (-1.07, -0.63) ref -0.72 (-0.93, -0.51) ref
6.75 (5.80 – 7.80) -1.00 (-1.22, -0.78) 0.16 -0.88 (-1.10, -0.67) 0.13
9.15 (7.80 – 10.71) -1.22 (-1.45, -0.99) 0.001 -1.12 (-1.34, -0.89) 0.0003
15.15 (> 10.71) -1.18 (-1.43, -0.93) 0.006 -1.08 (-1.32, -0.84) 0.003
Model is stratified by center and adjusts for age, sex, race/ ethnicity, systolic blood pressure, body mass index, smoking status, history of diabetes, cardiovascular disease, use of angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker medications, serum total albumin, natural log-transformed high sensitivity C- reactive protein levels, baseline estimated glomerular filtration rate (eGFR), natural log-transformed proteinuria, and cause of kidney disease.
Random slope and intercept as random effects in the model.
P value calculated by comparing the eGFR slopes of the higher quartiles to the 1st quartile group
Supplemental Table 4:Risk of ESKD and eGFR decline according to carbamylated albumin quartile using different GFR estimation equations Carbamylated albumin, mmol/mol
(mean, range) Rate per
1,000 patient-year
Hazard Ratio
Unadjusted Model 1a Model 2b Model 3c Model 4d Model 5e
ESKD
Overall population 39.86 (37.41, 42.40) 1.92 (1.81, 2.04) 1.94 (1.83, 2.07) 1.26 (1.14, 1.40) 1.24 (1.11, 1.37) 1.24 (1.11, 1.37) 1.20 (1.09, 1.33) Quartile 1: 4.71 (<= 5.80) 15.70 (13.02, 18.72) Reference Reference Reference Reference Reference Reference Quartile 2: 6.75 (5.80 - 7.80) 27.89 (24.14, 32.01) 1.77 (1.41, 2.22) 2.06 (1.63, 2.60) 1.39 (1.08, 1.79) 1.46 (1.12, 1.88) 1.46 (1.12, 1.88) 1.43 (1.11, 1.86) Quartile 3: 9.15 (7.80 - 10.71) 48.14 (42.73, 54.00) 3.03 (2.44, 3.76) 3.40 (2.73, 4.24) 1.42 (1.10, 1.84) 1.48 (1.14, 1.91) 1.48 (1.14, 1.91) 1.41 (1.09, 1.82) Quartile 4: 15.15 (> 10.71) 88.54 (80.04, 97.62) 5.51 (4.48, 6.79) 6.53 (5.26, 8.10) 1.75 (1.33, 2.32) 1.78 (1.34, 2.37) 1.78 (1.34, 2.37) 1.63 (1.22, 2.16) ESKD or 50% eGFR decrease
Overall population 50.09 (47.28, 53.01) 1.88 (1.77, 2.00) 1.77 (1.67, 1.88) 1.23 (1.12, 1.35) 1.21 (1.10, 1.33) 1.21 (1.10, 1.33) 1.17(1.07, 1.29) Quartile 1: 4.71 (<= 5.80) 24.22 (20.79, 28.02) Reference Reference Reference Reference Reference Reference Quartile 2: 6.75 (5.80 - 7.80) 37.76 (33.25, 42.65) 1.55 (1.28, 1.89) 1.79 (1.47, 2.18) 1.24 (1.00, 1.53) 1.29 (1.04, 1.59) 1.30 (1.04, 1.61) 1.26 (1.02, 1.56) Quartile 3: 9.15 (7.80 - 10.71) 60.82 (54.55, 67.56) 2.49 (2.07, 2.99) 2.76 (2.29, 3.33) 1.31 (1.05, 1.62) 1.34 (1.07, 1.66) 1.35 (1.08, 1.68) 1.27 (1.02, 1.58) Quartile 4: 15.15 (> 10.71) 99.00 (89.81, 108.80) 4.03 (3.38, 4.82) 4.70 (3.91, 5.67) 1.55 (1.21, 1.97) 1.56 (1.22, 2.00) 1.56 (1.22, 2.00) 1.43 (1.12, 1.84) Abbreviations: ESKD, end stage kidney disease; eGFR, estimated glomerular filtration rate
a Model 1 is stratified by center and adjusts for age, sex, race, and ethnicity.
b Model 2 adjusted for Model 1 variables plus eGFR, natural log-transformed proteinuria, cause of kidney disease, and blood urea nitrogen level.
c Model adjusted for variables from Models 1 and 2 plus systolic blood pressure, body mass index, smoking status, history of diabetes, cardiovascular diseases, use of angiotensin- converting enzyme inhibitor or angiotensin II receptor blocker medications, serum total albumin, and natural log-transformed high sensitivity C- reactive protein levels.
d Model 4 was the same as the fully adjusted Model 3 but eGFR was calculated using the 2021 CKD-EPI Creatinine equation.
e Model 5 was the same as the fully adjusted Model 3 but eGFR was calculated using the 2021 CKD-EPI Creatinine- Cystatin C equation.
Supplemental Table 5: P-values for interaction testing with carbamylated albumin across different subgroups
Interaction testing with
carbamylated albumin P Value
Age 0.79
Sex 0.01
Race 0.94
Diabetes 0.05
Smoking status 0.68
Cardiovascular disease 0.61
eGFR 0.61
Serum urea nitrogen 0.40
Cystatin C 0.58
24 hours urinary protein 0.18
Serum albumin 0.28
Hemoglobin 0.42
Low-density lipoprotein 0.74
Supplemental Table 6: Model comparisons to show impact of adding carbamylated albumin to fully adjusted CKD outcomes models
Model A Model B
Model B
P value Model C
Model C P value
ESKD Outcome
C-statistic 0.88 0.89 0.0001 0.88 0.0003
NRI -- 0.14 <0.001 0.11 <0.001
IDI -- 0.012 <0.001 0.010 <0.001
Abbreviations: ESKD, End Stage Kidney Disease; NRI, net reclassification improvement; IDI, integrated discrimination improvement.
Models computed the 10-year risk for each participant for the primary outcome of ESKD using Cox proportional hazard models adjusted for age, sex, race/ ethnicity, systolic blood pressure, body mass index, smoking status, history of diabetes, cardiovascular diseases, use of angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker medications, serum total albumin, natural log-transformed high sensitivity C- reactive protein levels, eGFR, natural log-transformed proteinuria, and cause of kidney disease.
Model A is considered the base model and includes the covariates noted above.
Model B compares the addition of carbamylated albumin (continuous, log-transformed) to the base model.
Model C compares the addition of blood urea nitrogen (continuous) to the base model.
Supplemental Figure 1 Adjusted Hazard Ratios for Reaching End Stage Kidney Disease According to Baseline Carbamylated Albumin Value (C-Alb). We fit a restricted cubic spline function with four knots (at the 5th, 35th, 65th, and 95th percentiles) and plotted the association between C-Alb and risk for ESKD. The hazard ratios are per each unit increase in baseline C-Alb (mmol/ mol). The shaded area is the 95% confidence interval from the restricted-cubic-spline model. The mean C-Alb value (4.71) of the lowest quartile of C-Alb was used as the reference. The hazard ratios were adjusted for age, sex, race and ethnicity, systolic blood pressure, body mass index, smoking status, history of diabetes, cardiovascular disease, use of angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker medications, serum total albumin, natural log-transformed high sensitivity C- reactive protein levels, estimated glomerular filtration rate (eGFR), natural log-transformed proteinuria, cause of kidney disease, and blood urea nitrogen level