Stable advanced CKD patients who were older than 18 years old with eGFR below 15 mL/min/1.73m2, as calculated by the CKD-EPI equation, were recruited into the study. CKD was diagnosed and classified according to the KDIGO guideline. The study was conducted in an ambulatory setting and started at 08:00–09:00 AM in order to avoid the diurnal variations of the renal function. Patients having the following conditions were excluded from the study: acute deterioration of the renal function, amputation, bedridden state, infection, gastrointestinal bleeding, and heart failure or hospitalization. Women of childbearing age without a reliable contraceptive method, patients on RRT, and patients taking methyldopa, levodopa, ascorbic acid, cimetidine, trimethoprim, antibiotics, steroids, or flucytosine were also excluded.
Reference GFR measurement
The reference GFR was determined by collecting plasma from 10 different time points and was subjected to 99mTc- Diethyle Triamine Penta-acetic Acid (99mTcDTPA) plasma clearance method, which was performed at the Department of Radiology, Chulalongkorn University. 99mTc-DTPA was purchased from the Office of Atoms for Peace, Bangkok, Thailand, with a radiopurity of >95% and 99mTc-DTPA bound to plasma protein of <5%. The same protocol was applied to all participants. In brief, heparin lock was inserted into the arm to obtain blood samples to determine the radioactivity background and for serum creatinine assay. A single intravenous bolus of 99mTc-DTPA was injected into each participant. Blood specimens were collected to assess plasma radioactivity at 5, 10, 15, 20, 30, 60, 90, 120, 180, and 240 minutes post 99mTc-DTPA injection. Plasma radioactive activities were then plotted as a function of time to create a time-activity curve in order to calculate the GFR. The GFR equation was determined using bi-exponential fitting method that was utilized in a previous study8.
Calibration for serum creatinine assay and laboratory measurement of Cystatin C
Fasting serum creatinine was measured using CREA plus (11775642) enzymatic assay® on a COBAS INTEGRA 400 plus analyzer, Roche Diagnostics (Indianapolis, IN), traceable to isotope-dilution mass spectrometry, as recommended by the National Kidney Disease Education Program9. The IDMS reference serum creatinine (SRM 967) was purchased from the National Institute of Standards and Technology. The certified concentration values of serum creatinine were 0.847±0.018 mg/dL for level 1 and 3.877±0.082 mg/dL for level 2. Cystatin C was measured by Biovendor human cystatin C ELISA kit® (RD191009100) (Candler, NC) which has been recommended for in vitro diagnostic use by the European Union.
The eGFR values were calculated using the re-expressed MDRD equation10, CKD-EPI equation6, Thai eGFR equation8, and cystatin C-based eGFR equation11 (supplementary data table S1).
Body composition analysis
Bioelectrical impedance analysis (BIA) by Inbody® 720 Body Composition Analyzer (Biospace, Seoul, Korea) was used to measure the body composition such as weight, fluid, muscle, and fat. This method utilized the tetrapolar eight-point tactile electrode system. Thirty impedance measurements were gathered using six different frequencies (1, 5, 50, 250, 500, and 1000 kHz) at each of the five segments of the body: right arm, left arm, trunk, right leg, and left leg. Reactance was performed utilizing 15 impedance measurements using the three frequencies (5, 50, and 250 kHz) at each of the five segments of the body. Bioelectrical impedance measurements were performed after the blood samples were collected.
Bland–Altman plots were used to assess the agreement between eGFR from various equations and the reference GFR. The regression of the average and the difference between the reference GFR and the eGFR (reference GFR minus eGFR) were analyzed. Bias was defined as the mean absolute difference between the reference and eGFR. Precision was defined as the standard deviation value of the mean absolute difference. Accuracy was deﬁned as the proximity of the estimation compared with the reference and was calculated using combined root mean square error and the percentage of GFR within 30% of the reference GFR. Root mean square error (RMSE) was calculated to estimate the spread out of the predicting error. The accuracy of the equation was compared by the χ2 test. Statistical analysis was performed by using SPSS for windows version 22.0.
Ethics approval and informed consent
The Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (Bangkok, Thailand; IRB.644/59), approved the study. Information pertaining to the study was provided to all interested participants prior to obtaining their written informed consent.