The study was reviewed and approved by the Minia University Faculty of Medicine Research ethics committee (Approval NO. 186: 6/2016) and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all participants. This study was an observational hospital-based, cohort study to compare the accuracy of different glomerular filtration rate equations based on creatinine, cystatin C, and both as valuable tools in the detection of kidney disease in liver cirrhosis related to HCV in Egyptian patients; with the participants being recruited from the Internal Medicine Department of Minia University hospital. The study enrolled 120 patients with hepatitis C virus (HCV)-related liver cirrhosis, as well as 60 age- and sex-matched healthy controls. The diagnosis of cirrhosis was based on a combination of physical examination, laboratory tests, and abdominal ultrasonography. The inclusion criterion was HCV-related liver cirrhosis in adult patients > 18 years old of both genders. The exclusion criteria for patients and healthy controls were acute viral or bacterial infection, primary renal disease or hepatorenal syndrome (HRS), chronic obstructive pulmonary disease (COPD), diabetes mellitus, thyroid dysfunction, active GIT bleeding or gastrointestinal bleeding during the month before enrollment, hepatocellular carcinoma, congestive heart failure (CHF), amputation of whole or part-limb, medications use including corticosteroids, antiviral drugs, angiotensin-II receptor blockers, angiotensin-converting enzyme inhibitors, aminoglycosides, nonsteroidal anti-inflammatory drugs (NSAIDs), and l-ornithine-l-aspartate.
Primary outcome measures
Percentage of estimated glomerular filtration rate values within 30% of ‘true’ measured glomerular filtration rate were compared. The accuracy of glomerular filtration rate (GFR) estimating equations was commonly expressed as the P30 value, the percentage of estimated GFR values within 30% of ‘true’ GFR. The study estimated and compared the accuracy and precision of GFR-estimating equations based on Cockcroft-Gault formula, the modification of diet in renal disease (MDRD) equation, and three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations using either creatinine or cystatin C or a combination of both in patients with liver cirrhosis related to HCV expressed as the P30 value. Cockcroft-Gault Formula was calculated with the following formula: (140 – age) × weight/72 × Scr × 0.85 [if female]. MDRD creatinine equation was calculated with the following formula: 175 × plasma creatinine−1.154 × age−0.203 (× 0.742 if female; × 1.21 if black). CKD-EPI creatinine equation was calculated using the following formula: 141 × min(Scr/κ, 1)α × max(Scr/κ, 1)-1.209 × 0.993Age × 1.018 [if female] × 1.159 [if black], where Scr is serum creatinine, κ is 0.7 for females and 0.9 for males, α is − 0.329 for females and − 0.411 for males, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1. CKD-EPI cystatin C equation was calculated using the following formula: 133 × min(Scys/0.8, 1)−0.499 × max(Scys/0.8, 1)−1.328 × 0.996Age × 0.932 [if female], where Scys is serum cystatin C, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1. Creatinine-Cystatin C equation was calculated using the following formula: 135 × min(Scr/κ, 1)α × max(Scr/κ, 1)−0.601 × min(Scys/0.8, 1)−0.375 × max(Scys/0.8, 1)−0.711 × 0.995Age × 0.969 [if female] × 1.08 [if black] where Scr is serum creatinine, Scys is serum cystatin C, κ is 0.7 for females and 0.9 for males, α is − 0.248 for females and − 0.207 for males, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1.
Every participant was educated on proper techniques for 24-h urine collection and provided with a detailed patient education pamphlet. One day prior to visit 2, each subject completed a 24-h urine collection for timed urine creatinine clearance using the formula urine creatinine/serum creatinine multiplied by 24-h urine volume (UCr/PCr) × V. This was divided by 1440 to get the value in ml/min.
Measurement of serum cystatin C (CysC) using a two-site second-generation enzyme-linked immunosorbent assay (ELISA) kit (Wkea Med Supplies Corp, China) was done. The microtiter plate was coated with monoclonal purified human anti-cystatin C antibody. Fifty microliters of standards or samples are added to the appropriate microtiter plate wells and incubate for 30 min at 37 °C. Remove the liquid of each well; add 50 μl of a biotin-conjugated polyclonal goat anti-CysC antibody (Detection Reagent A) to each well and incubate for 30 min at 37 °C. Aspirate each well and wash with wash buffer, repeating the process three times for a total of three washes, followed by the addition of 100 μl of Avidin conjugated to horseradish peroxidase (HRP) (Detection Reagent B) to each microplate well and incubated for 1 h at 37 °C. Color development was achieved using a 90 μl TMB substrate solution is added to each well and incubated for 15 min at 37 °C. Only those wells that contain biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of 50 μl sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 ± 2 nm. Serial dilutions of human CysC were used to establish a standard curve. Laboratory measurements were performed at Clinical Pathology Laboratory at the Minia University Hospital. Assay range is 30 μg/L up to 800 μg/L with the intra-assay CV (%) and inter-assay CV (%) are less than 15%.
Demographic data, weight, height, BMI, detailed history, and thorough physical examination were obtained at enrollment. Urinalysis with microscopy was performed before the timed urine collection for GFR measurement. Complete blood count, liver function tests, renal function tests, thyroid function tests, fasting and postprandial plasma glucose, and serum alpha fetoprotein were obtained at enrollment. Complete blood count, liver function tests, urea, creatinine, and uric acid were measured using stored ethylenediaminetetraacetic acid (EDTA) plasma samples using standard techniques. CBC was obtained using Mindray BC-3200 auto hematology counter (Shenzhen Mindray Bio-Medical Electronics Co., Ltd. China). Serum albumin was measured using spectrophotometry. Liver function tests, urea, creatinine, and uric acid were measured using Dimension ES chemical auto-analyzer (Dade Behring, Marburg, Germany).
Serological tests for hepatitis virus B and C were done. Anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA) and detectable serum HCV–RNA by polymerase chain reaction for 6 months or more were needed to confirm the diagnosis of HCV infection. Abdominal ultrasound was done for all patients.
All statistical analyses were performed with Statistical Package for the Social Science (SPSS for windows version 25.0) (SPSS Inc., Tokyo, Japan). The continuous variables were expressed as mean ± SD which compared using chi square test. Pearson’s correlation coefficient was used to estimate the correlation between each two variables. Statistical significance was defined as a probability level of p < 0.05. Bias was defined as the mean difference between measured and estimated GFR; precision was defined as the SD of the difference between measured and estimated GFR. Both precision and bias were expressed as ml/min per 1.73 m2; accuracy was defined as the proportion of values that were within 10 or 30% of the measured GFR.