Study participants
The study was reviewed and approved by Minia University Faculty of Medicine Research ethics committee and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all study participants, and the study protocol was approved by the institute’s committee on human research. This study was an observational, prospective, hospital-based, case-controlled study with the participants recruited from the inpatient service of Internal Medicine Department, Minia University Hospital. The study enrolled one-hundred and sixty (160) patients with HCV-related liver cirrhosis. Patients were classified into three groups: cirrhotic patients without ascites used as controls (40 patients), cirrhotic patients with ascites but without renal impairment (60 patients), and cirrhotic patients with ascites and with renal impairment diagnosed as having HRS-AKI (formerly, HRS type 1) (60 patients). The inclusion criteria were cirrhotic patients who are HCV positive, with liver cirrhosis diagnosed on liver biopsy or through clinical, radiographic, and biochemistry investigations, and HRS-AKI patients were diagnosed using the revised diagnostic criteria of hepatorenal syndrome (HRS) with rapidly progressive decrease in kidney function, defined as absolute increase in serum creatinine ≥ 0.3 mg/dl within 48 h or relative increase in serum creatinine ≥ 50% from baseline within 7 days and/or decrease in urine output ≤ 0.5 ml/kg ≥ 6 h. The exclusion criteria were HRS-NAKI (formerly HRS type 2) defined as stable or slowly progressive decrease in kidney function with eGFR < 60 ml/min/1.73m2, serious comorbidities (functional class IV heart failure, O2-dependent COPD, advanced cancer), chronic kidney disease, other causes of liver cirrhosis, shock, documented established parenchymal kidney disease, obstructive uropathy, use of nephrotoxic drugs in the previous 30 days, urinary tract infection, anuria for 12 h, need for renal replacement therapy, or any solid organ transplantation.
Primary predictors and outcomes
The primary predictors were urinary concentrations of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and interleukin-18 (IL-18). Within 2 to 3 h of admission, 10 mL urine was collected from study participants and centrifuged at 2000g for 20 min, and the supernatant was frozen at −800 °C and retained for analysis. We measured urinary NGAL, KIM-1, by commercially available enzyme-linked immunosorbent assay (ELISA) kit (BOSTER Biological Technology Co. USA), according to the manufacturer’s instructions.
Urine NGAL assay was performed according to the following steps: the microtiter plate was coated with monoclonal anti-NGAL antibody. A total of 100 μl of standards or samples are added to the appropriate microtiter plate and incubated for 90 min at 37 °C. Remove the liquid of each well, add 0.1 ml of a biotin-conjugated polyclonal anti-NGAL antibody to each well, and incubate for 1 h 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) to each microplate and incubated for 30 min at 37 °C. Color development was achieved using a 90 μl TMB substrate solution is added to each well and incubated for 30 min in the dark 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 100 μl sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Serial dilutions of recombinant human NGAL were used to establish a standard curve. The detection limit of NGAL with this research assay is 15.63–1000 pg/ml. The intra-assay coefficients of variations (CVs) were 3.9% and 6.2% at 1246 ± 48.6 and 6827 ± 423.3 pg/ml, respectively. Inter-assay CVs were 8.1% and 6.9% at 1522 ± 123.3 and 7133 ± 492.2 pg/ml, respectively.
Urine KIM-1 assay was performed according to the following steps: the microtiter plate was coated with monoclonal anti-KIM-1 antibody. A total of 100 μl of standards and samples are added to the appropriate microtiter plate and incubated for 90 min at 37 °C. Remove the liquid of each well, add 100 μl of a biotin-conjugated polyclonal anti-KIM-1 antibody to each well, and incubate for 1 h 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 HRP to each microplate and incubated for 30 min at 37 °C. Color development was achieved using a 90 μl TMB substrate solution is added to each well and incubated for 30 min in the dark 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 100 μl sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Serial dilutions of recombinant human KIM-1 were used to establish a standard curve. The detection limit of KIM-1 with this research assay is 31.2–2000 pg/ml. The intra-assay CVs were 4.6% and 6.1% at 253 ± 11.64 and 1220 ± 74.42 pg/ml, respectively. Inter-assay CVs were 6.4% and 7.9% at 317 ± 20.3 and 1458 ± 115.2 pg/ml, respectively.
Urine IL-18 assay was performed according to the following steps; the microtiter plate was coated with monoclonal anti-IL-18 antibody. A total of 100 μl of standards and samples are added to the appropriate microtiter plate and incubated for 90 min at 37 °C .Remove the liquid of each well, add 100 μl of a biotin-conjugated polyclonal anti-KIM-1 antibody to each well, and incubate for 1 h 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 HRP to each microplate and incubated for 30 min at 37 °C. Color development was achieved using a 90 μl TMB substrate solution is added to each well and incubated for 30 min in the dark 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 100 μl sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Serial dilutions of recombinant human IL-18 were used to establish a standard curve. The detection limit of IL-18 with this research assay is 15.6–1000 pg/ml. The intra-assay coefficients of variations (CVs) were 4.3% and 5.5% at 135 ± 5.8 and 762 ± 41.9 pg/ml, respectively. Inter-assay CVs were 5.2% and 5.8% at 141 ± 7.3 and 776 ± 45.1 pg/ml, respectively.
Other measurements
Demographic data, history of any serious comorbidities, diuretic therapy (dose and duration), chronic kidney disease, other causes of liver cirrhosis, shock, documented established parenchymal kidney disease, obstructive uropathy, use of nephrotoxic drugs in the previous 30 days, urinary tract infection, anuria for 12 h, need for renal replacement therapy, solid organ transplantation, history of complications such as hematemesis, melena, hepatic encephalopathy, or spontaneous bacterial peritonitis, and urine output were obtained at enrollment. Complete blood count (CBC) was measured. Another sample on red topped plane tube was used. Serum was separated from whole blood by centrifugation at 3000 rpm for 10 min and was used for measuring albumin, calcium, phosphorus, total cholesterol, triglycerides, urea, creatinine, and uric acid levels. Anti-HCV and HBsAg were detected using ELISA (Sanofi Diagnostic Pasteur, Marne-la-coquette, France), with the use of BIOELISA HCV kit, (BIO kit, SA Barcelona). Complete urine analysis with examination of urine sediment and spot morning protein to creatinine ratio were performed to all patients. Abdominal ultrasonography was done using General Electric Ultrasound, USA, and transducer with a frequency of 3.5 megahertz (MHz).
Estimated GFR (eGFR) was derived using the Modification of Diet in Renal Disease (four variables MDRD equation). The fractional excretion of sodium (FeNa) was calculated by the formula (SCr × UNa)/(SNa × UCr). The fractional excretion of urea (FeUrea) was calculated by the formula (SCr × UUrea)/(SUrea × UCr). We assess the severity of the underlying liver disease by MELD-Na score and Child-Turcotte-Pugh (CPT) score. We assess organ dysfunction, morbidity, and mortality using the sequential organ failure assessment score (SOFA score), APACHE II score, and Glasgow coma scale (GCS).
Statistical analyses
All the analyses were performed with Statistical Package of Social Science (SPSS), version 25. Qualitative data were expressed as frequency (%), and quantitative data were summarized as mean ± standard deviation (SD). Qualitative data were analyzed by chi-square test (χ2) and Fisher exact test as appropriate, while quantitative results were compared with either Student t-test or analysis of variance (ANOVA) followed by Bonferroni correction. Kruskal-Wallis test for nonparametric quantitative data followed by Mann-Whitney test and Dunn-Bonferroni. Also, simple and multiple logistic regression analyses were used to detect independent predictors. For all analyses, statistical significance was defined as p-values ≤ 0.05.