Cirrhosis refers to a nodular disorder that affects the normal functioning of the liver due to the main preventable cases attributed to viral hepatitis, NAFLD, or extreme alcohol consumption [1]. Patients with progressive liver cirrhosis are at an increased hazard of developing renal dysfunction as they frequently use intravenous radiocontrast mediators and diuretics, undergo paracentesis, and suffer from gastrointestinal hemorrhages, such as variceal bleeding, or infection as spontaneous bacterial peritonitis [2-35].

Moreover, renal dysfunction usually progresses to hepatorenal syndrome with the development of liver cirrhosis and portal hypertension [3]. The progress of renal dysfunction is one of the greatest important prognostic features in these patients [4]. The prognosis is mostly poor once HRS develops [5].

An accurate assessment of renal function in these patients is essential to estimate the prediction and determine the accurate therapeutic interference (including drug dosing) and reaction [6]. Hepatorenal syndrome (HRS) is a reversible functional renal impairment that occurs in patients with advanced liver cirrhosis or those with fulminant hepatic failure, in the absence of a pre-existing renal pathology [7, 8].

Cystatin C is a nonglycosylated 13 kDa protein that is one of the cystatin superfamilies of cysteine protease inhibitors [10]. It is produced at a constant degree in all nucleated cells and spontaneously crosses the glomerular membrane, being reabsorbed and broken down in the proximal tubular cells of the kidney, without extrarenal elimination. Contrasting creatinine levels, cystatin C levels do not depend on muscle mass, age, and gender, and are not influenced by inflammatory disorders or malignancy [11-20].

Evaluation of cystatin C levels could be valued in the early prediction of renal dysfunction as they increase faster than creatinine levels as the GFR declines [12]. Thus in this study, we investigated the role of serum cystatin C as a diagnostic tool for renal dysfunction in cirrhotic patients with and without the hepatorenal syndrome.

After approval of the Research Ethical Committee and written consent from participants involved in the study, this case-control study was conducted on 60 patients admitted to the Tropical Medicine Department of Tanta University Hospital and 10 healthy volunteers from January 2018 till June 2019. The participants were divided into 3 groups. Group 1 included 30 patients with liver cirrhosis and normal kidney function, group 2 included 30 cirrhotic patients with hepatorenal syndrome type 2 according to the criteria for diagnosis of the hepatorenal syndrome [13], and group 3 included 10 people who served as healthy control volunteers.

The diagnosis of cirrhosis was based on a combination of physical examination, laboratory tests, and using imaging [14]. Cirrhotic patients with and without hepatorenal syndrome were included in the study. Patients with the following features were excluded from the study, such as patients with chronic renal failure or other causes of renal dysfunction apart from the hepatorenal syndrome, patients with heart failure, patients with metabolic syndrome diagnosed when the fasting glucose is ≥ 100 mg/dl (or receiving drug therapy for hyperglycemia), blood pressure ≥140/90 mm Hg (or receiving drug therapy for hypertension), triglycerides ≥ 150 mg/dl (or receiving drug therapy for hypertriglyceridemia), HDL-C <40 mg/dl in men or <50 mg/dl in women (or receiving drug therapy for reduced HDL-C), and waist circumference ≥102 cm (40 inches) in men or ≥88 cm (35 inches) in women.

All individuals were subjected to thorough history taking, with emphasis on the history of liver disease and drug history, including NSAIDs. Complete clinical examination with detection of signs of shock and hypotension was done. Laboratory investigations included CBC, blood urea, creatinine, liver function tests (total bilirubin, serum ALT, serum AST, and serum albumin), hepatitis markers (HCV-antibody, HBs-ag), blood electrolytes (sodium and potassium), blood sugar, glomerular filtration rate (GFR), and calculation of the scores were carried out. The Child-Pugh score was classified according to total serum bilirubin, serum albumin, INR, ascites status, and degree of hepatic encephalopathy [15].The MELD score was calculated according to the following formula; MELD score = 3.78 × ln (total bilirubin, mg/dl) + 11.2 × ln (INR) + 9.57 × ln (Cr, mg/dl) + 6.43 [16].

Estimation of cystatin C was performed using about 5 ml of the random blood sample taken from each patient and put into plan tubes and then centrifugated for about 2 minutes. The serum was collected and stored at -20°C till the time of analysis. Estimation of cystatin C by automated chemistry using Roche/Hitachi MODULAR analyzers and Cobas c. The C.f.a.s cystatin C is a liquid ready-to-use calibrator based on pooled human serum enriched with recombinant human cystatin C produced in E. coli. The concentration of the calibrator component had been adjusted to ensure optimal calibration of the appropriate Roche method on clinical chemistry analyzers. The calibrator values were determined using the method stated in the electronically available or enclosed value sheets. Determinations were performed under strictly standardized conditions on Roche analyzers using Roche system reagents and the Roche master calibrator.

Abdominal ultrasound was done on all participants using Toshiba Nemio XG apparatus by B-mode ultrasound at the Tropical Medicine Department; the patients were fasting at least for 6 hours and were asked to be in the supine position to assess liver echogenicity, surface, edge, size, portal vein diameter, attenuation of hepatic veins, size of the spleen, and presence of ascites.

In cases of hepatorenal syndrome, no pre-existing intrinsic renal disease should be reported. The kidneys should be in normal size with no evidence of atrophy, and good differentiation between cortex and medulla. Normal appearing kidneys on ultrasound are required for the diagnosis of hepatorenal syndrome according to the Hepatorenal Syndrome-AKI Guidelines issued by the International Club of Ascites.

All cases of group 1 and group 2 were of post hepatitis C cirrhosis. Considering lower limb edema, a significant difference was found among the three groups, which was found to be more prevalent in group 1 and group 2. Regarding ascites, there was a significant difference among the studied groups, 25 cases of moderate to severe in group 1 and 24 cases in group 2. In terms of hepatic encephalopathy, there were 5 cases in group 1 and 12 cases in group 2 with a significant difference between groups 1 and 2. The baseline demographic and clinical data are demonstrated in Tables 1 and 2. Kidney function tests and electrolytes in the studied groups are demonstrated in Table 3.

Child score was significantly worse in group 2 compared to group 1 (P = 0.011). The MELD score was significantly higher in group 2 compared to group 1 (P < 0.001), as shown in Table 4.

Regarding cystatin C, there was a significant difference among the three groups (P <0.001). A significant difference between group 1 and group 2 (P1 <0.001), between group 1 and group 3 (P2 = 0.029), and between group 2 and group 3 (P3 <0.001), was observed, as shown in Table 5.

The diagnostic performance of cystatin C for diagnosis of hepatorenal syndrome was evaluated using the ROC curve. The cutoff value of cystatin C was 1.9 mg/L, which showed a diagnostic sensitivity of 90%, specificity of 97.5%, positive predictive value (PPV) of 96.4%, and negative predictive value (NPV) of 92.9%. The area under the curve (AUC) was 0.972 and the P-value was <0.001 (i.e. good predictor for hepatorenal syndrome) (Tables 6 and 7).

There was a positive statistically significant correlation between cystatin C and MELD (r = 0.766, P <0.001). An insignificant correlation was found between cystatin C and Child score, and AST/ALT ratio.

Diagnosing HRS requires exclusion of other kidney disorders, absence of shock, no current or recent treatment with nephrotoxic drugs, and no improvement in serum creatinine after at least two days of diuretic withdrawal and volume expansion with albumin. HRS has a high mortality rate; therefore, early identification is important [17]. New biomarkers, including CysC, are known to be more accurate in estimating GFR and better correlate with the gold standard method of GFR estimation, such as DTPA scan or iohexol-based clearance [18]. In this study, GFR was found to be decreased in HRS patients (group 2) than cirrhotic patients (group 1). This may be due to severe renal vasoconstriction and decreased vasodilator factors acting on renal circulation. Our results were in agreement with the findings of a study conducted by Markwardt et al. [19], who showed that GFR was significantly lower in patients with HRS. Considering creatinine clearance, our results demonstrated that it was decreased in patients with HRS more than the cirrhotic patients compared to its normal level in controls. This met the alternative definition of HRS which is characterized by slow and progressive worsening of renal function, with serum creatinine clearance of > 40 ml/min{Arroyo, 1996 #1479}. Regarding the Child–Pugh score, we found that the score was worse in patients with HRS (most of them were Child class C) compared to cirrhotic patients (most of them were Child class B). In terms of cystatin C, a significant difference was found among the three groups as cystatin C was higher in patients with HRS compared to the cirrhotic group and healthy controls.

Our results were in agreement with the study conducted by Lei et al. [20] demonstrating that serum Cys C levels in the AKI group (2.4 ± 1.0 mg/L) were significantly higher than those in the non-AKI group (0.8 ± 0.1 mg/L) and the control group (0.7 ± 0.1 mg/L). Gomaa et al. [21] showed that the mean values of Cys C were higher in HRS patients than in the control group (2.62±0.45 and 0.74±0.08 mg/l, respectively) and cirrhotic patients with normal renal function (0.93±0.10 mg/l). Also, Markwardt et al. [19] in a study determining the plasma levels of CysC in 429 patients hospitalized for acute decompensation of cirrhosis in acute-on-chronic liver failure in cirrhosis (CANONIC) showed that CysC was significantly higher in patients with HRS. Our results were in agreement with the findings of Sharawey et al. [22], who found that cystatin C was higher in the HRS group than other cirrhotic patients (p < 0.001).

We found that the cut-off value of cystatin C, i.e., 1.9 mg/L, can be used for diagnosis of hepatorenal syndrome with a diagnostic sensitivity of 90%, specificity of 97.5%, PPV of 96.4%, and NPV of 92.9% with AUC of 0.972 and P-value <0.001 (i.e., good predictor for hepatorenal syndrome). These results were in agreement with the study conducted by Sharawey et al. [22], who revealed that the area under the curves (AUCs) for CysC was 0.86 with an optimal cut-off value of 1.8 mg/L CysC. The sensitivity and specificity for predicting the development of HRS were 88.8% and 67.6%, respectively. Near to our results were the findings of a study conducted by Gomaa et al. [21], who showed that Cys C at a cut-off value of 1.6 mg/l had a sensitivity of 73.3% and a specificity of 63.33%, PPV of 50%, NPV of 82.6, and AUCs of Cyst C was 0.72 (95% CI: 0.581–0.870; P=0.015). Also, Omar et al. [23] demonstrated that Cys C at a cut-off value of 1.2 mg/L had 89.6% sensitivity and 63.6% specificity in detecting early HRS. AUC was found to be 0.785. Also, Chung et al. [24] revealed that the sensitivity, specificity, PPV, and NPV of the cystatin C reference value in regard to a renal injury that occured within three months were found to be 66.7%, 86.4%, 50%, and 92.7%, respectively. The area under the ROC curve was 0.809 (95% CI, 0.671-0.947, p<0.001) in serum cystatin C. Also, Lei et al. [20] showed that the AUC of serum Cys C was 0.816 and P was < 0.001. Moreover, a cutoff value of serum Cys C concentration was 0.93 mg/L, and the sensitivity and specificity were 76.1% and 78.6%, respectively. Wang et al. [25] found that the optimal cut-off value of Cys C was 1.24 mg/l in the diagnosis of kidney impairment in hepatic cirrhosis patients with a sensitivity of 87.6% and a specificity of 93.1%. In our study, there was a positive significant correlation between cystatin C and serum creatinine, urea, GFR, and MELD score [25-28].

Orlando et al. [28] stated that, unlike creatinine, the plasma cystatin C concentration is not affected by decompensated cirrhosis and is a reliable GFR marker in such patients. Studying cirrhotic patients, creatinine failed to detect reduced renal function, with sensitivity being only 23%, whereas cystatin C exhibited good diagnostic sensitivity (88%). Also, Kim et al. [26] showed that serum CysC level was the only independent predictor for significantly decteting a renal impairment.

This study revealed that cystatin C is a very good predictor for hepatorenal syndrome. This study recommends adding cystatin C as an important part of the evaluation of cirrhotic patients with hepatorenal syndrome.

From our results, serum cystatin C is a good predictor for hepatorenal syndrome with a good correlation with serum creatinine, blood urea, GFR, and creatinine clearance.

Fathia El Sayed Asal designed the methodology of the study. Fathia El Sayed Asal, Mohamed Yousef, and Hend Atteya Abdelkhalek Abdraboh contributed to providing the study materials, recruiting patients, and collecting laboratory samples. Ahmed Abdelaziz Abdelaziz Shama and Mohamed Elbahnasawy analyzed the statistics of the study. Sherief Abd-Elsalam, Mohamed Elbahnasawy, Mohammed H Elnaggar, Hesham Ahmed Alsrogy, and Heba Elashry wrote the manuscript. Sherief Abd-Elsalam revised the manuscript.