Role of Serum Angiopoietin-2 in Prediction of Mortality in Cirrhotic Patients with Acute Kidney Injury

study


INTRODUCTION
Hepatorenal syndrome (HRS) and acute kidney injury (AKI) are serious complications that commonly occur in individuals with portal hypertension and end-stage liver disease.The main treatment approach for HRS involves supportive care to alleviate the clinical symptoms associated with splanchnic vasodilation, which is characterized by reduced effective circulating volume, systemic vasoconstriction, and decreased renal blood flow [1].However, less than half of the patients respond effectively to current treatments such as intravenous albumin and splanchnic vasoconstrictors like terlipressin, indicating a need for a better understanding of the underlying mechanisms [2].AKI, often caused by sepsis, is a prevalent and severe complication that can lead to critical illness, prolonged hospital stays, especially in intensive care units ICUs), and potentially fatal outcomes.
Prompt recognition of AKI and the implementation of effective management strategies are crucial for improving patient outcomes and reducing mortality.While extensive research has focused on acute tubular epithelial injury in sepsis-related AKI, the importance of endothelial dysfunction and injury has been somewhat overlooked [3].
The Angiopoietin/tyrosine kinase with Ig and EGF (epidermal growth factor) homology domains (Tie2) signaling axis plays a crucial role in regulating vascular integrity.Tie2 receptors are widely expressed on endothelial cells, and their activation strengthens inter-endothelial junctions while reducing the expression of adhesion molecules on leukocytes [4].Angiopoietin 1 (ANG1) acts as a Tie2 receptor agonist, promoting blood vessel formation and maturation.However, ANG2, originally known as a competitive antagonist of ANG1/Tie2, has recently been found to function as both a Tie2 agonist and antagonist, depending on factors such as inflammation triggered by infection or tumor necrosis factor alpha.In inflammatory conditions, ANG2 acts as a pro-inflammatory agent, increasing vascular permeability, promoting vascular inflammation by weakening adherens junctions, recruiting inflammatory cells, and contributing to abnormal blood clotting in small blood vessels [5].
Several studies have reported elevated levels of Ang-2 in the serum of patients with conditions such as hepatocellular carcinoma, advanced liver fibrosis, and kidney disease [6].These findings highlight the need to investigate the mechanisms related to inflammation and vascular function that may contribute to HRS and AKI in individuals with cirrhosis.However, there are limited studies exploring serum Ang-2 levels in cirrhotic patients, whether or not they have AKI.Therefore, this study aims to evaluate the role of serum Ang-2 levels, both as an independent marker and in conjunction with the Model for End-Stage Liver Disease (MELD) score, in the early detection of AKI and the prediction of allcause mortality in patients with decompensated cirrhosis.

SUBJECTS AND METHODS:
This cross-sectional study was conducted collaboratively between the Tropical Medicine department at the Faculty of Medicine, Menoufia University Hospital, and the Clinical Pathology department at Menoufia University.The study spanned from October 2019 to August 2021.Ninety patients were carefully selected from the Tropical Medicine outpatient clinics and inpatient wards at Menoufia University Hospitals.These patients were divided into three groups: Group I consisted of 30 patients with compensated cirrhosis, Group II included 30 patients with decompensated cirrhosis but without acute kidney injury (AKI), and Group III comprised 30 patients with decompensated cirrhosis who were diagnosed with AKI.
The inclusion criteria involved patients aged 18 years or older, diagnosed with liver cirrhosis (compensated or decompensated), with or without AKI, based on a comprehensive evaluation including historical data, clinical examination, ultrasonography, and laboratory assessments.Exclusion criteria included individuals who had undergone renal transplant, those under the age of 18, and pregnant women.
Comprehensive clinical assessments were conducted, including a detailed medical history, age, gender, smoking history, smoking index, clinical examination findings, history of hypertension (HTN), medication intake, body mass index (BMI), and the presence of diabetes mellitus (DM).
Under strict aseptic conditions, 5 ml of venous blood was collected from each patient.Two milliliters were placed in a citrated tube for prothrombin time (PT) and international normalized ratio (INR) measurements.The remaining 3 ml were collected in a plain tube, and serum separation was achieved through centrifugation for subsequent biochemical laboratory investigations.
Radiological investigations, such as abdominal ultrasonography or CT scans, were conducted as needed.
The diagnosis of AKI was established based on a comprehensive evaluation of historical data, clinical examination, laboratory results, imaging studies, and therapeutic responses.AKI patients were classified into three subtypes: pre-renal, renal, and Hepato-renal Syndrome, based on specific criteria.AKI staging was performed according to the International Club of Ascites (ICA) criteria [30]; [31] The relationship between serum Ang-2 levels, both independently and in conjunction with MELD scores, and the severity of liver cirrhosis and AKI staging was analyzed systematically.
Statistical Analysis: Data collection and tabulation were conducted using IBMcompatible personal computers with the Statistical Package for the Social Sciences (SPSS) version 25 (SPSS Inc., Released 2015, IBM SPSS Statistics for Windows, v. 25.0, Armonk, NY: IBM Corp.) and MEDCALC V.19.6.1 software.The statistical analysis included descriptive statistics presented as mean, standard deviation (SD), median, range, and qualitative data presented as numbers (N) and percentages (%).Analytical statistics comprised the Mann-Whitney test (U), ANOVA (F) test, and Kruskal-Wallis test.A p-value <0.05 was considered statistically significant.

RESULTS
The study enrolled participants with a mean age of 54.30±11.50years, with 53.3% being male.The average body mass index (BMI) was 27.03±2.99kg/m².The Child Pugh-Turcotte classification (CTPC) had an average of 7.5±10.7,and the Model for End-Stage Liver Disease (MELD) score was 15.44±8.48.Among the patients, 43.3% were smokers, 36.7% had diabetes, and 13.3% had hypertension.Additionally, 93.3% tested positive for hepatitis C virus antibodies (HCV-Ab), and 3.3% were positive for hepatitis B surface antigen (HBs-Ag).Mean S. Ang-2 level was 1463.89±943.24(Table 1).Significant differences were observed among the study groups in terms of age, history of nephrotoxic drug use, BMI, portal vein (PV) diameter, CTPC, MELD score, and S. Ang-2 levels (p-value<0.001), with the highest values seen in GIII.There were also significant differences in HB levels, platelet count, S. albumin, INR, S. creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (p-value<0.001).Statistically significant differences were found in S. direct bilirubin and total leukocyte count (TLC), but no significant difference was observed in total bilirubin among the groups (Table 2).
Gender did not appear to have an impact on S. Ang-2 levels, and there were no significant associations with smoking history or disease severity.However, positive and statistically significant correlations were found between S. Ang-2 levels and diabetes mellitus (DM) and hypertension (HTN) (p-value 0.007 and 0.001, respectively).
Additionally, significant correlations were observed between S. Ang-2 and hepatomegaly, ascites, splenomegaly, anemia type, and urine analysis findings among the studied patients (Table 3).
When predicting hepatorenal syndrome (HRS), S. Ang-2 levels demonstrated a sensitivity of 97% and specificity of 95% at a cutoff level of 1810 ng/ml, with an area under the curve (AUC) of 0.989 and a 95% confidence interval (CI) of 0.97-1.00.In contrast, the traditional MELD score had a sensitivity of 80% and specificity of 92% at a cutoff level of 19.0, with an AUC of 0.883.However, when combined, S. Ang-2 and the MELD score had a sensitivity of 100% and specificity of 88% in the early detection of HRS, surpassing the performance of each individually.Furthermore, S. Ang-2 exhibited high sensitivity (95%) and specificity (90%) in predicting mortality at a cutoff point >2325 ng/ml, with p<0.001.The combination of S. Ang-2 and the MELD score demonstrated greater specificity (92%) in detecting mortality, outperforming each individually (Figure 1).Multiple logistic regression analysis identified creatinine, BUN, urine pus cell count >100, MELD score, and S.
Ang-2 as the most statistically significant factors associated with the detection of HRS (P<0.05).Conversely, total bilirubin and albumin showed the least statistically significant associations with the detection of HRS (P>0.05).Univariate logistic regression analysis revealed that creatinine, BUN, CTPC, MELD score, S. Ang-2, and pus cells >100 were predictors of mortality among the studied patients (P value <0.05) (Table 4).

CONCLUSION
The study concluded that Ang-2 levels were significantly higher in decompensated cirrhotic patients with AKI compared to those without AKI and compensated cirrhotic patients.
Combining Ang-2 levels with MELD score resulted in a sensitivity of 100% for early detection of HRS and a specificity of 92% for predicting mortality.There were no significant associations between Ang-2 levels and variables such as sex, smoking history, or smoking index.However, significant correlations were found between Ang-2 levels and anemia type, presence of urine pus cells exceeding 100, DM, and HTN within the study population.These findings highlight the potential clinical importance of Ang-2 levels in cirrhosis, particularly in relation to AKI, HRS detection, and mortality prediction.This information could be valuable in improving risk assessment and patient management strategies.

CONFLICT OF INTEREST: None.
ETHICAL APPROVAL: Informed written consent was obtained from all participants, and the study protocol received ethical approval from the Research Ethics Committee of Medical Research at the Faculty of Medicine, Menoufia University number and date 19819TROP9.

ACKNOWLEDGMENT
The authors would thank all colleagues who helped to conduct this study.We are also grateful to, Dr/ Atef Abu Elsoud Ali, Professor of Tropical Medicine, Faculty of Medicine, Menoufia University, Shebin Elkom, Egypt for reviewing the main manuscript file.

HIGHLIGHTS
 ANG2 acts as a pro-inflammatory agent, increasing vascular permeability, promoting vascular inflammation by weakening adherens junctions, recruiting inflammatory cells, and contributing to abnormal blood clotting in small blood vessels. Serum Ang-2 levels were significantly higher in decompensated cirrhotic patients with AKI compared to those without AKI and compensated cirrhotic patients. The combination of S. Ang-2 and the MELD score demonstrated greater specificity (92%) in detecting mortality, outperforming each individually.

Figure 1 .
Figure 1. S. Ang-2 levels alone or in combination with MELD score in early detection of HRS and mortality rate among the studied groups.

Table 1 .
Baseline characteristics of the studied patients.

Table 2 .
clinical presentation, U/S and Laboratory investigations finding among the studied groups.

Table 3 .
Correlation between MELD score and S. Ang-2 with all variables.

Table 4 .
Univariate and multivariate logistic regression analysis for prediction of mortality and HRS among the studied patients.