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Urinary Biomarkers of Acute Kidney Injury in Patients With Liver Cirrhosis

ABSTRACT Background and aim: Acute kidney injury is a common complication in cirrhotic patients. Serum creatinine is a poor biomarker for detection of renal impairment in cirrhotic patients. The aim of this study was to evaluate Urinary Neutrophils Gelatinase-Associated Lipocalin (NGAL) and Urinary interleukin-18 (IL-18) as early biomarkers of acute kidney injury in cirrhotic patients. Patients and methods: 160 cirrhotic patients was enrolled in this study divided into 3 main groups according to presence or absence of ascites and renal impairment. Results: Significant elevation of both Urinary NGAL and Urinary IL-18 in cirrhotic patients with renal impairment especially in patients with Acute tubular necrosis (ATN) was observed. AUROC was (0.909) with (sensitivity 95.5 %, specificity 76.1) for Urinary NGAL and AUROC was (0.975), with (sensitivity 95.5 %, specificity 91.3 %) for Urinary IL-18. Conclusion: both Urinary NGAL and Urinary IL-18 can act as urinary biomarkers of acute kidney injury in cirrhotic patient


INTRODUCTION
Egypt was blessed with the river Nile and ancient culture but was deemed with liver diseases. Liver cirrhosis is a common disease in Egypt as Egypt has the highest incidence rate of HCV infection worldwide (1).
Acute kidney injury (AKI) in patients with cirrhosis is common. Up to 20 % of hospitalized patients with cirrhosis develop AKI (2) and once AKI occurs there is a reported fourfold increased risk of mortality (3).
Typically, patients with decompensated liver cirrhosis have significant circulatory dysfunction which is characterized by a vasodilatory state, lower total peripheral resistance, activated renin-angiotensin-aldosterone system (RAAS) and finally renal arterial vasoconstriction (4).
In cirrhosis, AKI types include pre-renal azotemia, hepatorenal syndrome (HRS) and acute tubular necrosis (ATN) with prevalence rates of 68%, 25%, and 33% respectively but their effect on mortality risk varies (2). Unfortunately these forms of AKI are difficult to distinguish clinically as serum creatinine (sCr), the clinical standard to define kidney function, poorly discriminates AKI type in cirrhosis (5).
Furthermore, various factors can affect serum creatinine in cirrhotic patients such as age, gender, nutritional status, muscle mass, drug and volume distribution in addition deranged hepatic synthesis of the precursor (creatine) may contribute to impaired creatinine production in liver cirrhosis so utilization of serum creatinine con-centration for diagnosing renal dysfunction could be even more unsatisfactory (4).
Recently, in an effort to improve the definition of AKI and to highlight the importance of non-HRS kidney dysfunction in cirrhosis, the Acute Dialysis Quality Initiative (ADQI) and the International Ascites Club (IAC) jointly published a consensus statement regarding AKI classification (6), incorporating the Risk, Injury, Failure, Loss and End stage disease (RIFLE) and the Acute Kidney Injury Network (AKIN) guidelines. IAC definition of HRS (7) was classified as a specific form of AKI (8). Management of AKI in the setting of cirrhotic patients depends primarily on detection of the cause. HRS is treat ED pharmacologically whereas pre-renal azotemia is treated with plasma volume expansion which is not suitable in management of ATN (9,10). Neutrophils Gelatinase-Associated Lipocalin (NGAL) is a novel biomarker for diagnosing acute kidney injury (AKI). Several studies have demonstrated the utility of early NGAL measurements for predicting the severity and clinical outcomes of AKI (11,12,13,14).
In fact, urine IL-18 has been shown to serve as an accurate biomarker to differentiate ATN from other etiological factors of renal disease (15). Moreover, the prognostic role of urine IL-18 has been validated in general patient groups admitted to ICU (16).
Therefore, The study was to assess the ability of urinary NGAL and IL-18 as early biomarkers of AKI in patients with cirrhosis.

Study protocol
This is a cross sectional study included 160 patients with cirrhosis admitted to the Liver Units in Zagazig university hospitals from July 2012 to December 2012 with history of follow up in outpatients clinics. The majority of patients were hospitalized for treatment of complications of cirrhosis. Exclusion criteria include: a) patients with hepatocellular carcinoma or cholangiocarcinoma; b) liver transplant patients; c) chronic kidney diseases patients maintained on regular hemodialysis before admission; and d) kidney transplant patients.
Study design was approved by institutional review board (IRB) of faculty of medicine, Zagazig university. Patients included in the study gave informed written consents in accordance with the Declaration of Helsinki.
Clinical and biochemical data were collected at the time of admission to Liver Units in Zagazig university hospitals with reference to previous patients data done in outpatient clinics. Child-Pugh Score and MELD score were calculated on admission (17,18). Estimated GFR was calculated using MDRD equation (19). A fresh urine sample was taken to measure the urinary levels of sodium, creatinine, NGAL and IL-18. Venous blood samples were drawn from all participants, and sera were separated immediately.

Classification of patients
Patients were classified into three groups: a) non ascetic patients (n=42); b) ascetic patients without renal impairment (n=50), and c) ascetic patients with renal impairment (n=68).
This classification was used because it reflects the different stages of cirrhosis (20). Patients with renal impairment was further divided into four subgroups: a) pre-renal azotemia; b) Chronic kidney disease (CKD) (21); c) HRS (7); and d) ATN (22). The definitions for these different causes of renal impairment are shown in the Supplementary material Analytic procedures Urine samples for NGAL and IL-18 levels were immediately centrifuged, separated and stored at -80° C until further analysis. Urinary NGAL was measured using NGAL ELISA kit (BioVendor GmbH, Germany) in relation to urinary creatinine. Urinary IL-18 was measured using a human IL-18 enzyme-linked immunosorbent assay kit (Medical and Biologic Laboratory, Nagoya, Japan) in relation to urinary creatinine.
Routine biochemical parameters were measured by calorimetric methods (Spinreact, SA Ctra, and Santa Coloma, Spain).

Statistical analysis
Results for continuous variables were expressed as mean±SD. Counts and percentages were used for the description of the categorical variables. Comparisons among groups were made with analysis of variance for continuous normal-distributed variables and with chi-squared test for categorical variables. Correlations were evaluated by means of the non-parametric Spearman's coefficient. The significance level for all statistical tests is set at 0.05 two-tailed. All statistical analyses were performed using SPSS V 19 software.

Characteristics of the patient population
The demographic, clinical data and biochemical parameters of all patients are shown in Table 1. Patients with renal impairment were divided into the four subgroups as show in Table 2
The cut-off value of urinary NGAL that differentiate between patients with AKI and those with other causes of renal impairment was 286.3 µg/g creatinine (area under ROC curve is 0.909) with (sensitivity 95.5 %, specificity 76.1 %) with positive predictive value (PPV) of 65.6 and negative predictive value (NPV) of 99.2 ( Figure 3).
Urinary IL-18 levels were analyzed in renal impairment patients' subgroups. We observed that Patients with ATN had the highest values of urinary IL-18, while patients with pre-renal azotemia had the lowest values (1687±447 vs. 451.47±121.73 µg/g creatinine, respectively; p=0.0001). Patients with HRS had intermediate values (953±273 µg/g creatinine), which were significantly higher than those of patients with pre-renal azotemia (p=0.0015) and lower than those of patients with ATN (p=0.0001). Urinary IL-18 levels in patients with CKD (582±98.24 µg/g creatinine) were significantly different from those of patients with HRS (p=0.001) (Figure 2).
The cut-off value of urinary IL-18 that differentiate between patients with AKI and those with other causes of renal impairment was 1119. 6 Table 3. Simple linear regression of urinary IL-18 and urinary NGAL in renal impairment patients. β: regression coefficient

DISCUSSION
AKI is a common condition in cirrhotic patients admitted to ICU. It is now recognized that AKI can significantly impact patients' outcomes (21). In fact, ATN is an important precipitating factor for the development of hepatorenal syndrome (HRS) (2,18). It has been proposed that intense renal vasoconstriction in HRS, if prolonged, may lead to tubular ischemia and ultimately progress into ATN (24,25,26). Therefore, it may be difficult to distinguish HRS from ATN in clinical settings.
In general patients without liver cirrhosis, urine sediment and FeNa may help make the diagnosis of ATN. However, confounding results may be encountered in cirrhotic patients. Diamond et al. have shown that, in cirrhotic patients, FeNa may not correspond to the usual range for general patients with ATN (27). Therefore, FeNa is a less useful diagnostic tool for tubular injury in patients with advanced liver diseases, whose pre-existing hemodynamic states make them sodium-avid (2,18,27,28).
Our study demonstrated a significant difference of Urinary NGAL and IL-18 in each category of AKI: highest in ATN, intermediate in HRS and low in pre-renal disease. Moreover, Urinary NGAL and IL-18 levels in patients with pre-renal azotemia were similar to those with normal kidney function and stable CKD. In contrast, sCr was not different between patients with ATN and HRS.
The mechanism by which patients with HRS have intermediate Urinary NGAL and IL-18 levels remains unclear. HRS physiology is thought to be an extreme pre-renal state (29,30) with severe renovascular vasoconstriction and decreased GFR, but normal intrinsic kidney function. Kidney function can return to normal after improvement of hepatic hemodynamics (29,30,31), or after renal transplantation into a recipient with normal hepatic function (32,33).
However, pathologic investigations have reported subtle kidney tubular and glomerular damage in HRS kidneys, some seen only with electron microscopy (24,34), perhaps resulting from the cellular changes associated with chronic activation of angiotensin-aldosterone signaling (35). It is conceivable that profound renovascular constriction may cause sub-clinical tubular damage in at least a subset of nephrons, not detectable by urinary sodium, which is not sensitive enough to detect mild or patchy tubular epithelial damage. Our study also confirms previous studies demonstrating that a single Urinary NGAL or Urinary IL-18 measurement on hospital admission has the potential to assist in determining type of kidney dysfunction, perhaps improving patient management and outcomes (13,36,37). Urinary NGAL and Urinary IL-18 demonstrated an excellent discriminative power compared to serum creatinine (AUROC 0.909 for NGAL, 0.975 for IL-18, 0.622 for Cr) for discriminating ATN from HRS in our patients. This is in contrast to traditional measurements of kidney and liver disease severity including sCr and MELD score. Our data demonstrate that patients with cirrhosis are at a high risk for acute renal impairment (33.1 % of the patients, 41 % of them diagnosed as ATN and 26.4 % diagnosed as HRS).The present findings seem to be consistent with previous studies (3,38) which confirm that non-HRS types of AKI are common in patients with cirrhosis and further studies are needed to determine inciting factors of AKI in this population. This study has some limitations. First, sCr inaccurately measures kidney function in cirrhosis (5,39,40), and there is no widely available technique to accurately measure GFR in these patients. Second, Absence of kidney biopsy, which is rarely performed in this population, because of the high risk of complications and consequently lack a correlation between renal pathology and urinary NGAL and urinary IL-18.

CONCLUSION
Urinary NGAL and urinary IL-18 have the ability to differentiate between AKI types in patients with cirrhosis. This could improve risk stratification for patients admitted to the hospital with cirrhosis, perhaps leading to early ICU admission, transplant evaluation, prompt initiation of HRS therapy and early management of AKI. These findings, if confirmed in larger cohorts, could lead to the development of biomarker algorithms to rapidly identify patients with HRS and ATN and accurately predict prognosis.