The use of urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin for diagnosis of hepato-renal syndrome in advanced cirrhotic patients.

BACKGROUND: Chronic liver disease is a common and important clinical problem.Hepatorenal syndrome (HRS) is a life threatening complication. Serum creatinine (Cr) remains the only conventional indicator of renal function. However, the interpretation of serum Cr level can be confounded by malnutrition and reduced muscle mass often observed in patients with severe liver disease. Here, we present a cross-sectional study to explore the sensitivity and specificity of other markers as urinary KIM-1 and NGAL for cases of HRS. METHODS: Cross-sectional study was conducted on 88 patients who were admitted to Alexandria main university hospital. Enrolled patients were divided in two groups; group 1: patients with advanced liver cirrhosis (child B and C) who have normal kidney functions while group 2: patients who developed HRS. Stata© version 14.2 software package was used for analysis. RESULTS: Group 1 included 18 males and 26 females compared to 25 males and 19 females in group 2 (p = 0.135). Only the urinary KIM-1 showed a statistically significant difference between both groups in the multivariate logistic regression analysis adjusted for gender, serum bilirubin, serum albumin, INR, serum K, AST and ALT levels. CONCLUSION: In conclusion, our study aligns with prior research, as seen in the consistent findings regarding Urinary NGAL elevation in cirrhotic patients with AKI. Urinary KIM-1, independent of Urinary NGAL, may have a role in precisely distinguishing between advanced liver cirrhosis and HRS and merits further exploration.

Could protein content of Urinary Extracellular Vesicles be useful to detect Cirrhosis in Alcoholic Liver Disease?

Alcohol abuse has a high impact on the mortality and morbidity related to a great number of diseases and is responsible for the development of alcoholic liver disease (ALD). It remains challenging to detect and evaluate its severity, which is crucial for prognosis. In this work, we studied if urinary EVs (uEVs) could serve in diagnose and evaluate cirrhosis in ALD. To this purpose, uEVs characterization by cryo-electron microscopy (Cryo-EM), Nanoparticle Tracking Analysis (NTA) and Western blotting (WB) was performed in a cohort of 21 controls and 21 cirrhotic patients. Then, proteomics of uEVs was carried out in a second cohort of 6 controls and 8 patients in order to identify new putative biomarkers for cirrhosis in ALD. Interestingly, uEVs concentration, size and protein composition were altered in cirrhotic patients. From a total of 1304 proteins identified in uEVs, 90 of them were found to be altered in cirrhotic patients. The results suggest that uEVs could be considered as a tool and a supplier of new biomarkers for cirrhosis in ALD, whose application would be especially relevant in chronic patients. Yet, further research is necessary to obtain more relevant result in clinical terms.

Admission Urinary and Serum Metabolites Predict Renal Outcomes in Hospitalized Patients With Cirrhosis.

BACKGROUND AND AIMS: Acute kidney injury (AKI) has a poor prognosis in cirrhosis. Given the variability of creatinine, the prediction of AKI and dialysis by other markers is needed. The aim of this study is to determine the role of serum and urine metabolomics in the prediction of AKI and dialysis in an inpatient cirrhosis cohort. APPROACH AND RESULTS: Inpatients with cirrhosis from 11 North American Consortium of End-stage Liver Disease centers who provided admission serum/urine when they were AKI and dialysis-free were included. Analysis of covariance adjusted for demographics, infection, and cirrhosis severity was performed to identify metabolites that differed among patients (1) who developed AKI or not; (2) required dialysis or not; and/pr (3) within AKI subgroups who needed dialysis or not. We performed random forest and AUC analyses to identify specific metabolite(s) associated with outcomes. Logistic regression with clinical variables with/without metabolites was performed. A total of 602 patients gave serum (218 developed AKI, 80 needed dialysis) and 435 gave urine (164 developed AKI, 61 needed dialysis). For AKI prediction, clinical factor-adjusted AUC was 0.91 for serum and 0.88 for urine. Major metabolites such as uremic toxins (2,3-dihydroxy-5-methylthio-4-pentenoic acid [DMTPA], N2N2dimethylguanosine, uridine/pseudouridine) and tryptophan/tyrosine metabolites (kynunerate, 8-methoxykyunerate, quinolinate) were higher in patients who developed AKI. For dialysis prediction, clinical factor-adjusted AUC was 0.93 for serum and 0.91 for urine. Similar metabolites as AKI were altered here. For dialysis prediction in those with AKI, the AUC was 0.81 and 0.79 for serum/urine. Lower branched-chain amino-acid (BCAA) metabolites but higher cysteine, tryptophan, glutamate, and DMTPA were seen in patients with AKI needing dialysis. Serum/urine metabolites were additive to clinical variables for all outcomes. CONCLUSIONS: Specific admission urinary and serum metabolites were significantly additive to clinical variables to predict AKI development and dialysis initiation in inpatients with cirrhosis. These observations can potentially facilitate earlier initiation of renoprotective measures.

Discovery, validation and sequencing of urinary peptides for diagnosis of liver fibrosis-A multicentre study.

BACKGROUND: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease. METHODS: In a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis. FINDINGS: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89-0.97, p<0.0001). When adjusted for age, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89-0.97, p<0.0001) in chronic liver disease patients with (N = 19) or without (N = 17) liver fibrosis progression. In this prospective diagnostic phase III validation set, age-adjusted LivFib-50 showed 84.2% sensitivity (95% CI: 60.4-96.6) and 82.4% specificity (95% CI: 56.6-96.2) for detection of liver fibrosis. The sequence-identified peptides are mainly fragments of collagen chains, uromodulin and Na/K-transporting ATPase subunit γ. We also identified ten putative proteolytic cleavage sites, eight were specific for matrix metallopeptidases and two for cathepsins. INTERPRETATION: In liver fibrosis, urinary peptides profiling offers potential diagnostic markers and leads to discovery of proteolytic sites that could be targets for developing anti-fibrotic therapy.

Accurate non-invasive diagnosis and staging of non-alcoholic fatty liver disease using the urinary steroid metabolome.

BACKGROUND: The development of accurate, non-invasive markers to diagnose and stage non-alcoholic fatty liver disease (NAFLD) is critical to reduce the need for an invasive liver biopsy and to identify patients who are at the highest risk of hepatic and cardio-metabolic complications. Disruption of steroid hormone metabolic pathways has been described in patients with NAFLD. AIM(S): To assess the hypothesis that assessment of the urinary steroid metabolome may provide a novel, non-invasive biomarker strategy to stage NAFLD. METHODS: We analysed the urinary steroid metabolome in 275 subjects (121 with biopsy-proven NAFLD, 48 with alcohol-related cirrhosis and 106 controls), using gas chromatography-mass spectrometry (GC-MS) coupled with machine learning-based Generalised Matrix Learning Vector Quantisation (GMLVQ) analysis. RESULTS: Generalised Matrix Learning Vector Quantisation analysis achieved excellent separation of early (F0-F2) from advanced (F3-F4) fibrosis (AUC receiver operating characteristics [ROC]: 0.92 [0.91-0.94]). Furthermore, there was near perfect separation of controls from patients with advanced fibrotic NAFLD (AUC ROC = 0.99 [0.98-0.99]) and from those with NAFLD cirrhosis (AUC ROC = 1.0 [1.0-1.0]). This approach was also able to distinguish patients with NAFLD cirrhosis from those with alcohol-related cirrhosis (AUC ROC = 0.83 [0.81-0.85]). CONCLUSIONS: Unbiased GMLVQ analysis of the urinary steroid metabolome offers excellent potential as a non-invasive biomarker approach to stage NAFLD fibrosis as well as to screen for NAFLD. A highly sensitive and specific urinary biomarker is likely to have clinical utility both in secondary care and in the broader general population within primary care and could significantly decrease the need for liver biopsy.

Role of biomarkers as predictors of acute kidney injury and mortality in decompensated cirrhosis.

Evidence suggests that novel biomarkers predict acute kidney injury (AKI) development and outcome earlier than serum creatinine. The aim of this study was to determine the incidence and prognosis of AKI in decompensated cirrhotic patients, and also assess the usefulness of plasma cystatin C, urine neutrophil gelatinase associated lipocalin (NGAL), tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) in early prediction of AKI and mortality. Single-center, prospective observational study enrolling decompensated cirrhotic patients without AKI at the time of admission. Of 111 patients with decompensated cirrhosis, 45 (40.5%) developed AKI while hospitalized. Even with 53.3% being transient (stage 1), mortality was significantly higher in AKI than non-AKI patients (46.5% vs. 25%, p = 0.02). Plasma cystatin C and urine NGAL, but not urine [TIMP-2]·[IGFBP7] at the time of admission were found to be independent early predictors of AKI. Substitution of cystatin C for creatinine significantly improved the model for end-stage liver disease (MELD) score accuracy for mortality prediction. The incidence of AKI is high and is associated with high mortality in decompensated cirrhotic patients. Plasma cystatin C and urine NGAL are useful for early detection of AKI. MELD-cystatin C, rather than original MELD, improves predictive accuracy of mortality.

Urinary neutrophil gelatinase-associated lipocalin for diagnosis of spontaneous bacterial peritonitis.

Cirrhotic patients with ascites are at high risk of developing spontaneous bacterial peritonitis (SBP). After exclusion of patients with acute kidney injury (AKI) or other infections, urinary neutrophil gelatinase-associated lipocalin (NGAL) levels were compared between two matched groups of Egyptian cirrhotic patients with ascites, mostly secondary to hepatitis C infection (98%). Group 1 had SBP (n = 41) and group 2 did not (n = 45). By univariate analysis, urinary-NGAL, high total bilirubin, serum creatinine, international normalised ratio and the Model of End-Stage Liver Disease (MELD) score and low platelet count were all significantly correlated with the presence of SBP, but only urinary-NGAL could independently predict development of SBP (P = 0.001). Urinary-NGAL at a cut-off value of 1225 pg/mL, showed a sensitivity of 95% and a specificity of 76%, and is therefore a most useful tool.

Activation of RAAS in a rat model of liver cirrhosis: no effect of losartan on renal sodium excretion.

BACKGROUND: Liver cirrhosis is characterized by avid sodium retention where the activation of the renin angiotensin aldosterone system (RAAS) is considered to be the hallmark of the sodium retaining mechanisms. The direct effect of angiotensin II (ANGII) on the AT-1 receptor in the proximal tubules is partly responsible for the sodium retention. The aim was to estimate the natriuretic and neurohumoral effects of an ANGII receptor antagonist (losartan) in the late phase of the disease in a rat model of liver cirrhosis. METHODS: Bile duct ligated (BDL) and sham operated rats received 2 weeks of treatment with losartan 4 mg/kg/day or placebo, given by gastric gavage 5 weeks after surgery. Daily sodium and potassium intakes and renal excretions were measured. RESULTS: The renal sodium excretion decreased in the BDL animals and this was not affected by losartan treatment. At baseline the plasma renin concentration (PRC) was similar in sham and BDL animals, but increased urinary excretion of ANGII and an increase P-Aldosterone was observed in the placebo treated BDL animals. The PRC was more than 150 times higher in the losartan treated BDL animals (p < 0.001) which indicated hemodynamic impairment. CONCLUSIONS: Losartan 4 mg/kg/day did not increase renal sodium excretion in this model of liver cirrhosis, although the urinary ANGII excretion was increased. The BDL animals tolerated Losartan poorly, and the treatment induced a 150 times higher PRC.

Liquid biopsy biomarkers of renal interstitial fibrosis based on urinary exosome.

Renal interstitial fibrosis (RIF) is difficult to diagnose. This paper explored liquid biopsy markers in urinary exosomes derived from RIF patients. Urine samples from 32 patients with various degrees of RIF and 20 non-RIF patients were collected. The size and morphology of urinary exosomes isolated by polyethylene glycol were observed with electron microscopy. Protein biomakers of exosomes were analyzed by Western blot. qRT-PCR was used to detect the levels of biomarkers (miR-29c, miR-21, E-cadherin, and vimentin) of epithelial mesenchymal transition in urinary exosomes. The diagnostic value was detected with ROC curves. Results displayed successfully isolated urinary exosomes. The examined miRNAs and mRNAs were checked from all urinary exosomes samples, except for two cases of RIF which lacked E-cadherin mRNA. RNA levels were different in patients with diverse degrees of RIF. Urinary miR-29c was decreased with the progress of fibrosis. Levels of E-cadherin mRNA were first decreased and then increased. The contents of miR-21 and vimentin mRNA were also depended on the degrees of RIF. ROC curve analysis showed that the area under the curve (AUC) of miR-29c was 0.8621, statistically significant compared with the non-RIF group (P < 0.05). The miR-29c level within the urinary exosomes is a promising marker for the diagnosis of RIF.

Urine albumin-to-creatinine ratio is associated with the severity of liver disease, renal function and survival in patients with decompensated cirrhosis.

BACKGROUND AND AIMS: To investigate if urine albumin-to-creatinine ratio (UACR) is associated with the presence of glomerular filtration rate (GFR) <60 mL/min, severity of liver disease and survival in patients with stable decompensated cirrhosis. METHODS: We evaluated prospectively 220 patients (73 % male, age 52.8 ± 12 years). In each patient, assessment of GFR was based on 51chromium-EDTA. Random urine samples were obtained for measurement of UACR. RESULTS: Thirty-eight patients (17 %, group 1) had UACR ≥30 mg/g and 182 (83 %, group 2) had UACR <30 mg/g. Group 1, compared to group 2 patients, had significantly lower levels of "true" GFR (61 vs. 71 ml/min, p = 0.035). Patients with "true" GFR <60 mL/min (n = 93), compared to those with "true" GFR ≥60 mL/min (n = 127), had higher levels of UACR (16 vs. 11.3 mg/g, p = 0.023). In multivariate analysis, serum creatinine and UACR (ΟR 0.98, 95 % CI 0.95-0.99, p = 0.04) were independently associated with the presence of GFR <60 mL/min. Based on the area under the ROC curves, the best cut-off point for UACR was >16.51 mg/g giving a sensitivity 70 %, specificity 49 %, PPV 68 % and NPV 51 %. During the follow-up period [17 (6-52) months], the patients who died or underwent LT (n = 158), compared to those who remained alive (n = 62), had higher levels of UACR (41 vs. 13 mg/g, p = 0.025). Patients with UACR ≥30 mg/g had worse outcome, compared to those with UACR <30 mg/g (log rank p = 0.053). CONCLUSIONS: We showed for the first time that UACR ≥30 mg/g was associated with more severe liver disease, lower GFR and worse LT-free survival in patients with decompensated cirrhosis. However, further studies are needed to confirm these findings.

Urinary metabolic profiling by 1H NMR spectroscopy in patients with cirrhosis may discriminate overt but not covert hepatic encephalopathy.

To date urinary metabolic profiling has been applied to define a specific metabolic fingerprint of hepatocellular carcinoma on a background of cirrhosis. Its utility for the stratification of other complications of cirrhosis, such as hepatic encephalopathy (HE), remains to be established. Urinary proton nuclear magnetic resonance (1H-NMR) spectra were acquired and NMR data from 52 patients with cirrhosis (35 male; 17 female, median (range) age [60 (18-81) years]) and 17 controls were compared. A sub-set of 45 patients (33 male; 12 female, [60 (18-90) years, median model for end stage liver disease (MELD) score 11 (7-27)]) were fully characterised by West-Haven criteria, Psychometric Hepatic Encephalopathy Score (PHES) and electroencephalogram (EEG), and defined as overt HE (OHE, n = 21), covert HE (cHE, n = 7) or no HE (n = 17). Urinary proton nuclear magnetic resonance (1H-NMR) spectra were analysed by partial-least-squares discriminant analysis (PLS-DA). The results showed good discrimination between patients with cirrhosis (n = 52) and healthy controls (n = 17) (R2X = 0.66, R2Y = 0.47, Q2Y = 0.31, sensitivity-60 %, specificity-100 %) as the cirrhosis group had higher 1-methylnicotinamide with lower hippurate, acetate, phenylacetylglycine and N-methyl nicotinic acid levels. While patients with OHE could be discriminated from those with no HE, with higher histidine, citrate and creatinine levels, the best models lack robust validity (R2X = 0.65, R2Y = 0.48, Q2Y = 0.12, sensitivity-100 %, specificity-64 %) with the sample size used. Urinary 1H-NMR metabolic profiling did not discriminate patients with cHE from those without HE, nor discriminate subjects on the basis of PHES/EEG result or MELD score. In conclusion, patients with cirrhosis showed different urinary 1H-NMR metabolic profiles compared to healthy controls and those with OHE may be distinguished from those with no HE although larger studies are required. However, urinary 1H-NMR metabolic profiling did not discriminate patients with differing grades of HE or according to severity of underlying liver disease.

Chromatographic determination of some biomarkers of liver cirrhosis and hepatocellular carcinoma in Egyptian patients.

Metabolomics has been shown to be an effective tool for disease diagnosis, biomarker screening and characterization of biological pathways. A total of 140 subjects were included in this study; urine metabolomes of patients with liver cirrhosis (LC, n = 40), patients with hepatocellular carcinoma (HCC; n = 55) and healthy male subjects (n = 45) as a control group were studied. Gas chromatography/mass spectrometry-based urine metabolomics profiles were investigated for all participants. Diagnostic models were constructed with a combination of marker metabolites, using principal components analysis and receiver operator characteristic curves. A total of 57 peaks could be auto-identified of which 13 marker metabolites (glycine, serine, threonine, proline, urea, phosphate, pyrimidine, arabinose, xylitol, hippuric acid, citric acid, xylonic acid and glycerol) were responsible for the separation of HCC group from healthy subjects. Also, eight markers metabolites (glycine, serine, threonine, proline, citric acid, urea, xylitol and arabinose) showed significant differences between the LC group and healthy subjects. No significant difference was detected between HCC and LC groups regarding all these metabolites. Metabolomic profile using GC-MS established an optimized diagnostic model to discriminate between HCC patients and healthy subjects; also it could be useful for diagnosis of LC patients. However, it failed to differentiate between HCC and LC patients.

Urine Monocyte Chemoattractant Protein-1 Is an Independent Predictive Factor of Hospital Readmission and Survival in Cirrhosis.

UNLABELLED: MCP-1 (monocyte chemoattractant protein-1) is a proinflammatory cytokine involved in chemotaxis of monocytes. In several diseases, such as acute coronary syndromes and heart failure, elevated MCP-1 levels have been associated with poor outcomes. Little is known about MCP-1 in cirrhosis. AIM: To investigate the relationship between MCP-1 and outcome in decompensated cirrhosis. METHODS: Prospective study of 218 patients discharged from hospital after an admission for complications of cirrhosis. Urine and plasma levels of MCP-1 and other urine proinflammatroy biomarkers: osteopontin(OPN), trefoil-factor3 and liver-fatty-acid-binding protein were measured at admission. Urine non-inflammatory mediators cystatin-C, ß2microglobulin and albumin were measured as control biomarkers. The relationship between these biomarkers and the 3-month hospital readmission, complications of cirrhosis, and mortality were assessed. RESULTS: 69 patients(32%) had at least one readmission during the 3-month period of follow-up and 30 patients died(14%). Urine MCP-1 and OPN levels, were associated with 3-month probability of readmission (0.85 (0.27-2.1) and 2003 (705-4586) ug/g creat vs 0.47 (0.2-1.1) and 1188 (512-2958) ug/g creat, in patients with and without readmission, respectively; p<0.05; median (IQR)). Furthermore, urine levels of MCP-1 were significantly associated with mortality (1.01 (1-3.6) vs 0.5 (0.2-1.1) µg/g creat, in dead and alive patients at 3 months; p<0.05). Patients with higher levels of urine MCP-1 (above percentile 75th) had higher probability of development of hepatic encephalopathy, bacterial infections or AKI. Urine MCP-1 was an independent predictive factor of hospital readmission and combined end-point of readmission or dead at 3 months. Plasma levels of MCP-1 did not correlated with outcomes. CONCLUSION: Urine, but not plasma, MCP-1 levels are associated with hospital readmission, development of complications of cirrhosis, and mortality. These results suggest that in cirrhosis there is an inflammatory response that is associated with poor outcomes.

Urine neutrophil gelatinase-associated lipocalin: a diagnostic and prognostic marker for acute kidney injury (AKI) in hospitalized cirrhotic patients with AKI-prone conditions.

BACKGROUND: Acute kidney injury (AKI) is known to increase mortality in hospitalized cirrhotic patients; therefore early identification is utmost significance. There are only a few studies evaluating the cut-off level of urine neutrophil gelatinase-associated lipocalin (uNGAL) for diagnosing AKI and its prognostic value in cirrhotic patients. We aimed to determine the accuracy of uNGAL as a biomarker for early identification of AKI and to determine the cut-off level of uNGAL for diagnosing AKI in hospitalized cirrhotic patients; and (2) to explore the association of 30-day liver-related mortality with uNGAL level. METHODS AND MATERIAL: We prospectively enrolled cirrhotic patients admitted at the King Chulalongkorn Memorial Hospital during May 1, 2011 to Dec 31, 2013. UNGAL levels were measured within 24 h after admission. Clinical and laboratory data were obtained. Patients were followed up to 30 days. RESULTS: Of 137 cirrhotic hospitalized patients, 121 cirrhotic patients (88.3 %) with AKI-prone conditions were included with mean age of 57.3 ± 14.7 years. Thirty-five patients (29 %) developed AKI within 72 h of admission. The causes of AKI were prerenal azotemia (68.6 %), acute tubular necrosis (25.7 %), hepatorenal syndrome (5.7 %), respectively. The mean uNGAL level was significantly higher in the patients who developed AKI compared with those who did not (290.6 ± 356.3 vs. 54.4 ± 73.7 ng/mL; P = 0.0001). The AUC of uNGAL for diagnosing AKI was 0.83 (95 % [CI]: 0.76-0.91) with the optimal cut-off level of 56 ng/mL, providing 77.1 % sensitivity and 73.3 % specificity. Fourteen percent of subjects died during the 30-day follow-up period. The mean uNGAL levels were significantly higher in the mortality group. The AUC of uNGAL in predicting mortality was 0.75 (95 % [CI]: 0.66-0.85), with a best cut-off level of 72 ng/mL providing 70.6 % sensitivity and 69.2 % specificity. However, in multivariate logistic regression analysis, uNGAL is not an independent factor for 30-day liver-related mortality prediction. CONCLUSIONS: uNGAL is a valid marker for the early detection of AKI in hospitalized cirrhotic patients with AKI-prone conditions; however, its level could not independently predict 30-day liver-related mortality.

Analysis of a urinary biomarker panel for clinical outcomes assessment in cirrhosis.

BACKGROUND: Biomarkers are potentially useful in assessment of outcomes in patients with cirrhosis, but information is very limited. Given the large number of biomarkers, adequate choice of which biomarker(s) to investigate first is important. AIM: Analysis of potential usefulness of a panel of urinary biomarkers in outcome assessment in cirrhosis. PATIENTS AND METHODS: Fifty-five patients with acute decompensation of cirrhosis were studied: 39 had Acute Kidney Injury (AKI) (Prerenal 12, type-1 HRS (hepatorenal syndrome) 15 and Acute Tubular Necrosis (ATN) 12) and 16 acute decompensation without AKI. Thirty-four patients had Acute-on-chronic liver failure (ACLF). A panel of 12 urinary biomarkers was assessed, using a multiplex assay, for their relationship with ATN, ACLF and mortality. RESULTS: Biomarker with best accuracy for ATN diagnosis was NGAL (neutrophil-gelatinase associated lipocalin): 36 [26-125], 104 [58-208] and 1807 [494-3,716] µg/g creatinine in Prerenal-AKI, type-1 HRS and ATN, respectively; p<0.0001 (AUROC 0.957). Other attractive biomarkers for ATN diagnosis were IL-18, albumin, trefoil-factor-3 (TFF-3) and glutathione-S-transferase-π (GST-π) Biomarkers with less accuracy for ATN AUCROC<0.8 were ß2-microglobulin, calbindin, cystatin-C, clusterin and KIM-1 (kidney injury molecule-1). For ACLF, the biomarker with the best accuracy was NGAL (ACLF vs. No-ACLF: 165 [67-676] and 32 [19-40] µg/g creatinine; respectively; p<0.0001; AUROC 0.878). Interestingly, other biomarkers with high accuracy for ACLF were osteopontin, albumin, and TFF-3. Biomarkers with best accuracy for prognosis were those associated with ACLF. CONCLUSIONS: A number of biomarkers appear promising for differential diagnosis between ATN and other types of AKI. The most interesting biomarkers for ACLF and prognosis are NGAL, osteopontin, albumin, and TFF-3. These results support the role of major inflammatory reaction in the pathogenesis of ACLF.

Assessment of 6-sulfatoxymelatonin rhythms and melatonin response to light in disease states: lessons from cirrhosis.

Circadian rhythmicity and non-visual sensitivity to light can be assessed, in healthy subjects, by measuring the rhythm of the urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) and by determining the response of plasma melatonin to nocturnal retinal light exposure, respectively. However, the validity of these techniques has not been assessed in disease states in which disruption of the circadian rhythm is known or suspected to occur. Thus, the aims of this study were as follows: (i) to assess the reliability of circadian aMT6s profile estimates derived from 36 h versus 56 h urine collections and (ii) to test different models for calculating melatonin suppression in response to light in healthy volunteers and patients with cirrhosis. Twenty patients with biopsy-proven cirrhosis and 10 matched healthy volunteers undertook: (i) separate 36 - and 56-h urine collections, under controlled conditions, for cosinor analysis of the urinary aMT6s profile; (ii) a melatonin suppression test, comprising of a baseline night, during which subjects were woken and asked to sit in front of a switched off light sphere, and an experimental night, identically executed, except that the light sphere was switched on and the subjects were exposed to white light (4.1 × 10(14) photons/cm(2)/s) for 30 min. Alternative approaches to the calculation of melatonin suppression were taken, with/without inclusion of the baseline night. Eighteen patients and eight healthy volunteers had matched analysable 36 - and 56-h urinary samples. Cosinor analysis showed a significant fit in 88% of the remaining 56 h collections, and 48% of the remaining 36-h collections. Thus, eight patients and five healthy volunteers had matched analysable samples for cosinor analysis. In the healthy volunteers, aMT6s profile indices obtained using the 36 - and the 56-h collections did not differ significantly. In contrast, considerably more variability was observed in patients [i.e. the difference in the aMT6s peak time was 0.5 ± 1.7 h (limits of agreement: -3.9; +2.9 h)]. No difficulties were encountered in obtaining suppression estimates by use of the experimental night only. In contrast, suppression estimates obtained by use of both nights were considered inaccurate in one (11%) healthy volunteer and in 5 (28%) patients, primarily because: (i) melatonin concentrations at the beginning of light administration were significantly different on baseline and experimental night; (ii) the rise in melatonin was inconsistent on baseline night; and (iii) the shape of the rising phase of melatonin was different on baseline and experimental night. In conclusion, shorter urine collections lead to a higher number of profiles with no significant cosinor fit, and differences in cosinor indices obtained from the 36 - and 56-h collections were considerable, especially in patients. Thus, 56-h collections are probably advisable. Use of both baseline and experimental nights to calculate melatonin suppression often resulted in increased variation and confounding, due to point oscillations in melatonin concentration and lack of repeatability of the melatonin profiles on the two nights. Thus, use of the experimental night only is probably advisable.

Development of urinary pseudotargeted LC-MS-based metabolomics method and its application in hepatocellular carcinoma biomarker discovery.

Hepatocellular carcinoma (HCC) is one of the pestilent malignancies leading to cancer-related death. Discovering effective biomarkers for HCC diagnosis is an urgent demand. To identify potential metabolite biomarkers, we developed a urinary pseudotargeted method based on liquid chromatography-hybrid triple quadrupole linear ion trap mass spectrometry (LC-QTRAP MS). Compared with nontargeted method, the pseudotargeted method can achieve better data quality, which benefits differential metabolites discovery. The established method was applied to cirrhosis (CIR) and HCC investigation. It was found that urinary nucleosides, bile acids, citric acid, and several amino acids were significantly changed in liver disease groups compared with the controls, featuring the dysregulation of purine metabolism, energy metabolism, and amino metabolism in liver diseases. Furthermore, some metabolites such as cyclic adenosine monophosphate, glutamine, and short- and medium-chain acylcarnitines were the differential metabolites of HCC and CIR. On the basis of binary logistic regression, butyrylcarnitine (carnitine C4:0) and hydantoin-5-propionic acid were defined as combinational markers to distinguish HCC from CIR. The area under curve was 0.786 and 0.773 for discovery stage and validation stage samples, respectively. These data show that the established pseudotargeted method is a complementary one of targeted and nontargeted methods for metabolomics study.

Urinary biomarkers of acute kidney injury in patients with liver cirrhosis.

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.

Study of urinary steroid hormone disorders: difference between hepatocellular carcinoma in early stage and cirrhosis.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Discovery of novel biomarkers for early HCC from other liver diseases such as cirrhosis is of great clinical benefit. In this study, a novel steroid hormone metabolomic method based on liquid chromatography-mass spectrometry combined with logistic regression analysis was applied to study the steroid hormone disorders and to screen potential urinary steroid hormone biomarkers of early HCC. Thirty-six urinary steroid hormones were detected and quantified in healthy controls, cirrhotic patients, and early HCC patients. Heat map analysis and multivariate statistical analysis suggested severe disorders of steroid hormone network and holistically decreased urinary steroid hormone pattern in cirrhotic and early HCC patients. Logistic regression analysis reveals that a panel of two urinary steroid hormones (epitestosterone and allotetrahydrocortisol) displayed excellent diagnostic capability for distinguishing early HCC from cirrhosis with area under the curve (AUC) = 0.938 of receiver operating characteristic (ROC) analysis. These results help to overcome the disadvantage of lower sensitivity and specificity of alpha-fetoprotein for distinguishing early HCC from cirrhosis. Our work shows that steroid hormone metabolomics is a promising biomarker tool for biomarker study of early HCC.