Neutrophil gelatinase-associated lipocalin level is a prognostic factor for survival in rat and human chronic liver diseases

Abstract

Chronic liver disease patients often have complications, such as hepatocellular carcinoma (HCC) and acute bacterial infection. Model for end-stage liver disease and Child-Pugh scores are useful prognostic factors for chronic liver diseases but not for all chronic conditions, such as HCC. Our investigative aim targeted the prognostic abilities of neutrophil gelatinase-associated lipocalin (NGAL) in rat and human chronic liver diseases. Blood NGAL levels were measured by enzyme-linked immunosorbent assay in rats with cirrhosis and 96 patients with chronic liver disease and HCC. We examined the correlation between blood NGAL levels and liver functions as well as survival. In our rat model, liver NGAL expression was assessed by immunostaining, real-time quantitative polymerase chain reaction, and immunoblot. In rats with cirrhosis, blood NGAL levels were continuously and significantly elevated in the deceased group and were significantly correlated with liver functions. Liver NGAL, toll-like receptor 4, and interleukin-6 levels were increased in the deceased group compared to the survival group. Blood NGAL levels were significantly correlated with liver NGAL levels, indicating blood NGAL was derived from the liver. In patients with chronic liver disease, blood NGAL levels were associated with liver function and renal function. Blood NGAL levels were significantly increased in patients with chronic liver disease with HCC compared to without HCC. For the survival group, 38 out of 96 patients were dead in the average follow-up period of 9.9 months. The patients with blood NGAL ≤119 ng/mL had significantly longer rates of survival compared to patients with blood NGAL >119 ng/mL. Conclusion: Blood NGAL predicts the survival rate in rat and human chronic liver diseases. Our findings suggest blood NGAL may be prognostic of survival in chronic liver diseases complicated by HCC. (Hepatology Communications 2017;1:946-956).

Figure 1

Blood NGAL levels were correlated with liver and kidney function in rats with cirrhosis. (A,B) Time course of (A) blood NGAL or (B) Alb levels in deceased and survival groups of rats with cirrhosis. **P <  0.01, *P <  0.05. (C,D) Correlation between blood NGAL levels and ALT at (C) 9 weeks or (D) 13 weeks in rats with cirrhosis administered CCl₄. (E,F) Correlation between blood NGAL levels and Cr at (E) 9 weeks or (F) 13 weeks in rats with cirrhosis. (G) Correlation between blood NGAL levels and Alb at 9 weeks in rats with cirrhosis. Values are mean ± SEM.

Figure 2

Liver NGAL, TLR4, and IL‐6 levels were elevated in deceased group of rats with cirrhosis, and liver NGAL levels were correlated with blood NGAL levels. (A) Immunohistochemical staining of liver sections from rats with cirrhosis specific for NGAL or MPO (neutrophils) at 5 weeks (control) or 21 weeks of CCl₄ administration. Scale bar, 100 μm. Arrows point to MPO‐positive cells/area. (B) Protein expression of NGAL and β‐actin in whole liver, using immunoblotting. (C) Bar graph shows quantification of NGAL normalized with β‐actin. (D,F,G) Gene expression of (D) NGAL, (F) TLR4, or (G) IL‐6 genes as measured by qPCR. All gene expression levels were normalized to housekeeping control β2 microglobulin and shown relative to the expression levels of control rats. ***P <  0.001, **P <  0.01, *P <  0.05. (E,H) Correlation between liver NGAL mRNA levels and (E) TLR4 mRNA levels or (H) blood NGAL levels. Values are mean ± SEM. Abbreviations: Cont, control; MPO, myeloperoxidase; qPCR, quantitative PCR.

Figure 3

Blood NGAL levels in human chronic liver disease were correlated with liver and kidney functions and were elevated in patients with chronic liver disease complicated with HCC. (A‐E) Correlation of blood NGAL levels with (A) Alb, (B) BUN, (C) Cr, (D) Na, or (E) eGFR. (F) Bar graph shows blood NGAL levels in human chronic liver diseases complicated with or without HCC. ***P <  0.001. Values are mean ± SEM.

Figure 4

Survival was improved in human chronic liver diseases guided by several factors: blood NGAL levels, blood Alb levels, blood Na levels, or complication with HCC. (A‐D) Survival curve in human chronic liver diseases with (A) blood NGAL levels, (B) Alb levels, (C) Na levels, or (D) complicated with or without HCC. ***P <  0.001.