Plasma Aldo-Keto Reductase Family 1 Member B10 as a Biomarker Performs Well in the Diagnosis of Nonalcoholic Steatohepatitis and Fibrosis

Abstract

We found several blood biomarkers through computational secretome analyses, including aldo-keto reductase family 1 member B10 (AKR1B10), which reflected the progression of nonalcoholic fatty liver disease (NAFLD). After confirming that hepatic AKR1B10 reflected the progression of NAFLD in a subgroup with NAFLD, we evaluated the diagnostic accuracy of plasma AKR1B10 and other biomarkers for the diagnosis of nonalcoholic steatohepatitis (NASH) and fibrosis in replication cohort. We enrolled healthy control subjects and patients with biopsy-proven NAFLD (n = 102) and evaluated the performance of various diagnostic markers. Plasma AKR1B10 performed well in the diagnosis of NASH with an area under the receiver operating characteristic (AUROC) curve of 0.834 and a cutoff value of 1078.2 pg/mL, as well as advanced fibrosis (AUROC curve value of 0.914 and cutoff level 1078.2 pg/mL), with further improvement in combination with C3. When we monitored a subgroup of obese patients who underwent bariatric surgery (n = 35), plasma AKR1B10 decreased dramatically, and 40.0% of patients with NASH at baseline showed a decrease in plasma AKR1B10 levels to below the cutoff level after the surgery. In an independent validation study, we proved that plasma AKR1B10 was a specific biomarker of NAFLD progression across varying degrees of renal dysfunction. Despite perfect correlation between plasma and serum levels of AKR1B10 in paired sample analysis, its serum level was 1.4-fold higher than that in plasma. Plasma AKR1B10 alone and in combination with C3 could be a useful noninvasive biomarker for the diagnosis of NASH and hepatic fibrosis.

Keywords: aldo-keto reductase family 1 member B10; biomarkers; diagnosis; nonalcoholic fatty liver disease.

Figure 1

Computational identification of the tentative secretome reflecting NAFLD progression that was available from the analyses of public gene datasets. (A) Workflow of secretome identification by analyzing public datasets of NAFLD- and HCC-related genes. Detailed information on the datasets is described in Section 4 and presented in Supplementary Table S1. (B) Heatmap of putative secretory biomarker genes. Stepwise upregulated (upper 4 listed genes) and downregulated common genes (lower 25 listed genes) according to disease progression. (C) Hepatic AKR1B10 expression according to the progression of NAFLD was divided into 3 categories of disease progression. ***, p < 0.001.

Figure 2

Hepatic expression of the AKR1B10 gene and protein in patients with NAFLD and HCC. (A) RNA sequencing data showing 4 upregulated common DEGs in study subjects with a spectrum of NAFLD progression based on the NAFLD activity score (NAS) and fibrosis stage (n = 12). TPM, transcripts per million. (B) Immunoblotting analysis and semiquantification of AKR1B10, ZNF468, annexin A2 (ANXA2), and CD24 protein expression in liver tissues from independently selected study subjects with a spectrum of NAFLD progression (n = 13). Note that protein expression levels normalized by β-actin expression were expressed relative to the corrected intensity values of the first lanes in each band. (C) Immunoblotting analysis and semiquantification of AKR1B10, ZNF468, ANXA2, and CD24 protein expression in nontumor and tumor tissues of liver samples from patients with HCC (n = 5). Band intensities of blots were normalized with respect to the signal intensities of the loading internal control (β-actin or GAPDH) detected on the same blots. *—p < 0.05; **—p < 0.01. Note: because commercial antibody to annexin A2P2 (ANXA2P2) was not available, immunoblotting on ANXA2 protein, a paralogous protein of ANXA2P2, was performed.

Figure 3

Correlations between major parameters and AUROC curves of biomarkers for the detection of NASH. (A) The results of Pearson’s correlation analyses between major parameters. (B–D), The predictive performance of AKR1B10 and other biomarkers for NASH vs. normal/NAFL.

Figure 4

Changes in biomarkers 6–12 months after bariatric surgery. Box-whisker plots are shown, with the bottom and top of the box representing the 25th and 75th percentiles, respectively, and the middle line representing the median. The whiskers extend to the 5th and 95th percentiles, and outliers are presented as dots. *—p < 0.05; **—p < 0.01; ***—p < 0.001; and n.s.—not significant.

Figure 5

Validation of plasma AKR1B10 as a NAFLD progression marker in an independent cohort with a broad range of eGFRs. (A) Plasma AKR1B10 levels according to CKD status (n = 195). (B) and (C) Plasma AKR1B10 levels according to the likelihood of hepatic steatosis and advanced fibrosis based on HSI and FIB-4 score systems (n = 195). (D) Comparison of paired plasma and serum measurements of AKR1B10 in selected patients across a range of eGFRs. *—p < 0.05; **—p < 0.01; and n.s.—not significant.