The mechanisms that drive nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. This large multicenter study characterized the transcriptional changes that occur in liver tissue across the NAFLD spectrum as disease progresses to cirrhosis to identify potential circulating markers. We performed high-throughput RNA sequencing on a discovery cohort comprising histologically characterized NAFLD samples from 206 patients. Unsupervised clustering stratified NAFLD on the basis of disease activity and fibrosis stage with differences in age, aspartate aminotransferase (AST), type 2 diabetes mellitus, and carriage of PNPLA3 rs738409, a genetic variant associated with NAFLD. Relative to early disease, we consistently identified 25 differentially expressed genes as fibrosing steatohepatitis progressed through stages F2 to F4. This 25-gene signature was independently validated by logistic modeling in a separate replication cohort (n = 175), and an integrative analysis with publicly available single-cell RNA sequencing data elucidated the likely relative contribution of specific intrahepatic cell populations. Translating these findings to the protein level, SomaScan analysis in more than 300 NAFLD serum samples confirmed that circulating concentrations of proteins AKR1B10 and GDF15 were strongly associated with disease activity and fibrosis stage. Supporting the biological plausibility of these data, in vitro functional studies determined that endoplasmic reticulum stress up-regulated expression of AKR1B10, GDF15, and PDGFA, whereas GDF15 supplementation tempered the inflammatory response in macrophages upon lipid loading and lipopolysaccharide stimulation. This study provides insights into the pathophysiology of progressive fibrosing steatohepatitis, and proof of principle that transcriptomic changes represent potentially tractable and clinically relevant markers of disease progression.

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic ( = 27) or hepatitis C (HCV, = 30) cirrhotic versus noncirrhotic (control) livers ( = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.

Protein expression of renal uptake and efflux transporters was quantified by quantitative targeted proteomics using the surrogate peptide approach. Renal uptake transporters assessed in this study included organic anion transporters (OAT1-OAT4), organic cation transporter 2 (OCT2), organic/carnitine cation transporters (OCTN1 and OCTN2), and sodium-glucose transporter 2 (SGLT2); efflux transporters included P-glycoprotein, breast cancer resistance protein, multidrug resistance proteins (MRP2 and MRP4), and multidrug and toxin extrusion proteins (MATE1 and MATE2-K). Total membrane was isolated from the cortex of human kidneys (N = 41). The isolated membranes were digested by trypsin and the digest was subjected to liquid chromatography-tandem mass spectrometry analysis. The mean expression of surrogate peptides was as follows (given with the standard deviation, in picomoles per milligram of total membrane protein): OAT1 (5.3 6 1.9), OAT2 (0.9 6 0.3), OAT3 (3.5 6 1.6), OAT4 (0.5 6 0.2), OCT2 (7.4 6 2.8), OCTN1 (1.3 6 0.6), OCTN2 (0.6 6 0.2), P-glycoprotein (2.1 6 0.8), MRP2 (1.4 6 0.6), MRP4 (0.9 6 0.6), MATE1 (5.1 6 2.3), and SGLT2 (3.7 6 1.8). Breast cancer resistance protein (BCRP) and MATE2-K proteins were detectable but were below the lower limit of quantification. Interestingly, the protein expression of OAT1 and OAT3 was significantly correlated (r > 0.8). A significant correlation was also observed between expression of multiple other drug transporters, such as OATs/OCT2 or OCTN1/OCTN2, and SGLT2/OCTNs, OCT, OATs, and MRP2. These renal transporter data should be useful in deriving in vitro to in vivo scaling factors to accurately predict renal clearance and kidney epithelial cell exposure to drugs or their metabolites.

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RNA-sequencing (RNA-seq) is a powerful technique to investigate the complexity of gene expression in the human brain. We used RNA-seq to survey the brain transcriptome in high-quality postmortem dorsolateral prefrontal cortex from 11 individuals diagnosed with bipolar disorder (BD) and from 11 age- and gender-matched controls. Deep sequencing was performed, with over 350 million reads per specimen. At a false discovery rate of <5%, we detected five differentially expressed (DE) genes and 12 DE transcripts, most of which have not been previously implicated in BD. Among these, Prominin 1/CD133 and ATP-binding cassette-sub-family G-member2 (ABCG2) have important roles in neuroplasticity. We also show for the first time differential expression of long noncoding RNAs (lncRNAs) in BD. DE transcripts include those of serine/arginine-rich splicing factor 5 (SRSF5) and regulatory factor X4 (RFX4), which along with lncRNAs have a role in mammalian circadian rhythms. The DE genes were significantly enriched for several Gene Ontology categories. Of these, genes involved with GTPase binding were also enriched for BD-associated SNPs from previous genome-wide association studies, suggesting that differential expression of these genes is not simply a consequence of BD or its treatment. Many of these findings were replicated by microarray in an independent sample of 60 cases and controls. These results highlight common pathways for inherited and non-inherited influences on disease risk that may constitute good targets for novel therapies.