Acute phosphate-salt exposure and expression of iron- and heme-related genes in HepaRG cells

Abstract Background Hepatic iron regulation involves iron transport, heme handling, and hepcidin regulation. We examined whether acute phosphate-salt exposure alters related transcripts in HepaRG cells. An earlier report on these data addressed albumin, HNF4A, and ERK activation. Methods and Results We reanalyzed a previously generated RNA sequencing (RNA-seq) dataset of HepaRG cells treated for 24 h with vehicle or phosphate (5.0 mM total; three samples per condition). Selected transcripts were measured by quantitative PCR (qPCR) in four to six independent cultures per target and compared by paired log-scale tests with Holm correction across nine targets. RNA-seq showed lower expression of hemojuvelin (HJV), transferrin (TF), hemopexin (HPX), and aminolevulinate dehydratase (ALAD) at a false discovery rate (FDR) below 0.05. A limma-voom reanalysis gave concordant results. By qPCR, HJV expression was lower in all six cultures (geometric mean phosphate-to-vehicle ratio 0.268; 95% confidence interval [CI] 0.157–0.458; Holm-adjusted p = 0.013). The ratios for TF, HPX, matriptase-2 (TMPRSS6), transferrin receptor 2 (TFR2), and ALAD were 0.396, 0.516, 0.425, 0.460, and 0.422, respectively, none significant after Holm adjustment. Estimates for ferritin heavy chain 1 (FTH1) and heme oxygenase 1 (HMOX1) were close to unity, and biliverdin reductase A (BLVRA) showed no clear change. Hepcidin (HAMP) measurements were insufficiently reliable for relative quantification. Albumin (ALB) and transthyretin (TTR) decreased in RNA-seq. Conclusions Acute phosphate-salt exposure was associated with reduced HJV expression and exploratory changes in other iron- and heme-related transcripts. These observations do not establish pathway specificity, hepcidin activation, or cellular iron retention.