Acute phosphate-salt exposure is associated with attenuation of the albumin-producing phenotype, HNF4A down-regulation, and ERK activation in HepaRG cells

Abstract

Hyperphosphatemia is a common complication of chronic kidney disease (CKD) and has been implicated in systemic inflammation, protein-energy wasting, anemia, and skeletal muscle wasting. Hypoalbuminemia is also frequent in CKD and is associated with poor clinical outcomes. Although CKD-associated hypoalbuminemia is generally interpreted in the context of malnutrition, inflammation, protein loss, or increased albumin catabolism, whether extracellular phosphate affects hepatocyte albumin-producing capacity remains unclear. HepaRG cells were treated with vehicle, inorganic phosphate (Pi), fibroblast growth factor 23 (FGF23), or Pi plus FGF23. Albumin, HNF4A, and IL6 expression levels were examined using RNA sequencing, and additional transcriptomic overview analyses, including TCC-based differential-expression visualization and TRRUST-based HNF4A target gene analysis, were performed. Albumin secretion into culture supernatants was measured by enzyme-linked immunosorbent assay. Cell viability was assessed by MTT assay. ERK phosphorylation after short-term Pi stimulation was evaluated by immunoblot analysis. Pi exposure reduced albumin expression in HepaRG cells, whereas FGF23 alone did not exert a comparable suppressive effect. Pi exposure was also associated with reduced HNF4A expression, a key transcriptional regulator of hepatocyte differentiation and liver-specific gene expression. Additional transcriptomic overview analyses, including TCC-based differential-expression visualization and TRRUST-based HNF4A target gene analysis, suggested attenuation of hepatocyte-function/HNF4A-associated gene programs in Pi-treated cells. Consistent with the transcriptomic findings, Pi, but not FGF23 alone, significantly reduced albumin secretion into the culture supernatant. This reduction was not attributable to decreased cell viability. Pi rapidly increased ERK phosphorylation. Notably, Pi-associated reductions in albumin and HNF4A were not accompanied by robust IL6 induction under the present experimental conditions. Under the present acute, supraphysiological exposure conditions, phosphate-salt treatment was associated with attenuation of the albumin-producing phenotype in HepaRG cells, accompanied by HNF4A downregulation and ERK activation. Because sodium/osmolality-matched controls and particle characterization were not included, these findings cannot be attributed specifically to dissolved phosphate. Further studies under physiologically relevant conditions are required to determine phosphate specificity and potential relevance to CKD-associated hypoalbuminemia.