Cyclosporin a inhibits albumin synthesis in Huh7 cells

Abstract

Background/aims: Hypoalbuminemia occurs frequently in renal transplant recipients immediately after renal transplantation. We studied the regulation of hepatic albumin synthesis by cyclosporin A (CsA) in Huh7 cells.

Methods: Huh7 cells were incubated with various concentrations of CsA for 4, 8, 16, and 24 hours. Albumin was measured in Huh7 cell-conditioned medium by sandwich enzyme-linked immunosorbent assay and Western blot. Albumin mRNA expression was analyzed by Northern blotting in CsA-treated cells.

Results: CsA (10(-7)-10(-4) M) inhibited albumin synthesis in Huh7 cells in a dose- dependent manner. A Western blot analysis for albumin in the conditioned medium released from CsA-treated (10(-7)-10(-5) M) cells also showed significant inhibition of albumin synthesis in a dose-dependent manner. Vehicle (olive oil) did not affect albumin synthesis. In contrast, a Northern blot analysis revealed no inhibition of albumin mRNA expression by CsA at any time point from 1-24 hours, indicating that the inhibition of albumin synthesis occurred at the translational level.

Conclusions: Our results suggest that inhibition of hepatic albumin synthesis by high dose CsA contributes to the hypoalbuminemia in renal transplant recipients.

Keywords: Albumins; Cyclosporin A; Hypoalbuminemia; Kidney transplantation.

Figure 1

Albumin concentration in the conditioned medium was measured in untreated- and cyclosporine A (CsA)-treated Huh7 cells. (A) Confluent cells were incubated with or without various doses of CsA for 24 hours. (B) The conf luent cells were incubated with or without CsA (10^-6 M) for the indicated time periods, and albumin was measured by enzyme-linked immunosorbent assay (ELISA). Olive oil was used as a vehicle. Data are expressed as mean ± SE. Values are means of four independent experiments performed in duplicate samples. ^*p < 0.01 vs. control. (C) Western blot analysis for albumin in Huh7-cell conditioned medium. The cells were incubated with or without various doses of CsA for 24 hours. Numbers on the left represent sizes in kilodaltons. Olive oil was used as a vehicle. Densitometric values are the mean of two experiments and are expressed as mean ± SE.

Figure 2

Northern blot analysis for albumin gene expression in Huh7 cells. (A) The confluent cells were incubated with or without various doses of cyclosporine A (CsA) for 24 hours, and total RNA was isolated. (B) Confluent cells were incubated with or without CsA (10^-6 M). Total RNA was isolated after 1, 2, 4, 8, 16, and 24 hours. The figures in both (A) and (B) are representative of three experiments. Densitometric values are the mean of three experiments, and are expressed as mean ± SE.