Ciclosporin reduces paracellin-1 expression and magnesium transport in thick ascending limb cells

Abstract

Background: Renal magnesium (Mg2+) wasting is one of the ciclosporin (CsA) tubular effects. The major site of Mg2+ transport is the thick ascending limb (TAL), where 70% of the ultrafiltrable Mg2+ is reabsorbed paracellularly. Paracellin-1 is a tight junction protein, which regulates the paracellular Mg2+ transport in the TAL. We hypothesize that CsA reduces the expression and function of paracellin-1 and accounts for the observed renal Mg2+ wasting.

Methods: We established an immortalized cultured cortical TAL (cTAL) cell line from L-PK/Tag1 transgenic mice by microdissection. The cultured cells expressed paracellin-1 and the characteristics of cTAL cells. Real-time PCR and western blotting were used to test the CsA effects on paracellin-1 expression of cultured cTAL cells. Cytosolic-free Mg2+ concentration [Mg2+]i change with time in a single cTAL cell was used as an indicator of transcellular Mg2+ transport and assessed by using fluorescence dye Mag-fura-2 AM. Paracellular Mg2+ transport was measured by cells grown in porous filters.

Results: The results showed that CsA significantly reduced paracellin-1 mRNA and protein expression in a dose-dependent manner. CsA (100 ng/ml) incubation for 24 h induced a decrease of paracellin-1 mRNA by 89.4% and paracellin-1 protein by 75.4%. CsA (100 ng/ml) did not change transcellular Mg2+ transport, but paracellular Mg2+ transport was decreased in CsA-treated cTAL cells by 74.4%.

Conclusion: These results suggested that reduced PCLN-1 expression and paracellular Mg2+ transport might play a role in the renal Mg2+ wasting in the CsA tubular effect.