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

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.