INSERM UMR-S 999, LabEx LERMIT, University Paris-Sud, Faculty of Pharmacy, Centre Chirurgical Marie Lannelongue, 92350 LePlessis-Robinson, France.
J Pharm Pharm Sci. 2012;15(3):389-98. doi: 10.18433/j3qk57.
Hypomagnesemia with urinary magnesium (Mg) wasting is a well acknowledged side effect of cyclosporin A (CsA) and tacrolimus (FK506) treatments. TRPM6, TRPM7 and MagT1 are involved in the active transcellular Mg transport processes in intestine and kidney. Since Mg homeostasis is tightly controlled by the dynamic action of intestinal absorption of dietary Mg and renal excretion of Mg, we investigated whether CsA and FK506 in commercially available solutions for clinical use decrease the expression and the function of TRPM6, TRPM7 or MagT1 in the intestinal epithelial cell line Caco2.
Changes of intracellular free Mg concentrations were measured by Mag-fura-2 imaging in Mg-free medium after the addition of 1 mM MgCl2. TRPM6, TRPM7 and MagT1 were evidenced in cells by immunofluorescence. Proteins and mRNAs were quantified after 18 hours of treatment with CsA or FK506 by western-blot and real-time RT-PCR analyses, respectively.
TRPM6 and MagT1 were evidenced on all cell membranes, TRPM7 only on the inner membranes. CsA was responsible for a profound decrease in Mg2+ influx in intestinal epithelial cells, which may result in a decrease of intestinal Mg absorption, whereas FK506 was responsible for a marked increase in Mg2+ influx. Neither CsA nor FK506 altered TRPM6, TRPM7 or MagT1 mRNA levels or MagT1 protein level.
In Caco2 cells, Mg2+ influx was inhibited by CsA solutions whereas enhanced by FK506 solutions, without alteration of MagT1, TRPM6 and TRPM7 expression, leading to the conclusion that CsA and FK506 have opposite effects in the functional activity of the Mg transporters herein examined. In clinical use, FK506 should be preferred for patients at risk for hypomagnesemia.
低镁血症伴尿镁排泄增加是环孢素 A（CsA）和他克莫司（FK506）治疗的公认副作用。TRPM6、TRPM7 和 MagT1 参与肠和肾中的主动细胞间镁转运过程。由于镁的体内平衡受肠道吸收膳食镁和肾脏排泄镁的动态作用的严格控制，我们研究了市售的 CsA 和 FK506 溶液在临床上的使用是否会降低肠上皮细胞系 Caco2 中 TRPM6、TRPM7 或 MagT1 的表达和功能。
在添加 1mM MgCl2 后的无镁培养基中，通过 Mag-fura-2 成像测量细胞内游离镁浓度的变化。用免疫荧光法在细胞中检测 TRPM6、TRPM7 和 MagT1。用 CsA 或 FK506 处理 18 小时后，通过 Western blot 和实时 RT-PCR 分析分别定量蛋白质和 mRNA。
TRPM6 和 MagT1 可见于所有细胞膜上，TRPM7 仅见于内膜上。CsA 导致肠上皮细胞中镁内流明显减少，可能导致肠道镁吸收减少，而 FK506 导致镁内流明显增加。CsA 和 FK506 均未改变 TRPM6、TRPM7 或 MagT1 的 mRNA 水平或 MagT1 蛋白水平。
在 Caco2 细胞中，CsA 溶液抑制镁内流，而 FK506 溶液增强镁内流，而 MagT1、TRPM6 和 TRPM7 的表达不变，这表明 CsA 和 FK506 在本研究中检查的镁转运体的功能活性中具有相反的作用。在临床使用中，对于有低镁血症风险的患者，应优先选择 FK506。
