Background: Disease-associated claudin-16 mutants cause mislocalization and decrease reabsorption of Mg 2ϩ in the kidney. Results: Knockdown of syntaxin 8 in kidney cells decreased the tight junctional localization of claudin-16 in parallel with a decrease in Mg 2ϩ permeability. Conclusion: Syntaxin 8 controls the localization of claudin-16. Significance: Our findings provide a new insight into the trafficking mechanism of tight junctional proteins.

Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used in treatments for transplantation and cancer. Rapamycin causes hypomagnesemia, although precisely how has not been examined. Here, we investigated the effect of rapamycin on the expression of transient receptor potential melastatin 6 (TRPM6), a Mg2+ channel. Rapamycin and LY-294002, an inhibitor of phosphatidilinositol-3 kinase (PI3K) located upstream of mTOR, inhibited epidermal growth factor (EGF)-induced expression of the TRPM6 protein without affecting TRPM7 expression in rat renal NRK-52E epithelial cells. Both rapamycin and LY-294002 decreased EGF-induced Mg2+ influx. U0126, a MEK inhibitor, inhibited EGF-induced increases in c-Fos, p-ERK, and TRPM6 levels. In contrast, neither rapamycin nor LY-294002 inhibited EGF-induced increases in p-ERK and c-Fos levels. EGF increased p-Akt level, an effect inhibited by LY-294002 and 1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor). Akt inhibitor decreased TRPM6 level similar to rapamycin and LY-294002. These results suggest that a PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 expression. Rapamycin inhibited the EGF-induced increase in TRPM6 mRNA but did not inhibit human TRPM6 promoter activity. In the presence of actinomycin D, a transcriptional inhibitor, rapamycin accelerated the decrease in TRPM6 mRNA. Rapamycin decreased the expression and activity of a luciferase linked with the 3'-untranslated region of human TRPM6 mRNA. These results suggest that TRPM6 expression is up-regulated by a PI3K/Akt/mTOR pathway and rapamycin reduces TRPM6 mRNA stability, resulting in a decrease in the reabsorption of Mg2+.

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.