The impact of dietary magnesium restriction on magnesiotropic and calciotropic genes.

BACKGROUND: Magnesium (Mg(2+)) is an essential electrolyte with important physiological functions. Consequently, hypomagnesaemia, an electrolyte disorder frequently diagnosed in critically ill patients, can have life-threatening consequences. The kidney plays a central role in the regulation of the Mg(2+) balance. The present study investigated the molecular consequences of dietary Mg(2+) restriction on renal Mg(2+) transporters. METHODS: Two groups of 10 mice were fed a Mg(2+)-deficient diet or a Mg(2+)-enriched diet for 2 weeks. Serum and urine electrolyte concentrations were assayed. Next, renal mRNA expression levels of Mg(2+)-related genes were measured to determine their sensitivity to the dietary Mg(2+) content. Subsequently, parvalbumin (PV) and the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), both co-expressed in the distal convoluted tubule (DCT) with TRPM6, were further analysed at the protein level using immunoblotting and immunohistochemistry. RESULTS: Serum and urine electrolyte measurements revealed that dietary Mg(2+) restriction resulted in significant reduction of serum Mg(2+) and Ca(2+) levels, and that the urinary excretion of these ions was also markedly reduced, while phosphate (Pi) excretion was significantly increased. In addition, the serum FGF23 level was markedly increased, whereas Pi was not significantly changed in the Mg(2+)-restricted mouse group. The renal abundance of hepatocyte nuclear factor 1 homeobox B (HNF1B) and the epithelial Mg(2+) channel TRPM6 were increased in response to dietary Mg(2+) restriction, whereas other magnesiotropic transporters were not affected. PV abundance was upregulated, while NCC was significantly downregulated. Furthermore, the expression levels of the epithelial Ca(2+) channel TRPV5 and calbindin-D28K were markedly reduced in the low Mg(2+) group. CONCLUSIONS: Our data indicate an essential adaptive role for DCT during hypomagnesaemia since TRPM6, HNF1B, PV and NCC expression levels were adjusted. Moreover, hypomagnesaemia resulted in severe changes in Ca(2+) and Pi reabsorption and expression levels of calciotropic proteins.

Cisplatin-induced injury of the renal distal convoluted tubule is associated with hypomagnesaemia in mice.

BACKGROUND: Cisplatin is an effective anti-neoplastic drug, but its clinical use is limited due to dose-dependent nephrotoxicity. The majority of cisplatin-treated patients develop hypomagnesaemia, often associated with a reduced glomerular filtration rate (GFR), polyuria and other electrolyte disturbances. The aim of this study is to unravel the molecular mechanism responsible for these particular electrolyte disturbances. METHODS: Two groups of 10 mice were injected intraperitoneally three times, once every 4 days, with cisplatin (5 mg/kg body weight,) or vehicle. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of distal convoluted tubule (DCT) genes epithelial Mg(2+) channel TRPM6, the Na(+)-Cl(-) cotransporter (NCC), and parvalbumin (PV), as well as marker genes for other tubular segments were measured by real-time qPCR. Subsequently, renal protein levels of NCC, PV, aquaporin 1 and aquaporin 2 were determined using immunoblotting and immunohistochemistry (IHC). RESULTS: The cisplatin-treated mice developed significant polyuria (2.5 ± 0.3 and 0.9 ± 0.1 mL/24 h, cisplatin versus control, P < 0.05), reduced creatinine clearance rate (CCr) (0.18 ± 0.02 and 0.26 ± 0.02 mL/min, cisplatin versus control, P < 0.05) and a substantially reduced serum level of Mg(2+) (1.23 ± 0.03 and 1.58 ± 0.03 mmol/L, cisplatin versus control, P < 0.05), whereas serum Ca(2+), Na(+) and K(+) values were not altered. Measurements of 24 h urinary excretion demonstrated markedly increased Mg(2+), Ca(2+), Na(+) and K(+) levels in the cisplatin-treated group, whereas Pi levels were not changed. The mRNA levels of TRPM6, NCC and PV were significantly reduced in the cisplatin group. The expression levels of the marker genes for other tubular segments were unaltered, except for claudin-16, which was significantly up-regulated by the cisplatin treatment. The observed DCT-specific down-regulation was confirmed at the protein level. CONCLUSIONS: The present study identified the DCT as an important cisplatin-affected renal segment, explaining the high prevalence of hypomagnesaemia following treatment.

Increased expression of renal TRPM6 compensates for Mg(2+) wasting during furosemide treatment.

BACKGROUND: Furosemide is a loop diuretic, which blocks the Na(+), K(+), 2Cl(-) cotransporter (NKCC2) in the thick ascending limb of Henle (TAL). By diminishing sodium (Na(+)) reabsorption, loop diuretics reduce the lumen-positive transepithelial voltage and consequently diminish paracellular transport of magnesium (Mg(2+)) and calcium (Ca(2+)) in TAL. Indeed, furosemide promotes urinary Mg(2+) excretion; however, it is unclear whether this leads, especially during prolonged treatment, to hypomagnesaemia. The aim of the present study was, therefore, to determine the effect of chronic furosemide application on renal Mg(2+) handling in mice. METHODS: Two groups of 10 mice received an osmotic minipump subcutaneously for 7 days with vehicle or 30 mg/kg/day furosemide. Serum and urine electrolyte concentrations were determined. Next, renal mRNA levels of the epithelial Mg(2+) channel (TRPM6), the Na(+), Cl(-) cotransporter (NCC), the epithelial Ca(2+) channel (TRPV5), the cytosolic Ca(2+)-binding protein calbindin-D28K, as well parvalbumin (PV), claudin-7 (CLDN7) and claudin-8 (CLDN8), the epithelial Na(+) channel (ENaC) and the Na(+)-H(+) exchanger 3 (NHE3) were determined by real-time quantitative polymerase chain reaction. Renal protein levels of NCC, TRPV5, calbindin-D28K and ENaC were also measured using semi-quantitative immunohistochemistry and immunoblotting. RESULTS: The mice chronically treated with 30 mg/kg/day furosemide displayed a significant polyuria (2.1 ± 0.3 and 1.3 ± 0.2 mL/24 h, furosemide versus control respectively, P < 0.05). Furosemide treatment resulted in increased serum concentrations of Na(+) [158 ± 3 (treated) and 147 ± 1 mmol/L (control), P < 0.01], whereas serum K(+), Ca(2+) and Mg(2+) values were not significantly altered in mice treated with furosemide. Urinary excretion of Na(+), K(+), Ca(2+) and Mg(2+) was not affected by chronic furosemide treatment. The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K. CONCLUSIONS: During chronic furosemide treatment, enhanced active reabsorption of Mg(2+) via the epithelial channel TRPM6 in DCT compensates for the reduced reabsorption of Mg(2+) in TAL.

Investigation into the mechanism regulating MRP localization.

The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.