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

van Angelen AA, van der Kemp AW, Hoenderop JG, Bindels RJ - Clin Kidney J (2012)

Bottom Line: 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.The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology , Radboud University Nijmegen Medical Centre , Nijmegen , the Netherlands.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Effect of chronic furosemide treatment on renal NCC, calbindin-D28K and αENaC protein expression levels. The effect of furosemide (30 mg/kg/day for 7 days) on protein expression levels of the Na+–Cl− cotransporter (NCC) (A), calbindin-D28K (B) and the alpha-subunit of the epithelial Na+ channel (αENaC) (C), determined by immunoblotting. The upper part of each figure shows the immunoblot, with on the left side the molecular mass (in kDa) and the lower parts depict the expression levels as percentage of control. Values are presented as average ± SEM (n = 4), while experiments are performed in duplo. *P < 0.05 compared with control.
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SFS140F5: Effect of chronic furosemide treatment on renal NCC, calbindin-D28K and αENaC protein expression levels. The effect of furosemide (30 mg/kg/day for 7 days) on protein expression levels of the Na+–Cl− cotransporter (NCC) (A), calbindin-D28K (B) and the alpha-subunit of the epithelial Na+ channel (αENaC) (C), determined by immunoblotting. The upper part of each figure shows the immunoblot, with on the left side the molecular mass (in kDa) and the lower parts depict the expression levels as percentage of control. Values are presented as average ± SEM (n = 4), while experiments are performed in duplo. *P < 0.05 compared with control.

Mentions: Ultimately, to confirm our data obtained by real-time qPCR and IHC, we performed immunoblotting for NCC, calbindin-D28K and αENaC. Quantification of the immunoblots revealed that NCC (165 ± 15 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 5A) and calbindin-D28K (180 ± 23 and 100 ± 12%, furosemide versus control, P < 0.01) (Figure 5B) were markedly upregulated in the furosemide-treated group, confirming the results obtained using IHC. Moreover, the immunoblot for ENaC further substantiated the result obtained on the mRNA level, showing that αENaC protein expression is not affected by furosemide treatment as well (122 ± 17 and 100 ± 14%, furosemide versus control, P > 0.2) (Figure 5C).Fig. 5.


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

van Angelen AA, van der Kemp AW, Hoenderop JG, Bindels RJ - Clin Kidney J (2012)

Effect of chronic furosemide treatment on renal NCC, calbindin-D28K and αENaC protein expression levels. The effect of furosemide (30 mg/kg/day for 7 days) on protein expression levels of the Na+–Cl− cotransporter (NCC) (A), calbindin-D28K (B) and the alpha-subunit of the epithelial Na+ channel (αENaC) (C), determined by immunoblotting. The upper part of each figure shows the immunoblot, with on the left side the molecular mass (in kDa) and the lower parts depict the expression levels as percentage of control. Values are presented as average ± SEM (n = 4), while experiments are performed in duplo. *P < 0.05 compared with control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400563&req=5

SFS140F5: Effect of chronic furosemide treatment on renal NCC, calbindin-D28K and αENaC protein expression levels. The effect of furosemide (30 mg/kg/day for 7 days) on protein expression levels of the Na+–Cl− cotransporter (NCC) (A), calbindin-D28K (B) and the alpha-subunit of the epithelial Na+ channel (αENaC) (C), determined by immunoblotting. The upper part of each figure shows the immunoblot, with on the left side the molecular mass (in kDa) and the lower parts depict the expression levels as percentage of control. Values are presented as average ± SEM (n = 4), while experiments are performed in duplo. *P < 0.05 compared with control.
Mentions: Ultimately, to confirm our data obtained by real-time qPCR and IHC, we performed immunoblotting for NCC, calbindin-D28K and αENaC. Quantification of the immunoblots revealed that NCC (165 ± 15 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 5A) and calbindin-D28K (180 ± 23 and 100 ± 12%, furosemide versus control, P < 0.01) (Figure 5B) were markedly upregulated in the furosemide-treated group, confirming the results obtained using IHC. Moreover, the immunoblot for ENaC further substantiated the result obtained on the mRNA level, showing that αENaC protein expression is not affected by furosemide treatment as well (122 ± 17 and 100 ± 14%, furosemide versus control, P > 0.2) (Figure 5C).Fig. 5.

Bottom Line: 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.The present study shows specific renal upregulation of TRPM6, NCC, TRPV5 and calbindin-D28K.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology , Radboud University Nijmegen Medical Centre , Nijmegen , the Netherlands.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus