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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 expression levels. Real-time quantitative PCR was used to determine the mRNA level of the thiazide-dependent Na+, Cl− cotransporter (NCC) in the kidneys of mice chronically treated with furosemide (30 mg/kg/day for 7 days). Expression levels are corrected for GAPDH and presented as relative percentage of expression in control mice (A). NCC protein abundance was determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (B). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
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SFS140F2: Effect of chronic furosemide treatment on renal NCC expression levels. Real-time quantitative PCR was used to determine the mRNA level of the thiazide-dependent Na+, Cl− cotransporter (NCC) in the kidneys of mice chronically treated with furosemide (30 mg/kg/day for 7 days). Expression levels are corrected for GAPDH and presented as relative percentage of expression in control mice (A). NCC protein abundance was determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (B). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.

Mentions: The effect of furosemide treatment on the renal expression level of TRPM6 was determined by real-time quantitative PCR (qPCR). A significant increase in renal TRPM6 mRNA expression was observed in mice chronically treated with furosemide (129 ± 9 and 100 ± 6%, furosemide versus control, P < 0.05) (Figure 1). Next, the renal mRNA expression level and protein abundance of the thiazide-sensitive NCC were determined. Chronic furosemide treatment had no effect on mRNA expression of NCC (98 ± 4 and 100 ± 5%, furosemide versus control, P > 0.2) (Figure 2A). The renal protein abundance of NCC was examined by IHC. In order to semi-quantify the protein expression, the amount of immunopositive tubules in the total kidney cortex was determined for each experimental group. The averaged values of the furosemide-treated group are presented as relative percentage of expression in control mice. In contrast with the mRNA level, the protein abundance of NCC was substantially increased (176 ± 9 and 100 ± 8%, furosemide versus control, P < 0.01) (Figure 2B).Fig. 1.


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 expression levels. Real-time quantitative PCR was used to determine the mRNA level of the thiazide-dependent Na+, Cl− cotransporter (NCC) in the kidneys of mice chronically treated with furosemide (30 mg/kg/day for 7 days). Expression levels are corrected for GAPDH and presented as relative percentage of expression in control mice (A). NCC protein abundance was determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (B). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

SFS140F2: Effect of chronic furosemide treatment on renal NCC expression levels. Real-time quantitative PCR was used to determine the mRNA level of the thiazide-dependent Na+, Cl− cotransporter (NCC) in the kidneys of mice chronically treated with furosemide (30 mg/kg/day for 7 days). Expression levels are corrected for GAPDH and presented as relative percentage of expression in control mice (A). NCC protein abundance was determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (B). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
Mentions: The effect of furosemide treatment on the renal expression level of TRPM6 was determined by real-time quantitative PCR (qPCR). A significant increase in renal TRPM6 mRNA expression was observed in mice chronically treated with furosemide (129 ± 9 and 100 ± 6%, furosemide versus control, P < 0.05) (Figure 1). Next, the renal mRNA expression level and protein abundance of the thiazide-sensitive NCC were determined. Chronic furosemide treatment had no effect on mRNA expression of NCC (98 ± 4 and 100 ± 5%, furosemide versus control, P > 0.2) (Figure 2A). The renal protein abundance of NCC was examined by IHC. In order to semi-quantify the protein expression, the amount of immunopositive tubules in the total kidney cortex was determined for each experimental group. The averaged values of the furosemide-treated group are presented as relative percentage of expression in control mice. In contrast with the mRNA level, the protein abundance of NCC was substantially increased (176 ± 9 and 100 ± 8%, furosemide versus control, P < 0.01) (Figure 2B).Fig. 1.

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