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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 TRPV5 and calbindin-D28K expression levels. Real-time qPCR was used to determine the mRNA expression levels of the epithelial Ca2+ channel TRPV5 (A) and the cytosolic Ca2+-binding protein calbindin-D28K (B) 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. TRPV5 and calbindin-D28K protein abundance were determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (C and D). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
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SFS140F3: Effect of chronic furosemide treatment on renal TRPV5 and calbindin-D28K expression levels. Real-time qPCR was used to determine the mRNA expression levels of the epithelial Ca2+ channel TRPV5 (A) and the cytosolic Ca2+-binding protein calbindin-D28K (B) 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. TRPV5 and calbindin-D28K protein abundance were determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (C and D). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.

Mentions: To determine the specificity of the upregulation of TRPM6 and NCC in response to furosemide treatment, renal mRNA expression levels of other genes specifically expressed in the DCT and CNT were quantified. To evaluate the effect of furosemide treatment on Ca2+ handling, renal expression levels of TRPV5 and the cytosolic Ca2+-binding protein calbindin-D28K in the late DCT (DCT2) and CNT were determined. Furosemide significantly increased the mRNA expression levels of TRPV5 (125 ± 6 and 100 ± 6%, furosemide versus control, P < 0.01) (Figure 3A) and calbindin-D28K (153 ± 14 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 3B). Subsequently, the renal protein abundance of TRPV5 and calbindin-D28K was examined by IHC. In accordance with the mRNA levels, the protein levels of TRPV5 (171 ± 18 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 3C) and calbindin-D28K (173 ± 13 and 100 ± 8%, furosemide versus control, P < 0.01) (Figure 3D) were markedly increased. Subsequently, the mRNA abundance of parvalbumin (PV), a DCT-specific Ca2+- and Mg2+-binding protein [29], the tight junction proteins claudin-7 (CLDN7) and claudin-8 (CLDN8) expressed along the entire aldosterone-sensitive part of the nephron [30] and the epithelial Na+ channel (ENaC) in the CNT and collecting duct (CD) were determined by real-time qPCR. Furosemide treatment had no significant effect on the mRNA expression levels of these genes; PV (122 ± 9 and 100 ± 8%) (Figure 4A), CLDN7 (115 ± 6 and 100 ± 11%) (Figure 4B), CLDN8 (103 ± 9 and 100 ± 8%) (Figure 4C) and ENaC (122 ± 13 and 100 ± 7%) (Figure 4D), for all four of these genes; furosemide versus control, P > 0.05. Finally, the mRNA expression level of the Na+–H+ exchanger 3 (NHE3) in the proximal tubule (PT) and to a minor extent in TAL [31, 32] was determined to define whether chronic furosemide treatment increased Na+ reabsorption in the PT. Furosemide treatment did not substantially change the renal mRNA level of NHE3 (125 ± 17 and 100 ± 6%, furosemide versus control, P > 0.1) (Figure 4E).Fig. 3.


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 TRPV5 and calbindin-D28K expression levels. Real-time qPCR was used to determine the mRNA expression levels of the epithelial Ca2+ channel TRPV5 (A) and the cytosolic Ca2+-binding protein calbindin-D28K (B) 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. TRPV5 and calbindin-D28K protein abundance were determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (C and D). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
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SFS140F3: Effect of chronic furosemide treatment on renal TRPV5 and calbindin-D28K expression levels. Real-time qPCR was used to determine the mRNA expression levels of the epithelial Ca2+ channel TRPV5 (A) and the cytosolic Ca2+-binding protein calbindin-D28K (B) 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. TRPV5 and calbindin-D28K protein abundance were determined by computerized analysis of immunohistochemical images and presented as relative percentage of control mice (C and D). Values are presented as means ± SEM (n = 10). *P < 0.05 compared with control.
Mentions: To determine the specificity of the upregulation of TRPM6 and NCC in response to furosemide treatment, renal mRNA expression levels of other genes specifically expressed in the DCT and CNT were quantified. To evaluate the effect of furosemide treatment on Ca2+ handling, renal expression levels of TRPV5 and the cytosolic Ca2+-binding protein calbindin-D28K in the late DCT (DCT2) and CNT were determined. Furosemide significantly increased the mRNA expression levels of TRPV5 (125 ± 6 and 100 ± 6%, furosemide versus control, P < 0.01) (Figure 3A) and calbindin-D28K (153 ± 14 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 3B). Subsequently, the renal protein abundance of TRPV5 and calbindin-D28K was examined by IHC. In accordance with the mRNA levels, the protein levels of TRPV5 (171 ± 18 and 100 ± 10%, furosemide versus control, P < 0.01) (Figure 3C) and calbindin-D28K (173 ± 13 and 100 ± 8%, furosemide versus control, P < 0.01) (Figure 3D) were markedly increased. Subsequently, the mRNA abundance of parvalbumin (PV), a DCT-specific Ca2+- and Mg2+-binding protein [29], the tight junction proteins claudin-7 (CLDN7) and claudin-8 (CLDN8) expressed along the entire aldosterone-sensitive part of the nephron [30] and the epithelial Na+ channel (ENaC) in the CNT and collecting duct (CD) were determined by real-time qPCR. Furosemide treatment had no significant effect on the mRNA expression levels of these genes; PV (122 ± 9 and 100 ± 8%) (Figure 4A), CLDN7 (115 ± 6 and 100 ± 11%) (Figure 4B), CLDN8 (103 ± 9 and 100 ± 8%) (Figure 4C) and ENaC (122 ± 13 and 100 ± 7%) (Figure 4D), for all four of these genes; furosemide versus control, P > 0.05. Finally, the mRNA expression level of the Na+–H+ exchanger 3 (NHE3) in the proximal tubule (PT) and to a minor extent in TAL [31, 32] was determined to define whether chronic furosemide treatment increased Na+ reabsorption in the PT. Furosemide treatment did not substantially change the renal mRNA level of NHE3 (125 ± 17 and 100 ± 6%, furosemide versus control, P > 0.1) (Figure 4E).Fig. 3.

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