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René J. M. Bindels - One of the best experts on this subject based on the ideXlab platform.

  • Van gen naar ziekte; mutaties in het SLC12A3-gen als oorzaak van het syndroom van Gitelman
    Nederlands Tijdschrift voor Geneeskunde, 2005
    Co-Authors: Elisabeth A. M. Cornelissen, René J. M. Bindels, Lies H. Hoefsloot, Nine V.a.m. Knoers
    Abstract:

    Gitelman's syndrome is characterised by persistent hypokalaemia, hypomagnesaemia and Hypocalciuria (OMIM 263800). This rare autosomal recessive disorder is caused by renal Na+, Cl-, K+ and Mg2+ wasting. Other typical features include Hypocalciuria and an intact renal concentrating ability. Gitelman's syndrome is caused by mutations in the SLC12A3 gene, encoding the thiazide-sensitive sodium-chloride co-transporter (NCC). NCC is located in the distal convoluted tubule of the kidney, a segment known to play an important role in active magnesium reabsorption in the nephron. The exact mechanisms underlying hypomagnesaemia and Hypocalciuria in Gitelman's syndrome are still poorly understood, but point to enhanced proximal Na+ and Ca2+ reabsorption and apoptosis of distal convoluted tubule cells.

  • enhanced passive ca2 reabsorption and reduced mg2 channel abundance explains thiazide induced Hypocalciuria and hypomagnesemia
    Journal of Clinical Investigation, 2005
    Co-Authors: Tom Nijenhuis, Volker Vallon, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying Hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced Hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced Hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of Hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

  • thiazide induced Hypocalciuria is accompanied by a decreased expression of ca2 transport proteins in kidney
    Kidney International, 2003
    Co-Authors: Tom Nijenhuis, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced Hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and Hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced Hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

  • genetic renal disorders with hypomagnesemia and Hypocalciuria
    Journal of Nephrology, 2003
    Co-Authors: Nine V.a.m. Knoers, Joke C De Jong, Iwan C Meij, Lambert P Van Den Heuvel, René J. M. Bindels
    Abstract:

    : The combination of hypomagnesemia and Hypocalciuria is the phenotypic signature of two distinct genetic renal tubular transport disorders: Gitelman's syndrome and autosomal dominant isolated renal magnesium wasting. In the past 5 years the genetic defects underlying these disorders have been elucidated through positional candidate cloning approaches. The defective proteins involved in both diseases are located within the distal convoluted tubule (DCT), a segment of the nephron known to play an important role in active magnesium reabsorption in the nephron. The introduction outlines the magnesium handling in the body in general and, in particular, in the kidney, followed by a detailed discussion of Gitelman's syndrome and isolated renal magnesium wasting, including the clinical and biochemical symptoms, genetic aspects and pathophysiology.

Tom Nijenhuis - One of the best experts on this subject based on the ideXlab platform.

  • enhanced passive ca2 reabsorption and reduced mg2 channel abundance explains thiazide induced Hypocalciuria and hypomagnesemia
    Journal of Clinical Investigation, 2005
    Co-Authors: Tom Nijenhuis, Volker Vallon, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying Hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced Hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced Hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of Hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

  • thiazide induced Hypocalciuria is accompanied by a decreased expression of ca2 transport proteins in kidney
    Kidney International, 2003
    Co-Authors: Tom Nijenhuis, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced Hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and Hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced Hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

Hibbard E Williams - One of the best experts on this subject based on the ideXlab platform.

  • is hydrochlorothiazide induced Hypocalciuria due to inhibition of prostaglandin e2 synthesis
    Clinical Science, 1990
    Co-Authors: Lorenzo A Calo, S Cantaro, F Marchini, Giovanni Gambaro, B Baggio, Angela Dangelo, Sandro Giannini, R Castrignano, A Antonello, Hibbard E Williams
    Abstract:

    : 1. Since prostaglandin E2 could play a role in idiopathic hypercalciuria, and considering the well-established hypocalciuric action of hydrochlorothiazide, we have evaluated the effect of 15 days' treatment with hydrochlorothiazide in 10 hypercalciuric male stoneformers on urinary Ca2+ and prostaglandin E2, as well as on plasma bicyclo-prostaglandin E2, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone. 2. In addition to lowering urinary Ca2+ (P less than 0.001), hydrochlorothiazide also promoted a significant fall in urinary prostaglandin E2 (P less than 0.001), plasma bicyclo-prostaglandin E2 (P less than 0.001) and 1,25-dihydroxyvitamin D (P less than 0.01), and an increase in plasma parathyroid hormone (P less than 0.025), whereas plasma 25-hydroxyvitamin D was unchanged. 3. A positive correlation between urinary Ca2+ and prostaglandin E2 was present before (P less than 0.00005), but not after, hydrochlorothiazide. Plasma bicyclo-prostaglandin E2 and plasma 1,25-dihydroxyvitamin D were positively correlated both before (P less than 0.005) and after (P less than 0.005) hydrochlorothiazide, as was also the percentage change in each induced by the drug (P less than 0.05). Furthermore, the changes in plasma 25-hydroxyvitamin D and plasma 1,25-dihydroxyvitamin D after hydrochlorothiazide were negatively correlated (P less than 0.05). 4. It is suggested that a block of prostaglandin E2 synthesis plays a role in the effect of hydrochlorothiazide on Ca2+ metabolism, most probably through an inhibition of 1 alpha-hydroxylase activity.

Johannes Loffing - One of the best experts on this subject based on the ideXlab platform.

  • enhanced passive ca2 reabsorption and reduced mg2 channel abundance explains thiazide induced Hypocalciuria and hypomagnesemia
    Journal of Clinical Investigation, 2005
    Co-Authors: Tom Nijenhuis, Volker Vallon, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying Hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced Hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced Hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of Hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

  • Altered renal distal tubule structure and renal Na(+) and Ca(2+) handling in a mouse model for Gitelman's syndrome.
    Journal of The American Society of Nephrology, 2004
    Co-Authors: Johannes Loffing, Volker Vallon, Joost G J Hoenderop, Dominique Loffing-cueni, Fintan Aregger, Kerstin Richter, Laurence Pietri, May Bloch-faure, Gary E. Shull, Pierre Meneton
    Abstract:

    Gitelman's syndrome, an autosomal recessive renal tubulopathy caused by loss-of-function mutations in the thiazide-sensitive NaCl co-transporter (NCC) of the distal convoluted tubule (DCT), is characterized by mild renal Na(+) wasting, Hypocalciuria, hypomagnesemia, and hypokalemic alkalosis. For gaining further insights into the pathophysiology of Gitelman's syndrome, the impact of NCC ablation on the morphology of the distal tubule, on the distribution and abundance of ion transport proteins along its length, and on renal tubular Na(+) and Ca(2+) handling in a gene-targeted mouse model was studied. NCC-deficient mice had significantly elevated plasma aldosterone levels and exhibited Hypocalciuria, hypomagnesemia, and compensated alkalosis. Immunofluorescent detection of distal tubule marker proteins and ultrastructural analysis revealed that the early DCT, which physiologically lacks epithelial Na(+) (ENaC) and Ca(2+) (TRPV5) channels, was virtually absent in NCC-deficient mice. In contrast, the late DCT seemed intact and retained expression of the apical ENaC and TRPV5 as well as basolateral Na(+)-Ca(2+) exchanger. The connecting tubule exhibited a marked epithelial hypertrophy accompanied by an increased apical abundance of ENaC. Ca(2+) reabsorption seemed unaltered in the distal convolution (i.e., the DCT and connecting tubule) as indicated by real-time reverse transcription-PCR, Western blotting, and immunohistochemistry for TRPV5 and Na(+)-Ca(2+) exchanger and micropuncture experiments. The last experiments further indicated that reduced glomerular filtration and enhanced fractional reabsorption of Na(+) and Ca(2+) upstream and of Na(+) downstream of the DCT provide some compensation for the Na(+) transport defect in the DCT and contribute to the Hypocalciuria. Thus, loss of NCC leads to major structural remodeling of the renal distal tubule that goes along with marked changes in glomerular and tubular function, which may explain some of the clinical features of Gitelman's syndrome.

  • thiazide induced Hypocalciuria is accompanied by a decreased expression of ca2 transport proteins in kidney
    Kidney International, 2003
    Co-Authors: Tom Nijenhuis, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced Hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and Hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced Hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

Joost G J Hoenderop - One of the best experts on this subject based on the ideXlab platform.

  • enhanced passive ca2 reabsorption and reduced mg2 channel abundance explains thiazide induced Hypocalciuria and hypomagnesemia
    Journal of Clinical Investigation, 2005
    Co-Authors: Tom Nijenhuis, Volker Vallon, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying Hypocalciuria and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced Hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced Hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of Hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.

  • Altered renal distal tubule structure and renal Na(+) and Ca(2+) handling in a mouse model for Gitelman's syndrome.
    Journal of The American Society of Nephrology, 2004
    Co-Authors: Johannes Loffing, Volker Vallon, Joost G J Hoenderop, Dominique Loffing-cueni, Fintan Aregger, Kerstin Richter, Laurence Pietri, May Bloch-faure, Gary E. Shull, Pierre Meneton
    Abstract:

    Gitelman's syndrome, an autosomal recessive renal tubulopathy caused by loss-of-function mutations in the thiazide-sensitive NaCl co-transporter (NCC) of the distal convoluted tubule (DCT), is characterized by mild renal Na(+) wasting, Hypocalciuria, hypomagnesemia, and hypokalemic alkalosis. For gaining further insights into the pathophysiology of Gitelman's syndrome, the impact of NCC ablation on the morphology of the distal tubule, on the distribution and abundance of ion transport proteins along its length, and on renal tubular Na(+) and Ca(2+) handling in a gene-targeted mouse model was studied. NCC-deficient mice had significantly elevated plasma aldosterone levels and exhibited Hypocalciuria, hypomagnesemia, and compensated alkalosis. Immunofluorescent detection of distal tubule marker proteins and ultrastructural analysis revealed that the early DCT, which physiologically lacks epithelial Na(+) (ENaC) and Ca(2+) (TRPV5) channels, was virtually absent in NCC-deficient mice. In contrast, the late DCT seemed intact and retained expression of the apical ENaC and TRPV5 as well as basolateral Na(+)-Ca(2+) exchanger. The connecting tubule exhibited a marked epithelial hypertrophy accompanied by an increased apical abundance of ENaC. Ca(2+) reabsorption seemed unaltered in the distal convolution (i.e., the DCT and connecting tubule) as indicated by real-time reverse transcription-PCR, Western blotting, and immunohistochemistry for TRPV5 and Na(+)-Ca(2+) exchanger and micropuncture experiments. The last experiments further indicated that reduced glomerular filtration and enhanced fractional reabsorption of Na(+) and Ca(2+) upstream and of Na(+) downstream of the DCT provide some compensation for the Na(+) transport defect in the DCT and contribute to the Hypocalciuria. Thus, loss of NCC leads to major structural remodeling of the renal distal tubule that goes along with marked changes in glomerular and tubular function, which may explain some of the clinical features of Gitelman's syndrome.

  • thiazide induced Hypocalciuria is accompanied by a decreased expression of ca2 transport proteins in kidney
    Kidney International, 2003
    Co-Authors: Tom Nijenhuis, Annemiete W C M Van Der Kemp, Johannes Loffing, Joost G J Hoenderop, René J. M. Bindels
    Abstract:

    INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced Hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and Hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced Hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.