Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney.
BACKGROUND
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.


CONCLUSIONS
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.
Nijenhuis
Tom
T
Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Hoenderop
Joost G J
JG
Loffing
Johannes
J
van der Kemp
Annemiete W C M
AW
van Os
Carel H
CH
Bindels
René J M
RJ
D000818:Animals; D002118:Calcium / Q000652:urine*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D004232:Diuretics; D004573:Electrolytes / Q000097:blood / Q000652:urine; D006852:Hydrochlorothiazide / Q000494:pharmacology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000652:urine; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology*; D049992:Sodium Chloride Symporters; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D050916:TRPV Cation Channels
D015220:Calcium Channels; D004232:Diuretics; D004573:Electrolytes; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; D019831:Sodium-Calcium Exchanger; D027981:Symporters; D050916:TRPV Cation Channels; C492241:TRPV5 protein, rat; C110374:sodium-calcium exchanger 1; D006852:Hydrochlorothiazide; D012964:Sodium; D002118:Calcium