The hypocalciuric effect of thiazides: subcellular localization of the action

Abstract

The acute administration of thiazides results in a decrease in the urinary Ca²⁺/Na⁺ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca²⁺ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca²⁺ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately.

Preincubation of microdissected distal tubules in hypotonic solution containing 500 μM hydrochlorothiazide (HCTZ) did not influence the Ca²⁺-dependent ATP hydrolysis (Ca²⁺=1 μM) nor the Mg²⁺-dependent ATP hydrolysis (Mg²⁺=100 μM). Similarly 100 μM HCTZ did not change the Ca²⁺ ATPase activity in intact proximal and distal tubule suspensions, at Ca²⁺ concentrations ranging from 0.05 μM to 1 μM.

ATP-dependent Ca²⁺ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 μM HCTZ did not influence this transport. A Na⁺/ Ca²⁺ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 μM HCTZ significantly increased the Ca²⁺ uptake by these membranes. This increase in Ca²⁺ uptake, in the presence of Na⁺, was dose-dependent; the minimal and the maximal effects of the diuretic were observed at concentrations of 25 μM and 100 μM respectively. HCTZ increased the V _maxCa²⁺ from2.5±0.3 pmol μg⁻¹ (10 s⁻¹) to 3.7±0.6 pmol μg⁻¹ (10 s⁻¹) (P<0.01), but did not influence the K _m (1.43±0.25 mM and 1.37±0.1 mM Ca²⁺ in experimental and control membranes, respectively). Na⁺ was necessary for this effect. Na⁺ per se decreased Ca²⁺ uptake in a concentration-dependent manner and HCTZ partially reestablished Ca²⁺ uptake to the levels observed in a Na⁺-free medium. The anion of the Na⁺ salt also modulated the effect of HCTZ on Ca²⁺ transport. While Cl⁻ and SCN⁻ permitted HCTZ to enhance Ca²⁺ uptake, the SO ²⁻₄ anion did not. It is therefore concluded that (a) the hypocalciuric effect of thiazides is primarily due to an increase in the Ca²⁺ uptake of the luminal membrane from the distal tubule, (b) Na⁺ and Ca²⁺ transports are tightly related in the distal luminal membrane, (c) HCTZ modulates this interrelationship by decreasing the inhibitory effect of Na⁺ on Ca²⁺ uptake. Whether the Ca²⁺ and Na⁺ carriers are the same molecule or different entities needs further investigation.