Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia

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.

Figure 1

Effect of chronic HCTZ treatment on renal mRNA expression levels of Ca²⁺ and Na⁺ transport proteins in Trpv5^+/+ and Trpv5^–/– mice. Renal mRNA expression levels of the epithelial Ca²⁺ channel Trpv5 (A), calbindin-D_28K (B), NCX1 (C), and NKCC2 (D) were determined by real-time quantitative PCR analysis and are depicted as the ratio to hypoxanthine-guanine phosphoribosyl transferase (HPRT). Black bars, vehicle-treated; white bars, HCTZ-treated, 25 mg/kg/d during 6 days. n = 9 animals per treatment group. Data are presented as mean ± SEM. ^#P < 0.05 versus vehicle-treated Trpv5^+/+.

Figure 2

Effect of chronic HCTZ treatment on renal protein abundance of Ca²⁺ and Na⁺ transport proteins in Trpv5^+/+ and Trpv5^–/– mice. Protein abundance was determined by computerized analysis of immunohistochemical images and is presented as integrated optical density (IOD; AU) for the epithelial Ca²⁺ channel Trpv5 (A), calbindin-D_28K (B), NCC (C), and NHE3 (D). Black bars, vehicle-treated (CTR); white bars, HCTZ-treated, 25 mg/kg/d during 6 days; +/+ CTR and +/+ HCTZ, vehicle- and HCTZ-treated Trpv5^+/+ mice, respectively; –/– CTR and –/– HCTZ, vehicle- and HCTZ-treated Trpv5^–/– mice, respectively. n = 9 animals per treatment group. Data are presented as mean ± SEM. *P < 0.05 versus respective vehicle-treated controls; ^#P < 0.05 versus vehicle-treated Trpv5^+/+.

Figure 3

Effect of chronic HCTZ treatment on renal mRNA expression and protein abundance of the epithelial Mg²⁺ channel Trpm6. (A) Renal mRNA expression levels of the epithelial Mg²⁺ channel Trpm6 were determined by real-time quantitative PCR analysis and are depicted as the ratio to HPRT. (B) Trpm6 protein abundance was determined by computerized analysis of immunohistochemical images and is presented as integrated optical density (AU). HCTZ, 25 mg/kg/d HCTZ for 6 days. n = 9 per treatment group. Data are presented as mean ± SEM. *P < 0.05 versus vehicle-treated controls.

Figure 4

Renal mRNA expression and protein abundance of the epithelial Mg²⁺ channel Trpm6 in NCC^+/+ and NCC^–/– mice. (A) Renal mRNA expression levels of the epithelial Mg²⁺ channel Trpm6 were determined by real-time quantitative PCR analysis and are depicted as the ratio to HPRT. (B) Trpm6 protein abundance was determined by computerized analysis of immunohistochemical images and is presented as integrated optical density (AU). n = 6 animals per genotype. Data are presented as mean ± SEM. *P < 0.05 versus NCC^+/+ mice.

Figure 5

Time-dependent effects of HCTZ on urinary Na⁺ and Ca²⁺ excretion in Trpv5^+/+ and Trpv5^–/– mice. Trpv5^+/+ and Trpv5^–/– mice were housed in metabolic cages, and urine was sampled 6, 12, and 24 hours after the intraperitoneal administration of vehicle or 25 mg/kg HCTZ. Arrowhead indicates administration of a single dose of 25 mg/kg HCTZ. Filled squares, vehicle-treated Trpv5^+/+; open squares, HCTZ-treated Trpv5^+/+; filled triangles, vehicle-treated Trpv5^–/–; open triangles, HCTZ-treated Trpv5^–/–. n = 9 animals per treatment group. Data are presented as mean ± SEM; absolute values were normalized to respective vehicle-treated controls. *P < 0.05 versus vehicle-treated Trpv5^+/+ mice. ^#P < 0.05 versus vehicle-treated Trpv5^–/– mice.

Figure 6

Effect of chronic HCTZ treatment in mice on Ca²⁺ transport along the single nephron assessed by in vivo free-flow micropuncture. (A) Fractional reabsorption of Na⁺, fluid, and Ca²⁺ up to the last surface loop of the proximal tubule. (B) Relation between K⁺ concentration in tubular fluid of distal convolution and fractional Ca²⁺ delivery to these sites. Low K⁺ concentrations indicate early puncture sites, and high K⁺ concentrations indicate late puncture sites of distal convolution. Black bars and filled circles, vehicle-treated controls; white bars and open circles, HCTZ, 25 mg/kg/d during 6 days. n = 10 nephrons for distal collections and n = 17–21 nephrons for proximal collections in 5–6 mice per treatment group. Data are presented as mean ± SEM. *P < 0.05 versus vehicle-treated controls.