Action of Thiazide on Renal Interstitial Calcium

Abstract

Background

Although thiazides increase urinary sodium excretion, they also decrease urinary calcium excretion. Recent studies in our laboratory have shown that increased dietary salt significantly reduces interstitial fluid calcium in Dahl salt-sensitive (DS) rats, and this was associated with a rise in blood pressure and increased urinary calcium excretion. Owing to the vasorelaxant actions of increased extracellular fluid calcium, we reasoned that the antihypertensive action of hydrochlorothiazide (HCTZ), a commonly used thiazide, may be the result of increased interstitial fluid calcium as a consequence of decreased urinary calcium excretion.

Methods

To test this hypothesis, DS and Dahl salt-resistant (DR) rats were given high salt alone or in combination with HCTZ for 1 week. Renal cortical interstitial fluid calcium was determined by the zero net flux method.

Results

High salt decreased cortical interstitial fluid calcium (1.69 ± 0.25 vs. 1.13 ± 0.05 mmol/l; P < 0.05) in DS rats as previously reported; thiazide treatment had no effect on the high salt interstitial fluid calcium response in salt-sensitive animals. However, thiazide decreased interstitial fluid calcium in DS on a normal salt diet. Cortical interstitial fluid calcium was unchanged by dietary salt in DR rats, and thiazide did not alter this interstitial fluid calcium response.

Conclusion

We interpret these data to mean that (i) short-term thiazide treatment does not reduce blood pressure by restoring renal cortical interstitial fluid calcium concentration and (ii) a decrease in renal cortical interstitial fluid calcium may not contribute to the increased renal vasoconstriction seen in salt-sensitivity.

Our investigation of the calciuretic action of salt loading¹ reveals that Dahl salt-sensitive (DS) rats have higher basal renal cortical interstitial calcium (ISF_Ca²⁺) compared to Dahl salt-resistant (DR) rats and that a high salt diet decreases renal cortical ISF_Ca²⁺ in DS rats but not DR rats. The decrease in renal cortical ISF_Ca²⁺ was associated with an increase in urinary calcium excretion and blood pressure rise following the high salt diet. When coupled with the vasodilatory action of increased extracellular calcium,^2,3 these findings suggest that a high salt diet may modulate blood pressure secondary to a disruption in a calcium-dependent vasodilatory effect normally occurring in the cortical region of the kidney.

This study continues our investigation of the ISF_Ca²⁺ and calciuretic effect of increased dietary salt. Thiazides have been shown to be quite effective in lowering blood pressure, particularly within the African-American population.⁴ Although the effectiveness of diuretics is often ascribed to blood volume reduction, it is known that the subacute and long-term action of thiazides may not be subsequent to fluid loss but, rather, vasodilation.⁵ The mechanism of this vasodilatory action is unknown.

In light of the potential for alterations in extracellular calcium modulation of vascular resistance,^2,3 the excretion of calcium may be extremely important in the pathogenesis of hypertension development. Of interest is the finding that thiazides, in addition to increasing urinary sodium loss, significantly reduce urinary calcium excretion.^6,7 Therefore, this study was designed to determine whether thiazide treatment altered renal ISF_Ca²⁺ in salt-sensitive hypertension. To accomplish this, we measured renal cortical ISF_Ca²⁺ in DS and DR rats following 1 week of treatment with hydrochlorothiazide (HCTZ) while on control or high salt diet. We report that short-term treatment with HCTZ had no effect on renal cortical ISF_Ca²⁺ in DS rats on a high salt diet; however, HCTZ significantly reduced cortical ISF_Ca²⁺ in DR rats on a normal salt diet. These findings suggest that the short-term antihypertensive action of HCTZ is not due to increases in ISF_Ca²⁺.

METHODS

Animals and treatments (study protocol)

Male DS (n = 32) and DR (n = 30) outbred (John Rapp) rats (6–8 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN) or Charles River Laboratories (Wilmington, MA). All animals were housed in separate ventilated cages (Animal Care Systems, Littleton, CO) maintained at constant temperature and humidity. During acclimation animals were maintained on regular chow (Harlan Teklad Laboratories, Madison, WI) and water for a period of 7–10 days. Following acclimation, the animals were placed in one of four treatment groups: regular chow (0.45% NaCl) plus vehicle (0.002% ethanol), regular chow plus HCTZ (15 mg/kg/day), high salt (8% NaCl) plus vehicle, and high salt plus HCTZ. HCTZ stock was prepared by dissolving HCTZ in ethanol and constant stirring overnight on a heated stir plate. The previous day’s water consumption was used for estimating the HCTZ dosing. The Institutional Animal Care and Use Committee of North Carolina Central University approved all protocols used.

Blood pressure measurement

Systolic blood pressure (SBP) was measured by tail-cuff plethysmography (Visitech Systems BP 2000; Apex, NC). Rats were placed in holding units maintained at 37 °C. The animals were trained by placing them into the holding units twice for a total period of 30 min before taking an initial blood pressure. A basal blood pressure was obtained over the period of a week; blood pressure measurements were performed again on the last day of the diet to determine post-diet blood pressure.

In situ microdialysis

Rats were anesthetized using 4% isoflurane (Abbott Laboratories, Chicago, IL) and 70% nitrogen and 30% oxygen gas mix (Linden Gas, Morrisville, NC) (0.5 ml/min) for the initial induction of anesthesia and maintained with 2% isoflurane (0.5 ml/min). Once the animals were completely anesthetized, an incision was made from the lower abdominal region to approximately an inch below the xiphoid process to expose the kidney. Once the left kidney was exposed, a 5-mm linear microdialysis probe (Bioanalytical Systems, West Lafayette, IN) was inserted into the cortical region of the left kidney and set in place using veterinary bonding glue (3M Animal Care Products, St Paul, MN). The microdialysis probe was then perfused with 120 mmol/l NaCl in 20 mmol/l HEPES buffer at a rate of 1 μl/min for a 90-min equilibration period. After the equilibration period, the probe was then perfused at a rate of 1 μl/min with perfusate containing 0 mmol/l Ca²⁺ for 35 min, followed by a 15-min collection period. This was repeated for each of the remaining increasing concentrations (0.5, 1.0, 1.5, 2.0, 3.0 mmol/l) of calcium, i.e., a 35 min acclimation period followed by a 15-min collection period for a total of six collected dialysate samples. Each sample was collected on ice and stored at 4 °C for later analysis. Probe placement verification was done following killing of the animal by sectioning the kidney to visualize the probe. Only those animals with correct probe placement were used for data analysis.

Zero net flux analysis was used to determine renal interstitial fluid calcium concentration. Zero net flux is determined by plotting the difference between the calcium concentration of the dialysate and perfusate (y-axis) vs. the calcium concentration of the perfusate (x-axis). Linear regression analysis was used to determine the best curve or line fit. From this curve, the concentration at which the difference between perfusate and dialysate is zero is the concentration of calcium in the interstitial fluid.

Urine collection

Overnight urine was collected at baseline and on the evening of the seventh day of the diet. For each collection, animals were placed in metabolic cages with free access to water (for baseline collection) or water containing vehicle or HCTZ (for post-treatment collection) overnight for a period of 10 h. Animals were then returned to their cages, and urine samples were stored at 4 °C for analysis later.

Urine calcium measurements

Urinary and in situ microdialysis collections for net zero flux determination calcium concentrations were measured using a colorimetric assay (Calcium Assay kit; Diagnostic Chemical, Oxford, CT).

Statistical analysis

Values are expressed as mean ± s.e.m. Within-strain diet affects were assessed by t-test or paired t-test analysis. Diet and HCTZ effects within each strain were analyzed using one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison test. The statistical software used was Sigma Stat 3.5 (SPSS; Chicago, IL).

RESULTS

Blood pressure

The blood pressure response to high salt alone or in combination with HCTZ is shown in Table 1. One week of 8% NaCl increased SBP in DS rats but not DR rats as expected (162 ± 6 vs. 128 ± 2 mm Hg, P < 0.01, DS; 132 ± 3 vs. 139 ± 2 mm Hg, P = nonsignificant, DR). The rise in SBP in DS rats was not associated with a greater gain in body weight compared to DR rats; in the setting of the 8% salt diet, both strains had an increase in body weight of ~36 g (Table 1). HCTZ prevented the rise in blood pressure in DS rats following a high salt diet (Table 1; 130 ± 4 vs. 162 ± 6 mm Hg, P < 0.01) but had no effect on SBP in DS rats on control salt diet. There was no effect of high salt or HCTZ on blood pressure in the DR rats (Table 1).

Table 1.

Changes in systolic blood pressure and body weight following dietary salt and HCT Z interventions

Strain	Treatment	Time point	Systolic blood pressure (mm Hg)	BW (g)
DS	0.45% NaCl (n = 7)	Baseline	129 ± 2	196 ± 10
		After	125 ± 3	282 ± 16
	8% NaCl (n = 6)	Baseline	129 ± 2	236 ± 10
		After	162 ± 6*	272 ± 8
	0.45% NaCl + HCTZ (n = 8)	Baseline	128 ± 3	201 ± 12
		After	131 ± 4	269 ± 13
	8% NaCl + HCTZ (n = 10)	Baseline	128 ± 2	245 ± 9
		After	130 ± 4†	270 ± 8
DR	0.45% NaCl (n = 7)	Baseline	132 ± 1	197 ± 10
		After	132 ± 3	258 ± 18
	8% NaCl (n = 11)	Baseline	136 ± 2	207 ± 7
		After	139 ± 2	243 ± 10
	0.45% NaCl + HCTZ (n = 5)	Baseline	131 ± 1	202 ± 5
		After	136 ± 5	209 ± 11
	8% NaCl + HCTZ (n = 7)	Baseline	134 ± 2	233 ± 9
		After	134 ± 5	260 ± 5

Values represent mean ± s.e.

DR, Dahl salt-resistant; DS, Dahl salt-sensitive.

^*P < 0.01 vs. baseline;

^†P < 0.01 vs. 8% without hydrochlorothiazide (HCTZ).

Renal cortical interstitial fluid calcium

Figure 1 depicts the net zero calcium plots for DS rats on control (0.45%) and high (8%) salt diets without HCTZ (Figure 1a) and DS rats on control salt diet with and without HCTZ (Figure 1b). As previously reported, high salt caused a significant decrease in renal cortical in DS rats (Figures 1a and 2a; 1.69 ± 0.2 vs. 1.13 ± 0.05 mmol/l Ca²⁺; n = 5 and n = 6, P < 0.05, one-way ANOVA, Student–Newman–Keuls). Interestingly, HCTZ caused a significant decrease in cortical ISF_Ca²⁺ in DS rats on a control salt (0.45%) diet (Figures 1b and 2a; P < 0.05, one-way ANOVA). Specifically, ISF_Ca²⁺ in DS rats on a normal salt diet was 1.69 ± 0.2 without HCTZ and 1.23 ± 0.13 with HCTZ treatment (P < 0.05; one-way ANOVA and Newman–Keuls). However, HCTZ did not alter the high salt (8%) diet affect on cortical ISF_Ca²⁺ (Figure 2a). Unlike what was observed with DS rats, renal cortical ISF_Ca²⁺ was not affected by increased dietary salt in DR rats (Figure 2b), with DR cortical ISF_Ca²⁺ values following 0.45 and 8% NaCl diets being of 1.41 ± 0.2 and 1.16 ± 0.1 mmol/l, respectively (one-way ANOVA, P = nonsignificant); HCTZ treatment did not significantly alter cortical ISF_Ca²⁺ in DR rats on either control or high salt diets (one-way ANOVA, P = nonsignificant).

Figure 1.

Effect of salt on renal cortical interstitial fluid calcium (ISF_Ca²⁺). (a) Net calcium flux with increasing perfusate calcium concentration in Dahl salt-sensitive (DS) rats on control (open circles; 0.45% NaCl) or high salt (filled circles; 8% NaCl) diet in the absence of hydrochlorothiazide (HCTZ) and (b) ISF_Ca²⁺ in DS rats on control (0.45% NaCl) salt diet with (filled circles) and without (open circles) HCTZ.

Figure 2.

Renal cortical interstitial fluid calcium (ISF_Ca²⁺) in (a) Dahl salt-sensitive (DS) and (b) salt-resistant (DR) rats on control (0.45% NaCl) salt diet (dark gray bars) or high (8% NaCl) salt diet (light gray bars) without or with hydrochlorothiazide (HCTZ; 15 mg/kg/day). Numbers within parentheses represent number of animals. *P < 0.05 vs. 0.45% —HCTZ; one-way analysis of variance with Student–Newman–Keuls multiple comparison analysis.

Urinary excretion

Figure 3 shows the change in urinary sodium excretion in DS and DR rats on a high salt diet with and without HCTZ. There was a tendency for DS rats to excrete less sodium compared to DR rats following a high salt diet (from a baseline of 0.6 ± 0.3 to 1.2 ± 0.3 in DS vs. 0.4 ± 0.07 to 1.6 ± 0.3 μEq/10 h in DR). The change in U_NaV following a high salt diet was 1.39 ± 0.3 and 1.23 ± 0.4 mEq/10 h for DS and DR rats, respectively, with HCTZ treatment (Figure 3) compared to 0.6 ± 0.3 and 1.24 ± 0.3 mEq/10 h in DS and DR rats without HCTZ, respectively. There was no significant effect of HCTZ on the change in urinary sodium excretion in either strain (one-way ANOVA and t-test).

Figure 3.

Change in urinary sodium excretion in Dahl salt-sensitive (DS) and salt-resistant (DR) rats following 1 week on a high (8%) salt diet with and without hydrochlorothiazide (HCTZ). Number of animals in each group is shown in parentheses. Numbers within parentheses represent number of animals. There was no statistical difference among the treatment groups (one-way analysis of variance and t-test).

Figure 4a displays the ratio of urinary sodium excretion and calcium excretion in DS rats on a high salt diet with and without HCTZ treatment. In DS rats the U_NaV/U_CaV ratio was unchanged after 1 week on 8% NaCl diet without HCTZ (2.07 ± 1.2 before the diet and 2.99 ± 1.27 after the diet, P = nonsignificant paired t-test). In DS given 8% NaCl and HCTZ, the U_NaV/U_CaV ratio increased from 1.45 ± 0.58 to 11.9 ± 4.67, P = 0.05, paired t-test. This increase in the U_NaV/U_CaV ratio is due to an increase in urinary sodium excretion (0.38 ± 0.05 baseline vs. 1.78 ± 0.37 mEq/10 h after 8% NaCl plus HCTZ, P < 0.02 paired t-test; Figure 3) with a concomitant decrease in urinary calcium excretion (8.9 ± 3.1 at baseline vs. 4.0 ± 1.7 μmol/10 h after 8% NaCl plus HCTZ; P = 0.11). Interestingly, urinary calcium excretion was positively correlated with SBP in DS rats on high salt diet with and without HCTZ (Figure 4b; P < 0.01). Urinary calcium excretion was not significantly decreased by HCTZ in DR rats on a high salt diet (2.5 ± 0.07 vs. 4.4 ± 1.1 mmoles/10 h, baseline vs. HCTZ, respectively; P = 0.158).

Figure 4.

Ratio of urinary sodium excretion and calcium excretion (a; *P = 0.05, paired t-test) and correlation of urinary calcium excretion to change in systolic blood pressure (b; linear regression analysis) in Dahl salt-sensitive rats on a high (8%) salt diet without hydrochlorothiazide (−HCTZ) or with HCTZ (+HCTZ). Numbers within parentheses represent number of animals.

DISCUSION

We report the novel findings of decreased renal cortical interstitial fluid calcium ISF_Ca²⁺ following HCTZ treatment in DS rats on control salt diet but not high salt diet; although HCTZ prevented the rise in blood pressure in DS rats following a high salt diet, it did not prevent the fall in renal cortical ISF_Ca²⁺ following increased dietary salt in DS rats, despite a reduction in urinary calcium excretion. These data suggest that an antihypertensive dose of thiazide modulates ISF_Ca²⁺ in control normotensive conditions but not in acute salt-induced hypertension. Furthermore, modulation of renal ISF_Ca²⁺ does not appear to account for the blood pressure–lowering effects of acute thiazide treatment.

We previously reported that increased dietary salt caused a decrease in renal cortical ISF_Ca²⁺ in DS rats, and this was associated with a rise in SBP.¹ Therefore, the current study was designed to determine whether the fall in renal cortical ISF_Ca²⁺ in DS rats following a high salt diet accounted for the rise in blood pressure. We used HCTZ to address this for several reasons. First, previous reports have shown that HCTZ reduces urinary calcium excretion while lowering blood pressure^6–8 and that the antihypertensive action of HCTZ includes a vasodilatory component.⁵ Second, studies have shown that increased extracellular or interstitial calcium causes vessel relaxation and this in turn could lead to a decrease in blood pressure.^2,9 Therefore, we expected that as HCTZ reduced urinary calcium excretion, cortical ISF_Ca²⁺ would remain unaltered in DS rats during a high salt diet. However, this was not the case; despite normalization of blood pressure and decreased urinary calcium excretion with HCTZ, cortical ISF_Ca²⁺ decreased as previously observed in DS rats. These results suggest that the antihypertensive action of HCTZ does not involve modulation of renal cortical extracellular calcium and its subsequent vasodilatory action. This lack of blood pressure and ISF_Ca²⁺ association occurs despite a positive correlation between urinary calcium excretion and blood pressure.

It has been shown that the antihypertensive action of long-term thiazide treatment is due more to a vasodilation component than to volume contraction.⁵ In this study, HCTZ was administered for only 1 week; therefore, it may require >1 week before a measurable change in cortical interstitial fluid calcium and its accompanying vasodilation will be observed. Thus, the blood pressure–lowering effect of the 1-week HCTZ regimen appears to be independent of interstitial calcium changes, suggesting that the antihypertensive actions are due to volume changes. However, it is important to note that in this study, as in other studies, thiazide did not alter urinary sodium excretion in DS rats following a high salt diet (Figure 3). Specifically, DS rats on a high salt diet tended to excrete less sodium compared to DR rats, and HCTZ did not alter this difference between DS and DR rats. Therefore, urinary sodium excretion in DS rats remained less than that of DR rats even in the presence of HCTZ. This failure to increase sodium excretion with HCTZ treatment is consistent with other reports that demonstrate that HCTZ decreases sodium excretion.¹⁰ Perhaps an increase in sodium excretion occurred prior to day seven of the treatment as has been observed by others.¹⁰ Interestingly, Nijenhuis and associates found that during the first 6 h following HCTZ urinary calcium was unchanged, while urinary sodium excretion was increased. However, the increased sodium excretion is short-lived, decreasing during subsequent hours. Indeed, hypocalciuria did not occur until sodium excretion decreased during the subsequent 12–24 h.^10–12 It is of particular interest to note that Titze and associates have proposed that sodium retention is a compensatory mechanism to minimize calcium loss.¹³ Although we did not perform sodium metabolic balance in this study, the failure to observe an increase in urinary sodium excretion with a concomitant decrease in calcium excretion is consistent with what others have reported with HCTZ. More importantly, we demonstrated that urinary calcium excretion is directly related to SBP in treated and untreated DS rats (Figure 4). These findings would suggest that the acute antihypertensive action of HCTZ might involve prevention of calcium loss. More studies are needed to determine whether chronic HCTZ and long-term calcium retention will ultimately result in increases in extracellular calcium concentration.

The question that remains to be answered is what is the relationship between ISF and blood pressure, if any. The fall in ISF_Ca²⁺ following a high salt diet in DS rats may be an important compensatory response to salt loading. Previous studies suggest that redistribution of renal blood flow occurs following increased salt loading. Cortical blood flow increases or is autoregulated with chronic saline infusion in Sprague–Dawley and DR control animals, respectively.¹⁴ In contrast, cortical blood flow decreases in response to volume loading in DS rats.¹⁵ The change in cortical blood flow seen in non–salt-sensitive animals may be due to an influx of extracellular calcium to intracellular compartments via sodium hydrogen (Na⁺/H⁺) exchanger activation.¹⁰ Perhaps in salt-sensitive animals, the mechanisms involved in calcium mobilizations are enhanced in an attempt to activate vasoactive agents known to be impaired in salt-sensitivity.^16–18 Increased intracellular calcium may be required to enhance the actions of calcium-dependent vasoactive agents.¹⁹ More studies are required to determine the primary initiating agent or agents responsible for the alterations in ion transporters that ultimately lead to alterations in extracellular calcium.

However, HCTZ significantly decreased cortical ISF_Ca²⁺ in DS during control dietary salt intake. It is unclear how and why HCTZ alters ISF_Ca²⁺ under control normotensive conditions. Of interest is the finding that HCTZ decreases sodium chloride cotransporter, subsequently, causing an increase in the sodium calcium exchanger.^20,21 Consequently, an increase in the sodium calcium exchanger would lead to a decrease in extracellular calcium.^20,21 Investigators have demonstrated passive calcium reabsorption in proximal tubule following HCTZ as a result of increased Na⁺/H⁺/exchanger.²¹ Therefore, the effects of HCTZ in DS rats on control salt diet may be the result of HCTZ modulation of ion transporters.

In summary, renal cortical ISF_Ca²⁺ decreases in DS rats on a high salt diet Although an antihypertensive dose of HCTZ decreases urinary calcium excretion, the antihypertensive dose did not prevent the fall in ISF_Ca²⁺ seen in this rat model of hypertension. However, the failure of HCTZ to prevent the fall in renal cortical extracellular calcium may underlie the importance of this compensatory response to salt loading in salt-sensitive hypertension. Indeed, acute actions of HCTZ may facilitate the movement of calcium from the extracellular compartment to the intracellular compartment. However, more studies are needed to determine whether chronic HCTZ may increase extracellular calcium as a result of its hypocalciuric actions.