Lack of thiazide diuretic inhibition of agonist constriction of mouse mesenteric arterioles ex vivo

Abstract

The chronic reduction of arterial blood pressure by thiazide diuretics (TZD) in hypertensive patients is mediated through an extra-renal mechanism. It is widely held that this extra-renal mechanism is a direct TZD inhibition of vasoconstriction. This study tested whether the TZD, hydrochlorothiazide (HCTZ), inhibited agonist constriction of mesenteric arterioles ex vivo. Mice deficient in the kidney distal convoluted tubule Na⁺/Cl⁻ cotransporter (NCC), i.e., the target of thiazide inhibition–mediated diuresis, and wild type (WT), were subjected to Na⁺-restricted diet. Mesenteric arterioles from NCC knockout and WT mice were then isolated, placed under constant pressure, and the inhibitory effects of HCTZ (100 μM) on phenylephrine constriction determined. HCTZ did not inhibit phenylephrine constriction of arterioles from NCC knockout and wild type (WT) mice subjected to Na⁺-restricted diet. This study suggests that future investigations to identify the extra-renal site of chronic TZD treatment should (1) focus on indirect inhibition of vascular constriction and (2) be determined under clinically relevant conditions. These conditions include chronic TZD at relevant concentrations in hypertensive animals.

Introduction

The known target of the commonly used antihypertensive agents, the thiazide diuretics (TZD), is the kidney distal convoluted tubule Na⁺/Cl⁻ cotransporter (NCC; Hughes 2004; Duarte and Cooper-DeHoff 2010; Pourafshar et al. 2018). Inhibition of Na⁺/Cl⁻ cotransporter (NCC) by TZD results in diuresis and consequent decreased plasma volume, thereby lowering arterial blood pressure (Tobian and Coffee 1964; Shah et al. 1978; Hughes 2004; Ellison and Loffing 2009; Duarte and Cooper-DeHoff 2010; Shahin and Johnson 2016; Pourafshar et al. 2018).

However, the therapeutic efficacy of chronic TZD treatment is actually independent of diuresis and decreased plasma volume (Hughes 2004; Ellison and Loffing 2009; Duarte and Cooper-DeHoff 2010; Shahin and Johnson 2016; Pourafshar et al. 2018). Further, it is widely considered that the clinically relevant lowering of arterial pressure by TZD is mediated through an extra-renal action (Hughes 2004; Duarte and Cooper-DeHoff 2010; Pourafshar et al. 2018). Although this extra-renal target remains unidentified, there is evidence that TZD directly inhibit vasoconstriction (Hughes 2004; Ellison and Loffing 2009; Duarte and Cooper-DeHoff 2010; Shahin and Johnson 2016; Pourafshar et al. 2018; Table 1).

Table 1.

Effect of thiazide diuretics on vascular constriction ex vivo

Species	Vessel	Method	Inhibition	Reference
Guinea pig	Mesenteric artery	Isometric	+	Calder etal. 1992, 1993, 1994
	Mesenteric artery	Isometric	+	Pickkers et al. 1999
	Mesenteric artery	Isometric	+	Pickkers and Hughes 1995
Human	Subcutaneous artery	Isometric	+	Calder etal. 1992
	Mesenteric artery	Isometric	+	Colas et al. 2001
hypertensive	Mesenteric artery	Isometric	+	Colas et al. 2001
Mouse	Aorta	Isometric	−	Alshahrani etal. 2017
	Mesenteric arterioles	Pressurized	−	Rapoport et al. 2018
Rabbit	Aorta	Isometric	−	Daniel and Nash 1965
Rat	Aorta	Isometric	+/−a	Abrahams etal. 1996, 1998
	Aorta	Isometric	−	Colas et al. 2000b
	Aorta	Isometric	+	Zhu et al. 2005
	Femoral artery	Isometric	+	Sladkova et al. 2007
	Mesenteric artery	Isometric	+/−a	Abrahams etal. 1996
	Portal vein	Isometric	−	Abrahams etal. 1996
	Pulmonary artery	Isometric	+/−a	Abrahams etal. 1996
spontaneously hypertensive	Aorta	Isometric	+	Colas et al. 2000a, b, c

^aInhibition requires plasma or albumin; +, inhibition; −, no effect

While our recent finding that HCTZ decreased arterial pressure in NCC knockout (KO) mice subjected to Na⁺-restricted diet supports the presence of a non-renal TZD target, HCTZ failed to inhibit agonist constriction of aorta from NCC KO mice subjected to Na⁺-restricted diet (Alshahrani et al. 2017; Table 1). Although these findings suggest that the extra-renal TZD target is not the vasculature, the possibility remains that, in contrast to a conduit vessel such as aorta (Alshahrani et al. 2017; Table 1), TZD inhibit constriction in a resistance type vessel.

Indeed, demonstrations of TZD inhibition of constriction of isolated vessels are limited because only non-resistance-type vessels were used (Table 1). Additionally, the methodology utilized to investigate the inhibitory effects of the TZD on constriction, i.e., isometric constriction, is less physiologic than, e.g., vessel diameter measurements in pressurized vessels (Table 1). We tested, therefore, whether HCTZ inhibited constriction to the α₁ adrenergic receptor agonist, phenylephrine, in second-order branches under constant pressure, from mesenteric arterial bed of NCC KO and wild type (WT) mice subjected to Na⁺-restricted diet.

Methods and materials

General

Wild type (WT, n = 8) and NCC KO (n = 8) mice (C57BL6, male and female; Soleimani et al. 2012) were subjected to0.55% Na⁺ diet for 20.3 ± 0.6 days. Secondary branches of the mesenteric vascular bed were then excised and placed in an ex vivo perfusion apparatus at 60 mmHg for diameter measurements (Nevitt et al. 2016).

Physiologic characteristics of WT and NCC KO mice/mesenteric arteriole

1. Age (days): WT 168.6 ± 8.8 (8), KO 150.5 ± 3.3 (8);

2. Weight (g): WT 27.9 ± 1.5 (8), KO 38.4 ± 2.7 (8);

3. Weight gain on low Na⁺ diet day 14 compared to day 0 (%): WT 3.2 ± 1.5 (6), KO 7.7 ± 1.7 (6), p = .0521;

4. Basal diameter mesenteric arteriole (μm): WT + DMSO (vehicle) 154.7 ± 10.2 (7), WT + HCTZ (100 μM) 155.1 ± 10.1 (7), NCC KO + DMSO 136.1 ± 10.7 (7), NCC KO + HCTZ 134.3 ± 10.1 (7);

5. Basal width mesenteric arteriole (μm): WT 25.6 ± 1.8 (8), NCC KO 28.4 ± 1.0 (8)

Constriction

Mesenteric arterioles were initially tested for their ability to constrict and the presence of functional endothelium through respective challenge with phenylephrine followed by acetylcholine. Acetylcholine (10 μM) relaxed the phenylephrine constriction of arteriolar segments from WT and NCC KO subjected to Na⁺-restricted diet by 90.9 ± 3.6% (5) and 70.4 ± 12.8% (8), respectively (p = 0.21). Cumulative phenylephrine concentrations were then added to mesenteric arterioles exposed to DMSO (vehicle) and, after wash, 100 μM HCTZ added followed by phenylephrine. Amount of myogenic tone of WT and NCC KO arterioles from mice subjected to Na⁺-restricted diet, determined by exposure to Ca²⁺-free solution and supramaximal sodium nitroprusside concentration, was not significantly different (5.5% ± 0.9% (6) and 6.2% ± 1.3(7) of basal diameter, respectively).

Histology/immunohistochemistry

Mesenteric arterioles were placed in 10% formalin and histology (hematoxylin-eosin, Verhoeff-Van Gieson, and Masson’s trichrome staining) and immunohistochemistry (smooth muscle actin) were performed according to standard procedures. Evaluation included blinded observer.

Calculations and statistical analysis

Acetylcholine relaxation, myogenic tone, and phenylephrine constriction were calculated as percent relaxation of phenylephrine constriction, percent relaxation of basal tone, and percent basal tone, respectively. Differences between two means and concentration-constriction curves were determined with unpaired, two-tailed t test and two-way, repeated measures ANOVA, respectively. Statistical significance was accepted at p ≤ 0.05. Shown are mean ± SE (n), where n represents the number of mice.

Materials

HCTZ, phenylephrine, acetylcholine, and sodium nitroprus-side were from Sigma-Aldrich and 0.55% Na⁺ diet from PMI Nutrition International. DMSO served as vehicle for HCTZ.

Results

Constriction

HCTZ (100 μM) did not cause a rightwards shift of the phenylephrine concentration-constriction curve of secondary branches of the mesenteric vascular bed from WT and NCC KO mice subjected to Na⁺-restricted diet (Fig. 1). Phenylephrine concentration-constriction curves did not differ in arteriolar segments from WT and NCC KO mice subjected to Na⁺-restricted diet (Fig. 1).

Fig. 1.

Effect of HCTZ on phenylephrine cumulative concentration-constriction curv6es of pressurized mouse mesenteric arterioles in vitro. WT (circles) and NCC KO mice (squares) were subjected to restricted Na⁺ diet. Mesenteric arterioles were then removed, pressurized, and exposed to DMSO (vehicle) followed by cumulative phenylephrine concentrations (closed symbols). Following phenylephrine washout, 100 μM HCTZ and then phenylephrine (open symbols) were added. WT: n = 6 and 3 at ≤ 1 μM and 3 μM phenylephrine, respectively; NCC KO: n = 6. The lower n with 3 μM phenylephrine was due to absence of 3 μM phenylephrine challenge, exclusion of an outlier (≈ two standard deviations from the mean) and when 3 μM phenylephrine failed or minimally constricted the unmaintained constriction to 1 μM phenylephrine

Histology/immunohistochemistry

Comparison of hematoxylin-eosin, Verhoeff-Van Gieson, and Masson’s trichrome staining, as well as immunohistochemistry of smooth muscle actin, of mesenteric arterioles from WT and NCC KO subjected to low Na⁺ diet did not reveal any differences (data not shown).

Discussion

The major finding of this study is that HCTZ, even at the high concentration of 100 μM, did not inhibit phenylephrine constriction in mouse pressurized arterioles from NCC KO, as well as WT mice subjected to Na⁺-restricted diet. These findings are consistent with the lack of effect of HCTZ inhibition of agonist constriction (isometric) of aorta from NCC KO and WT mice subjected to Na⁺-restricted diet (Alshahrani et al. 2017; Table 1). Also, structural changes were not observed in mesenteric arterioles, consistent with lack of structural changes in aorta from these mice (Alshahrani et al. 2017).

The inability of HCTZ to directly inhibit agonist constriction of isolated mesenteric arterioles contrasts with numerous findings of TZD, including HCTZ, inhibition of vasoconstriction of isolated vessels (Table 1). While an explanation for these contrasting findings is not entirely clear, demonstrations of TZD inhibition of constriction of isolated vessels were performed (1) in vessels derived from species other than the mouse; (2) in conduit vessels and arteries rather than resistance type vessels; and (3) under isometric conditions, i.e., conditions less physiologic than the presently used constant pressure (Table 1).

An explanation for the lack of HCTZ inhibition of constriction may have been due to possible bell-shaped, concentration-inhibition curve. This explanation is based on the use of a single, high HCTZ concentration of 100 μM. In fact, clinical HCTZ plasma concentrations were 0.26 μM (median level; Sigaroudi et al. 2018).

On the other hand, HCTZ demonstrated typical sigmoidal concentration-inhibition curves with maximal concentration of 300 μM and 1000 μM, and with approximate IC₅₀’s of 80–100 μM (Abrahams et al. 1996, 1998; Mironneau et al. 1981; Table 1). Additionally, 100 μM HCTZ inhibited constriction of isolated vessels (Sládková et al. 2007; Table 1). Differences between preparations, including agonist potency, may also influence the findings (Dunn et al. 1994), as well as greater duration of TZD exposure.

This study suggests that future investigations to identify the extra-renal site of chronic TZD treatment should (1) focus on indirect inhibition of vascular constriction and (2) be determined under clinical conditions including chronic TZD at relevant doses in hypertensive animals.