The non-diuretic hypotensive effects of thiazides are enhanced during volume depletion states

Figures

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  Fig 1. Intravascular volume depletion in Pendrin/NCC dKO mice. (a) Northern hybridization shows a significant enhancement in mRNA expression levels of renin in kidneys of pendrin/NCC dKO mice compared to WT, pendrin KO and NCC KO. (b) Immunofluorescent microscopic analysis of kidney sections indicates that the expression of renin is significantly up regulated in pendrin/NCC dKO, but not in WT mice. (c) Western blots confirmed the significant increase in renin expression in pendrin/NCC dKO kidney vs WT. (d) Olmesartan (1 mg/kg) treatment causes a more significant reduction in the systolic BP of pendrin/NCC dKO mice compared to WT mice, (n = 4 each group); paired t-test * P<0.05.

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  Fig 2. Effects of HCTZ on Pendrin/NCC dKO mice: Role of volume depletion. (a) The systolic BP of pendrin/NCC dKO mice dropped below detectable levels at 1 and 3 hours after HCTZ treatment but returned to baseline levels at 6 and 24 hours later; the systolic BP of WT mice was not significantly affected by HCTZ (#; below detectable level; n = 4). (b) Baseline (BL) blood pressure of pendrin/NCC dKO mice and WT mice was measured on normal (1% NaCl) diet. Then, mice were placed on high salt (7% NaCl) diets for up to 14 days. On day 14, animals’ baseline (BL) blood pressure was measured and then treated with HCTZ, and their BP was measured at 1, 3, 6, and 24 hours (h). The effect of HCTZ is abrogated in euvolemic pendrin/NCC dKO mice. (c) Treatment with a high salt diet reduces the renal expression of renin in pendrin/NCC dKO mice, indicating the correction of the volume depletion (n = 3).

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  Fig 3. Intra-arterial BP monitoring in anesthetized WT and pendrin/NCC dKO mice before and after HCTZ. Mean arterial BP was measured at baseline and after HCTZ injection in WT (n = 4) and pendrin/NCC dKO mice (n = 5) at 5, 10, 20 and 40 minutes. One-way repeated measures ANOVA was performed; * P< 0.05 vs baseline.

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  Fig 4. Echocardiography in anesthetized WT and pendrin/NCC dKO mice before and after HCTZ. (a) M-mode images of cardiac function of WT and pendrin/NCC dKO mice before and after HCTZ. (b) The examination of cardiac index as measured by echocardiography does not demonstrate any significant reduction in cardiac output of pendrin/NCC dKO mice after HCTZ treatment (n = 4 each group).

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  Fig 5. Effect of HCTZ on systemic BP of NCC KO mice on a salt restricted diet. NCC KO mice were placed on low salt (0.1% NaCl) diet for 14 days and the effect of HCTZ on the systolic BP of was examined. (a) The baseline systolic BP is significantly lower in NCC KO mice fed low salt (0.1% NaCl) diet for two weeks compared to similarly treated WT. (b) Western blot analysis shows renin protein expression is increased in NCC KO mice placed on a 0.1% NaCl diet. (c) HCTZ lowers BP in NCC KO mice on low salt diet but not their littermate in regular diet. (n = 4 each group); * P< 0.05 vs baseline.

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  Fig 6. Role of BK channels in the effects of HCTZ on systemic BP. (a) Salt restricted NCC KO mice (1% NaCl, 2 weeks) were treated with HCTZ in presence of 8mg/kg of paxilline, paxilline failed to prevent the lowering BP effects at 1 hour after HCTZ; * P< 0.05. (b) HCTZ effects in BK KO mice. HCTZ lowers the BP of BK KO mice on 0.1% NaCl where it did not change their BP on normal diet (1% NaCl) (n = 4 each group). (c) HCTZ reduces the BP of BK/NCC dKO mice on 0.1% NaCl diet compared to the same treated WT mice (WT n = 4 vs BK/NCC dKO n = 6).

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  Fig 7. Effect of HCTZ on phenylephrine contraction of aorta in vitro from NCC KO mice with salt restricted (0.1%) and normal diet (1%). Cumulative concentration-contraction curves were elicited in aorta segments exposed to 100 μM hydrochlorothiazide (HCTZ) or DMSO (0.1%, vehicle control) from NCC KO mice with normal salt (a) and salt restricted diet (b). Contraction was calculated as mN/mg wet weight (c), and as percent of 50 mM KCl (d); * P< 0.05; n = 5 each group.