Representative wheel running rhythms from WT and <i>Kcnma1^−/−</i> mice.

<p>Mice were housed on a standard 12 hr light: 12 hr dark cycle for 18 days (light and dark bars above actogram), and then placed in constant darkness (DD) for 25 days (shaded bottom part of actogram). Tick marks denote wheel running and actograms are double-plotted to emphasize the behavioral rhythm. <i>A,</i> The WT mouse has a circadian period of 23.5 hr in DD, and the χ² circadian amplitude of the behavioral rhythm was 2390. <i>B,</i> Representative <i>Kcnma1^−/−</i> actogram (period = 24.2 hr and χ² circadian amplitude = 375).</p

SFR rhythms from WT and <i>Kcnma1^−/−</i> SCNs.

<p><i>A</i> and <i>B,</i> MEA recordings from 6 channels on cycle 2 within a WT (<i>A</i>) and a <i>Kcnma1^−/−</i> (<i>B</i>) SCN over 24 hr. Data were smoothed by applying a 1-hr moving window average and plotted to maximize any day-night differences in firing rate. Dotted lines denote CT6 (6 hours after lights on for the animal prior to slice harvest), the daytime peak for SFR. <i>C,</i> Individual firing frequencies from cycle 2. The daytime peak (CT5-7) and trough (CT12–14) SFR values were from 5 WT (<i>n</i> = 32 channels) and 4 <i>Kcnma1^−/−</i> (<i>n</i> = 30 channels) slices±s.e.m. *<i>p</i><0.05.</p

Multi-electrode array recordings of SFR rhythms from SCN.

<p><i>A,</i> Acute SCN slice (300 µm) on probe. Circles outline SCN. OT = optic tract. Electrode spacing = 100 µm. <i>B,</i> Oscilloscope view from slice in (<i>A</i>) showing spontaneous activity at each electrode (channel) on the probe. Red outlines channels within the SCN. 1 box = 2.5 sec/100 µV. <i>C,</i> Six active channels within the SCN showing a robust circadian regulation of the SFR over 40 hr. Light and dark bars indicate the prior entrainment of the animal before slice harvest. <i>D,</i> Perfusion of 1 µM tetrodotoxin (TTX) blocks spontaneous action potentials (2 channels shown). Washout was started 5 min after TTX addition. <i>E,</i> Synchronized rhythms over 3 circadian SFR peaks.</p

SFR rhythms from 3 circadian cycles from WT and <i>Kcnma1^−/−</i> SCNs.

<p><i>A,</i> Representative recordings over 3 cycles from a rhythmic WT (<i>left</i>), a rhythmic <i>Kcnma1^−/−</i> (<i>middle</i>), and an arrhythmic <i>Kcnma1^−/−</i> (<i>right</i>) channel within the SCN. <i>B,</i> Distribution of daily peaks for WT and <i>Kcnma1^−/−</i> SFR rhythms (± s.d.) on each day of recording.</p

Analysis of WT and <i>Kcnma1^−/−</i> SFR rhythms.

<p>Spontaneous activity was analyzed from channels within the SCN that had activity >1 Hz. R = the number of rhythmic channels (in parentheses: average time of the SFR peak±s.d.). AR = the number of arrhythmic channels (% of total). <i>N</i>-values are presented as the number of rhythmic and arrhythmic channels from 5 WT and 4 <i>Kcnma1^−/−</i> slices. Separate <i>n</i>-values (bottom of cell) are presented for peak-to-trough (PT) ratios because these values could not be extracted from every recording due to noise problems. Peak and trough frequencies were determined by averaging two hours of continuous raw data from rhythmic channels at CT 5–7 and CT 12–14, respectively. The PT ratio was calculated for each channel as (P–T)/P and is presented as the average±s.d. ^{*, #, §, †}<i>p</i><0.05.</p

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

<div><p>Thiazide derivatives including Hydrochlorothiazide (HCTZ) represent the most common treatment of mild to moderate hypertension. Thiazides initially enhance diuresis via inhibition of the kidney Na⁺-Cl^- Cotransporter (NCC). However, chronic volume depletion and diuresis are minimal while lowered blood pressure (BP) is maintained on thiazides. Thus, a vasodilator action of thiazides is proposed, likely via Ca²⁺-activated K⁺ (BK) channels in vascular smooth muscles. This study ascertains the role of volume depletion induced by salt restriction or salt wasting in NCC KO mice on the non-diuretic hypotensive action of HCTZ. HCTZ (20mg/kg s.c.) lowered BP in 1) NCC KO on a salt restricted diet but not with normal diet; 2) in volume depleted but not in volume resuscitated pendrin/NCC dKO mice; the BP reduction occurs without any enhancement in salt excretion or reduction in cardiac output. HCTZ still lowered BP following treatment of NCC KO on salt restricted diet with paxilline (8 mg/kg, <i>i</i>.<i>p</i>.), a BK channel blocker, and in BK KO and BK/NCC dKO mice on salt restricted diet. In aortic rings from NCC KO mice on normal and low salt diet, HCTZ did not alter and minimally decreased maximal phenylephrine contraction, respectively, while contractile sensitivity remained unchanged. These results demonstrate 1) the non-diuretic hypotensive effects of thiazides are augmented with volume depletion and 2) that the BP reduction is likely the result of HCTZ inhibition of vasoconstriction through a pathway dependent on factors present in vivo, is unrelated to BK channel activation, and involves processes associated with intravascular volume depletion.</p></div

Intravascular volume depletion in Pendrin/NCC dKO mice.

<p>(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.</p

Effect of HCTZ on systemic BP of NCC KO mice on a salt restricted diet.

<p>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.</p

Echocardiography in anesthetized WT and pendrin/NCC dKO mice before and after HCTZ.

<p>(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).</p

Effects of HCTZ on Pendrin/NCC dKO mice: Role of volume depletion.

<p>(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. (<b>c</b>) 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).</p