Bioimpedance spectroscopy for the estimation of body fluid volumes in mice.

Conventional indicator dilution techniques for measuring body fluid volume are laborious, expensive, and highly invasive. Bioimpedance spectroscopy (BIS) may be a useful alternative due to being rapid, minimally invasive, and allowing repeated measurements. BIS has not been reported in mice; hence we examined how well BIS estimates body fluid volume in mice. Using C57/Bl6 mice, the BIS system demonstrated <5% intermouse variation in total body water (TBW) and extracellular (ECFV) and intracellular fluid volume (ICFV) between animals of similar body weight. TBW, ECFV, and ICFV differed between heavier male and lighter female mice; however, the ratio of TBW, ECFV, and ICFV to body weight did not differ between mice and corresponded closely to values in the literature. Furthermore, repeat measurements over 1 wk demonstrated <5% intramouse variation. Default resistance coefficients used by the BIS system, defined for rats, produced body composition values for TBW that exceeded body weight in mice. Therefore, body composition was measured in mice using a range of resistance coefficients. Resistance values at 10% of those defined for rats provided TBW, ECFV, and ICFV ratios to body weight that were similar to those obtained by conventional isotope dilution. Further evaluation of the sensitivity of the BIS system was determined by its ability to detect volume changes after saline infusion; saline provided the predicted changes in compartmental fluid volumes. In summary, BIS is a noninvasive and accurate method for the estimation of body composition in mice. The ability to perform serial measurements will be a useful tool for future studies.

Alpha(2)-adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption.

Stimulation of alpha(2)-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses alpha(2)-receptors. We hypothesized that selective alpha(2)-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the alpha(2)-receptor agonist clonidine (10 nM) decreased chloride flux (J(Cl)) from 119.5 +/- 15.9 to 67.4 +/- 13.8 pmol. mm(-1). min(-1) (43% reduction; P < 0.02), whereas removal of clonidine from the bath increased J(Cl) by 20%. When NOS activity was inhibited by pretreatment with 5 mM N(G)-nitro-L-arginine methyl ester, the inhibitory effects of clonidine on THAL J(Cl) were prevented (81.7 +/- 10.8 vs. 71.6 +/- 6.9 pmol. mm(-1). min(-1)). Similarly, when the NOS substrate L-arginine was deleted from the bath, addition of clonidine did not decrease THAL J(Cl) from control (106.9 +/- 11.6 vs. 132.2 +/- 21.3 pmol. mm(-1). min(-1)). When we blocked the alpha(2)-receptors with rauwolscine (1 microM), we found that the inhibitory effect of 10 nM clonidine on THAL J(Cl) was abolished, verifying that alpha(2), rather than I(1), receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL J(Cl), indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 microM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J(Cl). In conclusion, alpha(2)-receptor stimulation decreases THAL J(Cl) by increasing NO release and stimulating guanylate cyclase. These data suggest that alpha(2)-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.

Alpha-2 and beta-adrenergic receptors mediate NE's biphasic effects on rat thick ascending limb chloride flux.

The sympathetic neurotransmitter norepinephrine (NE) influences renal sodium excretion via activation of adrenergic receptors. The thick ascending limb (THAL) possesses both alpha-2 and beta-adrenergic receptors. However, the role(s) different adrenergic receptors play in how isolated THALs respond to NE are unclear. We tested the hypothesis that both alpha-2 and beta-adrenergic receptors are responsive to NE in the isolated THAL, with alpha-2 receptors inhibiting and beta-receptors stimulating chloride flux (J(Cl)). THALs from male Sprague-Dawley rats were perfused in vitro, and the effects of 1) incremental NE, 2) the alpha-2 agonist clonidine, and 3) the beta-agonist isoproterenol on J(Cl) were measured. Low concentrations (0.1 nM) of NE decreased J(Cl) from a rate of 114.2 +/- 8.1 to 93.5 +/- 14.6 pmol. mm(-1). min(-1) (P < 0.05), with the nadir occurring at 1 nM (67.7 +/- 8.8 pmol. mm(-1). min(-1); P < 0.05). In contrast, greater concentrations of NE significantly increased J(Cl) from the nadir to a maximal rate of 131.0 +/- 28.5 pmol. mm(-1). min(-1) at 10 microM (P < 0.05). To evaluate the adrenergic receptors mediating these responses, the THAL J(Cl) response to NE was measured in the presence of selective antagonists of beta- and alpha-2 receptors. A concentration of NE (1 microM), which alone tended to increase J(Cl), decreased THAL J(Cl) (from 148.9 +/- 16.4 to 76.2 +/- 13.6 pmol. mm(-1). min(-1); P < 0.01) in the presence of the beta-antagonist propranolol. In contrast, a concentration of NE (0.1 microM), which alone tended to decrease J(Cl), increased THAL J(Cl) (from 85.5 +/- 20.1 to 111.8 +/- 20.1 pmol. mm(-1). min(-1); P < 0.05) in the presence of the alpha-2 antagonist rauwolscine. To further clarify the role of different adrenergic receptors, selective adrenergic agonists were used. The alpha-2 agonist clonidine decreased J(Cl) from 102.4 +/- 9.9 to 54.0 +/- 15.7 pmol. mm(-1). min(-1), a reduction of 49.1 +/- 11.0% (P < 0.02). In contrast, the beta-agonist isoproterenol stimulated J(Cl) from 95.3 +/- 11.6 to 144.1 +/- 15.0 pmol. mm(-1). min(-1), an increase of 56 +/- 14% (P < 0.01). We conclude that 1) the sympathetic neurotransmitter NE exerts concentration-dependent effects on J(Cl) in the isolated rat THAL, 2) selective alpha-2 receptor activation inhibits THAL J(Cl), and 3) selective beta-receptor activation stimulates THAL J(Cl). These data indicate the response elicited by the isolated rat THAL to NE is dependent on the neurotransmitter concentration, such that application of NE in vitro biphasically modulates J(Cl) via differential activation of alpha-2 and beta-adrenergic receptors in a concentration-dependent manner.

Endothelin inhibits thick ascending limb chloride flux via ET(B) receptor-mediated NO release.

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ET(B) receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux (J(Cl)). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ET(B) receptors and release of NO. Exogenous ET-1 (1 nM) decreased J(Cl) from 118.3 +/- 15.0 to 62.7 +/- 13.6 pmol. mm(-1). min(-1) (48.3 +/- 8.2% reduction), whereas removal of ET-1 increased J(Cl) in a separate group of tubules from 87.6 +/- 10.7 to 115.2 +/- 10.3 pmol. mm(-1). min(-1) (34.5 +/- 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J(Cl), we examined the effect of inhibiting of NO synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) on ET-1-induced changes in J(Cl). L-NAME (5 mM) completely prevented the ET-1-induced reduction in J(Cl), whereas D-NAME did not. L-NAME alone had no effect on J(Cl). These data suggest that the effects of ET-1 are mediated by NO. Blockade of ET(B) receptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ET(B) receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J(Cl) (from 120.7 +/- 12.6 to 75.4 +/- 13.3 pmol. mm(-1). min(-1)). In contrast, ET(A) receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J(Cl) (from 96.5 +/- 10.4 to 69.5 +/- 8.6 pmol. mm(-1). min(-1)). Endothelin increased intracellular calcium from 96.9 +/- 14.0 to 191.4 +/- 11.9 nM, an increase of 110.8 +/- 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J(Cl) by activating ET(B) receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.

eNOS mediates L-arginine-induced inhibition of thick ascending limb chloride flux.

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Nitric oxide-induced inhibition of transport by thick ascending limbs from Dahl salt-sensitive rats.

The factor responsible for salt sensitivity of blood pressure in Dahl rats is unclear but presumably resides in the kidney. We tested the hypotheses that (1) thick ascending limbs of Dahl salt-sensitive rats (DS) absorb more NaCl than those of Dahl salt-resistant rats (DR) and (2) NO inhibits transport to a lesser extent in thick ascending limbs from DS. We found that basal chloride absorption (J(Cl)) by thick ascending limbs from DR was 105.8+/-10.0 pmol. mm(-1). min(-1) (n=6). Ten and 100 micromol/L spermine NONOate, an NO donor, decreased J(Cl) in DR to 65.8+/-8.5 and 46.8+/-7.0 pmol. mm(-1). min(-1), respectively. Basal J(Cl) in DS was 131.6+/-13.4 pmol. mm(-1). min(-1) (n=7). In DS, 10 and 100 micromol/L spermine NONOate decreased J(Cl) to 111.5+/-12.8 and 46.8+/-6.2 pmol. mm(-1). min(-1), respectively. No difference was observed in basal or NO-inhibited Na absorption by cortical collecting ducts or in basal or NO-inhibited oxygen consumption by inner medullary collecting ducts. Because NO acts via generation of cGMP, we measured cGMP production by thick ascending limbs from DS and DR to see whether a difference in cGMP production could account for the difference in basal or NO-inhibited transport. Basal rates of cGMP production were similar between the 2 strains. Although NO increased cGMP production by thick ascending limbs from both strains, no difference existed between DS and DR. We concluded that the reduced ability of NO to block transport in thick ascending limbs in DS may account for at least part of the salt sensitivity of blood pressure in this strain.

Nitric oxide, endothelin and nephron transport: potential interactions.

1. Nitric oxide (NO) is produced and/or regulates transport in many segments of the nephron, including the proximal convoluted tubule, proximal straight tubule, thick ascending limb, cortical collecting duct and inner medullary collecting duct. 2. Endothelin (ET) is produced and/or regulates nephron transport in many of the segments that produce NO or in which transport is regulated by NO. 3. Four potential interactions between NO and ET are: (i) NO and ET may be antagonistic; (ii) NO and ET may be complementary; (iii) the effects of ET may be mediated via NO; and (iv) the effects of NO may be mediated by ET. 4. In conclusion, direct studies examining the interactions between NO and ET are few. However, circumstantial evidence suggests there may be many interactions between NO and ET in the regulation of nephron transport. In particular, recent data obtained from the collecting duct and thick ascending limb indicate that the effects of ET may be mediated by the production of NO and stimulation of its second messenger cascade.

Endogenous nitric oxide inhibits chloride transport in the thick ascending limb.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. We first determined the effect of exogenously added NO on net chloride flux (JCl). The NO donor spermine NONOate (SPM; 10 microM) decreased JCl from 101.2 +/- 9.6 to 65.0 +/- 7.7 pmol. mm-1. min-1, a reduction of 35.5 +/- 6.4%, whereas controls did not decrease over time. To determine whether endogenous NO affects cortical TALH transport, we measured the effect of adding the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the substrate L-arginine (L-Arg), or its enantiomer D-arginine (D-Arg) on JCl. L-NAME and D-Arg did not alter JCl; in contrast, addition of 0.5 mM L-Arg decreased JCl by 40.2 +/- 10.4% from control. The inhibition of chloride flux by 0.5 mM L-Arg was abolished by pretreatment with L-NAME, indicating that cortical TALH NOS is active, but production of NO is substrate-limited in our preparation. Furthermore, cortical TALH chloride flux increased following removal of 0.5 mM L-Arg from the bath, indicating that the reductions in chloride flux observed in response to L-Arg are not the result of NO-mediated cytotoxicity. We conclude that 1) exogenous NO decreases cortical TALH JCl; 2) cortical TALHs produce NO in the presence of L-Arg, which decreases JCl; and 3) the response of cortical TALHs to L-Arg is reversible in vitro. These data suggest an important role for locally produced NO, which may act via an autocrine mechanism to directly affect TALH sodium chloride transport. Thus TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO observed in vivo.

Fluorescent determination of chloride in nanoliter samples.

BACKGROUND: Measurements of Cl- in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl- indicator, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable. METHODS: This is a simple procedure that relies on the selectivity of the dye and the fact that Cl-quenches its fluorescence. To measure millimolar quantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mm SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl- injected. RESULTS: The method yielded a linear response with Cl- concentrations from 5 to 200 mm NaCl. The minimum detectable Cl- concentration was approximately 5 mm. The coefficient of variation between 5 and 200 mm was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mm and between 50 and 200 mm was 1 mm and 2.6 mm, respectively. Furosemide, diisothiocyanostilbene-2,2'-disulfonic acid and other nonchloride anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, whereas 150 mm NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl- did not vary with pH within the physiological range. CONCLUSION: We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.

Long-term increases in renal sympathetic nerve activity and hypertension.

1. Essential hypertensive patients have been characterized by increased sympathetic nerve activity, increased peripheral vascular tone, decreased plasma volume and normal cardiac output when compared with normotensive subjects. Bilateral renal denervation reduces the magnitude or delays the onset of the blood pressure response in numerous models of experimental hypertension regardless of the aetiology of the elevation in arterial pressure. 2. Using a servocontrolled intrarenal infusion system, we have elevated intrarenal noradrenaline concentration via intermittent renal artery infusion without decreasing renal blood flow as a method of simulating selective elevation of renal sympathetic outflow. 3. Chronic intrarenal adrenergic stimulation increased arterial pressure within 24 h and this hypertension persisted for 28 consecutive days. The elevated arterial pressure was not associated with sustained increases in plasma renin activity, aldosterone, circulating catecholamines, arginine vasopressin or significant renal vasoconstriction. Urinary sodium excretion was chronically elevated and the dogs remained in negative sodium balance for the duration of the intrarenal noradrenaline infusion. 4. After 2 weeks of elevated intrarenal neurotransmitter coupled with hypertension, renal vascular reactivity to further adrenergic stimulation was significantly increased because the hypertension was maintained during continual reductions in the daily dosage of neurotransmitter allowed to be infused by the servocontroller. After only 28 days of noradrenaline infusion, renal vascular hypertrophy developed in vessels from 150-300 microns. 5. We conclude that selective and intermittent increases in intrarenal adrenergic neurotransmitter are sufficient to elicit chronic hypertension in the absence of volume expansion. This intrarenal neuroadrenergic hypertension is closely associated with the haemodynamic parameters which characterize a major subset of human essential hypertensives.

Chronic renal neuroadrenergic hypertension is associated with increased renal norepinephrine sensitivity and volume contraction.

Individuals with essential hypertension have been characterized by increased renal sympathetic vascular tone with decreased plasma volume and normal cardiac output compared with normotensive individuals. We used a servo-controlled intrarenal infusion system to evaluate the hemodynamic, renal excretory, and plasma hormonal responses to 28-day, low-level elevations in the intrarenal adrenergic neurotransmitter norepinephrine. In uninephrectomized dogs (n = 6), servo-controlled norepinephrine infusion increased mean arterial pressure from 95.6 +/- 3.1 to 115.7 +/- 4.9 mm Hg on day 1 without concomitant reductions in renal blood flow. Arterial hypertension was sustained and renal vascular resistance increased during the 28 days of servo-controlled norepinephrine infusion despite significant decreases in the daily dose of intrarenal norepinephrine (1.49 +/- 0.23 to 0.47 +/- 0.25 mg/d) necessary to maintain renal blood flow constant. Arterial pressure returned to control values with the cessation of servo-controlled norepinephrine, whereas renal blood flow and renal vascular resistance remained slightly decreased and increased, respectively. Cumulative sodium balance exhibited a net 177 +/- 37 mmol sodium loss over the 28 days of norepinephrine infusion, indicating that the hypertension did not result from sodium retention or expansion of extracellular fluid volume. Intrarenal norepinephrine did not change plasma epinephrine, norepinephrine, or vasopressin concentrations. Atrial natriuretic factor, however, increased at 7 and 14 days of servo-controlled norepinephrine, and plasma renin activity increased on day 14 of norepinephrine infusion. We conclude that low-level elevation of intrarenal adrenergic neurotransmitter produces sustained arterial hypertension that is independent of expansion in extracellular fluid volume, increases in circulating catecholamines or plasma renin activity, or reductions in renal blood flow. This hypertension may be associated with increased renal vascular sensitivity to norepinephrine and/or other renal vasoactive factors.