Effects of renal nerves and plasma epoxyeicosatrienoic acids on blood pressure, renal hemodynamics and excretion in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display deficiency of epoxyeicosatrienoic acids (EETs). Their possible interaction with renal sympathetic nerves remains unexplored; synthesis of EET-A [disodium (S)-2-(13-(3-pentyl)ureido)-tridec-8(Z)-enamido)succinate], a stable 14,15-EET analog, helps clarify the issue. In anesthetized SHR, untreated or pretreated with EET-A, we assessed early responses of blood pressure (MAP), renal hemodynamics and excretion, and indices of nitric oxide (NO) activity, to bilateral noninvasive renal denervation (DNX). DNX significantly decreased MAP, with or without EET-A pretreatment. Renal perfusion decreased in EET-A treated but not in control rats. After EET-A pretreatment DNX decreased renal excretion of sodium and total solutes, compared to increasing tendency in untreated rats. In EET-A treated but not in untreated SHR denervation reduced the excretion of NO metabolites. Antihypertensive action of EET-A in anesthetized SHR was not clearly dependent on renal nerve activity. On the other hand, DNX unmasked the unexpected effect of EET-A to lower renal perfusion. The mechanism of this novel finding is unclear, as is also the simultaneous post-denervation decrease in renal excretion, again, observed only under EET-A treatment. Possibly, the decrease was secondary to falling MAP and renal perfusion. Increased renal excretion of nitric oxide metabolites under EETs elevation strongly suggests facilitation of NO release; the effect that was observed only with intact renal nerve activity.

Rapid blood pressure increase after renal denervation in anaesthetized rats: interaction of oxidative stress and neuronal nitric oxide synthase.

We showed previously that in anaesthetized rats acute noninvasive renal denervation (DNX) induced an increase in arterial blood pressure (MABP), unlike the usual hypotensive effect. Here we aimed to establish the background of such unusual response, especially the role of oxidative stress as suggested by an earlier study. The contribution of oxidative stress was explored by studying the effects on DNX-induced MABP increase of pretreatment with 4-hydroxy-3-methoxyacetophenone (apocynin, APO), a powerful antioxidant and antihypertensive agent, and N(omega)-propyl-L-arginine (L-NPA), a blocker of neuronal nitric oxide synthase (nNOS). In anaesthetized Wistar rats maintained on standard (STD) or high-salt (HS) diet sequential right- and left-side DNX was performed. MABP responses were examined without pretreatment and after APO (20 mg/day on two preceding days) and L-NPA (1 mg/kg/h throughout experiment), given alone or combined. In untreated rats, bilateral DNX increased MABP by 6% on STD and 15% on HS diet (P < 0.01 or less); the difference between MABP responses was highly significant (P = 0.002). In STD rats APO or APO + L-NPA treatment failed to alter post-DNX MABP increases whereas L-NPA alone reversed the response and a significant 7% decrease occurred. In HS rats APO and L-NPA given alone reversed the MABP response and significant decreases of 14% (P = 0.001) and 8% (P = 0.01), were seen. Surprisingly, with L-NPA + APO pretreatment only abolishment (not reversal) of post-DNX pressure increase occurred. The results suggest that both systemic, intrarenal and brain oxidative stress, and excessive nNOS activity, mostly in the brain, determine the unexpected post-DNX pressure increase.

Arginine and tetrahydrobiopterin supplementation in rats with salt-induced blood pressure increase: minor hypotensive effect but improvement of renal haemodynamics.

High salt (HS) intake can lead to hypertension, probably the result of the predominance of vasoconstrictor reactive oxygen species over vasodilator nitric oxide (NO). We aimed to examine if the supposed NO deficiency and the resultant blood pressure increase could be corrected by supplementation of L-arginine, the substrate, and tetrahydrobiopterin (BH4), a co-factor of NO synthases. Wistar rats without known genetic background of salt sensitivity were exposed to HS diet (4%Na) for 10 or 26 days, without or with supplementation with oral L-arginine, 1.4 mg/kg b.w. daily, alone or together with intraperitoneal BH4, 10 mg/kg daily. Systolic blood pressure (SBP, tail-cuff method) was measured repeatedly and found to increase ~40 mmHg after 26 days; L-arginine and BH4 did not significantly attenuate this increase. At the end of chronic studies, in anaesthetized rats the diet- and treatment-induced changes in renal haemodynamics were assessed. HS diet selectively decreased (-30%, P < 0.03) the inner medullary blood flow (IMBF, laser-Doppler flux) without changing total or cortical renal perfusion. Arginine supplementation tended to raise all renal circulatory parameters, and distinctly increased IMBF, to 61% above the HS diet level (P < 0.05). In conclusion, unlike in confirmed genetically determined salt-dependent hypertension, L-arginine and BH4 supplementation failed to attenuate the SBP increase observed after exposure to HS diet. On the other hand, arginine increased total and regional renal perfusion, especially IMBF. This suggests that the delivery of arginine increased intrarenal NO synthesis, an action of renoprotective potential which presumably countered the harmful influence of the local tissue oxidative stress.

Effects of high and low sodium diet on blood pressure and heart rate in mice lacking the functional grainyhead-like 1 gene.

Hypertension is a major health problem throughout the world because of its high prevalence and its association with increased risk of cardiovascular disease. Two independent studies discovered a locus conferring susceptibility to essential hypertension on chromosome 2, in the 2p25 region, but the causative gene remains unknown. Grainyhead-like 1 (GRHL1) is one of the genes located in this region. Our experiments determined that the Grhl1 -null mice, when fed standard diet, have the same blood pressure as their wild type littermate controls. However, we discovered that blood pressure of these mice increases following high sodium diet and decreases when they are fed low sodium diet, and similar effects were not observed in the control wild type littermates. This suggests that the Grhl1 -null mice are sensitive to the development of salt-sensitive hypertension. Thus it is possible that the GRHL1 gene is involved in the regulation of blood pressure, and it may be the causative gene for the locus of susceptibility to essential hypertension in the 2p25 region.

High salt intake increases blood pressure in normal rats: putative role of 20-HETE and no evidence on changes in renal vascular reactivity.

UNLABELLED: Background/Aims . High salt (HS) intake may elevate blood pressure (BP), also in animals without genetic salt sensitivity. The development of salt-dependent hypertension could be mediated by endogenous vasoactive agents; here we examined the role of vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). METHODS: In conscious Wistar rats on HS diet systolic BP (SBP) was examined after chronic elevation of EETs using 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB), a blocker of soluble epoxide hydrolase, or after inhibition of 20-HETE with 1-aminobenzotriazole (ABT). Thereafter, in acute experiments the responses of renal artery blood flow (Transonic probe) and renal regional perfusion (laser-Doppler) to intrarenal acetylcholine (ACh) or norepinephrine were determined. RESULTS: HS diet increased urinary 20-HETE excretion. The SBP increase was not reduced by c-AUCB but prevented by ABT until day 5 of HS exposure. Renal vasomotor responses to ACh or norepinephrine were similar on standard and HS diet. ABT but not c-AUCB abolished the responses to ACh. Conclusions . 20-HETE seems to mediate the early-phase HS diet-induced BP increase while EETs are not engaged in the process. Since HS exposure did not alter renal vasodilator responses to Ach, endothelial dysfunction is not a critical factor in the mechanism of salt-induced blood pressure elevation.

Oxidative stress and neuronal NOS activity: putative determinants of rapid blood pressure increase after renal denervation in anesthetized rats.

Long-term effects of renal denervation (DNX) commonly include a decrease in blood pressure (BP), observed in both normotensive animals and various models of hypertension. On the other hand, short term BP responses vary. We examined how post-DNX increase in BP observed in this study depends on baseline metabolic and functional status of animals, with a special interest for the role of oxidative stress. Anesthetized Wistar rats on standard (STD), low-sodium (LS) or high-sodium (HS) diet were used, untreated or pre-treated with tempol, a superoxide scavenger, or N(omega)-propyl-L-arginine (L-NPA), an inhibitor of neuronal NOS (nNOS). Early BP and renal hemodynamic responses were examined to right- and then left-side DNX performed using an own relatively non-invasive technique. Left kidney cortical, outer- and inner-medullary blood flows (CBF, OMBF, IMBF) were continuously recorded as laser-Doppler fluxes. Sequential denervations significantly increased BP to final 19 %, 12 %, and 6 % above control level in HS, LS, and STD groups, respectively. CBF, a measure of total renal perfusion, increased in LS and STD but not in HS rats. Tempol pretreatment prevented the post-denervation BP increase on each diet. Selective inhibition of nNOS prevented BP increase in STD and HS groups, a modest increase persisted in LS rats. We propose that enhanced afferent impulsation from intrarenal chemoreceptors related to oxidative stress in the kidney was the background for acute BP increase after DNX. The response was triggered by a release of brain sympatho-excitatory centers from inhibition by renal afferents, this was followed by widespread sympathetic cardiovascular stimulation.

Effects of ATP on rat renal haemodynamics and excretion: role of sodium intake, nitric oxide and cytochrome P450.

AIM: Adenosine-5'-triphosphate (ATP) affects intrarenal vascular tone and tubular transport via P2 receptors; however, the actual role of the system in regulation of renal perfusion and excretion remains unclear and is the subject of this whole-kidney study. METHODS: Effects of suprarenal aortic ATP infusion, 0.6-1.2 mg kg(-1) h(-1), were examined in anaesthetised rats maintained on low- (LS) or high-sodium (HS) diet. Renal artery blood flow (RBF, transonic flow probe) and the perfusion (laser-Doppler flux) of the superficial cortex (CBF) and outer and inner medulla (OM-BF, IM-BF) were measured, together with sodium and water excretion and urine osmolality. RESULTS: Adenosine-5'-triphosphate did not change arterial pressure, RBF or CBF while the effects on medullary perfusion depended on sodium intake. In LS rats ATP increased IM-BF 19 +/- 6%, the effect was prevented by inhibition of nitric oxide (NO) with N-nitro-l-arginine methyl ester. In HS rats ATP decreased OM-BF 16 +/- 3% and IM-BF (7 +/- 4%, not significant); previous inhibition of cytochrome P450 with 1-aminobenzotriazol blunted the OM-BF decrease and reversed the previous decrease of IM-BF to a 13 +/- 8% increase. Inhibition of P2 receptors with pyridoxal derivative (PPADS) abolished medullary vascular responses to ATP. In HS rats pre-treated with PPADS, ATP increased tubular reabsorption, probably via adenosine formation and stimulation of P1 receptors. CONCLUSION: The data indicate a potential role of ATP in the selective control of renal medullary perfusion, different in sodium depleted and sodium replete rats. The action of ATP appears to be mediated by the NO system and the cytochrome P450 dependent vasoactive metabolites.

Nitric oxide inhibition and the impact on renal nerve-mediated antinatriuresis and antidiuresis in the anaesthetized rat.

The contribution of nitric oxide (NO) to the antinatriuresis and antidiuresis caused by low-level electrical stimulation of the renal sympathetic nerves (RNS) was investigated in rats anaesthetized with chloralose-urethane. Groups of rats, n= 6, were given i.v. infusions of vehicle, l-NAME (10 microg kg(-1) min(-1)), 1400W (20 microg kg(-1) min(-1)), or S-methyl-thiocitrulline (SMTC) (20 microg kg(-1) min(-1)) to inhibit NO synthesis non-selectively or selectively to block the inducible or neuronal NOS isoforms (iNOS and nNOS, respectively). Following baseline measurements of blood pressure (BP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UV) and sodium excretion (U(Na)V), RNS was performed at 15 V, 2 ms duration with a frequency between 0.5 and 1.0 Hz. RNS did not cause measurable changes in BP, RBF or GFR in any of the groups. In untreated rats, RNS decreased UV and U(Na)V by 40-50% (both P < 0.01), but these excretory responses were prevented in l-NAME-treated rats. In the presence of 1400W i.v., RNS caused reversible reductions in both UV and U(Na)V of 40-50% (both P < 0.01), while in SMTC-treated rats, RNS caused an inconsistent fall in UV, but a significant reduction (P < 0.05) in U(Na)V of 21%. These data demonstrated that the renal nerve-mediated antinatriuresis and antidiuresis was dependent on the presence of NO, generated in part by nNOS. The findings suggest that NO importantly modulates the neural control of fluid reabsorption; the control may be facilitatory at a presynaptic level but inhibitory on tubular reabsorptive processes.

Effects of renal nerve stimulation on intrarenal blood flow in rats with intact or inactivated NO synthases.

AIM: We studied a possible action of nitric oxide (NO), an intrarenal vasodilator, to buffer a decrease in renal perfusion induced by electrical stimulation of renal nerves (RNS). METHODS: In anaesthetized rats RNS was performed (15 V, 2 ms pulse duration) for 10 s at the frequencies of 2, 3.5, 5 and 7.5 Hz. The total renal blood flow (RBF), an index of cortical perfusion, was measured using a Transonic probe on the renal artery. The outer and inner medullary blood flow (OMBF, IMBF) was measured by laser-Doppler flowmetry. The effect of RNS on RBF, OMBF and IMBF was determined in rats which were either untreated or pre-treated with L-NAME (0.6 or 1.8 mg kg(-1) i.v.), or S-methyl thiocitrulline (SMTC, 20 microg kg(-1) min(-1) i.v.), a selective inhibitor of neuronal NO synthase (nNOS). RESULTS: In untreated rats, RNS decreased IMBF significantly less than RBF and OMBF. High-dose L-NAME treatment significantly enhanced the RNS induced decrease of RBF but not of OMBF or IMBF. SMTC treatment significantly enhanced the decrease of IMBF, without affecting the response of RBF or OMBF. CONCLUSION: At intact NO synthesis the inner medullary circulation is not controlled by renal nerves to the extent observed for the outer medulla or cortex. NO generated by all NOS isoforms present in the kidney buffers partly the intrarenal vasoconstriction triggered by electrical RNS. The NO derived from nNOS seems of particular importance in the control of inner medullary perfusion, interacting with NO generated by endothelial NOS and renal nerves.

Role of prostaglandin cyclooxygenase and cytochrome P450 pathways in the mechanism of natriuresis which follows hypertonic saline infusion in the rat.

AIM: The prostaglandin cyclooxygenase (COX) and P450 cytochrome (CYP450) pathways of arachidonic acid metabolism are functionally interrelated and both engaged in control of sodium excretion; the study focused on their contribution to the natriuresis which follows hypertonic saline infusion in the rat. METHODS: In anaesthetized rats, clearance studies were conducted, supplemented with laser-Doppler measurements of the cortical and medullary blood flow (CBF, MBF), and measurement of medullary tissue admittance (Y), an index of interstitial ion concentration. RESULTS: Indomethacin (Indo), 5 mg kg(-1) i.v. paradoxically enhanced the natriuresis secondary to intra-aortic suprarenal 5% saline load, further increasing sodium excretion by 385 +/- 73% (P < 0.01). After acute clotrimazole, 10 mg kg(-1) i.v. an inhibitor of CYP450 epoxygenase, the increase in natriuresis was smaller and did not differ from that observed after the drug's ethanol solvent. In rats pre-treated with clotrimazole for 3 days, hypertonic saline loading increased sodium excretion (U(Na)V) to 0.94 +/- 0.22 micromol min(-1) , compared with a significantly greater (P < 0.05) increase to 2.76 +/- 0.48 micromol min(-1) measured in untreated controls. Indo increased U(Na)V twofold, similarly in the clotrimazole and in the control group; in the absence or presence of clotrimazole treatment, COX blockade significantly decreased MBF and increased Y. CONCLUSION: The data indicate that blockade of the CYP450 epoxygenase significantly impairs excretion of sodium in rats acutely loaded with hypertonic NaCl solution. The paradoxical post-Indo natriuresis is preserved in clotrimazole treated rats, which speaks against the role of CYP450 pathway in the response.

Early effects of renal denervation in the anaesthetised rat: natriuresis and increased cortical blood flow.

A novel method of renal denervation was developed based on electro-coagulation of tissue containing most of the sympathetic fibres travelling towards the kidney. Kidney tissue noradrenaline was decreased to 4.7 % of the content measured in the contralateral innervated kidney when studied 3 days postdenervation. The method was utilised in anaesthetised rats to examine the effects of denervation within the heretofore unexplored first 75 min period postdenervation. Sodium excretion (UNaV) increased significantly (+82 %, P < 0.03) over the 25-50 min after denervation. In a parallel group, with a lower baseline UNaV, there was also a significant increase in UNaV (+54 %, P < 0.03) within the first 25 min. The renal perfusion pressure was maintained at a constant value and the glomerular filtration rate did not change after denervation. Renal cortical and medullary blood flows (CBF, MBF) were estimated as laser Doppler flux and medullary tissue ion concentration was estimated as electrical admittance (Y). Following denervation, in both groups CBF increased significantly within the first 25 min (+12 %, P < 0.01 and +8 %, P < 0.05, respectively) while MBF did not change or decreased slightly; Y did not change. The data document the development of natriuresis within the first 25-50 min after denervation. The increase in CBF indicated that, prior to denervation, the cortical, but not medullary, circulation was under a tonic vasoconstrictor influence of the renal nerves. Such a dissociation of neural effects on the renal cortical vs. medullary vasculature has not been previously described.

Exaggerated volume expansion natriuresis in rats preloaded with hypertonic saline: a paradoxical enhancement by inhibition of prostaglandin synthesis.

In preliminary experiments rats preinfused with hypertonic saline showed exaggerated natriuresis after an additional small volume expansion (SVE). This was systematically studied in anaesthetized Wistar rats prepared for clearance studies of the left kidney and measurements of medullary blood flow (MBF, laser-Doppler technique) and tissue electrical admittance (Y ), an index of interstitial ion concentration. The rats were preinfused i.v. with 3 mL of 5% NaCl during 90 min. A subsequent injection of isotonic saline, 0.5% of body weight, increased sodium excretion (UNaV ) from 2.1 +/- 0.5 to 4.5 +/- 1.1 micromol min-1 and urine flow (V ) from 12.0 +/- 2.3 to 24.3 +/- 5.6 microL min-1 (P < 0.02). The same volume of whole blood increased UNaV from 5.0 +/- 1.4 to 8.7 +/- 1.7 micromol min-1 and V from 22.3 +/- 5.1 to 37.4 +/- 5.9 microL min-1 (P < 0.01). The glomerular filtration rate, MBF and Y did not change. In rats preinfused with 0.9% saline no natriuresis was observed after SVE. To examine if prostaglandins (PG) were involved in SVE natriuresis, indomethacin (Indo), 5 mg kg-1 or sodium meclophenamate (Meclo), 7.5 mg kg-1, were added to the injected 0.9% saline. Paradoxically, both PG synthesis inhibitors enhanced natriuresis to SVE. After Indo UNaV increased from 2.0 +/- 0.6 to 7.6 +/- 1.3 micromol min-1, significantly more than after SVE alone (P < 0.001). At higher baseline UNaV, the increase with Meclo from 4.5 +/- 1.2 to 13.5 +/- 1.8 micromol min-1 was significantly higher than after whole blood infusion (P < 0.001). MBF decreased and Y increased after both inhibitors. Further studies are required to explain the enhancement of natriuresis after blockade of PG synthesis.

Osmotic hypertonicity of the renal medulla during changes in renal perfusion pressure in the rat.

1. The relationship between renal perfusion pressure (RPP) and ion concentration in renal medulla was studied in anaesthetized rats. RPP was changed in steps within the pressure range 130-80 mmHg, while tissue electrical admittance (Y, index of interstitial ion concentration) and medullary and cortical blood flow (MBF and CBF; laser Doppler flowmetry) were measured, along with glomerular filtration rate (C in) and renal excretion. 2. With a RPP reduction from 130 to 120 mmHg, tissue Y remained stable; at 100 and 80 mmHg, Y was 5 and 17 % lower, respectively, than at 120 mmHg. 3. CBF fell less than RPP (partial autoregulation) in the range 130-100 mmHg only. MBF was autoregulated within 120-100 mmHg, but not above or below this range. 4. Each step of RPP reduction was followed by a decrease in sodium and water excretion (UNaV and V). The osmolality of excised inner medulla fragments was similar at 120 and 105 mmHg (586 +/- 45 and 618 +/- 35 mosmol (kg H2O)-1, respectively) but lower at 80 mmHg (434 +/- 31 mosmol (kg H2O)-1, P < 0.01); the ion concentration changed in parallel. 5. The data show that medullary hypertonicity was well preserved during RPP fluctuations within 130-100 mmHg, but not below this range. RPP-dependent changes of UNaV and V were not clearly associated with changes in solute concentration in medullary tissue.

Simultaneous recording of tissue ion content and blood flow in rat renal medulla: evidence on interdependence.

The relationship of renal medullary tissue ion concentration and medullary blood flow (MBF) has never been closely evaluated because of limitations of available measuring methods. In an attempt to overcome this difficulty, an integrated probe was developed for simultaneous recording in rat renal medulla of tissue electrical admittance (Y), an index of interstitial ion concentration, and tissue perfusion with blood (laser-Doppler method). During spontaneous-selective MBF variations tissue Y showed inverse changes (r = -0.77, P < 0.001). The inverse correlation of the two variables was also seen after MBF has been reduced (-43%) by indomethacin, 5 mg/kg body wt iv (r = -0.77, P < 0.01). A modest selective MBF reduction (15%) induced by glibenclamide, an inhibitor of ATP-dependent K channels, did not alter medullary tissue admittance. The data support experimentally the concept that the rate of medullary tissue perfusion with blood is one determinant of interstitial solute concentration; however, changes in the latter were demonstrable only with major alterations of the MBF.

Reinvestigation of water diuresis in the rat: role of anesthesia and acute surgery.

Renal effects of water loading on urine flow rate (V) and concentration (Uosm) were studied in Wistar rats subjected to minor or extensive surgery, under different kinds and level of anesthesia. In the explanted kidney of chronically prepared rats, under light chloralose anesthesia, 1 h.i.v. hypotonic fluid load increased V of the experimental (left) kidney 11 fold while Uosm decreased from 1181 +/- 142 to 210 +/- 80 mosm kg/H2O. In the acutely exposed kidney, under Inactin anesthesia, V increased 5 fold and Uosm decreased from 785 +/- 170 to 204 +/- 57 mosm/kg H2O. In similar experiments under Nembutal anesthesia V increased 4 fold but the urine did not become hypotonic. After acute surgery under Inactin anesthesia water diuresis can be induced but urine concentration before water loading is impaired. Urine hypotonicity after water loading is not achieved in acute experiments under Nembutal--an unsuitable approach to studies of water diuresis. Thus, a transition from antidiuresis to urine dilution can best be accomplished in the chronic explanted kidney model under light anesthesia and without invasive surgery.

Access to rat kidney with minimal anesthesia and surgery: a new experimental model.

Chronic surgical explantation of the left rat kidney out of the abdominal cavity under the flank skin enabled easy access to the organ and ipsilateral urine collection under light chloralose anesthesia without virtually any surgical intervention. The glomerular filtration rate, urine flow and osmolality, sodium excretion, and medullary tissue hypertonicity were similar in the explanted and in the contralateral kidney, whereas p-aminohippurate clearance was 14% lower. The function of the explanted kidney was also compared with that of the kidney acutely exposed in rats under thiobutabarbital anesthesia and rendered euvolemic by isoncotic albumin infusion. Again, in both preparations renal function was comparable except that over time urine osmolality remained stable in the former and fell from 1,385 +/- 195 to 835 +/- 167 mosmol/kgH2O (P < 0.02) in the latter, indicating deterioration of urine concentration. Laser-Doppler probes could be easily applied in the explanted kidney to measure cortical and medullary blood flow. The new experimental model offers some advantages, both over studies using conscious rats and over experiments involving deep anesthesia and acute surgery.

Glucagon increases medullary interstitial electrolyte concentration in rat kidney.

In anesthetized rats, tissue electrical admittance of the inner medulla (a measure of total ion concentration in the interstitium), medullary blood flow (laser Doppler technique), and renal clearances were measured simultaneously before and during i.v. infusion of glucagon at 110 and 330 ng.min-1.kg-1 body weight. Admittance increased modestly, 5.4% after a large glucagon dose (p < 0.01), whereas medullary blood flow was stable. Glomerular filtration rate increased transiently and then fell during high-dose glucagon infusion. The increase in tissue electrolyte (mostly NaCl) concentration in the medulla observed with stable medullary blood flow and decreasing glomerular filtration rate indicates that stimulation of NaCl reabsorption in the medullary ascending limb of Henle's loop by glucagon was the mechanism underlying augmentation of medullary ionic hypertonicity. This suggests that glucagon can contribute to the urine concentration process.