Effect Of Sodium Depletion Research Articles

Acute body sodium depletion induces skin sodium mobilization in female Wistar rats.

What is the central question of this study? Can Na+ depletion mobilize Na+ from the skin reservoir in ovariectomized rats? Does oestrogen replacement change the amount and the dynamics of skin Na+ storage? Is the reduced salt appetite after Na+ depletion in ovariectomized rats with oestrogen replacement related to changes in the skin Na+ ? What is the main finding and its importance? This work demonstrated that acute body Na+ depletion induced by frusemide mobilized the osmotically inactive skin Na+ reservoir to become osmotically active. Oestrogen treatment decreased the induced Na+ intake in ovariectomized rats but did not modulate the inactive Na+ reservoir in control conditions or its mobilization induced by Na+ depletion. Oestradiol, which is an important hormone for water and electrolyte balance, also has a role in the inhibition of induced Na+ appetite. Sodium can be stored in the skin in osmotically active or inactive forms, and this skin Na+ reservoir may be involved in the control of body Na+ levels during physiopathological challenges. In this study, we investigated whether the effect of sodium depletion by frusemide can mobilize Na+ from the skin reservoir and whether oestradiol replacement changes or mobilizes the Na+ reserves in the skin. Ovariectomized Wistar rats were treated with vehicle or oestradiol for 7days to evaluate the effects of oestrogen on the hydroelectrolyte balance, intake responses and skin Na+ and water content in basal conditions. Furthermore, the effects of oestrogen were evaluated after 24 h frusemide-induced whole-body Na+ depletion. Oestradiol-replaced rats exhibited reduced water intake without any significant changes in salt intake, Na+ excretion or water and Na+ skin content in basal conditions. After sodium depletion, both vehicle- and oestradiol-treated rats exhibited an increase in the osmotically active skin Na+ , which was associated with a decrease of the inactive skin Na+ reservoir. Oestrogen decreased the hypertonic saline intake induced by Na+ depletion, but it was not associated with any significant changes in the skin Na+ reservoir. Thus, sodium depletion is able to change the inactive-active skin Na+ reservoir balance. However, the oestrogenic modulation of sodium appetite after Na+ depletion is probably not related to the action of this hormone in the skin Na+ reservoir balance.

The effect of sodium depletion with or without concomitant dehydration on Fos immunoreactivity in the different subregions of the subfornical organ and organum vasculosum of the lamina terminalis of the rat

In rats, each of the two forebrain sensory circumventricular organs, the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) can be categorised anatomically and functionally into subregions. The main divisions of the SFO are its outer shell and its ventromedial core, while the OVLT has a lateral zone, dorsal cap and a central vascular plexus. The aim of these experiments was to determine the effect of Na depletion for 24 hours by furosemide (20 mg/kg s.c.) treatment and low Na diet on rats (4 per group) with or without water available to drink on Fos immunoreactivity (Fos‐IR) in the subregions of the SFO and OVLT. We also examined the effect after 2 hours on these responses of correcting the Na deficit by drinking either isotonic 0.15M or hypertonic 0.3M NaCl. After these treatments, rats were killed by Na pentobarbitone (100 mg/kg i.p.) and standard immunohistochemical methods used to identify Fos in cell nuclei of CVO neurons. Whereas control untreated rats on low Na diet for 24 h exhibited minimal Fos‐IR in any subregions of the SFO and OVLT, rats subjected to sodium depletion with water available to drink exhibited many Fos‐labelled cells in the SFO that were almost exclusively confined to its ventromedial core, while in the OVLT, Fos expression was observed predominantly in the lateral zone with little Fos‐IR in the dorsal cap. Fos‐IR was not observed in the supraoptic nucleus (SON), signifying that the rats were not dehydrated. Treatment with the angiotensin AT1 receptor antagonist valsartan (5 mg/kg s.c. × 2 doses) inhibited the Fos response in the ventromedial core of the SFO. If water was withheld from Na‐depleted rats, some Fos‐IR was now observed in the outer shell, although most Fos‐IR was still observed in the ventromedial core; in the OVLT, Fos‐IR was observed in both its dorsal cap and lateral zone and the SON expressed Fos‐IR. Correction of the Na‐deficit in water‐replete but Na‐depleted rats by drinking 0.15M NaCl reduced the intensity, but did not abolish the Fos‐IR in the ventromedial core of the SFO or the lateral zone of the OVLT by 2 h, whereas water‐replete, Na‐depleted rats that drank hypertonic 0.3M NaCl exhibited additional Fos‐IR in the outer shell region of the SFO and in the dorsal cap of the OVLT as well as the less intense Fos‐IR in the ventromedial core of SFO and lateral zone of OVLT. These data confirm that the ventromedial core of SFO and lateral zone of the OVLT are activated by loss of body Na, whereas neurons in the outer shell of the SFO and dorsal cap of OVLT are more attuned to loss of body water and hypertonicity.Support or Funding InformationSupported by G. Harold and Leila Y. Mathers Charitable Foundation

Effect of sodium depletion by frusemide on tissue concentrations and metabolism of VIP.

Effect of sodium depletion by frusemide on tissue concentrations and metabolism of VIP.

EFFECTS OF SODIUM DEPLETION ON TISSUE CONCENTRATIONS OF THE NATRIURETIC HORMONE VASOACTIVE INTESTINAL PEPTIDE

1. Variations in dietary sodium intake have been shown to affect the plasma concentration, the metabolic clearance rate and secretion rate of vasoactive intestinal peptide (VIP). In this study we sought to determine the effect of sodium depletion on the concentration of VIP in plasma and in three tissues, namely heart, lung and kidney. 2. Male Sprague-Dawley rats were placed on low or normal sodium diets and drinking water ad libitum. A third group was placed on a low salt diet and in addition were given frusemide, 1mg/kg per day in the drinking water. After 7 days the rats were killed, a blood sample collected and tissues harvested. VIP concentrations were determined by radioimmunoassay on unextracted plasma and in tissue after extraction. 3. There were significant differences between the three groups in the concentration of VIP in the lung (P < 0.0005), kidney (P < 0.005) and plasma (P < 0.025) but not the heart. In the group that received frusemide and the low sodium diet, VIP in the lung was significantly lower than the low sodium (P < 0.005) and normal sodium (P < 0.0001) groups. Similar differences were noted in the kidney (frusemide vs low sodium, P < 0.001; frusemide vs normal, P < 0.01) and plasma (frusemide vs low sodium P < 0.001, frusemide vs normal P < 0.05). 4. We conclude that sodium depletion decreases the concentration of VIP in plasma and in its metabolizing tissues.

Effects of sodium depletion on competition and summation between rewarding effects of salt and lateral hypothalamic stimulation in the rat.

The effect of sodium depletion on the value of lateral hypothalamic (LH) stimulation and on competition and summation between saline reward and LH reward was assessed. Sodium depletion did not alter curves relating the number of LH rewards earned per trial to the number of stimulation pulses per train, suggesting that negative sodium balance does not alter the reward value of the stimulation. In contrast, sodium depletion increased the ability of saline (0.9% NaCl) to compete with LH stimulation in a forced-choice preference test. Summation between the effects of the 2 rewards was also seen under sodium depletion. These findings imply that the rewarding effects of saline and LH stimulation are evaluated within a common system of measurement and that negative sodium balance modulates the value of the saline reward upstream from the point where the rewarding effects of saline and LH stimulation are combined.

Effect of Extracellular Sodium on Thyroid Hormone Uptake by Mouse Thymocytes*

In mouse thymocytes, a stereospecific saturable energy-dependent and ouabain-inhibitable system facilitates T3, but not T4, entry. We studied here the effect of sodium depletion on cellular uptake of thyroid hormones by mouse thymocytes. Time-course experiments indicated that extracellular sodium depletion reduced [125I]T3 uptake at each time studied. At equilibrium, the removal of extracellular sodium and its substitution with isoosmotic choline decreased saturable [125I]T3 uptake by 60 +/- 10%; this effect was dose dependent. The substitution of sodium with lithium, instead of choline, had no effect on the uptake process. [125I]T4 uptake was lower than that of [125I]T3 and not affected by sodium depletion. The half-maximal effect of sodium deprivation on [125I]T3 uptake was reached at an extracellular sodium concentration of about 40 mM. The variation of external pH influenced T3 accumulation by thymocytes. [125I]T3 progressively decreased from acid to alkaline pH under normal and sodium-depleted conditions; however, the sodium-dependent fraction was more than doubled at physiological pH compared to that at more acidic and more alkaline pH. The sodium ionophore monensin decreased T3 uptake by 51% at a concentration of 20 microM. These results indicated the existence of a sodium-related mechanism of T3 uptake into mouse thymocytes that does not operate for T4 uptake.

Regulation of receptors for atrial natriuretic peptide in the rat and human.

Atrial natriuretic factor or peptide (ANP) is a peptide recently isolated from mammalian atria with potent natriuretic, vasorelaxant, and aldosterone-inhibitory properties. ANP may play an important role in the regulation of blood pressure and body salt and fluid balance. The presence of binding sites for ANP in the vasculature and adrenal glomerulosa of rats and in platelets in humans has been demonstrated. These sites are involved in the mediation of the vasorelaxant effect of ANP and its inhibitory action on aldosterone secretion. The role of binding sites on platelets is unknown, but the availability of platelets makes them a useful model for investigating the regulation of receptors for atrial natriuretic factor in humans. The effect of sodium depletion and loading and mineralocorticoids on the density of rat vascular and adrenal sites for ANP was examined, as well as changes that occur after development of renovascular and DOCA-salt hypertension in rats. Sodium loading in the presence of reduced renal mass (unilateral nephrectomy) or mineralocorticoid administration produced renin suppression and resulted in down-regulation of vascular ANP receptors. In one-kidney, one-clip Goldblatt hypertensive rats and in DOCA-salt hypertensive rats, two models of volume-expanded, non-renin-dependent experimental hypertension, the density of ANP binding sites in the mesenteric arterioles was significantly decreased. The sensitivity to ANP of precontracted aorta from renovascular and mineralocorticoid hypertensive rats was significantly reduced. No consistent changes occurred in the density of ANP binding sites in the adrenal glomerulosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sodium depletion on peripheral vascular responses to heat stress in baboons.

The cutaneous vasodilation and renal vasoconstriction in baboons during environmental heating (EH) appear to be produced predominantly by sympathetic vasoconstrictor withdrawal and activation of the renin-angiotensin system, respectively. Since these mechanisms may be influenced differently by sodium depletion, this study examined the hypothesis that sodium depletion would have a differential effect on cutaneous and renal vascular responses to EH. Sodium depletion was produced in chronically instrumented baboons by placing them on low-salt intake for 8-19 days along with diuretic administration. EH consisted of exposing the baboon to an ambient temperature of 40-42 degrees C until core temperature (Tc) reached 39.8-40.0 degrees C. Both control plasma renin activity (PRA) and the rise in PRA with Tc during EH were considerably larger in sodium-depleted baboons. However, the magnitudes of the progressive increases in iliac vascular conductance (used as an index of hindlimb cutaneous vasodilation) and renal vascular resistance with rising Tc during EH were unaltered by sodium depletion. Therefore, neither cutaneous nor renal vascular responses to EH are influenced by elevated PRA and other changes accompanying sodium depletion in the baboon.

Effect of sodium depletion on pressor responsiveness in ACTH-induced hypertension in man.

1. Pressor responsiveness to angiotensin II (AII) and phenylephrine (PHE) was measured before and after 5 days of ACTH (1 mg i.m. daily) in six normal men maintained on low sodium diet (15 mmol daily). 2. ACTH caused a significant increase in systolic blood pressure of 9 mmHg. 3. There was no change in pressor responsiveness to AII with ACTH. 4. ACTH increased pressor responsiveness to PHE for both systolic and diastolic blood pressure, with a fall in the threshold for response from 0.9 to 0.6 micrograms/kg per min. 5. Increased pressor sensitivity to catecholamines during ACTH administration in man is not sodium-dependent.

The effect of sodium depletion and potassium loading on cortisol induced hypertension in sheep.

Glucocorticoid induced hypertension has been regarded as independent of sodium (Na), in contrast to mineralocorticoid induced hypertension, which is Na+-dependent. These studies compare the effect of Na+ depletion and potassium (K+) loading on glucocorticoid hypertension induced by cortisol in conscious sheep. Cortisol (480 mg/d) for 5 days, in sheep on a normal chaff diet (90-140 mmol/d Na+, 200-250 mmol/d K+) increased mean arterial pressure by 18 mmHg on day 5, increased plasma Na+ concentration, reduced plasma K+ concentration, and did not change urinary Na+ excretion. Following Na+ depletion (Na+ loss 603 +/- 49 mmol), cortisol increased mean arterial pressure from 70 +/- 1 mmHg to 76 +/- 3 mmHg on day 5 (P less than 0.001) and the increase in pressure was significantly less than the increase seen on the normal diet (P less than 0.05). Plasma Na+ increased and plasma K+ decreased. Urinary Na+ and K+ excretion was unchanged. KCl loading (700-900 mmol/day) for 10 days had no effect on the maximum rise in mean arterial pressure (+18 mmHg with cortisol in K+ loaded sheep). Plasma Na+ and K+ fell, and urinary Na+ excretion increased during the infusion. These studies show that Na+ depletion, but not KCl loading, reduced cortisol induced hypertension in sheep. These data show that glucocorticoid hypertension is not independent of Na+ status.

The effect of sodium depletion and potassium supplementation on vasopressin, renin and catecholamines in hypertensive men.

Seventeen 50-year-old hypertensive men (157 +/- 4/110 +/- 2 mmHg, mean +/- SE) were given low sodium diet for one week, which was supplemented with potassium the following week. The urinary Na+/K+ excretion ratio changed from 2:1 to 1:5 and 1:12, respectively, during dietary intervention. Arterial plasma vasopressin decreased by 3.4 +/- 1.7 ng/l (0.05 less than p less than 0.10) and urinary excretion of vasopressin was reduced by nearly 50% (p less than 0.001) during sodium depletion, while plasma noradrenaline increased by 38% (p less than 0.001) and plasma dopamine showed an increase by 58% (p less than 0.001). Plasma renin concentration increased four-fold during sodium depletion (p less than 0.001). With combined salt depletion and potassium supplementation, arterial plasma vasopressin decreased by 9.5 +/- 4.0 ng/l (p less than 0.05) compared to control. Urinary excretion of vasopressin together with plasma noradrenaline and dopamine were unchanged during the second week. The reduction of blood pressure was most marked during the first week (143 +/- 3/103 +/- 2 mmHg, p less than 0.05), but continued to fall also during the second week. Thus, during sodium restriction in middle-aged hypertensive men, blood pressure reduction occurs concomitantly with inhibited vasopressin release, despite enhanced renin and catecholamine release. Potassium supplementation during sodium restriction induces only minor changes in these variables.

Erythrocyte membrane transport in hypertensive humans and rats. Effect of sodium depletion and excess.

Erythrocyte membrane transport in hypertensive humans and rats. Effect of sodium depletion and excess.

Erythrocyte membrane transport in hypertensive humans and rats. Effect of sodium depletion and excess.

Sodium transport by erythrocyte membranes was studied in hypertensive and normotensive humans and in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The rate constants of sodium efflux were increased in both hypertensive humans and rats, and this increase was due mostly to an increase in the ouabain-resistant component of efflux. Both the furosemide-sensitive and furosemide-resistant components of efflux were increased. The ouabain-sensitive efflux was also increased, as confirmed by the ouabain-sensitive rubidium influx in rats. In rats, the intracellular sodium content was also increased in the SHR with respect to the WKY. The transport abnormalities of red cell membrane associated with hypertension were similar in humans and rats. In rats, sodium depletion failed to affect the transport abnormality, while sodium load made the difference in transport between SHR and WKY undetectable. Cross-incubation experiments, using plasma and erythrocytes of WKY and SHR, are more suggestive of a flux abnormality that is intrinsic to the cell membrane than of one that is humoral in nature.

Effect of sodium depletion on the release of [3H]norepinephrine from central and peripheral tissue of Wistar-Kyoto and spontaneously hypertensive rats.

To study the relationship between sodium intake, the sympathetic nervous system, and hypertension, we studied the effects of a 7-9 day dietary restriction of sodium in three different ages of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Field-stimulated [3H]norepinephrine ( [3H]NE) release was measured in portal vein, anterior hypothalamus, and the A2 region of the nucleus tractus solitarius (NTS) of 5- to 6-, 10- to 11-, and 28- to 30- week-old SHR and age-matched WKY. A low-sodium diet (0.05% Na+, control 0.5% Na+) significantly lowered stimulated [3H]NE release from portal vein and anterior hypothalamus in SHR and WKY at all three ages. However, release from the A2 region was not altered by sodium restriction. The results of the present study suggest that lowered dietary sodium can selectively alter norepinephrine release in both the peripheral and central sympathetic nervous system of SHR and WKY. The results also suggest that the SHR at 5-6 weeks are more sensitive to altered dietary sodium than are age-matched WKY.

Sodium depletion induces adrenergic potentiation at prejunctional site.

The objective of this study was to examine the effect of sodium depletion on basal sympathetic adrenergic vascular tone and vasoconstrictor responses to adrenergic nerve stimulation and norepinephrine in pentobarbital-anesthetized dogs. Mean arterial pressure, tibial blood flow, and tibial vascular resistance were not significantly different between sodium-replete and sodium-deplete dogs. However, plasma renin activity (PRA) was 11-fold higher in sodium-deplete dogs (P less than 0.01). Sympathetic denervation in sodium-replete and -deplete dogs resulted in a similar decrease in tibial vascular resistance. Nerve stimulation at 0.25 and 0.5 Hz caused 40 +/- 5 and 48 +/- 5% decreases, respectively, in tibial blood flow in sodium-replete dogs and 71 +/- 3 and 77 +/- 3% decreases, respectively, in sodium-deplete dogs (P less than 0.01). Responses to norepinephrine administered intra-arterially were similar in both groups. Neither captopril nor saralasin reduced the potentiated responses to nerve stimulation in sodium-deplete dogs. Bilateral nephrectomy 24 h prior to the experiment reduced PRA to an undetectable level in both sodium-replete and -deplete dogs and equalized the responses to nerve stimulation and norepinephrine in both groups. Because bilateral nephrectomy, but neither captopril nor saralasin, abolished the potentiated responses in sodium depletion, a renal hormone other than renin or vascular angiotensin II may be responsible for this prejunctional adrenergic potentiation.

The effect of sodium depletion on plasma norepinephrine kinetics in patients with essential hypertension.

The effects of sodium depletion on plasma norepinephrine (NE) kinetics were studied in 14 patients with essential hypertension using the steady state NE infusion technique on the theoretical basis of a two-compartmental open model. The calculated half time of rapid removal phase (t 1/2 alpha) of plasma NE was significantly delayed in the hypertensives (1.20 +/- 0.22 min, mean +/- SD) than in normal subjects (0.89 +/- 0.14) (p less than 0.01). The total clearance rates were comparable between the 2 groups (47 +/- 7 vs 48 +/- 11 ml/kg/min). The rates of the endogenous NE outflow into the circulation (RE-NE) were not significantly different between normal (5.29 +/- 1.68 ng/kg/min) and hypertensives (7.69 +/- 5.42), although 5 hypertensive patients (36%) showed an increased RE-NE above the normal range. The prolonged half time (t 1/2 alpha) in the hypertensives tended to be shortened after sodium depletion induced by either low sodium diet or diuretics (1.04 +/- 0.22, p less than 0.001). Both plasma NE and RE-NE were significantly increased after sodium depletion. A close correlation was observed between plasma NE and RE-NE (r = 0.907, p less than 0.001) in these subjects. These results suggest the slowed neuronal uptake of NE at the sympathetic nerve terminals in some patients with essential hypertension, occurring in part as a sodium-mediated consequence.

Evidence for the participation of sodium-sensitive pontine catecholaminergic neurons in the maintenance of DOCA-sodium hypertension in rats.

1. A reduced noradrenaline turnover rate has been previously demonstrated in the brain stem of deoxycorticosterone acetate (DOCA)-sodium hypertensive rats. In the present study, the turnover rate was measured in smaller brain regions and the effect of sodium depletion was studied on the turnover rate of these regions. 2. Catecholamine turnover rate was significantly reduced only in the pons, was slightly but not significantly reduced in the thoracic spinal cord and was normal in the upper and lower medulla oblongata, in the mesencephalon, in the hypothalamus and in the telencephalon. 3. The administration of a sodium-free diet for 3 weeks lowered significantly the blood pressure and concomitantly accelerated the turnover rate in the pons area of DOCA hypertensive rats. 4. It is concluded that pontine catecholaminergic fibres, sensitive to sodium balance, might be involved in the maintenance of DOCA-sodium hypertension in the rat.

Dissociated Renin – Aldosterone Response to Acute Sodium Depletion in Patients with a Previous History of Pregnancy – Associated Hypertension

The effect of sodium depletion on body weight, plasma renin activity (PRA) and plasma aldosterone concentration has been studied in 10 normotensive subjects with a previous history of pregnancy-associated hypertension. The results demonstrate an appropriate increase in PRA but a suppression of plasma aldosterone response to this form of acute sodium depletion. The significance of this dissociated renin-aldosterone response is discussed.

Sodium, potassium and water metabolism in the rabbit: the effect of sodium depletion and repletion.

1. Dietary sodium depletion and subsequent repletion was studied in rabbits. Potassium intake was maintained constant. 2. during sodium depletion and repletion blood pressure, packed cell volume, food consumption and body weight remained at control values. 3. Decreased sodium excretion was observed in both urine and faeces during sodium depletion and the physiological control of these changes is discussed in relation to the renin-angiotensin-aldosterone system. 4. Potassium excretion during sodium depletion initially fell as a result of reduced urine volume and gradually returned to normal. Urine potassium concentration remained constant. 5. Faecal excretion of potassium rose by 63% during sodium depletion and there was a rise from a control value of 17-25% in the proportion of total potassium excretion accounted for by the faecal component. 6. Water consumption and urine volume both decreased in the initial phase of sodium depletion and then returned to control levels. 7. It is important to consider both urinary and faecal excretion of sodium and potassium when calculating balance status for either ion. Faecal excretion, as well as kidney function, shows important physiological adaptations.

Effect of sodium depletion on the steroidogenic and pressor actions of angiotensin in the rat.

1. To investigate the relative roles of angiotensin II (AII) and des-Asp1-angiotensin II (angiotensin III) in the control of blood pressure and aldosterone release, the effects of seven angiotensin agonists on mean arterial blood pressure and serum aldosterone concentrations were compared in normal and sodium-depleted, conscious rats. 2. In normal rats, angiotensin I, α-Asp1-angiotensin II, β-Asp1-angiotensin II, and angiotensin II-amide were equipotent in elevating mean arterial blood pressure. Angiotensin III, des-Asp1-angiotensin I, and poly-O-acetylserine-angiotensin II were 25%, 25%, and 41% as potent as angiotensin II, respectively. After sodium depletion, pressor responses to these angiotensin peptides were reduced approximately 60–80% when compared with control responses. In contrast, pressor responses to noradrenaline were not significantly affected by sodium depletion. 3. Angiotensin II, β-Asp1-angiotensin II, angiotensin II-amide, and angiotensin III were equipotent in increasing serum aldosterone concentrations in normal animals. Angiotensin I was 59% and des-Asp1-angiotensin I only 5% as potent as angiotensin II in their abilities to release aldosterone. After sodium depletion, control serum aldosterone concentrations increased as did the slope of the dose—response curve for each angiotensin peptide. Angiotensin II was the most potent steroidogenic peptide in sodium-depleted rats with angiotensin III and β-Asp1-angiotensin II being 27%, angiotensin I 7%, angiotensin II-amide 3%, and des-Asp1-angiotensin I 1% as potent as angiotensin II in releasing aldosterone. Poly-O-acetylserine-angiotensin II has less steroidogenic effect than angiotensin II or III in both normal and sodium-depleted animals. 4. Infusions of the angiotensin II antagonist, Sar1-Ile8-angiotensin II, and the angiotensin III antagonist, Ile7-angiotensin III, enhanced aldosterone release in normal rats without altering blood pressure. After sodium depletion, Sar1-Ile8-angiotensin II decreased blood pressure without affecting aldosterone release whereas Ile7-angiotensin III diminished aldosterone release without altering blood pressure. 5. These data suggest that angiotensin II, independent of its conversion into angiotensin III, is an important regulator of steroidogenesis in the rat in normal sodium states. In sodium depletion, the octapeptide retains significant steroidogenic activity; however, the contribution of angiotensin III to its steroidogenic effects is increased.