Function and control of Na-K-ATPase in single nephron segments of the mammalian kidney.
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Adrenal Cortex Hormones↗
Biomedical subjects
A Doucet
Publications and source records attributed to A Doucet.
Sodium-independent in vitro induction of Na+,K+-ATPase by aldosterone in renal target cells: permissive effect of triiodothyronine.
The aim of this study was to develop an in vitro system in which we could study the causal relationship between short-term stimulation of Na+,K+-ATPase in the collecting tubule by aldosterone on the one hand and protein synthesis and changes in intracellular Na+ concentration on the other hand. Previous in vivo studies suggested that triiodothyronine might facilitate aldosterone-induced stimulation of Na+,K+-ATPase. Results show that when segments of cortical collecting tubules microdissected from collagenase-treated kidneys of adrenalectomized rats were incubated for 3 hr in the presence of either 10(-8) M aldosterone or 10(-8) M triiodothyronine alone Na+,K+-ATPase activity was not altered, whereas the addition of both hormones markedly stimulated the activity and the number of catalytic sites of Na+,K+-ATPase. This stimulation was abolished by actinomycin D and cycloheximide, whereas it was not altered in the absence of extracellular sodium or in the presence of the luminal Na+-channel blocker amiloride. Thus, triiodothyronine facilitates the in vitro induction of Na+,K+-ATPase synthesis by aldosterone. Aldosterone action on Na+,K+-ATPase is independent of Na+ availability.
Adrenalectomy↗
Aldosterone and sodium induce kidney Na-K-ATPase in vitro by two different mechanisms.
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Adrenalectomy↗
Effect of water intake on Na-K-ATPase in nephron segments of the desert rodent, Jaculus orientalis.
Na-K-ATPase activity was measured in individual pieces of nephron microdissected from collagenase-treated kidneys of jerboas, Jaculus orientalis. Na-K-ATPase activity was high in the distal convoluted tubule, intermediate in the thick ascending limb of the loop of Henle and low in the proximal and collecting tubule. When jerboas were adapted for several weeks to a hydrated diet and excreted a more diluted urine, Na-K-ATPase activity was altered in specific segments of the nephron: 1. In the proximal convoluted tubule, Na-K-ATPase activity decreased, especially in the juxtamedullary nephrons, suggesting that internephron heterogeneity was diminished; 2. In the medullary thick ascending limb, but not in the cortical portion, Na-K-ATPase activity decreased by 30%; 3. Na-K-ATPase was also diminished in the cortical collecting tubules (by 20%) but not in the medullary collecting tubule. Morphometric measurements also indicate that changes in Na-K-ATPase activity observed in the thick ascending limb are correlated to a cell atrophy, whereas in the collecting tubule, they occur independently of any visible morphological alteration. These differences in Na-K-ATPase activity are likely to be secondary to the changes in the plasma concentration of vasopressin previously described during such adaptation and to be involved in the control of water and sodium handling.
Animals↗
Mechanism of increased tubular Na-K-ATPase during streptozotocin-induced diabetes.
Since the mechanisms responsible for stimulation of kidney Na-K-ATPase during streptozotocin-induced diabetes are unknown, we studied the possible role(s) of kidney hyperfiltration and hypertrophy and of hyperaldosteronism on Na-K-ATPase induction. For this purpose, we studied the relationship between Na-K-ATPase activity in individual nephron segments and alterations of glomerular filtration rate during the early phase of diabetes. Within 2 days after streptozotocin administration, Na-K-ATPase activity markedly increased in the proximal convoluted tubule, medullary thick ascending limb and cortical and outer medullary collecting tubule, but not in the proximal straight tubule, cortical thick ascending limb and distal convoluted tubule. Streptozotocin administration also markedly enhanced the glomerular filtration rate but only after 4 days following initiation of treatment. Changes in Na-K-ATPase were specific since the activity of adenylate cyclase, another marker of basolateral membranes, was not altered. Finally, when animals were adrenalectomized prior to streptozotocin treatment, Na-K-ATPase stimulation was curtailed in the collecting tubule but not in more proximal segments. These results suggest that diabetes alters Na-K-ATPase activity in specific nephron segments independent of alterations of glomerular filtration rate and of kidney hypertrophy, and that the stimulation of collecting tubule Na-K-ATPase is secondary to hyperaldosteronism.
Adenylyl Cyclases↗
Alterations of enzymatic activities along rat collecting tubule in potassium depletion.
This study was designed to correlate morphological alterations induced in rat collecting tubule by potassium depletion with changes in the activity of enzymatic markers of the cell basolateral membrane. Results show the following responses. 1) Potassium depletion induced a huge and progressive hypertrophy of the outer medullary collecting tubule (MCT). Hypertrophy was paralleled by enhancements of vasopressin- and forskolin-dependent adenylate cyclase (AC) activities. Glucagon-sensitive AC was also increased, but with a different kinetics, whereas isoproterenol-dependent AC was only modestly stimulated. 2) In cortical (CCT) and papillary collecting tubules, AC response to hormones did not change. The concentrating defect of K-deprived rats, therefore, does not appear to result from an intrinsically defective adenylate cyclase system in any portion of the collecting tubule. Decreased AC response of the medullary thick ascending limb to vasopressin and glucagon, observed after 3-5 wk of K depletion, might account, at least in part, for reduced hypertonicity of medullary tissue. 3) Na+-K+-ATPase activity fell in CCT, probably in relation to decreased K secretion. Conversely, in MCT, Na+-K+-ATPase rose much more than tubular volume. The physiological significance of this latter observation remains to be established.
Adenylyl Cyclases↗
Characterization of K-ATPase activity in distal nephron: stimulation by potassium depletion.
Intercalated cells of the distal segments of the mammalian nephron are able to reabsorb K through an active mechanism, particularly during K depletion. However, the molecular basis of this transport is unknown. Therefore, we attempted to determine whether a K-ATPase similar to K-H-ATPase described in gastric mucosa and colon might be present in segments of the distal nephron and thereby account for active K reabsorption. K-stimulated ATPase activity was detected in microdissected segments of rabbit nephron: its activity was proportional to the density of intercalated cells, since it was highest in the connecting tubule, intermediate in the cortical collecting tubule, lowest in the outer medullary collecting tubule, and was not detectable in all other nephron segments. K-ATPase had a high affinity for K (Km approximately equal to 0.2-0.4 mM), was inhibited by vanadate and omeprazole, and was insensitive to ouabain, indicating that it is different from Na+-K+-ATPase but similar to K-H-ATPase. In the rat kidney, K-ATPase was also detected in the collecting tubule and its activity was markedly increased (+100-200%) following K depletion. This stimulation occurred before morphological alterations and might therefore be a primary event responsible for K conservation during K depletion. In summary, these results demonstrate the presence of a vanadate-sensitive, ouabain-insensitive K-ATPase activity in distal nephron segments of mammalian tubules. It is suggested that K-ATPase activity originates in intercalated cells where it might account, at least in part, for K reabsorption.
Adenosine Triphosphatases↗
Effect of adrenalectomy on NEM-sensitive ATPase along rat nephron and on urinary acidification.
An N-ethyl-maleimide (NEM)-sensitive ATPase that displays the properties of an electrogenic proton pump has been described in the different segments of the rat nephron where it mediates part of the active tubular proton secretion. Because corticosteroids are known to control kidney acidification, we evaluated whether or not NEM-sensitive ATPase is a target of corticosteroids in some nephron segments. For this purpose we measured NEM-sensitive ATPase activity in the different segments of nephron microdissected from normal and adrenalectomized rats. Results indicate that within 1 wk after adrenalectomy NEM-sensitive ATPase activity was markedly decreased in both cortical and outer medullary portions of the collecting tubule (cortex, from 398 +/- 12 (+/-SE) to 145 +/- 20; outer medulla, from 293 +/- 21 to 112 +/- 14 pmol X mm-1 X h-1); however, it was not altered in any other segment of the nephron. These results demonstrate that kidney NEM-sensitive ATPase is under the control of corticosteroids and suggest that mineralocorticoids rather than glucocorticoids are involved in this regulation that specifically occurs in mineralocorticoid-sensitive nephron segments. This paper also describes a new computerized method for the automatic determination of the length of single nephron segments.
Acid-Base Equilibrium↗
Triiodothyronine enhances renal response to aldosterone in the rabbit collecting tubule.
Since thyroid hormones and mineralocorticoids were observed to stimulate kidney Na-K-ATPase in similar sites and with similar time courses, this study was initiated to evaluate whether aldosterone is involved in the stimulation of Na-K-ATPase observed in collecting tubules 3 h after triiodothyronine (T3) administration to thyroidectomized (TX) rabbits. Results indicate that: Plasma aldosterone level decreased markedly in TX rabbits but was not restored 3 h after T3 injection; Early stimulation of Na-K-ATPase by T3 was abolished when plasma aldosterone level was suppressed by adrenalectomy or when aldosterone effects were blocked by spironolactone; Administration of aldosterone to TX rabbits mimicked the action of T3; Sensitivity of Na-K-ATPase to aldosterone markedly decreased after thyroidectomy. These results demonstrate an interaction between aldosterone and T3 in the control of Na-K-ATPase in the collecting tubule. Triiodothyronine enhances the sensitivity of Na-K-ATPase to aldosterone which, in turn, produces a stimulatory action despite the decreased plasma level observed during hypothyroidism.
Adrenalectomy↗
Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron.
This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.
Animals↗
Evidence for differences in the sensitivity to ouabain of NaK-ATPase along the nephrons of rabbit kidney.
To determine the possible intrarenal site of action of an endogenous ouabain-like natriuretic factor, we searched for the presence of NaK-ATPase highly sensitive to ouabain in the kidney, an organ previously reported to display a low sensitivity to ouabain. For this purpose, the sensitivity of NaK-ATPase to ouabain was determined at the level of single, well defined segments of nephron microdissected from rabbit kidney. Results indicated that NaK-ATPase activity is 10- to 30-fold more sensitive to ouabain in the collecting tubule, where final adjustments of sodium excretion take place, than in more proximal segments of the nephron. [3H]Ouabain binding experiments confirmed this finding as the affinity for ouabain increases from the proximal tubule to the collecting tubule. These results suggest that endogenous natriuretic factor may control sodium transport in the collecting tubule preferentially.
Animals↗
Kinetics of triiodothyronine action on Na-K-ATPase in single segments of rabbit nephron.
This study was initiated to define the dose- and time-dependence of triiodothyronine (T3) action on Na-K-ATPase in single microdissected nephron segments. For this purpose, the activity and the number of catalytic sites of Na-K-ATPase, as determined by the specific binding of 3H-ouabain, were measured following a single injection of T3 to rabbits thyroidectomized since 8-12 days. Triiodothyronine restored both the activity and the number of catalytic sites of Na-K-ATPase in a dose-dependent manner in all nephron segments where the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 micrograms/kg bw, T3 restored Na-K-ATPase activity and 3H-ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na-K-ATPase stimulation occurred after a 12 h period of latency and was completed within 24 h whereas in the collecting tubule, the stimulation was biphasic with a first increase within the first 3 h and a second increase concomitantly to that observed in the proximal tubule. These results indicate that thyroid hormones regulate Na-K-ATPase activity by altering the number of catalytic sites of the enzyme. This control depends on two different mechanisms which differ by their time-dependence.
Animals↗
Lack of stimulation of kidney Na-K-ATPase by thyroid hormones in long-term thyroidectomized rabbits.
The effects of long-term thyroidectomy and of subsequent triiodothyronine administration on kidney Na-K-ATPase were studied at the level of single nephron segments and were compared to the short-term effects previously reported. After 8-11 weeks, thyroidectomy resulted in a marked decrease in Na-K-ATPase activity in all the segments of the rabbit nephron, the proximal tubule, the thick ascending limb of Henle's loop, the distal convoluted tubule and the collecting tubule. Within this delay, thyroidectomy also decreased the ouabain-insensitive Mg-ATPase activity, the basal and hormone-stimulated adenylate-cyclase activity, and the volume of tubular epithelium in all the segments where these parameters were measured. Administration of 50 micrograms/kg body weight triiodothyronine to 8-11 weeks thyroidectomized rabbits did not restore Na-K-ATPase activity in any nephron segment within 48 h. These observations are different from those reported in animals thyroidectomized only 1 week before study since, within this latter delay, thyroidectomy altered specifically Na-K-ATPase activity, this action was observed on the proximal and collecting tubules exclusively and, triiodothyronine administration corrected Na-K-ATPase alterations after 48 h. Results of the present study indicate that in the long term, thyroidectomy has a wide spectrum of renal effects which involves the whole nephron and most cellular functions. The tubular involution induced by long-term thyroidectomy is probably responsible for the inability of kidney cells to quickly increase their Na-K-ATPase activity in response to hormonal stimulation.
Adenylyl Cyclases↗
Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: localization along the nephron and effect of corticosteroids.
To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO3-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO3-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO3-ATPase in kidney membrane fractions. HCO3-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO3-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO3-ATPase to vanadate indicates that it belongs to the F0-F1 class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO3-ATPase activity at the sites of urinary acidification.
Adenosine Triphosphatases↗
In vitro stimulation of Na-K-ATPase in rat thick ascending limb by dexamethasone.
Dexamethasone has been reported to stimulate Na-K-ATPase activity in the medullary thick ascending limb of adrenalectomized animals within a few hours. The present study was aimed at characterizing the mechanism of this action by investigating the stimulatory effect of the hormone in vitro. Dexamethasone (10(-8) M) added in vitro to segments of the medullary thick ascending limb of Henle's loop, which were microdissected from adrenalectomized rats, restored in a dose-dependent manner the depressed Na-K-ATPase activity within one h of incubation. This stimulation of Na-K-ATPase was inhibited by cycloheximide and actinomycin D. Dexamethasone also stimulated the component of oxidative metabolism coupled to sodium transport. These results, which confirm previous in vivo observations, demonstrate that dexamethasone-induced stimulation of Na-K-ATPase is a direct tubular action of the hormone mediated by protein synthesis. They suggest that this short-term effect of dexamethasone corresponds to the stimulation of sodium reabsorption by the dilution segment.
Adrenalectomy↗
Hormonal control of kidney functions at the cell level.
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Adenylyl Cyclases↗
Influence of chronic ADH treatment on adenylate cyclase and ATPase activity in distal nephron segments of diabetes insipidus Brattleboro rats.
The medullary thick ascending limb (MAL), but not the medullary collecting tubule (MCT), has been shown to have an impaired adenylate cyclase (AC) responsiveness to ADH and a selective hypoplasia in Brattleboro diabetes insipidus (DI) rats. Since chronic ADH administration has been found to increase epithelium volume and basolateral membrane surface area in MAL but not in MCT, we investigated whether chronic ADH infusion would affect the hormone-sensitive AC and the Na-K-ATPase activity--two markers of the basolateral membrane--in single micro-dissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC responses to in vitro AVP and glucagon and Na-K-ATPase activity increased to the same extent as did epithelium volume (60-80%); 2. changes in the other segments were independent of any morphological alteration. In the cortical thick ascending limb, AVP and glucagon-sensitive AC decreased by 30-40% whereas Na-K-ATPase activity did not change. In the collecting tubule, AC response to in vitro AVP was not altered by ADH-treatment but glucagon-sensitive AC dropped by 50% and Na-K-ATPase activity doubled, independently of any variation in plasma aldosterone and glucagon levels. These results show that, in the MAL, the ADH-induced variations in enzyme activity are a reflection of the enlargement of the basolateral membrane surface area. Further studies are needed to clarify the origin of enzymatic alterations in the other segments.
Adenosine Triphosphatases↗
Sites of thyroid hormone action on Na-K-ATPase along the rabbit nephron.
Although Ismail-Beigi and Edelman demonstrated in 1971 that thyroid hormones control the activity of Na-K-ATPase in the mammalian kidney, the actual site of this regulation inside the organ was not located. We therefore decided to study the relationship between thyroid hormones and Na-K-ATPase activity in individual nephron segments obtained by microdissection of collagenase-treated rabbit kidneys. For this purpose, the changes in the activity and number of catalytic sites of Na-K-ATPase in response to thyroidectomy or triiodothyronine administration were examined. Eight to 12 days after thyroidectomy, Na-K-ATPase activity had dropped by 40 to 80% in the convoluted and straight portions of the proximal tubules, and in the cortical and outer medullary collecting tubules, but not in the thick ascending limbs of Henle's loops or distal convoluted tubules. The apparent number of catalytic sites for Na-K-ATPase, as measured by specific binding of 3H-ouabain, decreased in parallel with Na-K-ATPase activity, and therefore this enzyme's specific activity was not altered. Fourty eight hours after injection of thyroidectomized animals with a single dose of either 100 or 500 micrograms/kg triiodothyronine, Na-K-ATPase activity in target segments was restored to the level measured in control animals. These effects of thyroid hormone were specific for Na-K-ATPase, since the activity of adenylate cyclase, another marker of the basolateral membrane, was not altered by thyroidectomy. The results obtained indicate that triiodothyronine controls Na-K-ATPase activity in specific nephron segments, by altering the number of this enzyme's catalytic sites.
Adenylyl Cyclases↗