A. Doucet

Short-term effect of aldosterone on renal sodium transport and tubular Na-K-ATPase in the rat

The short-term effect of one single injection of aldosterone on the renal sodium transport on one hand, and the Na−K-ATPase activity on the other hand, was studied in chronic adrenalectomized rats. Sodium transport was estimated by clearances, and Na−K-ATPase was measured in microdissected fragments of the nephron, according to our microtechnique previously described. Five to eight days after adrenalectomy, only 30% of the initial enzyme activity was recovered in the cortical collecting tubule (CCT). Administration of aldosterone completely restored the ATP-ase activity within three hours. Adrenalectomy also curtailed by 20–45% the activity of other nephron segments but aldosterone had no stimulatory effect on them. Sodium-reabsorption also increased after the hormone injection, following the same time (0.5<t1/2<1 h) and dose dependencies (0.8<K1/2<0.9 μg/kg) as those observed for the enzyme activity in the CCT. It is concluded that the short-term stimulation of Na−K-ATPase in the collecting tubule, after an acute administration of aldosterone, may be responsible for the simultaneous increase in sodium transport.

Short-term effects of aldosterone and dexamethasone on Na−K-ATPase along the rabbit nephron

Both glucocorticoids and mineralcorticoids stimulate the renal Na−K-ATPase. However, the exact site of their respective action is not precisely determined and it is still unknown wheter these effects are cumulative or not.We studied the effects of dexamethasone and aldosterone on Na−K-ATPase activity in microdissected nephron segments from adrenalectomized rabbits. In proximal convoluted tubule (PCT) the enzyme activity was altered neither by adrenalectomy nor by any steroid replacement. In the medullary thick ascending limb of the loop of Henle (MAL) and the distal convoluted tubule (DCT), Na−K-ATPase activity decreased by 40% after adrenalectomy, and was restored to control level three hours after administration of dexamethasone (100 μg/kg) but not by aldosterone (up to 10 μg/kg). In the cortical (CCT) and medullary (MCT) collecting tubule the enzyme activity decreased by 75% after adrenalcetomy but in contrast with the MAL and the DCT, these two segments were sensitive to both dexamethasone (100 μg/kg) and aldosterone (10 μg/kg) and recovered their activities within 3 h after the hormone injection. These effects were not additive. Spironolactone (100 μg/kg) abolished the action of each of the two hormones on the CCT and MCT. In contrast, spironolactone did not curtail the effect of dexamethasone on MAL and DCT. These results indicate that whereas glucocorticoid action is localized in MAL, DCT, CCT and MCT, the mineralocorticoid effect is restricted to the CCT and MCT exclusively. They also suggest that, in the CCT and MCT, the two types of hormones share the same receptors.

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.

Specific activity of Na−K-ATPase after adrenalectomy and hormone replacement along the rabbit nephron

Both aldosterone and dexamethasone are known to stimulate renal Na−K-ATPase activity although their action is restricted to specific nephron segments: the collecting tubule, the target site for mineralocorticoids, and the thick ascending limb and distal convoluted tubule, the target sites for glucocorticoids. As this stimulation by corticosteroids is very fast, we attempted to establish whether it occurs through de novo synthesis of new Na−K-ATPase units or by increasing the specific activity of the Na−K-ATPase units already present.For this purpose we studied the effects of aldosterone and dexamethasone on Na−K-ATPase specific activity in microdissected nephron segments from adrenalectomized rabbits. This specific activity was determined by the ratio of ATPase activity over the apparent number of catalytic units, as measured by specific3H ouabain binding. In the proximal tubule, neither adrenalectomy nor steroid replacement altered Na−K-ATPase activity or the apparent number of catalytic sites. In other nephron segments, adrenalectomy reduced Na−K-ATPase activity and specific3H ouabain binding concomitantly, and therefore left this enzymes specific activity unaltered. In the cortical and outer medullary collecting tubules, 10 μg/kg aldosterone simultaneously restored both the activity and apparent number of catalytic units of Na−K-ATPase to their control levels, and therefore did not modify the specific activity of the pump. Conversely, 100 μg/kg dexamethasone increased Na−K-ATPase activity in the thick ascending limb and distal convoluted tubule without changing the apparent number of catalytic units. These results indicate that both mineraloand glucocorticoids control kidney Na−K-ATPase activity, but at different sites and by different mechanisms: Aldosterone induces appearance of new catalytic units in the collecting tubule, whereas dexamethasone increases the specific activity of pre-existing units in the thick ascending limb and distal convoluted tubule.

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 of3H-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 were the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 μg/kg bw, T3 restored Na−K-ATPase activity and3H-ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na−K-ATPase stimualtion 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 timedependence.

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 microdissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC resposes 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 glucagonsensitive 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.

Presence of two isoforms of Na, K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the α1 isoform of the catalytic subunit, whereas the collecting duct expresses an α3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 ≈ 5.10−6 M) which is recognized by an anti-α3 antibody and another moiety of lower affinity for ouabain (IC50 ≈ 5.10−4 M) which is recognized by an anti-α1 antibody. Whether these two subpopulations correspond to different isoforms of the α subunit of Na,K-ATPase (α1 and α3-like) remains to be determined.

REGULATION OF RENAL NA+,K+-ATPASE IN RAT THICK ASCENDING LIMB DURING K+ DEPLETION : EVIDENCE FOR MODULATION OF NA+ AFFINITY

1. NaCl reabsorption along the loop of Henle is reduced in K(+)‐depleted rats. Because Na+,K(+)‐ATPase energizes this transport and because K+ depletion is known to induce an upregulation of Na+,K(+)‐ATPase in most tissues, the regulation of this enzyme was investigated at the level of single thick ascending limbs of the loop of Henle freshly microdissected from rats fed either a normal (control rats) or a low‐K+ diet (LK rats). 2. Within 2 weeks of K+ depletion, Na+,K(+)‐ATPase activity and [3H]ouabain binding were increased by 30‐50% in the medullary portion of the thick ascending limb (MTAL). 3. Despite this increase in the number of Na+,K(+)‐ATPase units, the transport capacity of the Na+,K+ pump, determined by ouabain‐sensitive Rb+ uptake in the presence of an extracellular concentration of Rb+ mimicking the kalaemia determined in control (4.0 mM Rb+) and LK rats (2.3 mM Rb+), was reduced in MTAL from LK rats. 4. Inhibition of the Na+,K+ pump was not accounted for by changes in either extracellular K+ or intracellular Na+ concentrations, but by a decrease in the pump affinity for Na+. 5. Because this change in the apparent affinity of the Na+,K+ pump for Na+ was detectable in intact but not in permeabilized MTAL cells, it is probably induced by a rapidly reversible cytosolic factor.

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 Henles loops or distal convoluted tubules. The apparent number of catalytic sites for Na−K-ATPase, as measured by specific binding of3H-ouabain, decreased in parallel with Na−K-ATPase activity, and therefore this enzymes specific activity was not altered. Fourty eight hours after injection of thyroidectomized animals with a single dose of either 100 or 500 μg/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 enzymes catalytic sites.

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.