Serial microanalysis of renal transcriptomes.

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1, 000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.

BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.

Rat G protein-coupled receptor kinase GRK4: identification, functional expression, and differential tissue distribution of two splice variants.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round spermatids. GRK4B mRNA is poorly expressed in testis and most rat tissues but is heterogeneously distributed in the kidney, with 20-fold enrichment in the outer medulla. GRKs4A and B are both functional protein kinases, as demonstrated in a rhodopsin phosphorylation assay. The differential tissue distribution of GRKA4 and GRK4B suggests that individual GRK4 variants may serve distinct physiological functions.

Regulation of Na+, K(+)-ATPase in the rat outer medullary collecting duct during potassium depletion.

Because in outer medullary collecting ducts (OMCD) of K(+)-depleted rats, K+ secretion is abolished, whereas Na+, K(+)-ATPase, which energizes this secretion, is markedly stimulated, it has been proposed that Na+, K(+)-ATPase was mislocated to the apical cell membrane and energized K+ reabsorption. This hypothesis has been supported by paradoxical effects of ouabain in K(+)-depleted compared with normal rats. However, we have recently shown that ouabain inhibits not only Na+, K(+)-ATPase but also apical H+, K(+)-ATPase in the OMCD of K(+)-depleted rats. Therefore, this study was designed to evaluate whether previous observations were accounted for by Na+, K(+)-ATPase or by ouabain-sensitive H+, K(+)-ATPase. Na+, K(+)-ATPase was distinguished from H+, K(+)-ATPase by its insensitivity to Sch-28080. Results indicate that the hydrolytic and transport activities of Na+, K(+)-ATPase, the number of its functional units, and the expression of mRNA of its alpha 1 and beta 1 subunits were increased threefold or more in the OMCD of rats fed a K(+)-depleted diet for 2 wk. By immunofluorescence, Na+, K(+)-ATPase staining was strongly increased in K(+)-depleted rats but remained localized to the basolateral pole of OMCD principal cells. In conclusion, K+ depletion is associated with marked induction of functional Na+, K+ pumps at the basolateral pole of rat OMCD. Therefore, reduced K+ secretion might result from inhibition of apical K+ conductances and stimulation of basolateral K+ recycling. It is proposed that increased Na+, K(+)-ATPase participates in the increased Na+ reabsorption prevailing in collecting ducts of K(+)-depleted rats.

Cold- and ouabain-resistance of renal Na,K-ATPase in cold-exposed and hibernating jerboas (Jaculus orientalis).

The temperature dependence and the ouabain sensitivity of Na,K-ATPase was examined in the nephron of normal, cold-exposed, and hibernating jerboas. The transport and hydrolytic activity of renal Na,K-ATPase displayed similar temperature dependence in rats and normal jerboas. Cold-resistance of Na,K-ATPase appeared in cold-exposed jerboas and further increased during hibernation. Three subpopulations of Na,K-ATPase displaying very high (Ki approximately 10(-13) M), high (Ki approximately 10(-9) M) and low sensitivity to ouabain (Ki approximately 10(-6) M) were detected in the thick ascending limb and collecting duct of jerboas. In thick ascending limbs, the subpopulation of very high sensitivity to ouabain disappeared in cold-exposed animals, which accounted for the previously reported decrease in Na,K-ATPase activity. In collecting ducts of cold-exposed animals, the subpopulation of very high sensitivity to ouabain also disappeared, but the resulting decrease in activity was overbalanced by the appearance of the subpopulation of high sensitivity.

Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin.

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1). GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 micrograms protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 micrograms protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 +/- SEM; control: 23.8 +/- 1.8; GLP, 88 micrograms protein/ml: 8.2 +/- 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

Re-evaluation of the expression of the gastric H,K-ATPase alpha subunit along the rat nephron.

Under normal conditions, the rat collecting duct displays an H, K-ATPase activity with kinetic and pharmacological properties very close to those of the gastric H,K-ATPase. However, whether the collecting duct H,K-ATPase and the gastric enzyme are identical remains controversial. Therefore, we re-evaluated the expression of the mRNAs encoding the gastric H,K-ATPase alpha subunit in the rat nephron. For this purpose, gastric H,K-ATPase mRNAs were quantitated by RT-PCR at the level of microdissected nephron segments using known amounts of gastric H,K-ATPase cRNA as external standards. Results indicate that gastric H,K-ATPase mRNAs are undetectable (<1 copy per cell) in the glomerulus and along the proximal tubule, thick ascending limb of Henle's loop and collecting duct, although a faint expression ( approximately 400 copies per micro;g total RNA) is measurable in whole-kidney preparations. Gastric H,K-ATPase mRNA is also absent along the nephron of K-depleted rats and of rats with chronic metabolic acidosis and alkalosis. Taken with other data from the literature, these results suggest that the collecting duct of normal rats might express an H,K-ATPase similar, but not identical, to the gastric isoform.

Na, K-ATPase: a molecular target for Leptospira interrogans endotoxin

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1) GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC5O: 120-220 mug protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 mug protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ñ SEM; control: 23.8 ñ 1.8; GLP, 88 mug protein/ml: 8.2 ñ 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis.

K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct.

Two distinct Sch-28080-sensitive K-adenosine triphosphatases (K-ATPases) were previously described in the rat nephron: a ouabain-resistant K-ATPase (type I) present in collecting ducts (CD) and a ouabain-sensitive from (type II) located in proximal tubules (PT) and thick ascending limbs (TAL). In K-depleted rats, K-ATPase activity is increased in CD, whereas it is reduced in PT and TAL. Because expression of colonic H-K-ATPase is restricted to the CD of K-depleted rats, we hypothesized that K-ATPase from the CD of K-depleted rats might be different from types I and II. Indeed, type III K-ATPase displays higher sensitivities to ouabain and to Sch-28080 than type II, a lower sensitivity to Sch-28080 than type I, and, conversely to types I and II, it can be stimulated by Na+. Pharmacological differences between types II and III K-ATPases were confirmed by [3H]ouabain binding experiments. Thus the rat kidney expresses three K-ATPases that differ by their pharmacological and kinetic properties, their distribution profile along the nephron and their behavior during K depletion.

Na-K-ATPase along rat nephron after subtotal nephrectomy: effect of enalapril.

Tubular overwork is thought to be a promoter of the tubular hypertrophy and renal failure that occur in response to renal mass reduction. Because Na-K-adenosinetriphosphatase (Na-K-ATPase) is an index of tubular work, we evaluated the effects of subtotal nephrectomy and of enalapril therapy, which delays the evolution of renal lesions, on tubular hypertrophy and Na-K-ATPase activity along the rat nephron. Within 6 wk, 70% reduction of renal mass engendered hypertrophy of the proximal convoluted tubule (PCT), thick ascending limb (TAL), and collecting duct (CD), as well as parallel increments in Na-K-ATPase activity per millimeter tubule length (Na-K-ATPase activity per unit surface area was not modified by subtotal nephrectomy). Chronic enalapril therapy prevented part of the hypertrophy (but not Na-K-ATPase stimulation) of the PCT and the whole stimulation of Na-K-ATPase (but not hypertrophy) in the CD, whereas it had no effect on the TAL. Enalapril effect on Na-K-ATPase in CD might result from reduced bradykinin metabolism, as the reduction in urinary excretion of bradykinin observed in subtotally nephrectomized rats was prevented by enalapril therapy.

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.

Effects of cold exposure and hibernation on renal Na,K-ATPase of the jerboa Jaculus orientalis.

Changes in activity and abundance of renal Na,K-ATPase were evaluated during cold exposure and hibernation of the jerboa Jaculus orientalis by measuring the hydrolytic activity, the number of units and the transport activity of Na,K-ATPase in isolated nephron segments. As compared to controls, jerboas exposed to cold (6 degrees C) for 4-5 weeks displayed mild diuresis, decreased urinary osmolality and increased kaliuresis. In cold-exposed jerboas, Na,K-ATPase hydrolytic activity was reduced in the medullary thick ascending limb and enhanced in the cortical and outer medullary collecting duct, whereas it was not altered in other nephron segments. The number of Na,K-ATPase units and the activity of Na,K-pump, determined by [3H]-ouabain binding and by ouabain-sensitive rubidium uptake respectively, changed in parallel with the hydrolytic activity in the medullary thick ascending limb and cortical collecting duct. The maximal rate of activity (Vmax) of Na,K-ATPase was not modified further during hibernation. Thus, cold exposure, but not the onset of hibernation, induces segment-specific changes in the abundance and activity of Na,K-ATPase units which are likely to be related to the entry into hibernation, but not to the maintenance of some renal functions during deep hibernation.

Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion.

Because a ouabain-sensitive H-K-adenosinetriphosphatase (H-K-ATPase) has been identified recently in the amphibian bladder, we evaluated whether such an ATPase might exist also in the mammalian kidney, along with the ouabain-insensitive H-K-ATPase previously described in the collecting duct. For this purpose, we searched for an Na-independent, K-stimulated, ouabain- and Sch-28080-inhibitable ATPase activity in single segments of rat nephron. Ouabain-sensitive K-stimulated ATPase activity was detected in the absence of Na+ in rat proximal convoluted and straight tubules and in medullary and cortical thick ascending limbs of Henle's loop but not in collecting ducts. This K-ATPase differs from Na-K-ATPase by 1) its absence of requirement for Na, 2) its sensitivity to Sch-28080, 3) its higher sensitivity to ouabain, and 4) its absence in the collecting duct. It differs from the collecting duct H-K-ATPase by 1) its distribution along the nephron, 2) its sensitivity to ouabain, and 3) its lower sensitivity to Sch-28080. Furthermore, in rats fed a K-depleted diet for 2 wk, ouabain-sensitive K-ATPase activity was markedly reduced in both proximal tubules and thick ascending limbs, whereas collecting duct H-K-ATPase was upregulated.

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 alpha 1 isoform of the catalytic subunit, whereas the collecting duct expresses an alpha 3-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 approximately 5.10(-6) M) which is recognized by an anti-alpha 3 antibody and another moiety of lower affinity for ouabain (IC50 approximately 5.10(-4) M) which is recognized by an anti-alpha 1 antibody. Whether these two subpopulations correspond to different isoforms of the alpha subunit of Na,K-ATPase (alpha 1 and alpha 3-like) remains to be determined.

Inhibition of Na,K-ATPase by an endotoxin extracted from Leptospira interrogans: a possible mechanism for the physiopathology of leptospirosis.

Clinical manifestations of leptospirosis include disorders of the electrolytical balance which might be related to inhibition of Na,K-ATPase. Although the physiopathological cellular mechanism of leptospirosis remains unknown, a bacterial endotoxin has been incriminated. Therefore, we evaluated whether a glycolipoprotein fraction extracted from Leptospira interrogans and known to be cytotoxic might inhibit Na,K-ATPase. This glycolipoprotein fraction (GLP) inhibited Na,K-ATPase activity in rabbit kidney epithelial cells as well as Na,K-ATPase purified from rabbit kidney medulla. Inhibition was dose-dependent, and at maximum it almost abolished Na,K-ATPase activity whereas it had no effect on other enzymes. The GLP did not change the apparent affinity of Na,K-ATPase for potassium whereas it increased that for sodium, revealing a mechanism of inhibition different from that of ouabain. Finally, the inhibitory principle present in the GLP preparation was thermostable and was curtailed by the presence of albumin. In conclusion, a glycolipoproteic fraction extracted from Leptospira interrogans contains a specific inhibitor of Na,K-ATPase. This glycolipoproteic fraction which is present in diseased tissues might induce, through this inhibitor, cellular dysfunctions responsible for the symptoms, in particular those associated with electrolytical disorders such as disturbances of renal electrolyte handling, cardiac arrhythmia or diarrhoea.

Insulin unresponsiveness of tubular monovalent cation transport during fructose-induced hypertension in rats.

1. Hyperinsulinaemia is considered to be a pathogenic factor for human and experimental hypertension. Thus, the contribution of the known insulin-stimulated tubular sodium reabsorption to this aetiological process has to be discussed. 2. Rats fed a fructose-enriched diet develop hyperinsulinaemia and hypertension, providing a model for studying the regulation of the tubular sodium handling and its possible relationship to hypertension. For this purpose, the sodium transport capacity of isolated nephron segments from control rats and from rats fed a fructose-enriched diet was investigated by measurement of ouabain-sensitive 86Rb uptake and of the hydrolytic activity of Na,K-ATPase. The number and affinity of insulin receptors were estimated from the specific [125I]insulin binding. 3. In rats fed a fructose-enriched diet, mild hypertension developed during the 14-day fructose diet. There were no differences, along the nephron, in basal 86Rb uptakes and ATPase activities between control rats and fructose-induced hypertensive rats. In control rats, insulin stimulated 86Rb uptake in the proximal convoluted tubule and cortical collecting duct, but exhibited an inhibitory action in the medullary thick ascending limb. In contrast, in fructose-induced hypertensive rats, 86Rb influx remained unresponsive to insulin concentrations ranging from 10(-11) to 10(-7) mol/l in the proximal convoluted tubule and cortical collecting duct. In the medullary thick ascending limb, the threshold of inhibition was displaced from 10(-11) mol/l up to 10(-7) mol/l. Insulin binding to the proximal convoluted tubule, medullary thick ascending limb and collecting duct were similar in control rats and in rats fed a fructose-enriched diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Are there several isoforms of Na,K-ATPase alpha subunit in the rabbit kidney?

Previous pharmacologic and kinetic studies have demonstrated the axial heterogeneity of the rabbit kidney tubule with regard to Na,K-ATPase. To evaluate whether this heterogeneity might reflect the presence of distinct isoforms of the alpha subunit of Na,K-ATPase, we used two monoclonal antibodies, IIC9 and IIE2 (G8), specific for the alpha 1 and alpha 3 isoforms, respectively, as probes for changes in the specific activity of Na,K-ATPase at the level of successive segments of the rabbit nephron. Single, well defined nephron segments were obtained by microdissection of collagenase-treated kidney. Results indicate that IIC9 antibody inhibited Na,K-ATPase activity by > 90% in all the segments of the nephron except the collecting duct. Conversely, IIE2 (G8) antibody abolished Na,K-ATPase activity in the collecting duct, whereas it had no effect in other nephron segments. These findings suggest that the rabbit collecting duct preferentially expresses a distinct isoform of Na,K-ATPase catalytic subunit, which is presumably alpha 3-like, in agreement with previous pharmacologic and kinetic observations, whereas other nephron segments would express the alpha 1 isoform.

Short-term effect of aldosterone on vasopressin-sensitive adenylate cyclase in rat collecting tubule.

Because previous studies indicated that, in the rat collecting tubule, vasopressin (AVP)-sensitive adenylate cyclase (AC) is controlled by mineralocorticoids in the long term, the present study was designed to investigate whether such a control also exists in the short term. Therefore, we investigated the in vivo and in vitro effects of aldosterone on AC activity in cortical and outer medullary collecting tubules (CCD and OMCD, respectively) from adrenalectomized rats. Injection of aldosterone (10 micrograms/kg body wt) to adrenalectomized rats restored within 3 h AVP-sensitive AC activity in the CCD and OMCD up to the levels observed in the corresponding segments of adrenal intact rats. Similarly, incubating CCD or OMCD from adrenalectomized rats for 2.5 h in the presence of 10(-8) M aldosterone enhanced AVP-sensitive AC activity up to values similar to those found in normal rats. In vitro stimulation of AVP-sensitive AC activity was dose dependent with regard to aldosterone [apparent affinity constant (K0.5) approximately 10(-9) M], appeared after a 30-min lag period, and reached its maximum after 2-2.5 h. In addition, it was totally abolished by the antimineralocorticoid spironolactone, whereas the specific glucocorticoid antagonist RU 38486 had no effect. Finally, actinomycin D and cycloheximide totally abolished the in vitro action of aldosterone, demonstrating the involvement of protein synthesis in that process.

Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome.

The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.