Kidneys Of Adrenalectomized Rats Research Articles

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney.

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.

Aldosterone-Induced Serum and Glucocorticoid-Induced Kinase 1 Expression Is Accompanied by Nedd4-2 Phosphorylation and Increased Na+ Transport in Cortical Collecting Duct Cells

Aldosterone plays a central role in Na+ homeostasis by controlling Na+ reabsorption in the aldosterone-sensitive distal nephron involving the epithelial Na+ channel (ENaC). Part of the effects of aldosterone is mediated by serum and glucocorticoid-induced kinase 1 (Sgk1), a Ser/Thr kinase whose expression is rapidly induced by aldosterone and that increases in heterologous expression systems ENaC cell surface abundance and activity. Previous work in Xenopus laevis oocytes suggested that Sgk1 phosphorylates specific residues (Ser212 and Ser328) on the ubiquitin-protein ligase Nedd4-2, an enzyme that directly interacts with ENaC and negatively controls channel density at the plasma membrane. It further indicated that phosphorylation of Nedd4-2 led to impairment of ENaC/Nedd4-2 interaction and consequently to more channels at the cell surface. These data suggested a novel mode of aldosterone-dependent action, yet this was not demonstrated formally in epithelial cells that physiologically express ENaC. Here it is shown, with the use of an anti-phospho-Ser328-mNedd4-2 antibody, that 2 to 6 h of aldosterone treatment induces an increase in Nedd4-2 phosphorylation, both in a mouse cortical collecting duct cell line (mpkCCDcl4) and in kidneys of adrenalectomized rats. This augmentation, which is accompanied by a raise in Sgk1 expression and transepithelial Na+ transport, is sensitive to phosphatidylinositol-3 kinase inhibition, as is Sgk1 phosphorylation and Na+ transport. Hence, these data provide evidence in cortical collecting duct cells in vitro and in vivo that Sgk1-dependent phosphorylation of Nedd4-2 is part of the aldosterone response.

Differential Regulation of Angiotensin Peptides in Plasma and Kidney: Effects of Adrenalectomy and Estrogen Treatment

Eight angiotensin peptides [angiotensin-(1–7), angiotensin II, angiotensin-(1–9), angiotensin I, angiotensin-(2–7), angiotensin-(2–8), angiotensin-(2–9), and angiotensin-(2–10)] were measured in plasma and kidney of adrenalectomized rats and estrogen-treated rats. In comparison with sham-operated rats, adrenalectomy increased plasma renin levels by 50-fold and reduced plasma angiotensinogen levels by 67%. Adrenalectomy increased plasma angiotensin peptide levels by 9- to 30-fold, but the increases in renal angiotensin peptide levels were much less than those seen for plasma. In comparison with vehicle-treated rats, estrogen treatment increased plasma angiotensinogen levels by 3-fold and reduced plasma renin levels by 41%. Estrogen treatment decreased plasma angiotensin peptide levels, whereas renal angiotensin peptide levels increased by as much as 2- to 3-fold. These results confirm the differential regulation of angiotensin peptide levels in plasma and kidney, and provide further support for the essential role of angiotensinogen in modulating plasma and tissue angiotensin peptide levels.

Differences between aldosterone and its antagonists in binding kinetics and ligand-induced hsp90 release from mineralocorticosteroid receptor

We have previously reported that mineralocorticosteroid receptor (MR) is a 8–9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8–9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

ACTH immunoactivity in normal rat tissues: modulation by hypophysectomy and adrenalectomy.

This study investigates the effect of pertubation of the normal pituitary-adrenal axis on concentrations of adrenocorticotropin (ACTH)-like immunoactivity in peripheral tissues. We used a polyclonal antibody (West antibody) to measure ACTH-like immunoactivity in glacial acetic acid extracts of five tissues in adult male rats at increasing times (1, 7, 14, and 28 days) after hypophysectomy or adrenalectomy, and in normal control rats. Concentrations of ACTH-like immunoactivity were similar to those previously reported in liver, colon, heart, and small intestine and were not significantly affected by either hypophysectomy or adrenalectomy. While hypophysectomy also had no effect in the kidney, adrenalectomy resulted in a four-fold increase in extractable immunoactivity, first noticeable at seven days (p less than 0.005), but increasing progressively to 28 days (p less than 0.0005). Gel filtration showed that most of the increase in activity in kidneys of adrenalectomized rats corresponded to the 4.5 kD form comprising most of the serum ACTH immunoactivity and suggesting that the activity increase in kidney was largely due to ACTH derived from blood.

Mineralocorticoid and Renal Receptor Binding Activity of 21-Deoxyaldosterone

Since several aldosterone metabolites are known to be active, we have assessed the mineralocorticoid biological and renal receptor binding activities of the aldosterone metabolites, 21-deoxyaldosterone (21-deoxy-Aldo), 21-deoxytetrahydroaldosterone (21-deoxy-THAldo), and 3 alpha, 5 beta-tetrahydroaldosterone (THAldo). We synthesized these steroids by bioreduction of aldosterone with intestinal bacteria. Mineralocorticoid agonist activity of 21-deoxy-Aldo, 21-deoxy-THAldo and THAldo, determined by bioassay using adrenalectomized rats, was 1-5%, less than 0.01%, and 0.1-0.5% that of aldosterone, respectively. 21-Deoxy-Aldo showed no antagonist activity. The relative affinity in competing with [3H]aldosterone for binding to mineralocorticoid receptors in adrenalectomized rat kidney cytosols was 94%, less than 0.01%, and less than 0.01% that of aldosterone. The relative binding affinity for rat renal glucocorticoid receptors was 23%, less than 0.01%, and less than 0.01% that of dexamethasone, and for corticosteroid-binding globulin 17%, less than 0.01%, and less than 0.01% that of cortisol. These results show that the naturally occurring steroid, 21-deoxy-Aldo, possesses mineralocorticoid agonist activity which is equivalent to that of 11-deoxycorticosterone, and has substantial affinity for rat renal mineralocorticoid and glucocorticoid receptors. The results also implicate the pathophysiological role of 21-deoxy-Aldo as a potential mineralocorticoid in 21-hydroxylase deficiency, where urinary excretion of this steroid is invariably elevated.

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.

Increase in the formation of cyclooxygenase and lipoxygenase products in kidneys of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in rat kidneys after incubation with the Calcium-ionophore A23187 has been determined. The metabolites were isolated by HPLC. The main components formed are PGD2, PGE2, PGF22α LTB4, 5,12 di-HETE and 5-, 12- and 15-HETE. After adrenalectomy, an increase occurs in the formation of PG and LT, which is highest in that of PGD2 and 12-HETE. These effects are most probably related to a diminished formation or inactivation of lipocortin in adrenalectomized animals, a glucocorticosteroid induced peptide with phospholipase A2 inhibitory activity.

Effects of aldosterone on potassium recycling in the kidney of adrenalectomized rats.

Recent studies have suggested that potassium, like urea, undergoes medullary recycling. The present cortical and papillary micropuncture studies were designed to confirm the existence of medullary potassium recycling and to determine whether acute infusions of aldosterone affected this phenomenon. Thus, nephron segmental analysis of potassium and sodium transport was conducted in adrenalectomized Munich-Wistar rats and similarly prepared rats that received aldosterone acutely to achieve physiological blood levels. The clearance results demonstrated that aldosterone has an acute antinatriuretic and a kaliuretic effect, whereas the micropuncture studies demonstrated that 1) aldosterone increases potassium secretion between early and late distal tubule punctures; 2) aldosterone causes an increase in delivery of potassium to the papillary collecting duct; 3) aldosterone does not increase potassium secretion across the papillary collecting duct; and 4) aldosterone significantly increases medullary potassium recycling as evidenced by increased quantities of potassium present at the bend of the loop of Henle in response to aldosterone infusions. Thus, the studies confirm the existence of potassium recycling and suggest that this phenomenon is a feedback system that, in part, regulates urinary potassium excretion.

Mineralocorticoid receptors during normal kidney growth and compensatory renal hypertrophy

[ 3H]-Aldosterone and [ 3H]-dexamethasone binding characteristics in the kidney cytosol of adrenalectomized rats were assessed during normal growth and compensatory hypertrophy of the kidney. The nephrectomy was performed on one month old rats and the growth processes studied for four weeks. The models for the ligand interaction with the cytosolic receptors, the corresponding dissociation constants and the maximum number of sites for each class of receptor were all determined. The dissociation constants of aldosterone ( KD I ≈- 1.4 × 10 −9 mol/l, KD II ≅ 4.3 × 10 −8 mol/l) and dexamethasone ( KD ≅ 6.5 × 10 −9 mol/l) as well as the tissue concentrations of aldosterone binding sites (N 1 11 × 10 −13 mol/g tissue, N 2 ≅ 194 × 10 −13mol/g tissue) and dexamethasone binding sites (N ≅ 265 × 10 −13 mol/g tissue) remained unchanged during normal growth and compensatory renal hypertrophy and were found identical to those previously determined for the adult adrenalectomized rat kidney. These results indicate a correlation between the total number of binding sites and renal growth whether the growth is normal or related to a hypertrophy.

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney.

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.

Antihypertensive effect of riboflavin analogs in rats with mineralocorticoid-induced hypertension.

This study investigated whether the riboflavin analogs, 7,8-dimethyl-10-formylmethyl isoalloxazine (FMI) and 7,8-dimethyl-10-(2'-hydroxyethyl) isoalloxazine (HEI), are effective antihypertensive agents in mineralocorticoid-induced or deoxycorticosterone acetate (DOCA)-salt hypertension. These studies are based on our previous observation tht aldosterone enhances the biosynthesis of renal flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) from riboflavin, and that FMI and HEI competitively inhibit conversion of riboflavin to FMN and reabsorption of Na+ in the kidney of adrenalectomized rats. When 1.6 mg of FMI or HEI were administered simultaneously with 3.0 mg of DOCA, the tail systolic blood pressure (SBP) of unanesthetized rats rose only to 136 +/- 5 mm Hg (standard error of the mean, SEM) compared to 163 +/- 5 mm Hg during DOCA therapy alone (p less than 0.0005). This hypotensive effect of FMI or HEI was noted after the fourth week of treatment and persisted through the ninth week. The rats tolerated the medication well and had no signs of riboflavin deficiency. DOCA administration alone resulted in a 24% increase in iliopsoas muscle Na+ concentration (p less than 0.0005), and a 0.8% increase in the water content of the muscle (p less than 0.025), suggesting a positive Na+ balance. Administration of FMI or HEI blunted the ability of DOCA to increase muscle Na+ concentration (p less than 0.025), water content (p less than 0.01). HEI treatment of the Kyoto strain of spontaneously hypertensive rats (SHR) did not lower their mean SBP. Thus it appears that the hypotensive actions of FMI or HEI are closely associated with their ability to modify the effects of mineralocorticoids on NA+ balance.

Adrenalectomy alters acid-soluble leucine pool in rat kidney and heart

1. 1. After a continuous infusion of [ 3H]leucine into conscious rats the 3H count in the acid soluble extract was significantly higher in kidney, heart and liver of adrenalectomized rats than in the controls. However, the 3H counts remaining as [ 3H]leucine were similarly low in all three tissues from both groups of animals. 2. 2. Application of the 14C-dansylation method revealed that the specific activity (dpm/nmol) of leucine in the tissue extracts of adrenalectomized rats decreased by 30% in kidney and liver and by 12% in heart. due to corresponding increases in the free leucine pools. 3. 3. Adrenalectomy also increased the isoleucine and valine content in kidney and liver and lysine and proline in the liver, but decreased the glycine content in kidney. 4. 4. Aldosterone treatment of rats had no effect on any of the changes caused by adrenalectomy.

Aldosterone Effects on Renal Function in the Isolated Perfused Rat Kidney

The excretion of potassium and the reabsorption of sodium were studied in isolated perfused kidneys of adrenalectomized rats following the administration of aldosterone. Aldosterone (10<sup>-7</sup><i>M) </i>produced a rapid increase in fractional excretion of potassium, without affecting glomerular filtration rate, urine flow rate or sodium reabsorption. Spironolactone (10<sup>-4</sup><i>M) </i>completely abolished the kaliuretic effect of aldosterone, while actinomycin D had no inhibitory effect. In the absence of exogenous metabolic substrate, enhancement of potassium excretion was not seen following aldosterone. With either glucose, pyruvate, lactate or α-ketoglutarate in the perfusion medium, the kaliuretic effect of aldosterone was restored. The results confirm the direct action of aldosterone on renal potassium excretion and its dependency upon the availability of specific metabolic substrates.

Amplification of the action of aldosterone by 5 alpha-dihydrocortisol.

Elevated conversion rates of cortisol to 5α-dihydrocortisol (5α-DHF) have been found in a child with hypoaldosteronism, Na+ retention, hypokalemic alkalosis, and hypertension. Studies investigating possible etiological roles for 5α-DHF have shown in the rat urinary electrolyte bioassay in vivo that 5α-DHF (30–100 μg/100 g) has minimal mineralocorticoid activity when administered alone, but the same dose administered with aldosterone (1 μg/100 g) doubles the mineralocorticoid response. Under identical conditions, cortisol was without effect. In the adrenalectomized rat kidney, in vitro, 5α-DHF has 20% the affinity of dexamethasone for classical (type II) glucocorticoid receptors, 10% the affinity of corticosterone for (type III) corticosteroid-binding globulin-like glucocorticoid receptors, and 3% the affinity of aldosterone for mineralocorticoid receptors (type I). 5α-DHF has <0.1% the affinity of dihydrotestosterone in mouse kidney receptors and <0.1% the affinity of both estradiol and progesterone for their receptors in rat uterus. As 5α-DHF has low affinity for mineralocorticoid receptors and an effect (aldosterone amplification) totally outside the ambit of classical steroid effects, it may belong to a new physiological class of steroid hormones.

Relationship between mineralocorticoids and renal Na+-K+-ATPase: sodium reabsorption.

To evaluate the mechanism responsible for the effect of deoxycorticosterone acetate (DOCA) on renal Na+-K+-ATPase, we compared the relative contribution of this hormone and of increased absolute sodium reabsorption (TNa) to the restoration of the enzyme in kidneys of adrenalectomized rats. In study A, adrenalectomized animals maintained on a salt-free diet received 5 mg/kg per day DOCA i.m., while sham-operated and untreated adrenalectomized rats receiving the same diet served as controls. Absolute TNa and Na+-K+-ATPase specific activity in the cortex and outer medulla of DOCA-treated rats were similar to those measured in untreated adrenalectomized animals, but were significantly lower than in sham-operated controls. In study B, the adrenalectomized rats did not receive DOCA but were fed a high salt diet and received isotonic saline, 50 ml/kg per day s.c. Absolute TNa and cortical and medullary Na+-K+-ATPase specific activity were significantly higher in the salt-loaded group than in both adrenalectomized and sham-operated rats deprived of salt. These results suggest that absolute sodium reabsorption is a major determinant of renal Na+-K+-ATPase activity, and that the effect of DOCA on this enzyme is secondary to its stimulation of absolute tubular sodium transport.

Tryptophan force-feeding: Changes in the activities of acetylcholinesterase in various tissues of well-fed normal and adrenalectomized rats

The activity of acetylcholinesterase in the liver, heart, spleen, lungs and kidneys of well-fed normal and adrenalectomized rats was measured following a single tube-feeding of tryptophan. In well-fed normal rats, 30 min after tryptophan force-feeding, the enzyme activity in the heart and lungs was stimulated by 28 and 25% as compared to the water-fed control while in well-fed adrenalectomized rats acetylcholinesterase acticity in the heart, liver spleen and lungs was 40, 31, 22 and 15% increased, respectively over that of the corresponding control. In both groups of rats the enzyme activity in the kidney was unaffected by tryptophan. In the liver, spleen and heart of well-fed adrenalectomized rats the pattern of response for acetylcholinesterase to a tryptophan dose, over a period of 24-hr. was found to be biphasic. In well-fed adrenalectomized rats the tryptophan-mediated stimulation of acetylcholinesterase activity in the heart was found to be insensitive to actinomycin-D. The tryptophan-mediated stimulation of acetylcholinesterase activity in the heart of well-fed normal and adrenalectomized rats could not be related to the presence of an activator.

16α,18-dihydroxydeoxycorticosterone and the binding of aldosterone to mineralocorticoid receptors in kidney of adrenalectomized rats

It has been recently suggested that 16α,18-dihydroxydeoxycorticosterone (16α,18-diOHDOC) may act as a “positive allosteric effector” of the binding of aldosterone to mineralocorticoid receptors. To test this hypothesis, a series of in vitro and in vivo studies examining the effect of 16α,18-diOHDOC on tritiated aldosterone ( 3HA) binding to mineralocorticoid receptors was performed. Using kidney slices from adrenalectomized rats, in vitro incubations were made for 20′ at 37C, over a range of concentrations of 3HA plus tenfold dexamethasone to confine tracer binding to mineralocorticoid receptors. At no concentration of 3HA did 16α,18-diOHDOC enhance binding; at all tracer concentrations a slight competing effect was observed. When 3HA was injected into rats in vivo with and without 16α,18-diOHDOC, a similar insignificant displacement of 3HA binding was seen in renal cytoplasmic fractions from adrenalectomized test rats. Additional in vitro studies were performed in an attempt to elucidate the mechanism of postulated action of 16α,18-diOHDOC. Neither renal cytoplasmic binding of oestradiol, postulated as a secondary pathway for steroid influenced Na + retention, nor the binding of dexamethasone to renal glucocorticoid receptors, was altered by 16α,18-diOHDOC. Binding of tritiated 16α,18-iOHDOC in renal cyto-plasmic fractions was shown to be non specific, in that it could not be displaced by excess unlabelled 16α,18-diOHDOC. Finally, in a series of in vivo experiments using adrenalectomized rats, we could not show any effect of 16α,18-diOHDOC on urinary electrolyte excretion, either alone or in combination with low doses of aldosterone. Accordingly, we can find no evidence for 16α-diOHDOC having a direct effect on the kidney: in particular, there would appear as yet no molecular evidence for 16α,18-diOHDOC being a positive allosteric effector of aldosterone.

MECHANISM OF ACTION OF ALDOSTERONE IN ADRENALECTOMIZED MOUSE KIDNEY

Time-course of effect of aldosterone on the incorporation of 14C-orotic acid into kidney RNA from adrenalectomized and normal mice was examined with the following results.1. Stimulatory effect of aldosterone (0.55 μg/kg, s.c.) on 14C-orotic acid incorporation into RNA from kidney homogenate and its nuclear fraction was observed in early stages (1.53 hr) after aldosterone administration in adrenalectomized mice, however, the incorporation was not changed at 5 hr after aldosterone. RNA content in homogenate and subcellular fractions did not change in any cases.2. After the administration of larger dose of aldosterone (50μg/kg) to adrenalectomized mice, 14C-orotic acid incorporation into kidney RNA was not changed until 3 hr.3. Cortisol had no effect on RNA specific radioactivity and RNA amount in adrenalectomized mouse kidney. 4. 14C-orotic acid incorporation into kidney RNA was not changed until 5 hr between normal and adrenalectomized mice.5. 14C-orotic acid incorporation into kidney nuclear RNA from normal mice was increased until 3 hr after aldosterone (2 and 20μg/kg) administration and the maximum effect was observed at 1.5 hr later.6. The early effects of aldosterone on RNA synthesis in adrenalectomized mouse kidney are agreed with the general concept in “induction hypothesis” which has been offered by the study using adrenalectomized rat kidney and/or urinary bladder of toad.

Molecular modifications of anti-aldosterone compounds: Effects on affinity of spirolactones for renal aldosterone receptors

The currently accepted hypothesis of the anti-mineralocorticoid action of spirolactones is that of competition for specific aldosterone receptors in target tissues. Binding of aldosterone to cytoplasmic receptors was studied by incubating adrenalectomized rat kidney slices with 3H-aldosterone in the presence of 10-fold non-radioactive dexamethasone to prevent binding of tracer to glucocorticoid receptors. From the ability of a series of 24 spirolactone analogues to compete for 3H-aldosterone binding sites under these conditions, the relative affinities for this receptor have been determined. Affinity for the receptor is decreased by B ring unsaturation at the C-6/C-7 position, by γ-lactone unsaturation, or by γ-lactone ring opening with the formation of the water-soluble K + salt. Affinity is markedly increased by esterification, or thioesterification, at the C-7 position in the B ring. Various 19-nor spirolactones show greater, equivalent or lesser affinity vis-a-vis their parent compounds. These structure-affinity relationships should define one of the determinants of pharmacologic activity.