In order to test the proposal that the aldosterone specificity of mineralocorticoid receptors in the collecting duct depends on inactivation of glucocorticoids by the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD), we have assessed the effect of pharmacological inhibition of 11β-HSD on collecting duct Na + reabsorption in vivo . Adrenalectomized rats ( n = 14) were infused intravenously with high-dose corticosterone, and late-distal tubules were perfused orthogradely with artificial tubular fluid containing [ 14 C]inulin and 22 Na; urinary recoveries of the radioisotopes were monitored. Half of the rats received intravenous carbenoxolone to inhibit renal 11β-HSD activity. The urinary recovery of [ 14 C]inulin was complete in both groups of animals (101ŷ2% versus 101ŷ3%), but the recovery of 22 Na was lower in carbenoxolone-treated rats (34ŷ5%) than in the corticosterone-alone group (54ŷ4%, P < 0.01). These data, which provide the first demonstration of enhanced Na + reabsorption in the distal nephron during inhibition of renal 11β-HSD in vivo , strongly support the proposal that 11β-HSD normally prevents endogenous glucocorticoid from exerting mineralocorticoid-like effects.

In order to clarify the effects on sodium reabsorption in the loop of Henle of methazolamide (a carbonic anhydrase inhibitor), chlorothiazide and the loop diuretics frusemide and bumetanide, superficial loops were perfused in vivo in anaesthetized rats and the individual diuretics were included in the perfusate. Differentiation between effects in the pars recta and in the thick ascending limb of Henle (TALH) was achieved by comparing responses to the diuretics when using a standard perfusate, designed to mimic native late proximal tubular fluid, and a low-sodium perfusate, designed to block net sodium reabsorption in the pars recta. With the standard perfusate, methazolamide caused decreases in sodium reabsorption ( J Na ) and water reabsorption ( J V ); with the low-sodium perfusate, a modest effect on J Na persisted, suggesting that carbonic anhydrase inhibition reduces sodium reabsorption in both the pars recta and the TALH. The effects of chlorothiazide were very similar to those of methazolamide with both the standard and low-sodium perfusates, suggesting that chlorothiazide also inhibits sodium reabsorption in the pars recta and TALH, perhaps through inhibition of carbonic anhydrase. With the standard perfusate, both frusemide and bumetanide produced the expected large decreases in J Na , but J V was also lowered. With the low-sodium perfusate, the inhibitory effects of the loop diuretics, particularly those of frusemide, were substantially reduced, while net potassium secretion was found. These observations indicate that a significant component of the effect of frusemide (and possibly of bumetanide) on overall sodium reabsorption is located in the pars recta, and that loop diuretics induce potassium secretion in the TALH.

1. The type 2 isoform of 11β-hydroxysteroid dehydrogenase, an enzyme which converts cortisol or corticosterone to inactive 11-ketosteroid metabolites, is thought to be responsible for preventing access of endogenous glucocorticoids to mineralocorticoid receptors in the distal nephron; although direct in vivo evidence for this is still lacking. We have examined whether graded inhibition of renal 11β-hydroxysteroid dehydrogenase activities in vivo results in corresponding changes in urinary electrolyte excretion due to exposure of mineralocorticoid receptors to circulating endogenous glucocorticoids. 2. Anaesthetized rats were infused intravenously with vehicle alone or with one of three doses of carbenoxolone: 0.06, 0.6 or 6 ;mg/h. After measurement of renal electrolyte excretion, the kidneys were snap-frozen in liquid nitrogen and 11β-hydroxysteroid dehydrogenase activities were measured directly by enzyme assay in the presence of NAD + or NADP + . 3. A dose-dependent inhibition of renal 11β-hydroxysteroid dehydrogenase activities was observed: the low, intermediate and high doses of carbenoxolone causing approximately 50%, 80% and > 90% inhibition respectively. Only with the high dose was an effect on renal function observed (decreased fractional Na + excretion and urinary Na + /K + ratio). 4. The poor correlation between the extent of inhibition of renal 11β-hydroxysteroid dehydrogenase and altered urinary Na + excretion, apparent at the lower doses of carbenoxolone, suggests either that 11β-hydroxysteroid dehydrogenase has considerable functional reserve, or that it may not be the only mechanism determining mineralocorticoid receptor specificity in the distal nephron.

1. The nephron sites involved in the blunted natriuretic response to frusemide during sodium depletion were investigated using micropuncture techniques in anaesthetized rats. 2. Glomerular filtration rate was lower, and fractional sodium reabsorption in the proximal convoluted tubule higher, in sodium-depleted than in sodium-replete rats. Consequently, sodium delivery to the loop of Henle was reduced (by approximately 35%) in the sodium-depleted animals. Intravenous frusemide (2.5 mg h −1 kg −1 ; urinary water and electrolyte losses replaced) had no effect on glomerular filtration rate or proximal tubular sodium reabsorption in either group. 3. The inhibitory effect of intravenous frusemide on fractional sodium reabsorption in the nephron segments constituting the loop of Henle (measured by free-flow micropuncture) was attenuated during sodium depletion. However, when loops of Henle were microperfused at identical rates with artificial late proximal tubular fluid, no difference in the responses of sodium-depleted and sodium-replete rats to intraluminal frusemide (10 −5 mol/l) could be detected. 4. In sodium-replete animals, the increased load of sodium delivered from the loop of Henle during frusemide administration resulted in a lowering of fractional sodium reabsorption in the distal tubule. In contrast, in sodium-depleted rats given frusemide, fractional distal sodium reabsorption tended to increase, so that values in the two groups of frusemide-treated animals were markedly different (0.30 ±0.04 versus 0.51 ±0.03). 5. It is concluded that the blunted natriuretic response to frusemide during sodium depletion results from at least three factors: a reduced sodium delivery to the loop of Henle; a reduced inhibitory effect of frusemide on fractional sodium reabsorption in the loop of Henle, which may be a consequence of the reduced sodium load; and enhanced fractional reabsorption of sodium in the distal tubule, which partially buffers the diuretic-induced increase in sodium delivery from the loop.

1. The possible natriuretic and kaliuretic effects of a single dose of lithium, as used in lithium clearance studies, were investigated in 15 healthy subjects on fixed sodium (100 mmol/24 h) and potassium (70 mmol/24 h) intakes. Lithium carbonate (300 mg or 600 mg) or placebo tablets were administered, double-blind and in random order, midway through a 48 h urine collection (divided into six 8 h periods), at 23.00 hours. 2. During the three 24 h periods which preceded the administration of lithium or placebo (control days), rates of sodium and potassium excretion followed normal circadian patterns, but no differences in excretion rates between the 3 control days were observed. Placebo tablets did not affect excretion rates. 3. After the 300 mg dose of lithium carbonate, 24 h sodium excretion increased by approximately 17 mmol ( P < 0.05); almost all of the natriuretic effect occurred during the first two 8 h periods. No effect on potassium excretion was observed. 4. After the 600 mg dose of lithium carbonate, 24 h sodium excretion increased by approximately 48 mmol ( P < 0.001) and 24 h potassium excretion increased by approximately 19 mmol ( P < 0.01). These effects were confined to the first two 8 h periods and thus occurred before and during the usual lithium clearance period. 5. Plasma renin activity, measured in 10 subjects, increased after the 600 mg dose of lithium carbonate ( P < 0.005), but plasma concentrations of aldosterone and atrial natriuretic peptide were not significantly affected. Neither the 300 mg dose of lithium carbonate nor the placebo tablets affected hormone levels. 6. It is recommended that the test dose of lithium carbonate for use in lithium clearance studies should not exceed 300 mg.

1. Aldosterone is suppressed by sodium loading. We studied the contribution of this decrease in plasma aldosterone to the natriuresis after acute sodium loading in healthy volunteers. 2. Two litres of saline [0.9% (w/v) NaCl] were infused during the second hour of a 6 h infusion of aldosterone (3 pmol min −1 kg −1 ) or placebo in eight healthy young men. On the placebo day, plasma aldosterone decreased by 30 min after the start of saline infusion and remained suppressed. During aldosterone infusion, plasma aldosterone was maintained at around 400 pmol/l. 3. Urinary sodium excretion, lithium clearance and plasma atrial natriuretic peptide increased and plasma renin activity decreased after saline infusion, whether or not aldosterone was infused. However, from 60 to 240 min after saline infusion, natriuresis was significantly less during aldosterone infusion than on the placebo day. In addition, saline loading led to a progressive increase in the ratio of sodium clearance to lithium clearance, used as an index of the fractional distal tubular rejection of sodium, and in the ratio of urinary sodium to potassium. These increases were prevented by the infusion of aldosterone. 4. This study suggests that there are differences in the mechanisms determining the early and the later responses to an acute sodium load. Suppression of aldosterone may explain much of the later increase in natriuresis after saline infusion. In addition, the results are consistent with a role for atrial natriuretic peptide in the immediate increase in sodium excretion after saline loading.

1. In order to investigate the effects of K + depletion on renal function, micropuncture studies were performed on anaesthetized rats which had been kept on a K + -deficient diet for 2 weeks; results were compared with those from control animals. 2. In the K + -depleted animals, values for total glomerular filtration rate and single-nephron filtration rate were significantly lower than in controls. Urine osmolality was also reduced; this was associated with reductions in the osmolality, Na + concentration and K + concentration of papillary interstitial fluid. No significant difference between urine and papillary osmolality was observed. 3. Fractional reabsorption by the proximal convoluted tubule was enhanced in the K + -depleted animals; end-proximal fluid delivery was markedly reduced. 4. Absolute, but not fractional, delivery of K + to the beginning of the distal tubule was reduced in the K + -depleted animals. In contrast to observations in control rats, no net secretion of K + into the distal tubule occurred and there was indirect evidence of K + reabsorption in the collecting duct. 5. K + depletion was associated with reductions in the delivery of Na + and water to early and late regions of the distal tubule, whereas excretion rates of Na + and water were unaffected. 6. It is suggested that the reduction in Na + delivery to the loop of Henle (arising from the changes in filtration rate and proximal tubular reabsorption) might contribute to the reduced medullary osmotic concentration observed during K + depletion. Reductions in fractional reabsorption of Na + and water in the collecting duct might result from lowered plasma aldosterone levels and the reduced medullary osmolality.

1. Hydrochlorothiazide was administered at two doses to Long-Evans rats for 7–10 days. Both doses resulted in an initial natriuresis and diuresis. After 1 day of treatment the natriuresis abated, but the diuresis persisted. 2. The mechanisms responsible for these chronic effects were investigated by performing clearance and micropuncture studies on all animals; collections were made from late proximal tubules and from early and late regions of distal tubules. 3. Values for total glomerular filtration rate and single-nephron filtration rate in thiazide-treated rats were not significantly different from those in control animals. 4. The delivery of sodium to the end of the proximal convoluted tubule was considerably reduced in each group of thiazide-treated rats. Although sodium delivery to the early distal tubule was also significantly lower than in control animals, the difference had disappeared by the late distal tubule. 5. It is concluded that the return of sodium excretion to control levels during chronic hyrochlorothiazide administration is a consequence of increased fractional reabsorption by the proximal tubules, secondary to a thiazide-induced sodium depletion. This results in less sodium being delivered to the nephron site at which thiazides exert their major inhibitory effect. 6. Fluid delivery to the end of the proximal convoluted tubule and to the early distal tubule was significantly reduced in thiazide-treated rats; in animals given the higher dose of diuretic it was also significantly reduced at the end of the distal tubule. Nevertheless, in both thiazide-treated groups urine flow rate was elevated, suggesting that reabsorption of water from the collecting ducts is reduced during chronic thiazide administration.

1. Brattleboro rats with hereditary diabetes insipidus were maintained in metabolism cages for 12–14 days. During the final 5–7 days hydrochlorothiazide was added to the food of half the animals, resulting in a sustained antidiuresis. At the end of this time all rats were anaesthetized and their renal function was investigated. 2. Water, sodium and potassium excretion rates during anaesthesia were similar to their respective values during the final period in metabolism cages. 3. Total glomerular filtration rate and superficial nephron filtration rate were similar in untreated and thiazide-treated animals. Fractional fluid reabsorption in proximal convoluted tubules, as measured by tubular fluid/plasma inulin concentration ratios in late surface convolutions, was moderately increased in the thiazide-treated rats, and was associated with a small reduction in the volume of fluid delivered to more distal nephron segments. 4. The osmolality of renal papillary interstitial fluid from thiazide-treated rats was considerably greater than that from untreated animals. There was also a small increase in papillary fluid sodium concentration. 5. It is concluded that the mechanism of the sustained antidiuresis during chronic hydrochlorothiazide administration in diabetes insipidus differs from that of the acute response. The changes in proximal tubular function during chronic thiazide treatment only partially account for the reduction in urine volume; it seems probable that the raised papillary osmolality, by enhancing water reabsorption at sites beyond the proximal tubule, makes a greater contribution to the antidiuresis.

1. The antidiuretic effect of hydrochlorothiazide in diabetes insipidus was investigated in rats with the hereditary hypothalamic form of the disease (Brattleboro rats). 2. Administration of hydrochlorothiazide in the food resulted in a marked fall in urine volume and a corresponding rise in osmolality. These effects persisted throughout the period of treatment (6–7 days). 3. Body weight and extracellular volume were significantly reduced in the thiazide-treated rats. 4. Hydrochlorothiazide caused an increase in urinary sodium excretion only on the first day of treatment. The resulting small negative sodium balance (in comparison with untreated rats) remained statistically significant for 2 days only. Thiazide-treated rats gradually developed a potassium deficit which was statistically significant from the fourth day of treatment. 5. Total exchangeable sodium, measured after 7 days of thiazide treatment, was not significantly different from that of untreated rats. However, plasma sodium was reduced in thiazide-treated animals, whereas erythrocyte sodium concentration was elevated. 6. It is concluded that the antidiuresis resulting from chronic hydrochlorothiazide administration is associated with a reduction in extracellular volume, but not with a significant overall sodium deficit. Hydrochlorothiazide appears to cause a redistribution of the body's sodium such that the amount of sodium in the extracellular fluid compartment is reduced.

1. Renal function in anaesthetized Brattleboro rats with hereditary hypothalamic diabetes insipidus was studied with micropuncture techniques before, and 1–3 h after, a single injection of hydrochlorothiazide. 2. In rats given hydrochlorothiazide and kept in sodium and water balance, total glomerular filtration rate and superficial nephron filtration rate were similar to values in control animals, whereas fractional fluid reabsorption in the proximal tubule (as evidenced by tubular fluid/plasma inulin concentration ratios) was slightly, but significantly, reduced. This suggests that hydrochlorothiazide may have a small direct inhibitory effect on proximal tubular reabsorption. 3. When rats were given hydrochlorothiazide and the resultant extra urinary sodium losses were not replaced, there was a marked antidiuresis. In these animals total glomerular filtration rate was reduced by 23% and superficial nephron filtration rate by 27% when compared with values in control rats. Fractional proximal tubular fluid reabsorption increased significantly whereas absolute proximal fluid reabsorption was unaffected. 4. It is concluded that the reduction in body sodium which follows acute hydrochlorothiazide administration over-rides any inhibitory effect of the drug on proximal tubular reabsorption, and leads instead to an increase in fractional fluid reabsorption at this site. This effect, combined with the fall in glomerular filtration rate, results in a greatly reduced delivery of fluid to the more distal nephron segments, and is probably largely responsible for the observed antidiuresis.

1. The mechanism of the antidiuretic effect of hydrochlorothiazide in diabetes insipidus was studied in anaesthetized Brattleboro rats with the hereditary hypothalamic form of the disease. 2. The antidiuresis caused by acute administration of hydrochlorothiazide followed an increase in sodium excretion and was associated with a significant fall in the plasma sodium concentration. There were concomitant falls in effective renal plasma flow and glomerular filtration rate. 3. When sodium depletion was prevented by adjusting the infusion of sodium chloride, the falls in plasma sodium concentration, effective renal plasma flow and glomerular filtration rate were abolished. Under these circumstances there was an increase in urine volume, which suggests that hydrochlorothiazide may inhibit fractional fluid reabsorption in the proximal convoluted tubule. 4. The results indicate that the antidiuresis caused by hydrochlorothiazide in diabetes insipidus results, at least in part, from falls in effective renal plasma flow and glomerular filtration rate. These in turn seem to be entirely secondary to the drug-induced sodium depletion.