Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias.

Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane are closest to the mitochondria. These results establish that most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.

Sodium- and potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water.

The activity of adenosine triphosphatase activated by sodium and potassium ions is greatly increased in the gill and pseudobranch of the euryhaline killifish, Fundulus heteroclitus, after its adaptation to seawater. Adenosine triphosphatase activity in gills of fish in salt water is reduced by hypophysectomy. The data suggest that this enzyme is involved in the excretion of sodiumions by the gill and that the adaptive increase which occurs in seawater is influenced by the hypophysis.

Polyene toxicity in renal medulla: injury mediated by transport activity.

Polyene antibiotics such as amphotericin and nystatin increase membrane permeability and thus increase the amount of oxygen consumed in active electrolyte transport. In isolated perfused rat kidneys, the polyenes produced extensive injury to the medullary thick ascending limb, a segment of the nephron with limited oxygen supply. This damage was prevented if reabsorptive transport was inhibited by ouabain. Cell death under these circumstances thus appears to be mediated by increased oxygen demand for transport activity.

The role of sodium-potassium-activated adenosine triphosphatase in the reabsorption of sodium by the kidney.

In order to evaluate the possible role of sodium- and potassium-activated adenosine triphosphatase in the active transport of sodium by the renal tubules, we examined the effect of large changes in the tubular reabsorptive load of sodium on the Na-K-ATPase activity of rat kidney homogenates. Glomerular filtration and tubular reabsorption of sodium per gram of kidney tissue increased progressively after contralateral uninephrectomy. This was paralleled by an increase in Na-K-ATPase per milligram of protein in a microsomal fraction of kidney cortex. The importance of this change is underlined by the absence of simultaneous increases in other microsomal enzymes such as glucose-6-phosphatase and Mg(++)-dependent ATPase, or in succinic dehydrogenase or glutaminase. Similar increases in Na-K-ATPase were observed when the net tubular reabsorption of sodium was increased by feeding the animals a high-protein diet or after injection of methylprednisolone. On the other hand, Na-K-ATPase was lowered when tubular transport of sodium was reduced by bilateral adrenalectomy. The results of these experiments show that renal Na-K-ATPase changes in an adaptive way when renal reabsorption of sodium is chronically increased or diminished and support the hypothesis that this enzyme system is involved in the process by which sodium is actively transported across the renal tubule.

The importance of aerobic metabolism in the renal concentrating process.

The extent to which the concentrating function of the kidney depends on oxidative processes was investigated by infusing cyanide into one renal artery of dogs undergoing mild mannitol diuresis while receiving an infusion of vasopressin. This produced an abrupt fall in concentrating capacity (T(c) (H2O)) that was reversed when the cyanide infusion was stopped. The change could not be accounted for by the accompanying solute diuresis, since it was not reproduced by increasing the rate of mannitol infusion. The reduction in T(c) (H2O) induced by cyanide did not result from increased delivery of dilute urine to the collecting ducts, since free water clearance (C(H2O)), studied in other dogs during water diuresis, was unchanged or decreased by cyanide. Cyanide produced renal vasodilatation, as did intraarterial acetylcholine, but in contrast to the striking reduction in concentrating capacity evoked by cyanide, T(c) (H2O) was not significantly changed by acetylcholine. The data indicate that concentrating ability is closely tied to oxidative metabolism in the kidney, and it is suggested that the region where this is critically important is the red medulla and the thick ascending limb of Henle's loop.

Changes in proximal and distal tubular reabsorption produced by rapid expansion of extracellular fluid.

Acute infusions of isotonic saline in the rat cause an increase in glomerular filtration rate and in the excretion of salt and water. The kidney swells, due to expansion of tubular and interstitial volume. Despite the increase in tubular diameter, transit time through the proximal tubules and loops of Henle is decreased, presumably owing to a greatly accelerated rate of tubular flow. Proximal tubular reabsorption, measured in blocked tubules, is inhibited in a way that cannot be ascribed to changes in tubular diameter. The prolongation of proximal reabsorptive half-time is not affected by the administration of aldosterone. It occurs equally in rats chronically loaded with or deprived of salt, and it is therefore not likely that it is influenced by the renal content of renin. In contrast, reabsorption from the distal convoluted tubule is enhanced by saline infusion. This change is observed in segments of tubules blocked with oil and isolated from their glomeruli and thus appears to occur independently of changes in glomerular filtration or tubular flow.

The role of Na-K-activated adenosine triphosphatase in potassium adaptation. Stimulation of enzymatic activity by potassium loading.

The specific activity of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) in homogenates of rat kidneys increases when the dietary intake of potassium is chronically increased. The effect is seen first and is most prominent in the outer medulla, but large loads of potassium elicit an increase in the cortex as well. Levels of Na-K-ATPase in brian, liver, and muscle, by contrast, are unaffected by potassium loading. Although the changes in enzyme activity in the kidney resemble those reportedly produced by aldosterone, they are not induced by experimental sodium deprivation, and they can be evoked by potassium loading in the absence of the adrenal glands. The results suggest that Na-K-ATPase of renal tubular cells, presumably in the distal tubules and collecting ducts, plays an important role in the phenomenon of potassium adaptation and in the process by which potassium is excreted into the urine.

Mechanism of change in the excretion of sodium per nephron when renal mass is reduced.

As the population of nephrons is reduced, sodium excretion per nephron must increase if sodium balance is to be maintained. The mechanism of this adjustment was studied in rats in which 50% and approximately 85% of renal tissue was excised. Although glomerular filtration per remaining nephron rose after uninephrectomy, it did not rise further when more renal tissue was removed, even though sodium excretion per nephron mounted. Hyperfiltration does not, therefore, account for the stepwise increase in sodium excretion per nephron with progressive renal ablation. Proximal tubular absorption, estimated by reabsorption half-time, was unchanged by renal insufficiency, indicating that "third factor" did not produce the observed changes in sodium excretion per nephron. It seems likely that the earliest adjustments in sodium excretion in renal failure take place in the distal tubules of healthy nephrons, and that they are conditioned by changes in the osmotic load per nephron.

The distribution of sodium-potassium--activated adenosine triphosphatase in medulla and cortex of the kidney.

The activity of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) is considerably higher in homogenates of outer medulla than in the cortex or papilla of the kidney. The enzyme has similar kinetic characteristics in both cortex and medulla, and binds ouabain in the same proportion. The discrepancy in enzymatic activity is not paralleled by similar change in the activity of adenyl cyclase, 5'nucleotidase, glucose-6-phosphatase, or succinic dehydrogenase. Na-K-ATPase is also higher in distal convoluted tubules (ventral slices) than in the proximal tubules (dorsal slices) of the kidney of Amphiuma. The high concentration of Na-K-ATPase in the red medulla of the kidney is probably related to the presence here of the thick ascending limb of the loop of Henle, and this has important implications with regard to the mechanism of sodium reabsorption by different portions of the nephron.

Functional correlates of compensatory renal hypertrophy.

The functional correlates of compensatory renal hypertrophy were studied by micropuncture techniques in rats after the removal of one kidney. The glomerular filtration rate increased to roughly the same extent in the whole kidney and in individual surface nephrons, resulting in a greater amount of sodium delivered to the tubules for reabsorption. The fraction of the glomerular filtrate absorbed [determined from the tubular fluid-to-plasma ratio (TF/P) for inulin] remained unchanged in both proximal and distal portions of the nephron. The way in which the tubules adjusted to nephrectomy, however, differed in proximal and distal convolutions. After nephrectomy, the reabsorptive half-time, indicated by the rate of shrinkage of a droplet of saline in a tubule blocked with oil, was unchanged in the proximal tubule but significantly shortened in the distal convoluted tubule. Nevertheless, steady-state concentrations of sodium in an isolated raffinose droplet in the distal as well as the proximal tubule were the same in hypertrophied kidneys as in control animals. Possible reasons for this paradox are discussed. Transit time through the proximal tubules was unchanged by compensatory hypertrophy, but transit time to the distal tubules was prolonged. Changes in renal structure resulting from compensatory hypertrophy were also found to differ in the proximal and the distal protions of the nephron. Although tubular volume increased in both protions, the volume increase was twice as great in the proximal tubule as in the distal. In order, therefore, for net reabsorption to increase in the distal tubule, where the changes in tubular volume are not so marked, an increase in reabsorptive capacity per unit length of tubule is required. This increase is reflected in the shortening of reabsorptive half-time in the oil-blocked distal tubule that was actually observed.

Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney.

A specific anatomical lesion sharply localized to the cells of the medullary thick ascending limbs (mTAL) and characterized by mitochondrial swelling progressing to nuclear pyknosis and cell death is elicited reproducibly in isolated rat kidneys perfused for 15 or 90 min with cell-free albumin-Ringer's medium gassed with 5% CO2, 95% O2 (O2 content, 1.5 vol/100 ml). The lesion, involving about half of mTALs, appears first in mTALs removed from vascular bundles and near the inner medulla, areas most likely to be anoxic. Hypoxic perfusion (O2 content 0.12 vol/100 ml) exaggerates the lesion, wiping out gradations of damage and extending it to all mTALs. O2-enriched perfusions using rat erythrocytes (O2 content 7.1 vol/100 ml) completely eliminates the lesion (unless gassed with carbon monoxide). Similarly, supplementation of the perfusion medium with a purified hemoglobin (O2 content 5.8 vol/100 ml) prevents mTAL injury. Perfusion with a fluorinated hydrocarbon blood substitute, Oxypherol (O2 content 4.3 vol/100 ml) also attenuates the lesion. These findings suggest that the mTAL is exquisitely susceptible to anoxic damage because of low O2 supply imposed by the medullary vascular system and the high rate of metabolism mandated by active reabsorption of sodium chloride. The vulnerability of the mTAL to anoxic injury could play a key role in the pathogenesis of ischemic renal injury.

Sodium-potassium pump, ion fluxes, and cellular dehydration in sickle cell anemia.

We studied the role of the sodium-potassium pump in erythrocytes of 12 patients with sickle cell anemia (SS). Ouabain-binding sites per cell and pump-mediated Rb/K uptake were significantly higher in SS patients than in white or black controls. Ouabain-resistant Rb/K influx was also greater than in normal controls or patients with sickle cell trait. Deoxygenation of SS erythrocytes increased ouabain-sensitive Rb/K influx without altering ouabain binding, presumably as the consequence of an increase in the passive influx of sodium. Deoxygenation increased mean corpuscular hemoglobin concentration (MCHC) by 5.5%, and studies of the density distribution of SS cells indicated an increase in highly dense fractions known to contain sickled erythrocytes. Ouabain prevented the rise in MCHC and reduced the percentage of dense cells. These findings indicate a magnified role for the sodium-potassium pump in the pathophysiology of SS erythrocytes and suggest that its inhibition might prove useful in therapy.

Role of nitric oxide in renal medullary oxygenation. Studies in isolated and intact rat kidneys.

We investigated the role of the endothelial-derived relaxing factor nitric oxide (NO) in the homeostasis of O2 supply to the renal medulla, a region normally operating on the verge of hypoxia. Sensitive Clark-type O2 microelectrodes were inserted into renal cortex and medulla of anesthetized rats. The inhibitor of NO formation, L-NG-monomethylarginine (LNMMA), while increasing blood pressure and reducing renal blood flow, decreased medullary pO2 from 23 +/- 3 mmHg to 12 +/- 3 (P less than 0.001), with no change in the cortex. These responses were promptly reversed by L-arginine, which bypasses the LNMMA blockade. In isolated rat kidneys, LNMMA reduced perfusion flow without altering glomerular filtration rate, and augmented deep medullary hypoxic injury to thick ascending limbs from 68 to 90% of the tubules (P less than 0.02). These changes were prevented by L-arginine. Nitroprusside had a protective effect upon thick limb injury. Finally, in a previously reported model of radiocontrast nephropathy (1988. J. Clin. Invest. 82:401), LNMMA increased the severity of renal failure (final plasma creatinine from 2.3 +/- 2 mg% to 3.4 +/- 3, P less than 0.005) and the proportion of damaged thick limbs (from 24 +/- 6% to 53 +/- 9, P less than 0.01). Nitrovasodilatation may participate in the balance of renal medullary oxygenation and play an important role in the prevention of medullary hypoxic injury.

Potassium transport by the isolated perfused kidney.

Rat kidneys perfused outside of the body with an artificial medium are able to increase their fractional excretion of potassium in response to a rising concentration of potassium in the medium but never show net secretion of potassium. By contrast, isolated perfused kidneys from chronically potassium-loaded rats regularly secrete potassium in excess of the amount filtered. Ouabain completely blocks the secretion of potassium by these isolated kidneys, suggesting that Na-K-ATPase mediates potassium secretion by potassium-adapted rats. Neither sodium deprivation, pretreatment with deoxycorticosterone, nor pretreatment with methylprednisolone prepared the kidney to secrete potassium, despite stimulation of Na-K-ATPase activity in cortex or outer medulla. Potassium loading was the only maneuver tested that increased the activity of Na-Katpase in the inner medulla (white papilla) and also produced potassium secretion by the isolated kidney. Surgical ablation of the papilla abolished the net secretion of potassium normally seen in perfused kidneys of potassium-adapted rats, thus underlining the importance of the papilla in the process of potassium adaptation.

Elevated 1,25-dihydroxyvitamin D plasma levels in normal human pregnancy and lactation.

Plasma 1,25-dihydroxyvitamin D levels are elevated in early pregnancy and continue to increase throughout pregnancy. They remain elevated postpartum in lactating women. The elevated levels probably represent a physiological response to increased calcium requirements.

Acute renal failure with selective medullary injury in the rat.

Since human acute renal failure (ARF) is frequently the result of multiple rather than single insults, we used a combination of treatments to induce ARF in rats. Uninephrectomized, salt-depleted rats injected with indomethacin developed ARF after administration of radiocontrast. After 24 h, the plasma creatine rose from 103 +/- 3 to 211 +/- 22 mumol/liter (mean +/- SE) and the creatinine clearance dropped from 0.7 +/- 0.1 to 0.2 +/- 0.04 ml/min (P less than 0.001). Severe injury was confined to the outer medulla and comprised necrosis of medullary thick ascending limbs (mTALs), tubular collapse, and casts. Other nephron segments were free of damage except for the proximal convoluted tubules which showed vacuole formation originating from lateral limiting membranes that resembled changes reported in human contrast nephropathy. Cell damage to mTALs included mitochondrial swelling, nuclear pyknosis, and cytoplasmic disruption with superimposed calcification; these changes were most severe in the deepest areas of the outer medulla, away from vasa recta in zones remote from oxygen supply. The fraction of mTALs with severe damage was 30 +/- 7% (range 2-68) and the extent of injury was correlated with a rise in plasma creatinine (r = 0.8, P less than 0.001). Thus, the nature of mTAL injury was similar to the selective lesions observed in isolated kidneys perfused with cell-free medium and was shown to derive from an imbalance between high oxygen demand by actively transporting mTALs and the meager oxygen supply to the renal medulla. Combined multiple renal insults in the rat produce ARF that resembles the clinical syndrome of contrast nephropathy and is characterized by selective mTAL injury conditioned by medullary hypoxia.

Disparate mechanisms for hypoxic cell injury in different nephron segments. Studies in the isolated perfused rat kidney.

Hypoxic injury was evaluated morphologically in the proximal tubule and in the medullary thick ascending limb of isolated rat kidneys perfused for 90 min without O2 or with various metabolic inhibitors. Inhibition of mitochondrial respiration (with rotenone, antimycin, oligomycin) or of intermediary metabolism (with monofluoroacetate, malonate, 2-deoxyglucose) caused reduction in renal oxygen consumption, renal function, and ATP content comparable with those elicited by oxygen deprivation. Metabolic inhibition produced hypoxiclike injury in the first portions of the proximal tubule, S1 and S2 ("clubbing" of microvilli, mitochondrial swelling), and the extent of damage was correlated with the degree of ATP depletion. In the third portion of the proximal tubule, S3, hypoxiclike damage (cytoplasmic edema or fragmentation) occurred most consistently when both aerobic and anaerobic metabolism were inhibited simultaneously. In the medullary thick ascending limb, none of the metabolic or mitochondrial inhibitors used could reproduce the injury of oxygen deprivation. Thus, the proximal tubule and the thick ascending limb have markedly different responses to cellular energy depletion, suggesting disparate mechanisms for hypoxic injury along the nephron.

Effect of protein ingestion on urinary dopamine excretion. Evidence for the functional importance of renal decarboxylation of circulating 3,4-dihydroxyphenylalanine in man.

Since dietary protein increases urinary dopamine (DA) excretion in animals, this study was undertaken to assess the role of DA production in the acute changes in renal function following protein ingestion in man. Excretion of DA, sodium, potassium, water, solute, and creatinine were measured in six normal men in 30-min intervals over 5 h after oral ingestion of protein and/or carbidopa, an inhibitor of DA formation from 3,4-dihydroxyphenylalanine (DOPA). Overall, protein increased urinary DA 50% (P = 0.031) while carbidopa reduced it 70% (P less than 0.0001), although suppression of DA excretion by carbidopa was not uniform over the 5 h of observation. Carbidopa doubled the level of DOPA in venous plasma and greatly magnified the DOPA response to protein. Inhibition of decarboxylase activity reduced excretion of sodium, potassium, solute and water after protein ingestion. These results indicate that extraneuronal DOPA decarboxylation in kidney contributes to acute protein-induced changes in renal function in man and suggest a general role for the decarboxylation of circulating DOPA in the expression of dopaminergic effects on the kidney in vivo.

On the mechanism of lithium-induced diabetes insipidus in man and the rat.

The mechanism of lithium-induced diabetes insipidus was investigated in 96 patients and in a rat model. Polydipsia was reported by 40% and polyuria (more than 3 liter/day) by 12% of patients receiving lithium. Maximum concentrating ability after dehydration and vasopressin was markedly impaired in 10 polyuric patients and was reduced in 7 of 10 nonpolyuric patients studied before and during lithium therapy. Severe polyuria (more than 6 liter/day) was unresponsive to trials of vasopressin and chlorpropamide, but improved on chlorothiazide. Rats receiving lithium (3-4 meq/kg/day) developed massive polyuria that was resistant to vasopressin, in comparison to rats with comparable polyuria induced by drinking glucose. Analysis of renal tissue in rats with lithium polyuria showed progressive increase in the concentration of lithium from cortex to papilla with a 2.9-fold corticopapillary gradient for lithium. The normal corticopapillary gradient for sodium was not reduced by lithium treatment. The polyuria was not interrupted by brief intravenous doses of vasopressin (5-10 mU/kg) or dibutyryl cyclic AMP (10-15 mg/kg) capable of reversing water diuresis in normal and hypothalamic diabetes insipidus rats (Brattleboro strain). The present studies suggest that nephrogenic diabetes insipidus is a common finding after lithium treatment and results in part from interference with the mediation of vasopressin at a step distal to the formation of 3',5' cyclic AMP.

In vivo 24-hydroxylation of 25-hydroxyvitamin D3 in enterocolectomized rats.

To determine if the renal 25-hydroxyvitamin D3 24-hydroxylase is active in addition to the intestinal 25-hydroxyvitamin D3 24-hydroxylase in vivo in the rat, radiolabeled 25-hydroxyvitamin D3 was administrated to normocalcemic, vitamin D-replete, enterocolectomized rats. Substantial amounts of 24,25-dihydroxyvitamin D3 were detected and the identity of the isolated plasma 24,25-hydroxyvitamin D3 region in these animals were confirmed by coelution on high pressure liquid chromatography and periodate cleavage. This suggests that both intestinal and renal 25-hydroxyvitamin D3 24-hydroxylases are active in vivo in the rat.