Chloride-depletion metabolic alkalosis induces ECF volume depletion via internal fluid shifts in nephrectomized dogs.

We recently reported that chloride-depletion metabolic alkalosis (CDMA) results in renal losses of Na, K, and water. In these studies we investigated whether CDMA (induced using a new model that avoids external changes in Na and water balance) was also associated with internal Na and water shifts out of the ECF. CDMA was induced using haemofiltration in functionally nephrectomized dogs. Plasma ultrafiltrate was substituted quantitatively with a solution duplicating each dog's plasma electrolyte composition in control animals, and with a solution containing HCO3 as the sole anion in CDMA animals. ECF volume was estimated as the space of distribution of [3H]-mannitol. Plasma composition and [3H]-mannitol distribution space were unchanged in control dogs. In CDMA dogs metabolic alkalosis developed; despite the absence of external changes in Na and water balance, the space of distribution of [3H]-mannitol decreased by 335 +/- 46 ml (equivalent to 8% of baseline ECF volume), calculated chloride space fell by 304 +/- 50 ml, and haematocrit increased from 45.6 to 48.5 vol%. We conclude that CDMA causes an internal shift of fluid out of the ECF. The resulting ECF volume contraction appears to be an inherent feature of CDMA.

Transport and histochemical studies of bicarbonate handling by the alligator kidney.

The alligator excretes a persistently alkaline urine despite consuming an acid-residue diet. The amount of bicarbonate excreted is greater than the amount filtered, evidencing tubular secretion of bicarbonate. The parallel urinary excretion of ammonium maintains external acid balance. To investigate putative renal mechanisms responsible for the concurrent excretion of large quantities of ammonium bicarbonate, we used acridine orange fluorescence methodology in microvesicles prepared from the proximal tubule brush border to assess the activity of the Na+-H+ antiporter, and histochemical methods (cobalt sulfide precipitation) to assess carbonic anhydrase localization. We found no evidence for the presence of a functioning Na+-H+ antiporter, the protein known to be responsible for the majority of bicarbonate reabsorption in mammals; Na+-H+ exchange in vesicles from the alligator kidney failed to exhibit saturation kinetics, showed no affinity for lithium, and was not inhibited by amiloride. Sensitive histochemical techniques failed to reveal carbonic anhydrase activity anywhere in the proximal tubule but detected an abundance of enzyme activity in the basolateral membranes and nuclei of distal tubular cells. In the connecting segment and collecting duct, cells without carbonic anhydrase alternated with cells containing carbonic anhydrase; in the latter, the enzyme was localized to the basolateral and luminal membranes, the nucleus and, to a lesser extent, throughout the cytoplasm. We conclude that the proximal tubule of the alligator kidney is devoid of the machinery necessary for the transport of large amounts of bicarbonate. The principal site at which bicarbonate is added to the final urine appears to be the distal tubule, at which site carbonic anhydrase is widespread.

Acidemia alone does not stimulate rat renal Na+-H+ antiporter activity.

To examine whether systemic acidemia is the cause of the adaptive increase in renal brush-border membrane (BBM) Na+-H+ exchange activity seen in metabolic acidosis, we examined the time course of changes in Na+-H+ exchange activity in rats with chronic metabolic or respiratory acidosis. Metabolic acidosis was created by allowing rats free access to a 1.5% NH4Cl drinking solution. Respiratory acidosis was created by housing rats in a chamber designed to maintain ambient PCO2 levels at 10%. All rats were fed normal rat chow. Control and respiratory acidosis rats had free access to tap water. Rats from each group were studied 1, 3, 5, 7, and 14 days after onset of treatment. Na+-H+ exchange activity was examined in renal BBM vesicles using acridine orange. In both metabolic acidosis and respiratory acidosis, arterial blood [H+] increased markedly at day 1 and returned toward normal thereafter; at day 14, [H+] was elevated to a comparable degree in both groups. In metabolic acidosis, Na+-H+ exchange activity remained at control levels for 3 days but increased markedly thereafter. In contrast, in respiratory acidosis no adaptive increase in activity occurred at any time. Because no correlation was found between blood [H+] and renal BBM Na+-H+ exchange activity, we conclude that stimuli other than systemic acidemia are responsible for the adaptation seen in chronic metabolic acidosis.

Renal response to metabolic alkalosis induced by isovolemic hemofiltration in the dog.

We describe a new model of chloride-depletion alkalosis (CDMA), in which the method of induction of alkalosis does not itself cause a direct alteration in sodium and fluid balance. We have used this model, which is based on hemofiltration techniques in the dog, to study the immediate response of the kidney to the induction of CDMA. Normal dogs maintained with a NaCl-free diet for several days underwent hemofiltration of 50 ml/kg over a 35 minute period. The hemofiltrate was replaced ml for ml with a solution containing sodium and potassium in the same concentrations as found in each animal's plasma water. In control animals, the replacement solution contained chloride and bicarbonate in the same ratio as in the plasma; in the experimental (CDMA) animals the replacement solution contained bicarbonate as the only anion. In the control group, the procedure of hemofiltration coupled with isovolemic replacement caused no appreciable changes in plasma composition, urinary excretion rates, GFR, or tubular handling of bicarbonate. In the CDMA group, 106 +/- 8.4 mEq of chloride were removed in exchange for bicarbonate. A marked metabolic alkalosis resulted, plasma bicarbonate concentration increasing from 21.9 +/- 0.6 to 33.3 +/- 0.6 mEq/liter. The hemofiltration procedure itself, by design, did not alter sodium or fluid balance. Nevertheless, cumulative urinary sodium excretion increased over 2.5 hours by 23.0 +/- 6.4 mEq. A natriuresis of this magnitude is equivalent to a loss of ECF volume of approximately 200 ml. GFR did not change significantly. The rate of tubular reabsorption of bicarbonate increased significantly from 1209 +/- 82 to 1559 +/- 148 mu Eq/min in CDMA animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin synthesis by microsomes of circular and longitudinal gastrointestinal muscles.

Experiments were performed to determine whether longitudinal and circular muscles from various regions of stomach and small bowel had the capacity to convert arachidonic acid (AA) to prostaglandins (PGs). PG production by the microsomal fractions of isolated muscles was assayed by determining the conversion of [14C]AA to 14C-labeled 6-keto-PGF1 alpha, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane B2. Individual PGs were identified by thin-layer chromatography. The metabolism of [14C]AA to [14C]PGs was linearly related to substrate concentration, enzyme concentration, and incubation time at 37 degrees C and was inhibited in a dose-dependent manner by indomethacin. Longitudinal and circular muscles from all tested regions (corpus, fundus, antrum, pylorus, duodenum, jejunum, and ileum) synthesized PGs. In all regions the major end products of AA metabolism were 6-keto-PGF1 alpha, PGE2, and PGF2 alpha. The data indicate that circular and longitudinal muscles from all regions of the stomach and small bowel contain the enzymatic apparatus necessary to convert AA into prostaglandins.

Alkaline phosphatase in adaptation to low dietary phosphate intake.

Previous findings suggest that alkaline phosphatase (Alk Pase) may be involved in phosphate transport. Since phosphate reabsorption is enhanced in the kidney and duodenum of animals stabilized on a low-phosphorus diet (LPD), Alk Pase was measured in the kidney, small intestine, and other tissues in LPD rats. In particulate fractions from the renal cortex, intestine, renal medulla, liver, and heart ventricle from LPD rats the activity of Alk Pase was significantly increased but the activities of other plasma membrane enzymes were not different between control and LPD groups. The increased Alk Pase in the renal cortex was localized to the brush border of the proximal tubule histochemically and by measurement of Alk Pase in brush-border preparations. Also in the renal cortex, typical enzymes associated with mitochondria, lysosomes, and cytosol were unchanged with the exception of cytosolic adenosine 3',5' cyclic-monophosphate phosphodiesterase, which was increased in LPD rats. Alk Pase in the renal cortex and intestine may play a role in the enhanced phosphate reabsorption in LPD animals.

Renal adaptation to a low phosphate diet in rats.

The major renal adaptive changes in response to selective dietary phosphate restriction are a marked reduction in urinary excretion of phosphate and an increased urinary excretion of calcium; at the cellular level, there is selective increase in renal cortical brush border membrane phosphate uptake and increase in specific activity of alkaline phosphatase. In the present study we examined whether these functional and biochemical adaptive changes could be blocked by drugs known to inhibit protein synthesis. Administration of actinomycin D or cycloheximide to rats switched from a diet with normal phosphate content (0.7%) to a diet with low (0.07%) phosphate content either completely (actinomycin D) or partially (cycloheximide) prevented the expected decrease in urinary excretion of phosphate and increase in the urinary excretion of calcium. The specific activity of alkaline phosphatase measured in crude membrane fraction (washed 100,000 g pellet) from renal cortical homogenate in animals fed a low phosphate diet and treated with actinomycin D or with cycloheximide was significantly lower than in control animals also on a low phosphate diet receiving placebo; but there were no differences between treated and untreated animals in the activities of two other brush border enzymes, gamma-glutamyltransferase and leucine aminopeptidase. Actinomycin D administered to rats maintained on a normal phosphate diet throughout the course of the experiment caused an increase in the urinary excretion of phosphate on the last (6th) day of the experiment but did not change urinary excretion of calcium. In acute clearance experiments, infusion of actinomycin D to rats adapted to a low phosphate diet did not increase fractional excretion of phosphate. In separate experiments, using the same dietary protocol as above, brush border membrane fraction (vesicles) was prepared from renal cortex of rats sacrificed at the end of the experiment. In this preparation Na(+)-dependent (32)Pi and d-[(3)H]glucose uptake and activities of brush border enzymes membrane were determined. Brush border membrane vesicles prepared from rats fed a low phosphate diet showed significantly higher Na(+)-dependent (32)Pi uptake compared with rats fed a normal phosphate diet. This increase in (32)Pi uptake was completely prevented when rats on a low phosphate diet were simultaneously treated with actinomycin D. These differences were specific for (32)Pi transport as no differences were observed in d-[(3)H]glucose uptake among the three groups. There was a positive correlation (r = 0.82, P < 0.01) between (32)Pi uptake and specific activity of alkaline phosphatase measured in aliquots of the same brush border membranes, whereas no such correlation was observed with two other brush border membrane enzymes gamma-glutamyltransferase and leucine aminopeptidase. These observations show that actinomycin D prevents both the functional and cellular renal adaptive changes induced by a low phosphate diet. Taken together, these observations suggest that renal adaptation to a low phosphate diet could be prevented by inhibition of de novo protein synthesis.

Insulin inhibition of hormone-stimulated protein kinase systems of rat renal cortex.

Parathyroid hormone (PTH) and glucagon increase the urinary fractional excretion of phosphate, but insulin administration is associated with a decreased fractional excretion of phosphate. It was the purpose of this study to determine whether insulin will antagonize the effects of PTH and glucagon on cAMP levels and protein kinase activation of rat renal cortex. In situ incubation studies were performed on rat renal cortical slices exposed to insulin, PTH, and glucagon. Insulin alone did not affect the tissue cAMP and cGMP levels or the state of protein kinase activation. Preincubation of slices with insulin, however, did significantly inhibit increases in protein kinase activation induced by both PTH and glucagon. Insulin also significantly inhibited PTH-stimulated increases in tissue cAMP levels, but did not blunt the elevations of cAMP levels induced by glucagon. Insulin (10(-9) M) had no effect on either the in vitro activity of adenylate cyclase, basal or PTH-stimulated, or on the activities of low Km cytosolic or membrane-bound cAMP phosphodiesterase. The data show that insulin antagonizes activation of protein kinase by both PTH and glucagon in renal cortex. Separate mechanisms are probably involved for PTH and glucagon interaction. The antiphosphaturic effect of insulin in vivo may result in part from this antagonism at the cellular level.

Action of serotonin (5-hydroxytryptamine) on cyclic nucleotides in glomeruli of rat renal cortex.

Serotonin (5-hydroxytryptamine) is known to influence glomerular function and may have an important role in the pathogenesis of glomerulopathies. Because serotonin acts in nonrenal tissues through mediation of cyclic nucleotides, we investigated in vitro its effect on cAMP and cyclic guanosine monophasphate (cGMP) in tissue slices and isolated glomeruli from rat kidney. Serotonin increased cAMP 161 +/- 35% but not cGMP in renal cortex; it had no effect on cyclic nucleotides in medulla and papilla. In isolated glomeruli, serotonin elicited a dose-dependent (in the range of 10-7 to 10-4 M) increase in cAMP; the maximum increase over basal values was 376 +/- 45%. Serotonin increased cAMP either in the presence or in the absence of a cAMP phosphodiesterase inhibitor. In tubular fraction, serotonin elevated cAMP to a much lesser degree (82 +/- 15%). Neither in glomeruli nor in tubules did cGMP concentrations change in response to serotonin, but carbamylcholine, a known cGMP agonist, significantly increased cGMP concentrations. The increase in cAMP in response to serotonin was blocked (greater than 85% inhibition) by equimolar concentrations of serotonin antagonists methysergide and cinanserine. Results of this study demonstrate that interaction of serotonin with receptors in the kidney, particularly in the glomeruli, cause a striking increase in cAMP concentrations without detectable changes in cGMP concentrations. These findings suggest that serotonin, either synthesized in the kidney or released locally from platelets aggregated in glomeruli (for example, in association with immunopathologic injury) may exert of modulate its physiologic or pathologic effects via mediation of cAMP.

Modulation of cyclic nucleotides in islated rat glomeruli: role of histamine, carbamylcholine, parathyroid hormone, and angiotensin-II.

Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.Histamine caused a striking increase in cAMP in glomeruli (+Delta% 675+/-87) and, to a lesser degree, increased cAMP in tubules (+Delta% 103+/-25) or in tissue slices. This stimulation was dose-dependent in the range of 1 muM-1 mM histamine. Metiamide (an H(2)-antagonist), but not pyrilamine (an H(1)-antagonist) blocked the effect of histamine on cAMP, which indicates that histamine causes its effect via interaction with H(2) receptors. Histamine caused less extensive increases in cGMP in both glomeruli and tubules. Carbamylcholine caused a marked increase in cGMP in glomeruli (+Delta 295+/-7) and a much lower increase in tubules (+Delta% 70+/-20); these effects were blocked by atropine. Parathyroid hormone (1 mug/ml) increased cAMP and, to a much lesser degree, also cGMP in glomeruli. In tubules, parathyroid hormone caused much more extensive increases in cAMP than in glomeruli; no changes, or rather a small decline in cGMP, was observed. Angiotensin-II (2 muM) markedly lowered cAMP in glomeruli (-Delta% -45+/-8) and in tubules (-Delta% 33+/-7) but had no effect on cGMP. Bradykinin (20 muM) did not consistently influence either cAMP or cGMP in glomeruli or tubules. Present results demonstrate that cAMP and cGMP metabolism in glomeruli are controlled independently by humoral agents known to alter glomerular functions in vivo. Our findings are consistent with the view that histamine and cholinergic agents generated and (or) released locally in glomeruli or in their vicinity may play important roles as mediators of immunopathological injury of glomeruli, and that these effects are mediated by cAMP and (or) cGMP through interaction with H(2) receptors and muscarinic receptors. Likewise, our results suggest that the effects of angiotensin-II and parathyroid hormone on glomerular dynamics may be mediated by cyclic nucleotides.Thus, we surmise that extrarenal as well as intrarenal humoral agents may play an important role in the pathology and physiology of glomeruli through mediation of either cAMP, cGMP, or both.

Cyclic nucleotide phosphodiesterases in glomeruli of rat renal cortex.

The presence and properties of cyclic 3',5'-adenosine monophosphate phosphodiesterase (cAMP-PDIE) and cyclic 3',5'-guanosine monophosphate phosphodiesterase (cGMP-PDIE) were studied in glomeruli isolated from rat renal cortex by sieving and density gradient centrifugation. The specific activity of cGMP-PDIE was higher than the specific activity of cAMP-PDIE in glomeruli; in tubules and renal cortical slices, the specific activity of cAMP-PDIE was higher than that of cGMP-PDIE. In homogenates, X 100,000g supernate of homogenate (cytosol) and X 100,000g pellet (membrane fraction) from glomeruli, the specific activity of cGMP-PDIE was significantly higher than it was in analogous preparations from tubules or renal cortical slices. Cyclic 3',5'-GMP (10(-6)M to 10(-5)M) stimulated glomerular cAMP-PDIE, but it was without effect on cAMP-PDIE from tubules. Structural analogs of cyclic 3',5'-GMP or 5'-GMP did not stimulate glomerular cAMP-PDIE. Cyclic 3',5'-AMP slightly inhibited cGMP-PDIE from both glomeruli and tubules. N6-,2'-0-dibutyryl cyclic 3',5'-AMP inhibited cAMP-PDIE, but not cGMP-PDIE. The addition of calcium increased the activity of cGMP-PDIE, mainly in tubules, but was without effect on cAMP-PDIE. These results suggest the predominance of cyclic 3',5'-GMP catabolism in glomeruli in comparison with other cortical structures, and they demonstrate that both the specific activities and regulatory properties of cyclic nucleotide phosphodiesterase in glomeruli differ markedly from tubules or unfractionated renal cortical tissue.

Cellular interactions between vasopressin and prostaglandins in the mammalian kidney.

Current experimental evidence indicates that endogenous renal medullary prostaglandins modulate the antidiuretic response to vasopressin in the mammalian kidney. The predominant effect of prostaglandins is to attenuate the antidiuretic response to vasopressin; inhibition of prostaglandin synthesis potentiates the renal effect of vasopressin. Prostaglandins likely antagonize the renal effects of vasopressin at the cellular level of hormone-dependent cyclic adenosine 3,5-monophosphate metabolism, but the exact molecular mechanism is not known. Likewise, it is not known whether such modulatory effect is due to primary prostaglandins, prostaglandin precursors or to other metabolites of arachidonic acid. Vasopressin itself could stimulate intrarenal prostaglandin synthesis; this effect may represent a negative-feedback regulatory pathway for the antidiuretic response to the hormone. Recent experimental evidence suggests that modulatory effect of prostaglandin may be a factor in pathogenesis of some types of urinary concentrating defects.

Ribosomal proteins of mouse kidney: normal status and during compensatory renal hypertrophy.

Ribosomes were isolated from normal and growing kidney and the protein complement was examined by a two-dimensional gel electrophoretic procedure. Proteins were resolved in the first dimension on the basis of charge and, in the second dimension, on the basis of molecular weight. 60S and 40S ribosomal subunits from normal kidney contained respectively 42 and 31 proteins. 80S ribosomes contained 23 proteins not found with either sub-unit. Nineteen of these proteins were removed from the ribosomes when isolated ribosomes were washed in a high salt buffer. Six proteins of the 80S ribosome corresponded to proteins associated with both sub-units. 80S ribosomal proteins were also studied during compensatory renal hypertrophy after 4-96 h of induced growth. The protein complement displayed by electrophoresis was identical to the pattern seen from normal renal cells.

Cellular hypertrophy and renal function during compensatory renal growth.

Simultaneous measurements of biochemical and physiological events of compensatory renal hypertrophy were made in groups of white Spartan rats. Thirty hours following right unilateral nephrectomy, the left kidney had an increased rate of renal plasma flow, glomerular filtration rate, and potassium excretion when compared to sham-nephrectomized rats. Kidney cells were also hypertrophied as evidenced by an increase in the ratio of ribonucleic to deoxyribonucleic acid. The effects of a single injection of cycloheximide and actinomycin D on compensatory growth were studied. Both drugs prevented the rise in glomerular filtration rate and renal plasma flow 30 h postnephrectomy while having no inhibitory effect on cell hypertrophy. The maintenance of nephrectomized rats on a low-sodium diet also interfered with the physiological components of the response while having no effect on ribonucleic or deoxyribonucleic acid. These suggest that the physiological and biochemical aspects of compensatory renal hypertrophy may be under separate control systems.