Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = 300,000) by SDS PAGE . To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization . When a detergent extract of [35S] methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of -300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG . Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal antigp330 IgG failed to do so . When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences . When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule : maltase was concentrated (-90%) on the microvilli, and gp330 was concentrated (-90%) in the clathrin-coated apical invaginations located at the base of the microvilli . We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma .The luminal aspect of the kidney proximal tubule consists of a brush border that is differentiated into two distinct types of structures-microvilli and intermicrovillar apical invaginations (1) . The microvilli appear to closely resemble those of the intestine in their absorptive function and supporting cytoskeletal elements (2, 3). The apical invaginations are known to function in endocytosis (1) and have recently been shown to have extensive clathrin coats on their cytoplasmic surface (4), thus resembling clathrin-coated pits in other locations . At present there is relatively little information available on the comparative protein composition of these two proximal tubule structures. Microvillar fractions (which consist mainly ofvesicles derived from microvilli along with some THE JOURNAL OF CELL BIOLOGY " VOLUME 98 APRIL 1984 1505-1513 C The Rockefeller University Press -0021-9525/84/04/1505/09 $1 .00 contaminating intermicrovillar membranes), have been isolated and analyzed (5) and found to contain numerous proteins, some ofwhich are cytoskeletal and others are transmembrane glycoproteins identified as hydrolytic enzymes (aminopeptidases, disaccharidases, etc .) (5) or transport systems (e.g., for glucose and amino acids) (6) . Evide...

The glucocorticoid dexamethasone, but not the mineralocorticoid aldosterone, increased amiloride-sensitive Na'-H' exchange activity in rat proximal tubule brush border vesicles. Na' uptake, independent of amiloride, was not affected. The glucocorticoid decreased the Na' gradient-dependent phosphate uptake. Uptake in the absence of a Na' gradient was not inhibited. Dexamethasone did not affect the Na' gradient-dependent D-glucose uptake. METHODS AND MATERIALS Animals and Steroid Administration. Male Sprague-Dawley rats (295-345 g) were fed Purina rat chow pellets ad lib. Rats were adrenalectomized under light ether anesthesia. After surgery, normal saline was given to prevent volume depletion. Adrenalectomized animals were divided into three groups. One group received twice daily subcutaneous injections of 0.3 ml of 0.9% saline. The other two groups received twice daily subcutaneous injections (5 pLg/100 g of body weight) of dexamethasone or aldosterone in 0.9% saline, respectively. The quantity of dexamethasone administered daily as the glucocorticoid replacement was approximately equivalent to the amount of corticosterone secreted per day by the rat and was the dose that effected a return of Na+, K+, and water movements in the rat colon to control values after adrenalectomy (18). Likewise, the quantity of aldosterone administered daily was approximately equivalent to the amount ofaldosterone secreted per day by rats on a normal diet (18). Another set of rats underwent only bilateral flank incisions, without removal of adrenals, and subsequently were given tap water to drink. This set ofanimals was subdivided into two groups. One group, designated sham, received twice daily subcutaneous injections of 0.3 ml of 0.9% saline. The other group, designated sham with dexamethasone, received dexamethasone (60 ,g/100 g of body weight) on the same schedule. Transport studies were performed 2 days after surgery. Hormones were last administered 2 hr prior to sacrifice. Animals were killed between 10:00 and 10:30 a.m.At the time ofsacrifice, serum Na+ concentration was slightly higher in adrenalectomized rats given aldosterone relative to the other groups, but the value was not statistically different from that found in adrenalectomized animals (141.6 ± 1.0 vs. 139.2 ± 0.5 milliequivalents/liter; P > 0.05). Serum Pi was essentially similar in all groups, varying from 2.66 ± 0.04 to 2.90 + 0.08 mM, although sham animals that received high doses of dexamethasone had-a Pi concentration that was minimally lower statistically than that in sham animals (2.70 ± 0.05 vs. 2.90 + 0.08 mM; P < 0.05), and adrenalectomized rats given maintenance doses of glucocorticoid had a slightly lower serum Pi concentration than did the adrenalectomized animal (2.66 ± 0.04 vs. 2.81 ± 0.06 mM; P < 0.05). Serum Ca2+ ranged from 2.35 ± 0.04 to 2.56 ± 0.04 mM. Sham rats given high doses of dexamethasone had the highest serum Ca2+ level. This was significantly greater (P < 0.05) than the values found in the other groups, which did not significan...

Amiloride-sensitive Na+-H+ exchange activity in brush border membrane vesicles isolated from rat proximal tubule was increased in metabolic acidosis. The enhancement of exchange activity required an intact adrenal gland or glucocorticoid supplements. Ammonium and phosphate excretions were increased during acidosis and these were also largely dependent on an intact adrenal gland or glucocorticoid supplements. Amiloride-insensitive Na' uptake and passive H+ permeability were not altered by acidosis or the glucocorticoid status of the animal. These findings are consistent with glucocorticoids having an important regulatory role in the kidney by orchestrating the proximal tubular adaptation to metabolic acidosis.The kidney responds to metabolic acidosis by increasing secretion of acid, phosphate, and ammonium and by enhancing reabsorption of bicarbonate (1). Acid secretory processes provide a mechanism for bicarbonate reabsorption, 80%o of which occurs in the proximal tubule (2). This nephron segment is the locus of the Na+-H' exchanger (3). The carrier, found in the brush border membrane, mediates the electroneutral antiport of Na+ for H+ (4-6). Amiloride, at relatively high concentrations (Ki = 0.05 mM), competitively inhibits exchange activity (7).Metabolic acidosis is also associated with increased levels of adrenal corticosteroids (8-10). Adrenalectomy decreases renal net excretion of titratable acids and ammonium (8,9) and hyperglucocorticoid states are concomitant with metabolic alkalosis (11). Glucocorticoids increase endogenous acid production, stimulate acid secretion, enhance ammonium production, and induce phosphaturia (12)(13)(14). The site of action of glucocorticoids in decreasing phosphate reabsorption is largely confined to the proximal tubule (15), where glucocorticoid receptors have been found (16). In addition, we have recently reported that the glucocorticoid dexamethasone, but not the mineralocorticoid aldosterone, increases amiloride-sensitive Na+-H+ exchange activity and selectivity decreases Na+ gradient-dependent phosphate uptake in proximal tubule brush border membrane vesicles (17).These findings raise the possibilities that metabolic acidosis induces changes in renal brush border Na+-H+ exchange activity and phosphate and ammonium excretion and, further, that glucocorticoids may have a role in mediating these effects. The present communication addresses these questions.METHODS AND MATERIALS Animals: Dexamethasone Administration and Acid-Base Status. Male Sprague-Dawley rats weighing 200-320 g were fed Purina rat chow pellets ad libitum and had 0.9% saline in their drinking water for 6-8 days. Acidotic rats were given, in addition, 1% NH4Cl in the water. The adrenals of adrenalectomized animals were removed under light ether anesthesia on day 1. Dexamethasone-treated animals were given two injections (30 ,ug/100 g of body weight) of dexamethasone in 0.9% saline, 24 and 16 hr prior to sacrifice. The glucocorticoid dose approximated that reported to lead to a large increase in total...

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The enzyme trehalase, which hydrolytically splits trehalose (1-a-D-glucopyranosyl-a-D-glucopyranoside) to two glucose moieties, has been reported from yeast, fungi and other plants, and several phyla of invertebrate animals; it is ubiquitously found in insects.' Trehalose is the maj or blood sugar of insects.2-4 The "fat body" rapidly converts glucose and glycogen to trehalose, which is released to the hemolymph and is used to support a variety of energy-requiring functions. Trehalase is especially active in the insect gut,5-7 although the insect may never ingest trehalose. The enzyme is also prevalent in flight muscle,8-10 where the precipitous hydrolysis of the disaccharide at the initiation of flight is the rate-limiting reaction in glycolysis.10 These findings, plus others, led us to suggest that in insects trehalose is a vehicle of carbohydrate transport and that trehalase functions in the mechanism of sugar transport."In an investigation of digestive disaccharidases in rat intestine, Dahlqvist and Brun"2 have shown that maltose, sucrose, lactose, and trehalose are hydrolyzed. An intestinal trehalase, specific for trehalose, has been purified 20-fold.'3 The presence of trehalase in the mammalian intestine, which assumes added significance in light of our suggested role for the enzyme, prompted us to look for trehalase in other mammalian tissues and to see if trehalase may function in glucose transport in higher animals. In this communication we report a highly active trehalase in kidney as well as in intestine. The specific localization of the enzyme, plus the discovery in kidney and jejunum of the enzymes which in sequence lead to the synthesis of trehalose from glucose, support the hypothesis that the disaccharide has a role in the resorption of glucose by the kidney and the transport of the hexose across the intestinal mucosa. '4 Methods.-Trehalase was determined by either of two procedures, depending on the activity of the preparation. With more active preparations, the enzyme was assayed directly. The reaction mixture contained 0.5 Mmole of NADP, 0.5 Mmole of ATP, 12 Mmoles of trehalose, 5 /Amoles of MgCl2, 6 Mimoles of potassium phosphate, adjusted to pH 6.3, excesses of glucose-6-P dehydrogenase and hexokinase, and trehalase in a total volume of 0.62 ml. With less active or turbid preparations, tissue samples were incubated for 10 or 30 min in a reaction containing 12 mM trehalose and 10 mM potassium phosphate, pH 6.3, in a volume of 1.0 ml. The reaction was stopped with the addition of 0.1 ml of 8% perchloric acid. Glucose in the neutralized extract was determined as described previously. 10

A membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake of L-glutamate by the basolateral membrane vesicles was studied. A Na+ gradient (Na+]o greater than [Na+]i) stimulated the uptake of L-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na+-L-glutamate cotransport system in the basolateral membrane. A K+ gradient ([K+]i greater than [K+]o) increased the uptake additionally. This effect was specific for K+(Rb+). The action of the K+ gradient in enhancing the uptake of L-glutamate had an absolute requirement for Na+. In the presence of Na+, but in the absence of a Na+ gradient. i.e., [Na+]o = [Na+]i, the K+ gradient also energized the concentrative uptake of L-glutamate. This effect of the K+ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system for L-glutamate energized by both Na+ ([Na+]o greater than [Na+]i and K+ ([K+]o) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by which L-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.

Na+-H' exchange activity, i.e., amiloridesensitive Na' and H' flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na'-H' exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na' uptake.The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na' uptake in vesicles from hyperthyroid animals. Na'-dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na'-dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na' in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na+ reabsorption. Further, the regulation of transtubular Na+ flux has now been shown to be concomitant with modulation of the entry of Na' into the tubular cell across its luminal membrane, mediated by the exchange reaction, and with the previously reported control of the pumping of Na+ out of the cell across its basolateral membrane, mediated by the Na+,K+-ATPase.Thyroid hormones (thyroxine, T4; triiodothyronine, T3) have a significant role in controlling kidney growth and function. The hormones are important regulators of renal plasma flow, glomerular filtration rate, concentration and dilution of urine, oxygen consumption, and the reabsorption of phosphate, Ca2+, and Na+ (1, 2). Thyroid hormones stimulate Na+, K+-ATPase activity (3), and changes in renal Na+,K+-ATPase activity closely parallel alterations in net transport of Na+ (4). T3 is known to induce the synthesis of Na+, K+-ATPase (5). It also has been proposed that thyroid hormones augment renal Na+,K+-ATPase activity by an adaptive mechanism responding to changing resorptive Na+ loads (4). Both mechanisms, the induction of Na+ pump elements and the adaptive response to increased filtered Na+, could operate together in mediating the action of thyroid hormones on Na+ reabsorption (1). Another hypothesis, that thyroid hormones enhance the entry of Na+ from the filtrate to the tubular cell across the luminal membrane, merits consideration.Na+, at physiological concentrations, crosses the luminal brush border membrane of the proximal tubule mostly by Na+-H+ exchange (6, 7). The carrier mediates the electroneutral antiport of Na+ for H+, Na+ entering the cell as H+ migrates from cell to lumen (8-10). Amiloride at relatively high concentrations (Ki = 5 x 10-5M) competitively inhibits exchange activity (10). In contrast, the ouabainsensitive Na',K+-ATPase, which serves to pump Na' from cell to interstitium, is located on the basolateral membrane of the tubular cell (11).We have reported recently t...