Structural adaptation to altered electrolyte metabolism by cortical distal segments

The four different renal cell types in the cortical segments beyond the macula densa--distal convoluted tubule (DCT) cell, the connecting tubule (CNT) cell, the principal (P) cell, and the intercalated (I) cell--each respond to prolonged specific stimuli with changes in cell membrane area. Increases in basolateral cell membrane area, reflecting the transport capacity of the cell, are associated in DCT cells with luminal, chronically high sodium load, in CNT cells with mineralocorticoids and tubular solute load, and in P cells with mineralocorticoids. In I cells the luminal cell membrane area seems to be influenced by the local luminal environment of the cells as well as by the peritubular environment. These structural findings indicate that tubular fluid composition as well as peritubular factors (e.g., mineralocorticoid levels) may have a role in regulating the transport capacity of distal cell types in the kidney.     

Ultrastructure of distal nephron cells in rat renal cortex

Distal nephron segments in the rat renal cortex contain distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells). The present study was carried out to expand present knowledge on the ultrastructure of these cells. The cells were sampled from superficial cortex and analyzed by electron microscopy. Several morphometric parameters were determined and statistical comparison between cell types was performed. Significant structural differences between the cell types were demonstrated. DCT cells showed the highest volume density of mitochondria whereas the amplification of basolateral membranes was higher in CNT cells than in I and P cells. The surface density of the membrane that bounds intermediate vesicles in the apical cytoplasm was twofold higher in I cells than in the other cell types. The morphological differentiation found in the present study adds to available evidence indicating a functional differentiation between the cell types and provides a reference for structure-function correlations in these cells.     

Biochemical and histocytochemical studies on response of ammonia-producing enzymes for nh4cl-induced acidosis.

NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.     

Effect of acute changes in the PaCO2 on acid-base parameters in normal dogs and dogs with metabolic acidosis or alkalosis

There is a linear relationship between the PaCO2 and blood hydrogen ion concentration in normal dogs, but for theoretical reasons to be discussed, we questioned whether this relationship would apply in animals with metabolic acidosis or alkalosis. To study this in more detail, animals were divided into three groups: normal, metabolically acidotic, and metabolically alkalotic. Following anesthesia and bilateral ureteral ligation, dogs were intubated and ventilated to produce acute steady state PaCO2 values corresponding to the range observed during disease states. Changes in the volume and electrolyte composition of the gastrointestinal fluid and urine as well as the concentration and distribution of lactate were evaluated in all experiments. We observed the previously described linear relationship between the PaCO2 and blood hydrogen ion concentration in normal dogs, but the slope of the regression line differed significantly from those of dogs with metabolic acidosis and metabolic alkalosis. On the other hand, there was a consistent relationship between the ratio of the PaCO2 values, but not the absolute PaCO2, and the change in the plasma bicarbonate concentration over a wide range of PaCO2 values in all groups of dogs. The chemical basis for these observations will be discussed.     

Na+-H+ exchange in luminal-membrane vesicles from rabbit proximal convoluted and straight tubules in response to metabolic acidosis.

Na+-H+-exchanger activity of pars convoluta and pars recta luminal-membrane vesicles prepared from the proximal tubule of acidotic and control rabbits were assayed by a rapid-filtration technique and an Acridine Orange method. Both experimental approaches revealed the existence of an antiporter, sensitive to metabolic acidosis, in pars convoluta membrane vesicles. Kinetic data, obtained with the pH-sensitive dye, showed that the Km for Na+ transport was unchanged by acidosis, whereas Vmax. for exchanger activity was increased, on an average, by 44%. The fluorescence method, in contrast with the rapid-filtration technique, was able to detect exchanger activity in pars recta membrane vesicles. The Km value for the antiporter located in pars recta is comparable with that calculated for pars convoluta membrane vesicles. By contrast, the Vmax. of this exchanger is only about 25% of that found for pars convoluta. Furthermore, metabolic acidosis apparently does not increase Na+-H+-exchanger activity of pars recta luminal-membrane vesicles.     

Na+-H+ exchange in isolated renal brush-border membrane vesicles in response to metabolic acidosis. Kinetic effects

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

Distal tubule bicarbonate reabsorption during rebound metabolic alkalosis.

Rebound metabolic alkalosis is a transient alkalemia that is seen during recovery from NH4Cl-induced metabolic acidosis. The persistent elevation of plasma bicarbonate concentration is the result of continuing excretion of net acid by the kidney. Bicarbonate transport by inner medullary collecting ducts has been reported by others to proceed normally (i.e., bicarbonate reabsorption continues in this segment) during rebound metabolic alkalosis. No other segmental responses have been evaluated. Since the surface distal tubule of the rat is known to both reabsorb and secrete bicarbonate in vivo, it was of interest to determine the response of this segment. Our results show that the distal tubule microperfused in vivo during rebound metabolic alkalosis continues to reabsorb significant amounts of bicarbonate, despite the presence of systemic alkalemia that we have previously shown to be associated with distal tubule bicarbonate secretion.     

Acidotic alterations in oxidative metabolism influencing rat renal slice ammoniagenesis

We believe that two findings are interconnected and help to comprehend a major mechanism behind the regulation of renal ammonia production during acidosis. First, slices from acidotic compared to control and alkalotic rats produce more ammonia from glutamine. Second, inhibition of renal oxidative metabolism at various points by metabolic inhibitors augments slice ammoniagenesis. Based on this, our purpose was to determine whether enhanced renal ammoniagenesis during acidosis could occur through the same mechanism as the metabolic inhibitors. However, metabolic inhibitors (malonate; arsenite; 2,4-dinitrophenol) usually decrease while acidosis increases slice gluconeogenesis. There is one known exception. Fluorocitrate, which blocks citrate metabolism, simulates the acidotic condition by enhancing both ammonia and glucose production. Accordingly, a block of oxidative metabolism if located prior to citrate oxidation in the tricarboxylic acid cycle could theoretically augment ammoniagenesis during acidosis. Lactate, is a major renal fuel whose oxidative metabolism would be blocked by fluorocitrate. There, we concentrated on the effects of acidosis on lactate as well as glutamine metabolism. Lactate decarboxylation decreases in the face of increased glucose production during acidosis, and lactate inhibition of glutamine decarboxylation decreases in slices from acidotic rats. Also, we found lesser oxygen consumption in the presence of lactate by kidney slices from acidotic rats compared to control and alkalotic rats. We postulate that relatively less incorporation of lactate into the TCA cycle, causing decreased citrate formation and citrate oxidation during acidosis, contributes, at least in part, to acidotic adaptation of ammoniagenesis.     

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.     

Cells and intercellular contacts in glomeruli and tubules of the frog kidney

Summary By the use of thin sections and freeze-fracture replicas the glomerular and tubular structures of the kidney of the frog (Rana esculenta) were studied with special reference to intercellular junctions.In the glomerulus the filtration barrier is of very variable thickness, and frequent tight and gap junctional contacts occur between podocyte processes.Although structurally less elaborate, the proximal tubule resembles its mammalian counterpart. In the initial part the tight junctions are relatively shallow but become very broad in the mid and distal portions of the proximal tubule. The proximal tubular cells are extensively linked by gap junctions. In some animals the shapes of the cells in the proximal and distal portions of the proximal tubule were markedly different.The distal tubule consists of two segments which differ mainly in the pattern of interdigitations and the structure of the zonulae occludentes. Similarities with the tight junctional morphology of the mammalian distal tubule are striking. In the first part of the distal tubule (diluting segment) a narrow band of parallel tight junctions is found closely resembling that found in the mammalian straight distal tubule; in the more distal part of the distal tubule, however, a broad band of anastomosing tight junctional strands exists, like the zonula occludens of the mammalian convoluted distal tubule.The connecting tubule displays cellular dimorphism: its wall contains a mixture of light and dark (flask) cells. The luminal and basolateral membranes of the flask cells are covered with numerous rod-shaped particles. The tight junctions of the connecting tubule are broad and increase in depth and number of strands along its length; they are typical of a very tight epithelium.In spite of several dissimilarities with phylogenetically younger kidneys our findings suggest that many structural principles of the mammalian kidney are also represented in the kidneys of amphibians. The structural-functional relationships are discussed.     

Distal tubular segments of the rabbit kidney after adaptation to altered Na- and K-intake

Summary The baso-lateral cell-membrane area in kidney tubules appears to be associated with the capacity for electrolyte transport; in the rabbit, it decreases from the distal convoluted tubule (DCT-cells) over the connecting tubule (CNT-cells) to the cortical collecting duct (principal cells).Adaptation to low Na-, high K-intake changes this pattern: CNT-cells at the beginning of the connecting tubule have the highest membrane area, which decreases along the segment, but remains two-fold higher than in controls. Principal cells have a four-fold higher membrane area than in controls. Simultaneous treatment with the antimineralocorticoid canrenoate-K inhibits the structural changes in CNT-cells only in end-portions of the connecting tubule and in principal cells.After prolonged high Na-, low K-intake DCT-cells display a two-fold higher membrane area than controls, while CNT-cells and principal cells are not affected. Simultaneous treatment with DOCA does not affect the DCT-cells but provokes a moderate increase in membrane area in CNT-cells, and a 5.5-fold increase in principal cells.The data provide evidence that DCT-, CNT- and principal cells are functionally different cell types. The baso-lateral cell-membrane area, associated with electrolyte-transport capacity, appears to be influenced in DCT-cells mainly by Na-intake, in CNT-cells mainly by K-intake and in part also by mineralocorticoids, and in principal cells mainly by mineralocorticoids.Supported by SFB90, CARVAS, Heidelberg and Swiss National Science Foundation, Grant No. 3.900-0.79     

Ultrastructure of the Protonephridium in Acteonemertes bathamae Pantin (Rhynchocoela: Enopla: Hoplonemertini)

The protonephridial system consists of terminal cell, protonephridial capillary, protonephridial tubule and efferent duct. The terminal cell is an elongated, thin-walled, fenestrated basket containing a ciliary flame circumscribed by a palisade of straight microvilli. The filtration area is confined to the terminal cell and consists of slits bridged by a filtration membrane. The cilia, as well as the microvilli, projects into the proximal bell-shaped part of the thin-walled protonephridial capillary. The terminal cells are often found in pairs connected to the same capillary, which has a very narrow lumen. The proximal part of the thick-walled, convoluted protonephridial tubule is ciliated and shows characteristic foldings of the luminal plasma membrane and numerous small vesicles in the cytoplasm. The cells of the following, non-ciliated part of the tubule have interdigitating lateral surfaces and the bases deeply invaginated to form compartments with numerous mitochondria; in the cytoplasm are many large vesicles, possibly containing lipid droplets, and small amounts of glycogen. The distal protonephridial tubule resembles various epithelia with an osmoregulatory function, including the vertebrate nephron.     

Substrate utilization by the distal nephron in dogs with chronic metabolic acidosis: studies with ethacrynic acid

Glutamine and lactate oxidations provide the bulk of ATP required for sodium reabsorption in the dog kidney during chronic metabolic acidosis. Indirect evidence has suggested that glutamine is oxidized in the proximal convoluted tubule; if this is true, lactate should be the major fuel of the more distal nephron sites. The purpose of these experiments was to determine which substrates were metabolized by the acidotic dog kidney when a significant proportion of sodium chloride reabsorption was inhibited in the thick ascending limb of the loop of Henle. Ethacrynic acid, a loop diuretic, caused the fractional excretion of sodium to increase from 1 to 34%. The glomerular filtration rate declined somewhat, but there was no significant change in the renal blood flow rate. Renal oxygen consumption declined in conjunction with the natriuresis. However, when the data were examined at a constant filtered load of sodium (a constant rate of ATP turnover), there was no reduction in glutamine uptake or glutamine conversion to ATP in the presence of this natriuretic agent. The major change observed concerned lactate metabolism, in the presence of ethacrynic acid, there was no longer a significant rate of lactate extraction. These data are best explained by assuming that glutamine is the fuel of the proximal convoluted tubule of the acidotic dog kidney, whereas lactate oxidation occurs principally in the nephron sites where sodium reabsorption was inhibited by ethacrynic acid.     

A kinetically defined Na+/H+ antiporter within a mathematical model of the rat proximal tubule

The luminal membrane antiporter of the proximal tubule has been represented using the kinetic formulation of E. Heinz (1978. Mechanics and Engergetics of Biological Transport. Springer-Verlag, Berlin) with the assumption of equilibrium binding and 1:1 stoichiometry. Competitive binding and transport of NH+4 is included within this model. Ion affinities and permeation velocities were selected in a least-squares fit to the kinetic parameters determined experimentally in renal membrane vesicles (Aronson, P.S., M.A. Suhm, and J. Nee. 1983. Journal of Biological Chemistry. 258:6767-6771). The modifier role of internal H+ to enhance transport beyond the expected kinetics (Aronson, P.S., J. Nee, and M. A. Suhm. 1982. Nature. 299:161-163) is represented as a velocity effect of H+ binding to a single site. This kinetic formulation of the Na+/H+ antiporter was incorporated within a model of the rat proximal tubule (Weinstein, A. M. 1994. American Journal of Physiology. 267:F237-F248) as a replacement for the representation by linear nonequilibrium thermodynamics (NET). The membrane density of the antiporter was selected to yield agreement with the rate of tubular Na+ reabsorption. Simulation of 0.5 cm of tubule predicts that the activity of the Na+/H+ antiporter is the most important force for active secretion of ammonia. Model calculations of metabolic acid-base disturbances are performed and comparison is made among antiporter representations (kinetic model, kinetic model without internal modifier, and NET formulation). It is found that the ability to sharply turn off Na+/H+ exchange in cellular alkalosis substantially eliminates the cell volume increase associated with high HCO3- conditions. In the tubule model, diminished Na+/H+ exchange in alkalosis blunts the axial decrease in luminal HCO3- and thus diminishes paracellular reabsorption of Cl-. In this way, the kinetics of the Na+/H+ antiporter could act to enhance distal delivery of Na+, Cl-, and HCO3- in acute metabolic alkalosis.     

Comparison of the distribution of tissue kallikrein and esterase A, a kallikrein-like enzyme, in rat kidney using specific monoclonal antibodies

Tissue kallikrein (E.C. 3.4.21.35) and arginine esterase A, another closely related, kinin-generating serine protease, have been localized by immunocytochemistry in rat kidney, using monoclonal antibodies that do not crossreact with other kallikrein-related enzymes or with tonin. Kallikrein was present primarily in the apical cytoplasm of the connecting tubule and the cortical collecting duct. Esterase A, on the other hand, was present primarily in the basolateral region of both proximal and distal straight tubules in the outer medulla and medullary rays. In addition, esterase A was demonstrable in distal convoluted tubules and, to a lesser extent, in proximal convoluted tubules. The presence of different kinin-generating enzymes at these sites would permit the formation of kinins from appropriate substrates on both the vascular and luminal poles of separate segments of the kidney tubule.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.