Adenosinetriphosphatase activity in the cell membranes of kidney tubule cells

This cytochemical study demonstrates high levels of apparent ATPase activity in the infolded cell membranes of the proximal tubules (dog, rat, human, mouse, monkey, and opossum) and ascending loops of Henle (dog, rat, human and, to a variable degree, mouse). Electron microscopy has shown (see Rhodin (1)) that these membranes separate adjacent cells where they interlock with one another by multiple cytoplasmic lamellae containing oriented mitochondria. The significance of the high ATPase activity is considered in relation to possible movements of the membranes and to "active transport" believed to occur there. In the rat, enzyme activity in the proximal tubule membranes does not survive formol-calcium fixation, and it is therefore necessary to use unfixed sections for its demonstration. However, in edematous rats with experimental nephrosis (induced by the injection of aminonucleoside or with antikidney serum) marked ATPase activity is exhibited in these membranes even after formol-calcium fixation. When proximal tubule or Henle loop cells of the dog acquire an altered metabolism, as indicated by accumulated lipide spheres or by "droplets," the infolded ATPase-rich membranes are no longer demonstrable.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Quantitative immunogold localization of Na,K-ATPase along rat nephron

Summary Ultrastructural localization of Na, K-ATPase -subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against -subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per m of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6–3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Influence of L-thyroxine upon enzymatic activity in the renal tubular epithelium of the rat under normal conditions and in mercury-induced lesions. I. Histochemical studies of alkaline phosphatase, acid phosphatase, adenosine- tri-phosphatase and leucine-aminopeptidase.

HgC12-induced renal tubular lesions in the rat present histochemically with a transitory decrease of alkaline phosphatase, adenosinetriphosphatase (ATPase), and leucine-aminopeptidase activity. The toxic alterations of enzyme activity were more pronounced in the pars recta of the proximal tubule and in the loop of Henle, as compared with the tubulus contortus I. L-thyroxine treatment leads to an accelerated reversal of that enzymatic defect, followinga characteristic pattern, and to a differentiating increase of acid phosphatase and ATPase activity in certain parts of the normal renal tubule. The observations are discussed with reference to the specific mode of action of sublimate and l-thyroxine upon the tubular enzymes and to the well-known metabolic and functional influences of thyroid hormone on the kidney.     

Cyclic 3',5'-nucleotide phosphodiesterase; cytochemical localization in rat renomedullary interstitial cells.

The localization of cyclic 3', 5' -nucleotide phosphodiesterase activity in rat renal papillae was examined by utilizing cytochemical methods. Renal medullary interstitial cells had predictable phosphodiesterase activity predominantly on the cytoplasmic border of dilated cisternal membranes. Cells of the collecting tubule and loop of Henle contained diffuse reaction product. Capillaries had reaction product localized in pinocytic vesicles. Addition of theophylline resulted in no deposition of reaction product in interstitial cells and in cells of the collecting tubule and loop of Henle, suggesting an inhibition of phosphodiesterase activity. Since the membranes of dilated cisternae of renal medullary interstitial cells have been shown to be related to prostaglandin synthesis and probably to the anti-hypertensive function of these cells, the finding of phosphodiesterase activity on these membranes suggests a possible role of cyclic AMP in these two functions.     

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.     

Ectonucleotidases in the kidney

Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K _m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.     

Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney.

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO(3)(-) absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40-45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H(+)-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO(3)(-) absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

On the failure of calmodulin to activate Ca2+ pump ATPase of dog red blood cells

Ca2+-pump ATPase activities of membranes isolated from human and dog RBCs were compared under a variety of conditions. Specific activity of the dog enzyme was less than that of human. Unlike the human enzyme, the dog Ca2+-pump ATPase was not stimulated by exogenously added calmodulin (CaM) or oleate. The Ca2+ dependence of the dog Ca2+-pump ATPase resembled that of the CaM-activated form of the human enzyme. Cross-linking of Azido-125I-CaM to dog RBC membranes did not label a Ca2+-pump ATPase of molecular weight similar to that found in human RBC membranes. It is suggested that the Ca2+-pump ATPase in isolated dog RBC membranes exists in an activated state, not due to endogenous CaM, but possibly due to partial proteolysis.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Hypothalamic factor inhibits the (Na,K)ATPase from the extracellular surface. Mechanism of inhibition

We have characterized the effect of a stable small molecule isolated from bovine hypothalamus (Haupert, G. T., and Sancho, J. M. (1979) Proc. Natl. Acad. Sci. 76, 4658-4660) on mammalian (Na,K)ATPase. This hypothalamus-derived inhibitory factor, HIF, has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity (Haupert, G. T., Carilli, C. T., and Cantley, L. C. (1984) Am. J. Physiol. 247, F919-F924). In this report it is shown that HIF inhibits the ouabain sensitive component of 86Rb+ uptake into human red blood cells. HIF also inhibited (Na,K)ATPase activity of unsealed red cell membranes but not that of sealed inside-out vesicles, indicating that HIF is impermeant to red cell membranes and inhibits the (Na,K)ATPase from the extracellular side. In unsealed human red cell membranes, concentrations of HIF which caused 70% inhibition of the (Na,K)ATPase did not inhibit ATP hydrolysis by plasma membrane (Ca2+)ATPase or (Mg2+)ATPase. However, at a similar concentration, HIF was shown to inhibit rabbit muscle sarcoplasmic reticulum (Ca2+)ATPase. HIF also inhibited p-nitrophenylphosphatase activity of unmodified or fluorescein-5'-iso-thiocyanate labeled dog kidney (Na,K)ATPase. As judged by fluorescein fluorescence of the modified enzyme, HIF stabilized the low fluorescent "E2" conformation of the enzyme similar to that stabilized by ouabain. However, unlike ouabain, HIF blocked covalent phosphorylation of dog kidney (Na,K)ATPase by inorganic phosphate. These studies show that HIF is an inhibitor of (Na,K)ATPase which acts from the extracellular side of the membrane by a mechanism similar to but not identical to that of cardiac glycosides.     

3H]bumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na,K,Cl co-transport system

We have synthesized the radiolabeled "loop" diuretics [3H]bumetanide and [3H]benzmetanide (3-benzylamino-4-phenoxy-5-sulfamoylbenzoic acid) and have tested their potential as reversible labels of the Na,K,Cl co-transport system. These compounds bind with high affinity (Kd less than or equal to 30 nM, under optimal conditions) to membranes isolated from dog kidney; we found approximately 2 pmol/mg of sites in crude membranes from the outer medulla, and less than or equal to 0.5 pmol/mg in a similar preparation from kidney cortex. On sucrose gradient centrifugation, a peak of [3H]bumetanide binding activity (30 pmol/mg) is obtained at 37% (w/v) sucrose, distinct from the basolateral membranes in outer medulla and from brush borders in proximal tubule; our hypothesis is that this peak contains luminal membranes from the thick ascending limb of the loop of Henle. [3H]Bumetanide is displaced from its binding sites by various unlabeled loop diuretics at concentrations that have previously been shown to inhibit co-transport. Na+, K+, and Cl- (K1/2 congruent to 2, 1, and 1 mM, respectively) are required for [3H]bumetanide binding, and Cl- inhibits at higher concentrations. We interpret these data to demonstrate that the Na,K,Cl cotransport system is the site involved in [3H]bumetanide binding in kidney membranes.     

Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique

A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20-40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron.     

Evidence for a calmodulin-activated Ca2+ pump ATPase in dog erythrocytes

Previous work in several laboratories revealed little or no Ca2+ pump ATPase activity and little or no activation of the ATPase by calmodulin (CaM) in membranes isolated from dog red blood cells (RBCs). In the present work, intact RBCs from dogs were exposed to the ionophore, A23187, in the presence of Ca2+. A rapid, apparently first order, loss of ATP occurred under these conditions. The first order rate constant was 0.0944 min-1, or approximately 47% of that found in human RBCs under the same conditions. The anti-CaM drug, trifluoperazine, inhibited the loss of ATP and the Ca2+ activation curve of ATP loss in intact cells resembled that observed for CaM-activated Ca2+ pump ATPase in isolated human membranes. Taken together, these data are consistent with the interpretation that the dog RBC membrane contains a CaM-activated Ca2+ pump ATPase.     

Human renal 11beta-hydroxysteroid dehydrogenase 1 functions and co-localizes with COX-2

The local renal metabolism of glucocorticoids (GCs) by isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD1 and 11beta-HSD2) determines their biological effects. 11beta-HSD2, located in collecting duct epithelial cells of the mammalian and human kidney, serves as a putative "guardian" preventing GCs from binding to mineralocorticoid receptors. Various investigators have shown that both isoforms are present in kidney tissue from the rat, dog and other mammals. There is controversy as to whether 11beta-HSD1 exists and functions in human kidney. The current studies examine the locale and function of both isoforms in human kidney. The expression of 11beta-HSD1 was similar to that of 11beta-HSD2 by Western blot. Two distinct Lineweaver Burke plots could be drawn providing enzyme kinetics for both isoforms. The apparent Km for the NADP dependent 11beta-HSD1 enzyme was 0.42 muM while the apparent Km for the NAD dependent 11beta-HSD2 enzyme was 10.2 nM. Human renal 11beta-HSD1 appears to function as a dehydrogenase with no significant "reverse" reductase activity. Using immuno-histochemistry and Western blot analysis, 11beta-HSD1 was found to co-localize with COX-2 in proximal tubule cells; COX-2 was not seen with 11beta-HSD2 in cortical collecting duct. Thus, normal human kidney contains active 11beta-HSD1 and 11beta-HSD2. 11beta-HSD1 co-localizes with COX-2 in proximal tubule cells.     

Localization of carbonic anhydrase activity in the vertebrate nephron

Synopsis The localization of carbonic anhydrase activity in the vertebrate nephron has been examined with particular reference to the proximal tubule and collecting duct. In all species studied, activity was present in the proximal tubular epithelium. In the pigeon and turtle, distinctive and similar patterns of staining were observed in the glomerulus and first portion of the proximal tubule. In the rat and rhesus monkey, the entire proximal tubule exhibited activity; in these species it has been shown previously with micropuncture techniques that there is a high absorptive capacity of this nephron segment for bicarbonate. In contrast, large portions of the dog proximal tubule were inactive; similar studies in this animal have shown tubular concentrations of bicarbonate only slightly lower than plasma levels. In the rat and dog, the entire length of the collecting duct was diffusely and intensely active; in contrast, pigeon collecting duct showed no activity. An alternating pattern of inactive and intensely active cells was observed in the collecting ducts of the toad, turtle, rabbit and monkey. A similar pattern has been described in the turtle and toad bladder, tissues utilized forin vitro studies of ion transport and H⁺ secretion.