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.     

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.     

Renal carbonic anhydrase in the quail Coturnix coturnix japonica: I. Activity and distribution in male and female metanephros

Summary Carbonic anhydrase activity was studied in the quail metanephros by means of histochemical, histophotometrical and biochemical methods. Male and female samples were examined separately in order to show sex-related differences in enzyme activity and localization. The staining patterns revealed differential distribution of reaction product in the different, tubular segments. The initial portion of proximal tubules showed positivity on the brush border in female kidneys only.Extra situ investigations provided further evidence of sexual dimorphism resulting in higher values of enzyme activity for female than for male kidneys.In both sexes, marked staining was detected at the distal tubule level where histophotometric analysis confirmed the highest amount of reaction product. Moreover, the intracellular staining distribution at this site proved to be similar to that observed for mammalian proximal convoluted tubules.In the collecting ducts, a mosaic-like pattern was found with respect to both carbonic anhydrase staining and metachromatic properties.The functional significance of the presence of enzyme in the different renal tubules is discussed by comparison with the mammalian kidney. A model is proposed whereby the distal tubules represent the main sites of urinary acidification and bicarbonate reabsorption.     

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.     

Histochemical localization of carbonic anhydrase in the testis and epididymis of the boar.

The testis and epididymis of sexually mature, fertile boars were studied for localization of carbonic anhydrase (CA) using a cobalt precipitation technique. In the testis, cytoplasmic CA was found in the Sertoli cells and in the capillaries surrounding the seminiferous tubules. The epididymal duct was divided into initial, middle and terminal segments, and regional differences in CA activity were observed. The cell membranes of the basal cells were stained in the initial and middle segments. Strong cytoplasmic CA staining was present only in the apical cells in the initial and middle segments. The basolateral cell membranes were stained in the principal cells of the terminal segment and the ductus deferens. As a rule the capillaries surrounding the epididymal duct were unstained. The enzyme, specifically localized in regions of the male genitalia acting as sperm reservoirs, might be related to the quiescence of the stored spermatozoa by influencing the acid-base status of the epididymal fluid.     

Differential inhibition by acetazolamide on carbonic anhydrase distribution in the quail kidney: a proposal for a membrane-bound isoenzyme

Summary The effects of different concentrations of acetazolamide, a specific carbonic anhydrase inhibitor, have been investigated in the quail kidney. The histochemical patterns, interpreted by means of quantitative analyses proved that 0.1 m acetazolamide inhibited the enzyme activity in all the reactive tubular segments except for distal tubules. At this site, the reaction product disappeared from the cytoplasm but strong positivity persisted at the apical surface. The luminal staining was still present at higher inhibitor concentrations up to 0.8 m acetazolamide. Under histophotometric analyses, the residual reactivity proved to be nearly the same at the increasing inhibitor concentrations assayed. The validity of the results was checked by similar investigations in other control tissues.On the basis of the properties known for carbonic anhydrase in mammalian kidney, we conclude that the luminal membrane staining in the quail distal tubules might be due to a carbonic anhydrase isoenzyme that is similar, both in affinity for acetazolamide and in intracellular localization, to the membrane-bound enzyme purified from mammalian proximal convoluted tubules.     

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

Aquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1?4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1?4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Cross-reactivity of an antiserum to the α-subunit of the Na+, K+-ATPase of toad (Bufo marinus) kidney with basal and apical membranes of transporting epithelia of the rat

Summary An antibody to the 96 kD -subunit of the Na⁺, K⁺ -ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na⁺, K⁺ -ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Expression of the 56-kDa B2 subunit isoform of the vacuolar H(+)-ATPase in proton-secreting cells of the kidney and epididymis

B1 and B2 are two highly homologous isoforms of the vacuolar H⁺-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and B1. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis. vacuolar H⁺-ATPase B subunit; intercalated cells; clear cells; urogenital tract; immunofluorescence     

Renal carbonic anhydrase activity in DBA/2FG-pcy/pcy mice with inherited polycystic kidney disease.

DBA/2FG-pcy/pcy (D2-pcy) mice are a hereditary murine model of slowly progressive polycystic kidney disease (PKD) and characterized by the persistent excretion of acidic urine, in association with polyuria, after weaning. In this study, the activity of carbonic anhydrase (CA) and it histological distribution in the kidney of D2-pcy mice were investigated by immunohistochemistry. Significantly higher CA activity was detected in the cytosolic, but not membrane, fraction of kidney homogenates in 5-week-old D2-pcy mice than in age-matched, control DBA/2 (D2) mice, and a more rapid rate of urine acidification was noted in 11-week-old mice when acetazolamide, an inhibitor of the enzyme, was administered orally. By immunohistochemistry for the major renal CA isoenzyme (CA II), epithelial cells in the distal straight tubules and the cortical collecting ducts were stained intensely, whereas those of the proximal convoluted tubules had only weak and diffuse staining. The glomeruli, the proximal straight tubules and the ascending thin limb of Henle's loop were almost free from staining. In the cells lining cysts and/or dilated tubules, CA II activity was well preserved, although the staining intensity was considerably reduced in fully-flattened, lining cells of cysts, but no difference was found between D2-pcy and D2 mice in any segmental localization of renal CA II activity. From these results it seems that D2-pcy mice in the early stages of the cystic disease continue to secrete excess protons through the CA-mediated reaction that is stimulated for regulation of acid-base balance in the distal portion of the nephron and the collecting duct in kidney. It also suggests that monitoring urine pH may be useful in predicting the effects of early interventions on the progression of slowly developing renal cysts.     

The activity of mitochondrial alpha-glycerophosphate dehydrogenase in the rat nephron following triiodo-L-thyronine treatment: a histochemical study.

The effect of triiodo-L-thyronine (T3) treatment (15 mug/100 g body weight daily for 10 days) on the activity of mitochondrial alpha-glycerophosphate dehydrogenase (GPOX) in different nephron segments of the male rat was investigated by a histochemical staining method. The study showed marked segmental differences regarding the response to T3-treatment: 1. The first two proximal segments were unstained in the control rats and intensely stained following treatment. 2. The third proximal segments, the thick ascending limbs of Henle's loop and the distal convolted tubules showed a strong or moderate reaction in controls and a moderate increase after T3-treatment. 3. The high activity of collecting ducts in the cortex and outer zone of the medulla in controls was slightly increased by treatment. 4. Faintly reacting glomeruli and negative thin limbs of Henle's loop and collecting ducts in the inner medulla (papilla) were unaffected by T3-treatment. The results are discussed in relation to biochemical and physiological data.     

Histological and enzymatic studies on the nephron of the lungfish,Lepidosiren paradoxa

Nephron of South American lungfish was examined historically and enzyme-histochemically. Cells of the first and second proximal segments exhibited poor interdigitation forming narrow intercellular spaces, whereas the distal segment consisted of deeply interdigitated cells with wide intercellular spaces. Activities of aerobic enzymes (malate, isocitrate, NADH, and β-hydroxybutyrate dehydrogenases), Na-K-ATPase, and carbonic anhydrase were mostly detected in the distal segment. In contrast, hexokinase activity was mostly seen in the 1st and 2nd proximal segments. In the collecting tubule, two types of cells were distinguished by their histological and enzyme-histochemical features. One type showed deep interdigitation and intense carbonic anhydrase activity. The other did not have heavy interdigitation and carbonic anhydrase activity. However, both cell type exhibited intense activities of aerobic enzymes. These structures and enzyme distributions in the lungfish nephron indicate that the lungfish is more specialized in nephron than teleosteans and elasmobranchs, though, slightly similar to the latter.     

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.     

Morphometric parameters of nutria kidney structures in postnatal ontogeny

Data on the morphometric parameters of the renal corpuscle, renal tubules, and collecting ducts of male and female nutrias in postnatal ontogenesis were obtained. It was found that the area of the renal corpuscle, glomerulus, the cavity and lumen of the capsule, and the proximal tubule diameter in the right and left kidney of female and male nutrias in the first year of life increase. The distal tubule diameter also increases; however, the dynamics of its changes becomes sinuous after 4.5 months. The collecting duct diameter varies depending on gender, age, and renal topography. The nuclear-cytoplasmic ratio in the cells of proximal and distal tubules and collecting ducts changes in a sinuous manner and depends on the gender and age of nutrias. The minimum mean value of the nuclear-cytoplasmic ratio was found in the proximal tubule cells in the left kidney of 12-month-old female nutrias (0.162 ± 0.002), and the maximum value was found in the distal tubule cells in the left kidney of newborn male nutrias (0.435 ± 0.007).     

Selective Assembly of V-ATPase Subunit Isoforms in Mouse Kidney

The kidney plays vital roles in acid–base homeostasis, and the reabsorption of water, ions, and proteins. These processes are achieved through acidification of urine and endosomes of proximal tubule epithelial cells. Multisubunit vacuolar-type proton ATPase (V-ATPase) is one of the major acidification-machinery proteins that localizes to the apical or basolateral plasma membranes of intercalated cells in collecting ducts and the endosomal region at the base of brush border microvilli in proximal tubules. Multiple subunit isoforms of V-ATPase, which are expressed in kidney, have been identified. One obvious question is whether the pumps at different locations in the kidney have their own unique subunit identities. We have used a combination of methods to study this enzyme in kidney including immunocytochemical staining and immunoprecipitation analyses. The subunit isoforms of V-ATPase exhibited selective association/assembly in kidney: kidney-specific isoforms predominantly formed the intercalated cell proton pump, whereas the pump located in the brush border comprised ubiquitously expressed counterparts.     

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.     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.