Carbonic Anhydrase II-deficient Mice Research Articles

Deficiency of Carbonic Anhydrase II Results in a Urinary Concentrating Defect.

Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

Phenotypic Characteristics of Bone in Carbonic Anhydrase II-Deficient Mice

Carbonic anhydrase II (CAII)-deficient mice were created to study the syndrome of CAII deficiency in humans including osteopetrosis, renal tubular acidosis, and cerebral calcification. Although CAII mice have renal tubular acidosis, studies that analyzed only cortical bones found no changes characteristic of osteopetrosis. Consistent with previous studies, the tibiae of CAII-deficient mice were significantly smaller than those of wild-type (WT) mice (28.7 +/- 0.9 vs. 43.6 +/- 3.7 mg; p < 0.005), and the normalized cortical bone volume of CAII-deficient mice (79.3 +/- 2.2%) was within 5% of that of WT mice (82.7 +/- 2.3%; p < 0.05), however, metaphyseal widening of the tibial plateau was noted in CAII-deficient mice, consistent with osteopetrosis. In contrast to cortical bone, trabecular bone volume demonstrated a nearly 50% increase in CAII-deficient mice (22.9 +/- 3.5% in CAII, compared to 15.3 +/- 1.6% in WT; p < 0.001). In addition, histomorphometry demonstrated that bone formation rate was decreased by 68% in cortical bone (4.77 +/- 1.65 microm3/microm2/day in WT vs. 2.07 +/- 1.71 microm3/microm2/day in CAII mice; p < 0.05) and 55% in trabecular bone (0.617 +/- 0.230 microm3/microm2/day in WT vs. 0.272 +/- 0.114 microm3/microm2/day in CAII mice; p < 0.05) in CAII-deficient mice. The number of osteoclasts was significantly increased (67%) in CAII-deficient mice, while osteoblast number was not different from that in WT mice. The metaphyseal widening and changes in the trabecular bone are consistent with osteopetrosis, making the CAII-deficient mouse a valuable model of human disease.

Decreased Expression of Slc26a4 (Pendrin) and Slc26a7 in the Kidneys of Carbonic Anhydrase II-Deficient Mice

Background/Aims: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice. Methods: For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting. Results: Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice. Conclusion: CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family – Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.

Carbonic anhydrase in the gastrointestinal mucus of mammals—possible protective role against carbon dioxide

We show here that luminal mucus from the colon and the stomach of guinea pigs, mice and humans exhibits substantial carbonic anhydrase (CA) activity, by which the velocity of the CO 2 hydration reaction is accelerated 1000–2000-fold, approximately 1/10 of what is found in the red cell. Although this CA shares several properties with CA II, studies with CA II-deficient mice show that gastrointestinal mucus CA is not affected in these animals and thus does not appear to be CA II. We speculate that the mucus layer covering the luminal surface of gastrointestinal epithelium can, due to the presence of CA, maintain a normal tissue pCO 2 in the epithelium, even when the pCO 2 values in the lumen are much higher, as is known for stomach and colon. To test this hypothesis, we have developed a mathematical model which describes (a) diffusion of CO 2 and HCO 3 − across the mucus layer and (b) H + transport mediated by continuous secretion of mucus, which due to its high H + buffer capacity transports H + by convection towards the lumen. The model predicts that continuous transport of the reaction products of CO 2 towards the lumen, by diffusion and convection, protects the epithelium against high CO 2 partial pressures in the lumen.

Carbonic anhydrase in mouse testis and epididymis; transfer of isozyme IV to spermatozoa during passage.

Spermatozoa are subjected to major changes as they pass through the epididymal duct. The aim of the present study was to describe the distribution of carbonic anhydrase (CA) in the mouse testis and epididymis using a histochemical technique showing total catalytic activity, in combination with immunohistochemistry for the two important isoforms CAs II and IV. By comparing normal mice with CA II-deficient mice, we were able to study membrane-bound CA without influence from the ubiquitous cytoplasmic CA II. Spermatozoa, when studied in both the scanning electron and light microscope, were found to pickup membrane-bound CA IV during their passage through the epididymal duct. The transfer appeared to take place in the proximal part of the corpus, where the apical membrane and vesicles of principal cells were richly supplied with CA IV. In addition to CA IV, another membrane-bound isozyme was located in basolateral membranes of principal cells. Cytoplasmic CA II was found in varying amounts in apical/narrow cells and principal cells of the corpus in control animals. The significance of CA for pH-regulating processes vital for sperm storage and motility is discussed. A function in HCO3- transport during sperm capacitation at fertilization is suggested for the CA IV found in spermatozoa.

Postnatal disappearance of type A intercalated cells in carbonic anhydrase II-deficient mice

Despite chronic acidosis, collecting ducts in adult carbonic anhydrase II-deficient (CAD mice) are depleted of intercalated cells, including those of type A, which are acid-secreting cells. We hypothesized that this depletion could occur during postnatal development. Principal cells were identified by immunofluorescence using an antibody to rat aquaporin-2 (AQP-2), and type A intercalated cells using an antibody specific for anion exchanger (AE1). In CAD mice the proportion of AQP2-positive cells, normal at 11 days, increased progressively in the cortical (CCD) and outer medullary collecting duct (OMCD), to reach almost 100% in the OMCD in adults. The percentage of AE1-positive cells in the OMCD of CAD mice decreased by half by 6 weeks of age and further by adulthood. In controls, however, the proportion of AQP2-positive cells and that of AE1-positive cells in the OMCD remained stable after 10 days of age. AE1-positive cells accounted for the majority of intercalated cells in the OMCD. The mechanisms leading to selective postnatal cell depletion in the collecting duct in CAD mice remain to be determined.

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

Membrane-associated carbonic anhydrase (CA) activity is of importance for transepithelial transport of ions and fluid. Histochemical studies have indicated its presence in the brain, but the data are difficult to evaluate because of interference from cytoplasmic CA isozymes, of which CA II is the predominant one. CA II-deficient mice offer a possibility to study the location of membrane-associated CA-activity, without interference from CA II. The location of CA activity in the brain of CA II-deficient and normal mice was studied by the cobalt-phosphate histochemical method, and that of CA I, CA II and CA III by an immunocytochemical method. The brains of both types of mice lacked cytoplasmic isozymes CA I and CA III, and the CA II-deficient mice also lacked CA II. In the normal mice, oligodendrocytes and choroid epithelium stained for CA II in the cytoplasm. In normal and CA (II)D-mice there was an intense membrane associated histochemical CA activity in neuronal processes. Neuronal perikarya were not stained. Endothelial membranes of brain capillaries showed strong histochemical CA-activity. Choroid epithelial cells had histochemical CA activity in the cytoplasm and along apical and baso-lateral cell membranes. The results suggest that membrane-associated CA-activity found along neuronal processes probably modulates pH of the extracellular fluid and thus neuronal activity. CA II and the membrane-associated CA of choroidal epithelium are probably involved in the secretion of cerebrospinal fluid.

Use of carbonic anhydrase II-deficient mice in uncovering the cellular location of membrane-associated isoforms.

As long ago as 1960, plasma membranes and cell organelles were shown by Karler and Woodbury to exhibit carbonic anhydrase (CA) (carbonate hydrolyase; EC: 4.1.1.) activity. For tissues like kidney (Wistrand and Knuttilla, 1989), lung (Waheed et al., 1992), blood vessels (Ghandour et al., 1992), and skeletal muscle (Gros, 1991), this activity has since been shown to originate from a membrane-bound integral hydrophic isoform of CA, now designated as CA IV (cf. Sly and Hu, 1995). The CA IV activity of these tissues can be detected by the cobalt-phosphate histochemical method (cf. Ridderstrale, 1991). However, this method also detects CA activity associated with cell membranes of renal distal tubules, neuroretina, choroid epithelium, ciliary epithelium, cornea and brain (Ridderstrale et al., 1992, 1994; Ridderstrale and Wistrand, 1998), where CA IV has been claimed not to be present (Brown et al., 1990; Hageman et al., 1991; Ghandour et al., 1992). Moreover, the histochemical technique occasionally localizes CA-activity to cell nuclei, and mitochondria (cf. Ridderstrale, 1991).

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Correction of renal tubular acidosis in carbonic anhydrase II-deficient mice with gene therapy.

Carbonic anhydrase II (CAII) deficiency in humans is associated with a syndrome of renal tubular acidosis, osteopetrosis, and cerebral calcification. A strain of mice of CAII deficiency due to a point mutation also manifests renal tubular acidosis. We report here that retrograde injection of cationic liposome complexed with a CAII chimeric gene, using a cytomegalovirus (CMV) promoter/enhancer as an expression cassette to drive human CAII cDNA, into the renal pelvis of CAII-deficient mice results in expression of CAII in the kidney. The levels of both the CAII gene and its corresponding mRNA were highest by day 3 after treatment, diminishing thereafter, but remaining detectable by 1 mo. After gene therapy, CAII-deficient mice restored the ability to acidify urine after oral administration of ammonium chloride. The ability to acidify urine was maintained at 3 wk after gene therapy, and was eventually lost by 6 wk. Immunohistochemistry studies using anti-CAII antibodies showed that CAII was expressed in tubular cells of the outer medulla and corticomedullary junction. The gene therapy was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. To our knowledge, this is the first successful gene therapy of a genetic renal disease. Our results demonstrate the potential of gene therapy as a novel treatment for hereditary renal tubular defects.

Respiratory acidosis in carbonic anhydrase II-deficient mice.

To investigate the role of carbonic anhydrase (CA) II on pulmonary CO2 exchange, we analyzed arterial blood gases from CA II-deficient and normal control mice. CA II-deficient mice had a low arterial blood pH (7.18 +/- 0.06) and HCO3- concentration ([HCO3-]; 17.5 +/- 1.9 meq/l) and a high Pco2 (47.4 +/- 5.3 mmHg), consistent with mixed respiratory and metabolic acidosis. To eliminate the influence of metabolic acidosis on arterial blood gases, NaHCO3 (4 mmol/kg body weight) was given intraperitoneally, and arterial blood gases were analyzed 4 h later. Normal mice had a small increase in pH and were able to maintain Pco2 and [HCO3-]. The metabolic acidosis in CA II-deficient mice was corrected ([HCO3-], 22.9 +/- 2.4 meq/l), and respiratory acidosis became more profound (Pco2, 50.4 +/- 2.4 mmHg). These results indicate that CA II-deficient mice have a partial respiratory compensation for metabolic acidosis. We conclude that CA II-deficient mice have a mixed respiratory and metabolic acidosis. It is most likely that CO2 retention in these animals is due to CA II deficiency in both red blood cells and type II pneumocytes.

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level until the Purkinje cell population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

Increased susceptibility of brain slices from carbonic anhydrase II-deficient mice to low [Mg 2+] 0-induced seizures

Brain pH is thought to be an influential factor in determining susceptibility to seizures. We compared the susceptibility of brain slices from carbonic anhydrase II (CA II)-deficient mice to epileptiform activity induced by low extracellular [Mg 2+], with slices from normal littermates, both bathed in artificial cerebrospinal fluid at pH 7.3. In both entorhinal cortex and hippocampal field CA1, epileptiform activity started earlier in CA II-deficient slices. Raising extracellular [CO 2] (20%; extracellular pH, 6.7) reversibly blocked the epileptiform activity in normal, but not in CA II-deficient, slices. The data, combined with previous in vivo findings showing an increased resistance of mutants to seizures, suggest the presence of in vivo anticonvulsant acidosis with long-term compensatory changes that lead to in vitro ‘proconvulsant’ behavior in CA II-deficient slices clamped at pH 7.3.

Resistance of hippocampal synaptic transmission to hypoxia in carbonic anhydrase II-deficient mice

Mutant Car2 n/Car2 n mice deficient in carbonic anhydrase II (CA II; a major brain CA isozyme) suffer from systemic acidosis and are more resistant to experimental seizures than their normal littermates (+/+ or +/Car2 n). The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor has been shown to contribute to long-term potentiation (LTP) of synaptic transmission, hypoxic/ischemic neuronal injury and to be blocked by extracellular protons (acidosis). We compared the effects of hypoxia on synaptic transmission and LTP in field CA1 of hippocampal slices from CA II-deficient mice to their normal littermates. Slices were subjected to successive 5, 10 and 15 min-periods of hypoxia with 30 min-recoverry periods in between. Hippocampal slices from mutant, CA II-deficient mice, were more resistant to all periods of hypoxia tested than slices from normal littermates. In a separate set of mutant and normal slices, there were no differences in LTP of population spike amplitude. The relative resistance of CA II-deficient mice to hypoxia-induced damage may be a consequence of severe interstitial acidosis. The sustained influence of increased extracellular proton concentrations may change the characteristics of NMDA receptors resulting in an increased resistance of synaptic transmission in CA II-deficient mice to hypoxia compared to controls.

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.

Comparison of immunocytochemical staining of astrocytes, oligodendrocytes, and myelinated fibers in the brains of carbonic anhydrase II-deficient mice and normal littermates

Immunostaining for carbonic anhydrase (CA) was performed in paraffin sections from the brains of CA II-deficient mutant mice and their normal littermates. Double immunofluorescence staining showed CA in myelinated tracts and oligodendrocytes in the cerebellum of the normal but not the CA II-deficient mice, and also in astrocytes in the cerebral cortex of the normal mice but not the mutants. The data show that the CA in normal oligodendrocytes, astrocytes, and myelin is the II isoenzyme, because these structures in the mutants would be positively stained if the staining normally were due to a contaminant in the antiserum or an antibody against a different isoenzyme. The findings in normal gray matter also suggest that many neuronal cell bodies are surrounded by a network of fine, CA-positive astrocytic processes.