Actu Physiol Scund 1991, 141, 231-234
ADONIS 000 1677291000342
Membrane-bound carbonic anhydrase CA IV in the human kidney G. L O N N E R H O L M and P. J. W I S T R A N D Department of Medical Pharmacology, Uppsala University, Uppsala, Sweden G. & WISTRANDP. J. 1990. Membrane-bound carbonic anhydrase LONNERHOLM, CA IV in the human kidney. Actu Physiol Scund 141, 231-234. Received 22 May 1990, accepted 3 October 1990. ISSN 0001-6772. Department of Medical Pharmacology, Biomedical Centre Uppsala University, Sweden. The distribution of membrane-bound carbonic anhydrase, CA IV, was studied in human kidneys by an indirect immunoperoxidase method using a rabbit polyclonal antibody directed against human kidney CA IV. Clear staining of CA IV was found in the apical cell borders of some cells in the cortical and medullary segments of the collecting ducts, presumably the A type of intercalated cells. Weak staining for CA IV was located in the interior of a number of collecting duct cells and in the basolateral regions of the proximal convoluted tubules. However, no staining was found in the brush border of the same tubules. This is a surprising finding, since evidence for carbonic anhydrase activity has been found biochemically and histochemically both in isolated brush-border and baso-lateral membranes. Further work is needed to clarify this matter. The endothelium of the peritubular capillaries also stained for CA IV. Key words : Bicarbonate secretion, carbonic anhydrase, immunohistochemistry, cell membranes, kidney, renal tubular acidosis.
T h e location of carbonic anhydrase (CA, carbonate hydrolyase EC 4 . 2 . 1 . 1.) in the nephron is of considerable importance to the understanding of urinary acidification. W e have previously studied the distribution of CA in the human kidney by a histochemical method (Lonnerholm & Wistrand 1984). This method demonstrates total CA activity, as a catalyst in the CO, H,O = H,CO, system, but cannot identify the various isozymes of CA. CA activity was demonstrated in many parts of the kidney tubules, with marked differences in intracellular distribution of the enzyme. This study also included immunocytochemical work, but at that point of time only antisera against the two cytoplasmic isozymes C A I and CA I1 were available to us. CA I1 was found to be the only isozyme present in the kidney tubules (Lonnerholm & Wistrand 1984). However, another cytoplasmic isozyme CA 111, is also known to
+
Correspondence : Gudmar Lonnerholm, Dept of Medical Pharmacology, Box 593, Biomedicum, S-751 24 Uppsala, Sweden
exist in some mammalian tissues, i.e. skeletal muscle and liver (Carter et al. 1979). Furthermore, and most important, a membranebound isozyme designated CA IV has recently been isolated and purified from human kidneys (Wistrand & Knuuttila 1989). CA IV was isolated from renal microsomes, but the same form has been found in isolated apical and basolateral cell membranes from kidney tissue (Wistrand & Knuuttila 1989). CA IV is an hydrophobic membrane protein with a different amino acid composition, molecular weight and immunochemical reactivity, as compared to the cytoplasmic isozymes. However, CA IV resembles CA I1 with respect to substrate and inhibitor kinetics. CA IV is expressed in a range of fetal and adult human tissues, including the kidney, as evaluated by immuno-blotting analysis (Carter et al. 1990). T h e aim of the present work was to complete the previously described picture of CA in the human kidney by studying the distribution of CA 111 and CA IV. T h e latter form is of particularly great interest, since Lucci et al. 1983
23 1
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G. Lonnerholm and P . J . Wistrand
have demonstrated that it is the physiologically important form in bicarbonate reabsorption.
MATERIALS AND METHODS Human donor kidneys (n = 2) and renal tissue obtained at nephrectomy due to renal tumours ( n = 2) were used. Immediately after removal the donor kidneys were perfused with 50CL-1000ml of cold Ringer-dextran type of solution, followed by SO& 1000 ml of a 10% invertose solution to which sodium bicarbonate had been added to pH 7. These kidneys were stored at 4 "C and tissue preparation started within 24 h of removal. Specimens obtained at nephrectomy were selected from macroscopically normal renal tissue, and tissue preparation started immediately after removal. Dissected parts of the kidneys were fixed in Bouin's fluid for about 6 h or in 4 TI formaldehyde in phosphate buffer (pH 7) over night. This was followed by dehydration through graded ethanols and embedding in paraffin at 52-54 "C. Sections 4 km thick were put onto chrome gelatinated glass slides, deparaffinized and rehydrated through xylol and graded ethanols. The sections were exposed to specific or non-specific (control) antiserum over night at room temperature (22 "C). The sites of the enzyme were visualized by the avidin-biotinperoxidase-(ABC)-technique, using a Vectastain@ ABC kit (Vector Laboratories Inc., Burlingame, Ca, USA). A solution of 3-amino-9-ethylcarbazole, AEC, was used in the final step to visualize the reaction. The sections were mounted under coverslips in glyceringelatin. Control experiments included staining after blocking of endogenous peroxidase by exposure of sections to 0.3 yo H,O,. In other control experiments endogenous biotin was blocked using the blocking kit from the manufacturer. Renal membrane-bound CA IV was isolated from human donor kidneys and purified to homogeneity, and antiserum against CA IV was raised by immunization of rabbits as described by Wistrand & Knuuttila (1989). In an ELISA system this antiserum was tested and found to react with the non-denatured CA IV, but only weakly with CA 11, and not at all with CA I and CA 111. CA I1 antiserum was a generous gift from Behringwerke AG (Marburg, Lahn, GFR). In irnmunodiffusion tests it was shown to be monospecific and without cross-reactivity. CA I11 was isolated and purified from fresh extensor hallucis muscle taken from a patient after leg amputation. The purification was carried out according to the method of Carter et al. (1979) with minor modifications. The purified CA 111 was homogenous with respect to sedimentation in the ultracentrifuge, electrophoresis and isoelectric focusing. Antiserum against CA I11 was raised in New Zealand
white rabbits using standard immunological procedures. It did not cross-react with CA I and CA 11, as tested by double immunodiffusion. The antisera were diluted in PBS (0.8v41 NaCl in phosphate buffer pH 7.6) and tested as serial dilutions. Renal sections were stained for CA 11, 111 or CA IV. Some serial sections were stained alternatively with CA I1 or CA IV, so that the same tubular structures could be identified for a direct comparison of the distribution of the two isozymes. Nomenclature : The different parts of the kidney tubules were designated essentially as proposed by Tischer (1981), as described in detail elsewhere (Lonnerholm & Wistrand 1984).
RESULTS CA I11 was not found in the human kidney. CA IV on the other hand, was found to be present at distinct sites in the renal tissue. T h e distribution of CA IV in renal medulla and cortex is shown in Fig. 1 A and Fig. 2. Fig. 1 A and B are two serial sections stained for CA IV and 11, respectively, and show the marked difference in staining pattern for the two isozymes. T h e distribution of CA IV is described below. T h e glomeruli and Bowman's capsule were not stained and the convoluted part of the proximal tubule showed very weak or no staining. Faint staining appeared to be located at the deeply invaginating basolateral cell membranes, but further work is needed to confirm this. T h e straight part of the proximal tubule, the thin and thick limbs of Henle's loop and the distal convoluted tubules were all unstained. T h e collecting ducts were the only part of the kidney tubules showing clear staining for CA IV. T h e staining was located to the apical cell border facing the tubular lumen (Figs. 1A and 2). Both the cortical and the medullary segments of the collecting ducts contained unstained cells among stained ones. T h e epithelium of the collecting ducts is known to consist of two cell types, chief and intercalated cells (Tischer 1981). W e have previously shown by histochemical technique at the electron microscopic level (Lonnerholm & Wistrand 1984) that the whole intercalated cells stain for CA activity, while chief cells lack CA activity in the apical cell membranes. Thus, there is reason to assume that the cells observed here with apical staining for CA IV are intercalated cells. Some cells displayed weak staining
Renal membrane-bound carbonic anhydrase
233
Fig. 1. Human kidney, cortex. T w o serial sections stained for CA IV (A) and CA I1 (B), respectively. In 1 A the collecting duct (C) is the only tubular structure containing CA IV. T h e staining is restricted to the apical cell border facing the tubular lumen. Proximal (P) and distal (D) convoluted tubules and a glomerulus (G) are unstained. Peritubular capillaries show weak CA IV strain. 1B shows strong staining for CA I1 in a distal convoluted tubule (top) and in some cells in a collecting duct (C). Proximal convoluted tubules (P) are weakly stained for CA 11. G is an unstained glomerulus, containing a few CA I1 positive erythrocytes. Bar is 20 p m . Dilution of antiserum was 1 : 250 in A and 1 : 1000 in B
of the interior of the cell, in addition to the staining at the apical cell border (not shown). T h e detailed intracellular distribution of the CA IV stain could not be judged, however. T h e capillary endothelium was stained for CA IV in the peritubular capillaries. This is most clearly seen in Fig. 2, but capillary staining was found both in cortex and medulla. Glomeruli and larger vessels (arterioles, venules etc) lacked staining, and so did erythrocytes. DISCUSSION The renal tubular cells contain 0.8-1.3 mg CA I1 per g extractable protein in the cytoplasm (Wistrand 1980), and 0.8 mg CA IV per g extractable protein in the plasma membranes (Wistrand & Kinne 1977). However, of the cells’ total CA activity, only 3-5% originate from CA IV and the rest from CA I1 (Wistrand 1980). Since both forms are present in the same cells one could be concerned about possible crossreactivity against the abundant CA 11, when
using our antiserum for localizing CA IV. However, the cellular distribution of CA IV found here in the apical membranes was quite different from that (Lonnerholm & Wistrand 1984; Fig. 13)of CA I1 in the cytoplasm of the same cells, which testifyes to the specificity of the antiserum. CA I1 is considered to be a ‘marker ’ of the intercalated cells in the collecting duct (Lonnerholm & Wistrand 1984). Some of the intercalated cells containing CA I1 did not stain for CA IV. However, there are two types of intercalated cells. One type, called A cells, are believed to be responsible for H + secretion, whereas others, called B cells, may be involved in bicarbonate secretion (Madsen & Tischer 1986). We suggest that CA IV is located in the apical membranes of the A cells, where its function would be to dehydrate carbonic acid formed in the lumen during H+secretion, thereby avoiding the generation of steep pH-gradients across the luminal membrane. Some staining for CA IV was also found in the interior of a number of collecting duct cells,
234
G. Lonnerholm and P . J . Wistrand trations are only present in the collecting ducts and the capillaries. Further work is needed to clarify this matter. The authors thank Ms Christina Bergqvist for skilful technical assistance. This study was supported by the Swedish Medical Research Council grant no 2874 (to PJW) and no 5413 (to GL).
REFERENCES
Fig. 2. Human kidney, medulla. CA IV staining with antiserum 1 : 500. A collecting duct (C) shows distinct staining at the luminal cell border of most but not all cells. Capillaries are positively stained (two are denoted by arrows). Unstained tubules are thin and thick limbs of Henle’s loop. Bar is 20 pm.
although the staining was much less intense than at the luminal cell border. T h e detailed intracellular distribution could not be evaluated, but one possibility is that the isozyme is located to microsomal membranes, where it is synthesized before transport and integration into the plasma membranes. T h e weak and inconsistent staining in the proximal convoluted tubules indicates that the concentration of CA IV in this nephron segment must be considerably lower than in the collecting ducts. T h e faint staining seemed to be associated with basolateral cell membranes while no staining was found in the brush border region. This is somewhat surprising since CA activity has been found biochemically and histochemically in renal isolated brush border and basolateral cell membranes (Wistrand & Kinne 1977). One possible explanation is that the activity of our antiserum is so low that it can only detect very high local concentrations of CA IV and that such concen-
CARTER, N., JEFFERY, S., SHIELS,A., EDWARDS, Y., TIPLER, T. & HOPKINSON, D.A. 1979. Characterization of human carbonic anhydrase I11 from skeletal muscle. Biochem Genet 17, 837-853. CARTER, N.D., FRYER, A,, GRANT,A.G., HUMER., STRANGE, R.G. & WISTRAND, P.J. 1990. Membrane specific carbonic anhydrase (CA IV) expression in human tissues. Biochim Biophys Acta 1026, 113116. I,UCCI,M.S., TINKER, J.P., WEINER, I.M. & Du BOSE, T.D. JR. 1983. Function of proximal tubule carbonic anhydrase defined by selective inhibition. Am 3 Physiol 245, F443PF449. LONNERHOLM, G. & WISTRAND, P.J. 1984. Carbonic anhydrase in the human kidney : A histochemical and imrnunocytochernical study. Kidney Int 25, 886898. C.C. 1986. StructuralMADSEN,K.M. & TISCHER, functional relationships along the distal nephron. Am 3 Physiol 250 (Renal Fluid Electrolyte Physiol 19): F1-F15. TISCHER, C.C. 1981. Anatomy of the kidney. In: B.M. Brenner & F.C. Rector (eds) The Kidney, Vol. I, Chapt. I. WB Saunders Company, Philadelphia. WISTRAND, P.J. & KINNE,R. 1977. Carbonic anhydrase activity of isolated brush border and basallateral membranes of renal tubular cells. Pjtigers Arch 370, 121-126. WISTRAND,P.J. 1980. Human renal cytoplasmic carbonic anhydrase. Tissue levels and kinetic properties under near physiological conditions. Acta Ph,ysiol Scund 109, 239-248. WISTRAND, P J . & KNUUTTILA, K.-G. 1989. Renal membrane-bound carbonic anhydrase. Purification and properties. Kidney Int 35, 851-859.
N O T E ADDED I N P R O O F Brown et ul. (1990) found by immunolocalization that CAIV was localized in apical and basolateral plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle in the rat kidney. BROWN,D., ZHU, X. L. & SLY, W. S. 1990. Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells. Proc Nut1 Acad Sci 87, 7457-7461.
ADONIS 000 1677291000342
Membrane-bound carbonic anhydrase CA IV in the human kidney G. L O N N E R H O L M and P. J. W I S T R A N D Department of Medical Pharmacology, Uppsala University, Uppsala, Sweden G. & WISTRANDP. J. 1990. Membrane-bound carbonic anhydrase LONNERHOLM, CA IV in the human kidney. Actu Physiol Scund 141, 231-234. Received 22 May 1990, accepted 3 October 1990. ISSN 0001-6772. Department of Medical Pharmacology, Biomedical Centre Uppsala University, Sweden. The distribution of membrane-bound carbonic anhydrase, CA IV, was studied in human kidneys by an indirect immunoperoxidase method using a rabbit polyclonal antibody directed against human kidney CA IV. Clear staining of CA IV was found in the apical cell borders of some cells in the cortical and medullary segments of the collecting ducts, presumably the A type of intercalated cells. Weak staining for CA IV was located in the interior of a number of collecting duct cells and in the basolateral regions of the proximal convoluted tubules. However, no staining was found in the brush border of the same tubules. This is a surprising finding, since evidence for carbonic anhydrase activity has been found biochemically and histochemically both in isolated brush-border and baso-lateral membranes. Further work is needed to clarify this matter. The endothelium of the peritubular capillaries also stained for CA IV. Key words : Bicarbonate secretion, carbonic anhydrase, immunohistochemistry, cell membranes, kidney, renal tubular acidosis.
T h e location of carbonic anhydrase (CA, carbonate hydrolyase EC 4 . 2 . 1 . 1.) in the nephron is of considerable importance to the understanding of urinary acidification. W e have previously studied the distribution of CA in the human kidney by a histochemical method (Lonnerholm & Wistrand 1984). This method demonstrates total CA activity, as a catalyst in the CO, H,O = H,CO, system, but cannot identify the various isozymes of CA. CA activity was demonstrated in many parts of the kidney tubules, with marked differences in intracellular distribution of the enzyme. This study also included immunocytochemical work, but at that point of time only antisera against the two cytoplasmic isozymes C A I and CA I1 were available to us. CA I1 was found to be the only isozyme present in the kidney tubules (Lonnerholm & Wistrand 1984). However, another cytoplasmic isozyme CA 111, is also known to
+
Correspondence : Gudmar Lonnerholm, Dept of Medical Pharmacology, Box 593, Biomedicum, S-751 24 Uppsala, Sweden
exist in some mammalian tissues, i.e. skeletal muscle and liver (Carter et al. 1979). Furthermore, and most important, a membranebound isozyme designated CA IV has recently been isolated and purified from human kidneys (Wistrand & Knuuttila 1989). CA IV was isolated from renal microsomes, but the same form has been found in isolated apical and basolateral cell membranes from kidney tissue (Wistrand & Knuuttila 1989). CA IV is an hydrophobic membrane protein with a different amino acid composition, molecular weight and immunochemical reactivity, as compared to the cytoplasmic isozymes. However, CA IV resembles CA I1 with respect to substrate and inhibitor kinetics. CA IV is expressed in a range of fetal and adult human tissues, including the kidney, as evaluated by immuno-blotting analysis (Carter et al. 1990). T h e aim of the present work was to complete the previously described picture of CA in the human kidney by studying the distribution of CA 111 and CA IV. T h e latter form is of particularly great interest, since Lucci et al. 1983
23 1
232
G. Lonnerholm and P . J . Wistrand
have demonstrated that it is the physiologically important form in bicarbonate reabsorption.
MATERIALS AND METHODS Human donor kidneys (n = 2) and renal tissue obtained at nephrectomy due to renal tumours ( n = 2) were used. Immediately after removal the donor kidneys were perfused with 50CL-1000ml of cold Ringer-dextran type of solution, followed by SO& 1000 ml of a 10% invertose solution to which sodium bicarbonate had been added to pH 7. These kidneys were stored at 4 "C and tissue preparation started within 24 h of removal. Specimens obtained at nephrectomy were selected from macroscopically normal renal tissue, and tissue preparation started immediately after removal. Dissected parts of the kidneys were fixed in Bouin's fluid for about 6 h or in 4 TI formaldehyde in phosphate buffer (pH 7) over night. This was followed by dehydration through graded ethanols and embedding in paraffin at 52-54 "C. Sections 4 km thick were put onto chrome gelatinated glass slides, deparaffinized and rehydrated through xylol and graded ethanols. The sections were exposed to specific or non-specific (control) antiserum over night at room temperature (22 "C). The sites of the enzyme were visualized by the avidin-biotinperoxidase-(ABC)-technique, using a Vectastain@ ABC kit (Vector Laboratories Inc., Burlingame, Ca, USA). A solution of 3-amino-9-ethylcarbazole, AEC, was used in the final step to visualize the reaction. The sections were mounted under coverslips in glyceringelatin. Control experiments included staining after blocking of endogenous peroxidase by exposure of sections to 0.3 yo H,O,. In other control experiments endogenous biotin was blocked using the blocking kit from the manufacturer. Renal membrane-bound CA IV was isolated from human donor kidneys and purified to homogeneity, and antiserum against CA IV was raised by immunization of rabbits as described by Wistrand & Knuuttila (1989). In an ELISA system this antiserum was tested and found to react with the non-denatured CA IV, but only weakly with CA 11, and not at all with CA I and CA 111. CA I1 antiserum was a generous gift from Behringwerke AG (Marburg, Lahn, GFR). In irnmunodiffusion tests it was shown to be monospecific and without cross-reactivity. CA I11 was isolated and purified from fresh extensor hallucis muscle taken from a patient after leg amputation. The purification was carried out according to the method of Carter et al. (1979) with minor modifications. The purified CA 111 was homogenous with respect to sedimentation in the ultracentrifuge, electrophoresis and isoelectric focusing. Antiserum against CA I11 was raised in New Zealand
white rabbits using standard immunological procedures. It did not cross-react with CA I and CA 11, as tested by double immunodiffusion. The antisera were diluted in PBS (0.8v41 NaCl in phosphate buffer pH 7.6) and tested as serial dilutions. Renal sections were stained for CA 11, 111 or CA IV. Some serial sections were stained alternatively with CA I1 or CA IV, so that the same tubular structures could be identified for a direct comparison of the distribution of the two isozymes. Nomenclature : The different parts of the kidney tubules were designated essentially as proposed by Tischer (1981), as described in detail elsewhere (Lonnerholm & Wistrand 1984).
RESULTS CA I11 was not found in the human kidney. CA IV on the other hand, was found to be present at distinct sites in the renal tissue. T h e distribution of CA IV in renal medulla and cortex is shown in Fig. 1 A and Fig. 2. Fig. 1 A and B are two serial sections stained for CA IV and 11, respectively, and show the marked difference in staining pattern for the two isozymes. T h e distribution of CA IV is described below. T h e glomeruli and Bowman's capsule were not stained and the convoluted part of the proximal tubule showed very weak or no staining. Faint staining appeared to be located at the deeply invaginating basolateral cell membranes, but further work is needed to confirm this. T h e straight part of the proximal tubule, the thin and thick limbs of Henle's loop and the distal convoluted tubules were all unstained. T h e collecting ducts were the only part of the kidney tubules showing clear staining for CA IV. T h e staining was located to the apical cell border facing the tubular lumen (Figs. 1A and 2). Both the cortical and the medullary segments of the collecting ducts contained unstained cells among stained ones. T h e epithelium of the collecting ducts is known to consist of two cell types, chief and intercalated cells (Tischer 1981). W e have previously shown by histochemical technique at the electron microscopic level (Lonnerholm & Wistrand 1984) that the whole intercalated cells stain for CA activity, while chief cells lack CA activity in the apical cell membranes. Thus, there is reason to assume that the cells observed here with apical staining for CA IV are intercalated cells. Some cells displayed weak staining
Renal membrane-bound carbonic anhydrase
233
Fig. 1. Human kidney, cortex. T w o serial sections stained for CA IV (A) and CA I1 (B), respectively. In 1 A the collecting duct (C) is the only tubular structure containing CA IV. T h e staining is restricted to the apical cell border facing the tubular lumen. Proximal (P) and distal (D) convoluted tubules and a glomerulus (G) are unstained. Peritubular capillaries show weak CA IV strain. 1B shows strong staining for CA I1 in a distal convoluted tubule (top) and in some cells in a collecting duct (C). Proximal convoluted tubules (P) are weakly stained for CA 11. G is an unstained glomerulus, containing a few CA I1 positive erythrocytes. Bar is 20 p m . Dilution of antiserum was 1 : 250 in A and 1 : 1000 in B
of the interior of the cell, in addition to the staining at the apical cell border (not shown). T h e detailed intracellular distribution of the CA IV stain could not be judged, however. T h e capillary endothelium was stained for CA IV in the peritubular capillaries. This is most clearly seen in Fig. 2, but capillary staining was found both in cortex and medulla. Glomeruli and larger vessels (arterioles, venules etc) lacked staining, and so did erythrocytes. DISCUSSION The renal tubular cells contain 0.8-1.3 mg CA I1 per g extractable protein in the cytoplasm (Wistrand 1980), and 0.8 mg CA IV per g extractable protein in the plasma membranes (Wistrand & Kinne 1977). However, of the cells’ total CA activity, only 3-5% originate from CA IV and the rest from CA I1 (Wistrand 1980). Since both forms are present in the same cells one could be concerned about possible crossreactivity against the abundant CA 11, when
using our antiserum for localizing CA IV. However, the cellular distribution of CA IV found here in the apical membranes was quite different from that (Lonnerholm & Wistrand 1984; Fig. 13)of CA I1 in the cytoplasm of the same cells, which testifyes to the specificity of the antiserum. CA I1 is considered to be a ‘marker ’ of the intercalated cells in the collecting duct (Lonnerholm & Wistrand 1984). Some of the intercalated cells containing CA I1 did not stain for CA IV. However, there are two types of intercalated cells. One type, called A cells, are believed to be responsible for H + secretion, whereas others, called B cells, may be involved in bicarbonate secretion (Madsen & Tischer 1986). We suggest that CA IV is located in the apical membranes of the A cells, where its function would be to dehydrate carbonic acid formed in the lumen during H+secretion, thereby avoiding the generation of steep pH-gradients across the luminal membrane. Some staining for CA IV was also found in the interior of a number of collecting duct cells,
234
G. Lonnerholm and P . J . Wistrand trations are only present in the collecting ducts and the capillaries. Further work is needed to clarify this matter. The authors thank Ms Christina Bergqvist for skilful technical assistance. This study was supported by the Swedish Medical Research Council grant no 2874 (to PJW) and no 5413 (to GL).
REFERENCES
Fig. 2. Human kidney, medulla. CA IV staining with antiserum 1 : 500. A collecting duct (C) shows distinct staining at the luminal cell border of most but not all cells. Capillaries are positively stained (two are denoted by arrows). Unstained tubules are thin and thick limbs of Henle’s loop. Bar is 20 pm.
although the staining was much less intense than at the luminal cell border. T h e detailed intracellular distribution could not be evaluated, but one possibility is that the isozyme is located to microsomal membranes, where it is synthesized before transport and integration into the plasma membranes. T h e weak and inconsistent staining in the proximal convoluted tubules indicates that the concentration of CA IV in this nephron segment must be considerably lower than in the collecting ducts. T h e faint staining seemed to be associated with basolateral cell membranes while no staining was found in the brush border region. This is somewhat surprising since CA activity has been found biochemically and histochemically in renal isolated brush border and basolateral cell membranes (Wistrand & Kinne 1977). One possible explanation is that the activity of our antiserum is so low that it can only detect very high local concentrations of CA IV and that such concen-
CARTER, N., JEFFERY, S., SHIELS,A., EDWARDS, Y., TIPLER, T. & HOPKINSON, D.A. 1979. Characterization of human carbonic anhydrase I11 from skeletal muscle. Biochem Genet 17, 837-853. CARTER, N.D., FRYER, A,, GRANT,A.G., HUMER., STRANGE, R.G. & WISTRAND, P.J. 1990. Membrane specific carbonic anhydrase (CA IV) expression in human tissues. Biochim Biophys Acta 1026, 113116. I,UCCI,M.S., TINKER, J.P., WEINER, I.M. & Du BOSE, T.D. JR. 1983. Function of proximal tubule carbonic anhydrase defined by selective inhibition. Am 3 Physiol 245, F443PF449. LONNERHOLM, G. & WISTRAND, P.J. 1984. Carbonic anhydrase in the human kidney : A histochemical and imrnunocytochernical study. Kidney Int 25, 886898. C.C. 1986. StructuralMADSEN,K.M. & TISCHER, functional relationships along the distal nephron. Am 3 Physiol 250 (Renal Fluid Electrolyte Physiol 19): F1-F15. TISCHER, C.C. 1981. Anatomy of the kidney. In: B.M. Brenner & F.C. Rector (eds) The Kidney, Vol. I, Chapt. I. WB Saunders Company, Philadelphia. WISTRAND, P.J. & KINNE,R. 1977. Carbonic anhydrase activity of isolated brush border and basallateral membranes of renal tubular cells. Pjtigers Arch 370, 121-126. WISTRAND,P.J. 1980. Human renal cytoplasmic carbonic anhydrase. Tissue levels and kinetic properties under near physiological conditions. Acta Ph,ysiol Scund 109, 239-248. WISTRAND, P J . & KNUUTTILA, K.-G. 1989. Renal membrane-bound carbonic anhydrase. Purification and properties. Kidney Int 35, 851-859.
N O T E ADDED I N P R O O F Brown et ul. (1990) found by immunolocalization that CAIV was localized in apical and basolateral plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle in the rat kidney. BROWN,D., ZHU, X. L. & SLY, W. S. 1990. Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells. Proc Nut1 Acad Sci 87, 7457-7461.
