0022-1554/92/$3.30 “he J o u d of Histochcmisay and Cytochemisay Copyright 0 1992 by The Histochemical Society, Inc.
Vol. 40, NO. 10, pp. 1535-1545, 1992 Printed in C%S.A.
Original Article
Histochemical Carbonic Anhydrase in Rat Inner Medullary Collecting Duct’ J. G. KLEINMAN,’ J. L. W. BAIN, C. FRITSCHE, and D. A. RILEY Nephrology Division, Zublocki Veterans Afairs Medicai Center and Medical CoIIege of Wisconsin (JGK,CF),and the Department of Cell Biology and Anatomy, Medical College of Wisconsin (JLWB,DAR), Mdwaukee, Wisconsin.
Received for publication January 23, 1992 and in revised form April
20. 1992;
Rat inner medullary collecting duct (IMCD) seaetes substantial amounts of H’. However, carbonic anhydrase (CA), a concomitant of H’ seaetion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprisingthe IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportionhave features of intercalatedcells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third
cell type, previously called “IMCDcells,”appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these “IMCDcells” have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells dearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD. ( J Eiistdem Cytoc6em 40d535-1545, 1992) KEY WORDS:Rat kidney; H’secretion;Hansson technique; Intercalated cells; Principal cell; IMCD cell; Acetazolamide;Kidney ultrastructure.
Introduction Two types of epithelial cells involved in ion transport have been described in the mammalian collecting duct (CD): intercalated cells and principal cells. Because of morphological similarities to mitochondria-rich cells of turtle urinary bladder (a collecting duct analogue) and lectin binding characteristics, intercalated cells, particularly those of the cortical CD, have been further classified into A or a and B or fi subtypes (19). Intercalated cells contain the enzyme CA, and it has been proposed that the a-cells secrete H’ and the p-cells secrete HCO3- into tubule lumens (8,33). Rat IMCD has been reported to contain only small numbers of intercalated cells, presumably a type, yet this segment may be responsible for 50% of total acid secretion in acidosis (6,lOJl). Furthermore, isolated perfused segments of IMCD apparently lacking intercalated cells acidlfy their lumens (38). Principal cells of the outer medullary CD and the proximal third of the IMCD appear to be similar to those of the cortical CD (18).
’
Supported by the Department of Veterans Affairs, Merit Review Program and Career Development Award UGK)and by a NASA grant NAG 2-552 (DAR). Correspondence to: Jack G. Kleinman, MD, Nephrology Section111K, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295.
accepted May 1, 1992 (2A2568).
Principal cells may primarily mediate Na+,K’, water, and urea transport (18J9). CA activity, however, usually associated with H+or HCO3- transport, has been reported to be weak or entirely absent in principal cells with the possible exception represented by the rather generalized inner medullary CA staining at the light microscopic level reported in an early study by Rosen (17,22,25). For both morphological and functional reasons, the cells of the distal two thirds of the inner medullary CD have been considered to be a distinct type, termed “IMCD cell,’’ which is also reported to be negative for CA (18). These cells may transport urea and contain an unselective, conductive cation channel or Na+ channels (15,27,31). In addition, on the basis ofwork in cultured cells it has been proposed that IMCD has an Na+-H+exchanger (13,30,37), but the cell type in vivo that contains this transport system has not been identified. Because of incontrovertibleevidence for acetazolamide-sensitive acid secretion in segments of IMCD without recognizable intercalated cells (10,11,35,38), we reexamined the issue of the presence of CA in this segment with an improved histochemical technique and ultrastructural examination of morphology. We observed differential cell staining for CA activity without underlying morphological specificity throughout the IMCD. Moreover, there was variable staining among morphologically indistinguishable cells at the papillary tip and also among cells with features of both principal and intercalated cells located more proximally in the IMCD. 1535
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Materials and Methods Kidneys from six male rats weighing -125 g were fixed by perfusing 2.5% glutaraldehyde/O.l M phosphate buffer (pH 7.4) through the abdominal aorta, cardully maintaining a pressure of 110 mm Hg (12,21). Inner medullas, from the distal border of red medulla to papillary tip. were removed. For light microscopy, the tissue was post-fmed in perfusate for 2 hr at 4'C. rinsed, cryoprotected, frozen, cryostat-sectioned (10 pm thick), and stained for carbonic anhydrase (CA) activity for 8-10 min utilizing Riley and co-workers' modification of the Hansson histochemical technique (8.9.24).Kidney inner medullas processed for electron microscopy were vibratome-sectioned (20 pm thick) parallel to the long axis of the papillas and prepared as previously described (8.23). Kidney sections were otherwise unstained. Plastic sections of kidney were cut parallel to the long axis of the papillas. Thin sections were mounted on 4 x 2-mm colloidin-coated slot grids and examined in a JEOL 100 CX I1 electron microscope. Care was taken to restrict the analysis to sections from the most superficial S pm of the vibratome sections, at which depth diffusion of CO2 does not limit the staining reaction. Specificity of staining was verified by examining serial sections exposed to 0.01 mM acetazolamide during the histochemical reaction. In recent physiological studies, the IMCD has been divided, rather arbitrarily, into thirds (IMCDx-3) (27). Morphologists have generally failed to find distinguishing characteristics between the middle and last thirds of this segment. One group, in particular, has designated the first third as the initial IMCD and the two remaining thirds as the terminal IMCD (18). In the present report. the IMCDI-3 classification has been utilized to communicate the in vivo locations from which sections were examined. For the biochemical CA determinations, IMCD cells were isolated from
pooled renal papillas of five separate groups of rats, each consisting of six to 16 animals, by methods previously reported from this laboratory (13). The cells were washed with PBS, homogenized in 0.3 M sucrose, frozen in dry ice-acetone, and kept at - 70'C until analysis. CA activity was measured by Dr. Luc P. Brion of the Albert Einstein College of Medicine by the imidazole-Tris technique utilized by him and his co-workers (1). The results are stated as enzyme units (EU) per mg protein, where one enzyme unit is defined as the amount of homogenate necessary to halve the rate of color change of the p-nitrophenol indicator.
Results When reacted histochemically for 10 min. cells comprising the IMCD exhibited CA activity virtually to the papillary tip (Figure 1). Overall staining intensity and the proportion of stained cells diminished distally. However, intensely stained cells were observed in the ducts of Bellini and in tubules that were centrally situated within the papilla. The papillary surface epithelium also demonstrated significant CA activity. Specificity of staining for CA was shown by elimination of activity in an adjacent section after exposure to 0.01 mM acetazolamide (Figure 2). All cytoplasmic staining in the IMCD was blocked; persisting nuclear staining most likely represented nonspecific affinity of nucleic acids for cobalt (26). At the ultrastructural level, acetazolamide-treated tissue demonstrated virtual absence of reaction product except for a fine nonspecific precipitate along the apical cell membrane (Figure 3).
Figure 1. Low-power view of an entire inner medulla sectioned at the mid-thickness level of the papilla and reacted 10 min for CA activity. Arrows point to the terminal portion of a centrally located IMCD. Some of the IMCD cells are CA reactive. Original magnification x 96. Bar = 100 pm.
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Figure 2. Control section, adjacent to that in Figure 1, stained 10 min for CA in the presence of 0.01 mM acetazolamide. Specific staining of cells is inhibited; nonspecific COS tinting of nuclei persists. Original magnification x 96. Bar = 100 pm. Figure3. Electronmicrograph of cells of the IMCD, in a vibratome section reacted 10 min for CA in the presence of acetazolamide. There is absence of cytoplasmic and basolateral membraneassociatedstaining. A small amount of precipitate (arrows) is associated with the apical cell membrane; this is probably nonspecific binding of CO" to the glycocalyx. Original magnification x 8000. Bar = 1 pm.
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CA was also detected biochemically in homogenates of IMCD cells purified from rat papilla. Mean CA activity was 92 2 11 EUlmg protein (mean f SE; n = 5). The IMCD contains cells with three distinct ultrastructuralmorphologies: a cell with major characteristicsof collecting duct intercalated cells, a second whose predominating features are those of the collecting duct principal cell, and a third presumptively identical to the “IMCD cell” described by Madsen and co-workers(18). The distribution of these cell types varied with the IMCD subsegment, and the intensity of histochemical CA staining within each cell type varied. CA staining ranged from moderate to intense in intercalated-like cells and from little or none to intense in the principal and “IMCD cell” types. A cell comprising 10-20% of cells observed in IMCDl exhibited ultrastructural features of moderate to dark granular CAstaining cytoplasm, abundant centrally located mitochondria, and a substantial number of other tubulovesicularstructures in a largely subapical location (Figures4 and 5 ) . Compared with the luxuriant microplicated apical borders of the prototype for the intercalated cell, the turtle bladder mitochondria-rich or carbonic anhydraserich cell, the apical membranes of this cell are rather simplified (33). However, the rat kidney intercalated cells of the inner medulla also appear to have less complex apical cell borders than both the prototype and similar cells in the rat outer medulla (6,20). Nevertheless, to reflect this deviation in a cell with otherwise more characteristic intercalated cell features, we refer to it as intercalated-like. The intercalated-like cells in the IMCDi were interspersed among a majority population that displayed the morphology of principal cells (Figures 4 and 6), although they sometimesexhibited intense CA staining in substantial portions of their cytoplasm. In contrast to intercalated-likecells, they contained few mitochondria and subapical vesicles (Figure 6). A more conventional IMCDi principal cell is shown in Figure 7 . It presents a rather flat profile with finely granular cytoplasm containing few vesicular structures, a relatively small number of basally or laterally located mitochondria, short stubby apical microvilli, and a single long central cilium projecting from the apical membrane. There are also prominent basal infoldings, but less extensive lateral interlacing of cells than among the “IMCD cells” described below. The putative principal cell predominates in the IMCDl (80-90% of cells), becomes. less numerous and loses its central cilium in IMCD2, and disappears altogether in IMCD3. CA staining of widely varying intensity was observed among putative principal cells in both IMCDi and IMCD2. For example, the cell shown in the center of Figures 8 and 9 had the morphology described above, including the central cilium, and demonstrated
rather intense staining for CA. The immediately neighboring cells were morphologically indistinguishable, although central cilia were not present in the plane of this section. These neighboring cells showed a similar staining intensity in one case and, little, if any, staining in the other. The cell described above and shown in Fig. 7 , however, demonstrated identical morphology but much less staining, except for moderate reactivity of the basolateral infoldings. In IMCDl, at least half of cells with similar morphology exhibited moderate to intense CA staining. Moreover, this staining varied within the cytoplasm of such cells. Figure 10 shows gradients of staining for CA activity in the apical to basal direction within neighboring cells. In our hands, such gradients have been demonstrated not to be artifactual (9). In less intensely stained cells or areas of cells, punctate reaction product was observed in the cytoplasm and associated with apical microvilli (Figure 10) but little membrane associated staining was observed between cells, as in IMCD3 (Figure 13). The putative “IMCD cell” was low columnar to cubical, with a large basally located nucleus (Figure 11). As shown in Figures 12 and 13, mitochondria and subapical vesicular structureswere sparse to moderate in number. The apical plasma membrane had stubby microvilli with infrequent branching. The lateral and basal cell membranes, however, were highly branched and interlaced, with widely patent intercellular and subcellular spaces filled with amorphous material. The CA-reactive cytoplasm was moderately stained and granular in appearance. This cell type appeared in IMCDz and was the sole cell type present in IMCD3. In our hands, and in contrast to previous reports, the “IMCD cells” demonstrated histochemical staining for CA. In fact, a variable intensity of such staining was seen within both IMCD2 and IMCD3 subsegments. A low-power view (Figure 11)showed an intensely stained cell, several cells with moderate reaction product, and a few cells with little if any staining. The intensely stained cell in Figure 12 was not structurally distinguishable from its neighbors on the basis of mitochondria, subapical vesicles, and branched microvilli. Cells with less intense CA staining contained some punctate reaction product in their cytoplasm, along their apical processes (Figure ll), and prominently along their lateral cell membranes. Staining of the lateral membranes was sometimesheavy (Figure 13).
Discussion Histochemical CA activity in the rat IMCD was first demonstrated by Rosen (25). At the light microscopic level, the CA staining observed in the current study is similar to what was reported in the earlier report, especially if one considers a region such as shown
4
Figure 4. Low-power electron micrograph of an IMCQ containing two CA-reactive cells. The cell on the right has intercalated-likecell morphology and the cell on the left exhibits principal-like cell structure. Original magnification x 4200. Bar = 1 pn. Figure 5. A higher magnification image of the intercalated-likecell in Figure 4, showing moderate to dark CA staining. The cell contains many subapical vesicles and large mitochondria characteristic of cartical and outer medullary intercalated cells. Reaction product is cytoplasmic, most pronounced near the cell margins. Mitochondria lack CA staining. CA staining associated with the apical membrane is seen in the cell with CA-negative cytoplasm at left. Original magnification x 14,800. Bar = 1 pn. Figure 6. This principal-like cell, seen also in Figure 4, has short apical processes, a paucity of mitochondria, and few subapical vesicles. There is intense CA staining throughout the cytoplasm except for the central portion of the cell. CA staining associated with the apical membrane is seen in the cell with CA-negative cytoplasm at right. Original magnification x 11,600. Bar = 1 pm.
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Figure 9. Higher magnification of the CA-reactive principal cell in Figure 8. The cell has a central cilium, short microvilli, small numbers of mitochondria, and a low incidence of subapical vesicles. Original magnification x 14.800. Bar = 1 pm. Figure 10. A portion of an IMCD, just inside the inner strip of the outer medulla. The leftmost principal-like cell (small arrow) exhibits marked subapical CA staining and low basal staining, whereas the rightmost principal-like cell (large arrow) shpws the opposite staining pattern. Original magnification x 5700.Bar = 1 pm.
Figure 7. A low-power view of an IMCD, showing a principal cell with a central cilium and expressing a low level of CA staining except for moderate activity in the basal processes. Some of the surrounding cells with principal cell morphology, although lacking cilia in this plane of section, are more heavily CA reactive. Original magnification x 7300.Bar = 1 pm. Figure 8. Portion of an IMCD, with a highly CA-reactive principal cell. The central cilium (arrow), with marked CA staining, was confirmed in serial section to originate from this cell. Original magnification x 7300. Bar = 1 pm.
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Figure 13. Papillary tip "IMCD"cells exhibit heavy CA reactivity (arrows) associated with the lateral cell membranes. Original magnification x 8000. Bar = 1 pm.
in the right-hand portion of Figure 1 which appears to be most comparable to the early to mid-IMCD shown by Rosen. Even in his study, which differs from ours in a number of technical details, however, there is a suggestion of some heterogeneity of CA activity among cells (see ref. 25, Figures 4c and 4d). More recent studies, including some employing ultrastructural techniques, report that most CA activity in the distal portions of the nephron is contained within intercalated cells (17,22). The reactive cells reportedly comprise approximately 10% of IMCDi and are absent in IMCD2-3 (4.6). In the present study, cells partially resembling intercalated cells were observed in IMCDi. These contained many mitochondria and subapical vesicles but lacked the characteristic apical plasma membrane complexity. In addition, the cells showed some range of CA staining intensity. If these cells are indeed variants of the intercalated cell type, they may be less typical because of having been less activated by the acid-base status
of the animal. Activation would be expressed morphologically by expansion and multiplication of the apical cell border (20) and, at least in turtle bladder, by extension of intense staining for CA activity throughout the entire cytoplasm (9). The physiologicalcorrelate of these changes in morphology would be an increased rate of H+secretion. In any event, since the acid-base status of the animals usually has not been specified beyond reporting that the rats ate standard chow and drank tapwater, it is not clear whether acid-base status accounts for the differences in cell morphology and staining among different studies. Many of the cells comprising the majority population in the IMCD, and which also demonstrate heterogeneous CA staining, are presumably principal cells. This presumption is based on the paucity of mitochondria, absence of apical vesicles, simple microvillus apical border, and, most tellingly, the presence of a single long central cilium (19). A central cilium was observed both in cells
Figure 11. IMCD, cells at the center of a papillarytip show a diversity of CA staining patterns. There is a highly reactive cell (large arrow), a number of moderately stained cells (small arrows), and lateral cell membrane staining (arrowheads). Original magnification x 4000. Bar = 1 pm. Figure 12. Higher-magnificationview of the intensely stained cell in Figure 11. This cell possesses apical microvillisimilar to those of the surrounding less reactive cells. Subapical vesicles are present (white arrows). The basolateral cell processesare interdigitated and branched and surround amorphous extracellular material. The less reactive bordering cells possess fine punctate reaction product (black arrows) in the cytoplasm and apical membranes. Original magnification x 10,800. Bar = 1 urn.
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intensely stained for CA and in structurallysimilar cells with little staining. Staining of principal cells has been reported in the rat papilla but has been stated to become weaker and disappear altogether in IMCD2-3 (17). Unexpectedly, the present investigation revealed many CApositive cells in IMCD2-3. These cells lacked typical intercalated cell features and were morphologically indistinguishable from other cells lacking CA enzymatic activity. Presumably, the reactive and unreactive cells all belong to the distinct “IMCD cell” type of the terminal IMCD (18). It is doubtful that the failure to find CA activity in previous studies of this nephron segment is related to differences in rats. In the present study, rats were the same strain, although somewhat younger by weight, as those utilized by Clapp, Madsen, and co-workers(5,6,18-20). In addition, sections were not taken from peripheral regions of papilla in which a few intercalated cells have been observed as deep as in IMCDz ( 5 ) . Our observation of CA staining in cells out to the papillary tip may reflect preservation of CA by glutaraldehydeperfusion-fixation, the somewhat longer incubation times (10 min) employed to detect low levels of activity, and the use of non-embedded cryostat (10 pm) and vibratome (20 pm) sections. Previous work localizing antibodies to CA in rat kidney IMCD has found diminished to rare immunostaining in the terminal two thirds (3) The authors themselves point out, however, that although the anti-CA antibody raised against rat erythrocyte CA-C (CA-11) enzyme is specific for the soluble, cytoplasmic isoenzyme, there are discrepancies between weak immunohistochemicalstaining and pronounced CA histochemical staining in the proximal tubules and thick ascending limbs of the loop of Henle. They offer possible explanations, the most plausible of which is that certain tubules possess a CA isoform that is antigenically distinct from erythrocyte CA-C. This is suggested by the direct demonstration of positive histochemical and negative immunochemicalstaining in the same tissue (29). Uniqueness of CA isoenzymes in different regions of the kidney was also suggested by others (28,41). In addition to CA-11, the kidney is also known to contain a membrane-associatedType IV CA, believed to reside primarily in the apical membranes of proximal tubules. Functional evidence for luminal CA activity in inner stripe of OMCD and initial IMCD has been presented (32,36). Apical membrane CA activity is difficult to discern in cells with heavy cytoplasmic CA staining. However, reaction product distributed along the apical membrane of CA-negative cells is evident in Figures 5 and 6. The prominent membrane-associated CA staining in the terminal IMCD (Figure 13) does not appear to be in contact with the tubular lumen. The kidney also contains a Type V CA primarily associated with cortical, presumably proximal tubular, mitochondria (7). In the present studies, however, histochemical CA activity was not observed in mitochondria of any cell type in the IMCD. Finally, despite initially negative findings of biochemical CA activity in homogenates of papillae, a micromethod with increased sensitivity has detected CA activity in rat papilla, as reported in a previous study (1) and in disaggregated IMCD cells from this laboratory. These observations support the results of the present histochemical studies. IMCD acidification has been generally considered to be mediated by an electrogenicH’ pump with characteristics of an H’-ATPase (16,34,35,42). Cells morphologically similar to those proposed to
contain this transporter in cortical and outer medullary CD, however, have only been found in small numbers in the initial portion of the IMCD (6,18,19,22). Furthermore, only 10% of cells at the beginning of the initial third of the IMCD show immunolabeling for an H’-ATPase, and this labeling vanishes over the course of this subsegment (2). Although it seems likely that these were intercalated cells, no morphological confirmation was reported. The absence of staining for H+-ATPasemore distally in the IMCD is curious in view of the present results. The present differences from prior reports may be due to a technical problem, such as loss of soluble CA from less well-fixed sections utilized for antibody staining, a lack of H+secretion in intercalated cells and CA-positive cells of IMCD2-3, or the existence of an alternative mechanism(s) for H’ transport. In view of the finding of CA activity in a variety of cells throughout the IMCD and the relative contribution of this segment to overall renal acidification, at least under some circumstances, it is reasonable to postulate that cells other than the 10% of intercalated cells observed in the IMCDi possess an H’ transport capability. Although assigningproperties to cells in intact tissues or organs by extrapolation from cultured cells is hazardous, the demonstration of H+pump activity in IMCD cultures not considered to contain intercalated cells provides suggestive evidence for this notion (13,30). Moreover, there is evidence for a gastrictype H+,K+-ATPasein outer medullary CD (39,40), and we have reported preliminary evidence for this form of proton secretion in IMCD cultures (14). It is reasonable to speculate that some CApositive cells in situ may secrete acid by this mechanism. In summary, the present investigation demonstrates the occurrence of histochemical CA activity in cells distributed throughout the rat IMCD. The wide distribution of CA in a variety of cells with divergent morphological characteristicscasts doubt on the specificity of CA histochemical staining for marking intercalated cells. In addition, the results are consistent with the notion that cell types other than intercalated cells participate in IMCD acid secretion. The methodologies employed exclusively in the present study will have to be combined with future physiological studies of transport to fully examine this issue.
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erogeneity along the rat inner medullary collecting duct. Lab Invest 60:219, 1989 6. Clapp WL, Madsen KM, Verlander JW, Tisher CC Intercalated cells of the rat inner medullary collecting duct. Kidney Int 31:1080, 1987 7. Dodgson SJ: Carbonic anhydrases in the kidney. In Tashian RE, Gros
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Vol. 40, NO. 10, pp. 1535-1545, 1992 Printed in C%S.A.
Original Article
Histochemical Carbonic Anhydrase in Rat Inner Medullary Collecting Duct’ J. G. KLEINMAN,’ J. L. W. BAIN, C. FRITSCHE, and D. A. RILEY Nephrology Division, Zublocki Veterans Afairs Medicai Center and Medical CoIIege of Wisconsin (JGK,CF),and the Department of Cell Biology and Anatomy, Medical College of Wisconsin (JLWB,DAR), Mdwaukee, Wisconsin.
Received for publication January 23, 1992 and in revised form April
20. 1992;
Rat inner medullary collecting duct (IMCD) seaetes substantial amounts of H’. However, carbonic anhydrase (CA), a concomitant of H’ seaetion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprisingthe IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportionhave features of intercalatedcells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third
cell type, previously called “IMCDcells,”appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these “IMCDcells” have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells dearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD. ( J Eiistdem Cytoc6em 40d535-1545, 1992) KEY WORDS:Rat kidney; H’secretion;Hansson technique; Intercalated cells; Principal cell; IMCD cell; Acetazolamide;Kidney ultrastructure.
Introduction Two types of epithelial cells involved in ion transport have been described in the mammalian collecting duct (CD): intercalated cells and principal cells. Because of morphological similarities to mitochondria-rich cells of turtle urinary bladder (a collecting duct analogue) and lectin binding characteristics, intercalated cells, particularly those of the cortical CD, have been further classified into A or a and B or fi subtypes (19). Intercalated cells contain the enzyme CA, and it has been proposed that the a-cells secrete H’ and the p-cells secrete HCO3- into tubule lumens (8,33). Rat IMCD has been reported to contain only small numbers of intercalated cells, presumably a type, yet this segment may be responsible for 50% of total acid secretion in acidosis (6,lOJl). Furthermore, isolated perfused segments of IMCD apparently lacking intercalated cells acidlfy their lumens (38). Principal cells of the outer medullary CD and the proximal third of the IMCD appear to be similar to those of the cortical CD (18).
’
Supported by the Department of Veterans Affairs, Merit Review Program and Career Development Award UGK)and by a NASA grant NAG 2-552 (DAR). Correspondence to: Jack G. Kleinman, MD, Nephrology Section111K, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295.
accepted May 1, 1992 (2A2568).
Principal cells may primarily mediate Na+,K’, water, and urea transport (18J9). CA activity, however, usually associated with H+or HCO3- transport, has been reported to be weak or entirely absent in principal cells with the possible exception represented by the rather generalized inner medullary CA staining at the light microscopic level reported in an early study by Rosen (17,22,25). For both morphological and functional reasons, the cells of the distal two thirds of the inner medullary CD have been considered to be a distinct type, termed “IMCD cell,’’ which is also reported to be negative for CA (18). These cells may transport urea and contain an unselective, conductive cation channel or Na+ channels (15,27,31). In addition, on the basis ofwork in cultured cells it has been proposed that IMCD has an Na+-H+exchanger (13,30,37), but the cell type in vivo that contains this transport system has not been identified. Because of incontrovertibleevidence for acetazolamide-sensitive acid secretion in segments of IMCD without recognizable intercalated cells (10,11,35,38), we reexamined the issue of the presence of CA in this segment with an improved histochemical technique and ultrastructural examination of morphology. We observed differential cell staining for CA activity without underlying morphological specificity throughout the IMCD. Moreover, there was variable staining among morphologically indistinguishable cells at the papillary tip and also among cells with features of both principal and intercalated cells located more proximally in the IMCD. 1535
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Materials and Methods Kidneys from six male rats weighing -125 g were fixed by perfusing 2.5% glutaraldehyde/O.l M phosphate buffer (pH 7.4) through the abdominal aorta, cardully maintaining a pressure of 110 mm Hg (12,21). Inner medullas, from the distal border of red medulla to papillary tip. were removed. For light microscopy, the tissue was post-fmed in perfusate for 2 hr at 4'C. rinsed, cryoprotected, frozen, cryostat-sectioned (10 pm thick), and stained for carbonic anhydrase (CA) activity for 8-10 min utilizing Riley and co-workers' modification of the Hansson histochemical technique (8.9.24).Kidney inner medullas processed for electron microscopy were vibratome-sectioned (20 pm thick) parallel to the long axis of the papillas and prepared as previously described (8.23). Kidney sections were otherwise unstained. Plastic sections of kidney were cut parallel to the long axis of the papillas. Thin sections were mounted on 4 x 2-mm colloidin-coated slot grids and examined in a JEOL 100 CX I1 electron microscope. Care was taken to restrict the analysis to sections from the most superficial S pm of the vibratome sections, at which depth diffusion of CO2 does not limit the staining reaction. Specificity of staining was verified by examining serial sections exposed to 0.01 mM acetazolamide during the histochemical reaction. In recent physiological studies, the IMCD has been divided, rather arbitrarily, into thirds (IMCDx-3) (27). Morphologists have generally failed to find distinguishing characteristics between the middle and last thirds of this segment. One group, in particular, has designated the first third as the initial IMCD and the two remaining thirds as the terminal IMCD (18). In the present report. the IMCDI-3 classification has been utilized to communicate the in vivo locations from which sections were examined. For the biochemical CA determinations, IMCD cells were isolated from
pooled renal papillas of five separate groups of rats, each consisting of six to 16 animals, by methods previously reported from this laboratory (13). The cells were washed with PBS, homogenized in 0.3 M sucrose, frozen in dry ice-acetone, and kept at - 70'C until analysis. CA activity was measured by Dr. Luc P. Brion of the Albert Einstein College of Medicine by the imidazole-Tris technique utilized by him and his co-workers (1). The results are stated as enzyme units (EU) per mg protein, where one enzyme unit is defined as the amount of homogenate necessary to halve the rate of color change of the p-nitrophenol indicator.
Results When reacted histochemically for 10 min. cells comprising the IMCD exhibited CA activity virtually to the papillary tip (Figure 1). Overall staining intensity and the proportion of stained cells diminished distally. However, intensely stained cells were observed in the ducts of Bellini and in tubules that were centrally situated within the papilla. The papillary surface epithelium also demonstrated significant CA activity. Specificity of staining for CA was shown by elimination of activity in an adjacent section after exposure to 0.01 mM acetazolamide (Figure 2). All cytoplasmic staining in the IMCD was blocked; persisting nuclear staining most likely represented nonspecific affinity of nucleic acids for cobalt (26). At the ultrastructural level, acetazolamide-treated tissue demonstrated virtual absence of reaction product except for a fine nonspecific precipitate along the apical cell membrane (Figure 3).
Figure 1. Low-power view of an entire inner medulla sectioned at the mid-thickness level of the papilla and reacted 10 min for CA activity. Arrows point to the terminal portion of a centrally located IMCD. Some of the IMCD cells are CA reactive. Original magnification x 96. Bar = 100 pm.
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Figure 2. Control section, adjacent to that in Figure 1, stained 10 min for CA in the presence of 0.01 mM acetazolamide. Specific staining of cells is inhibited; nonspecific COS tinting of nuclei persists. Original magnification x 96. Bar = 100 pm. Figure3. Electronmicrograph of cells of the IMCD, in a vibratome section reacted 10 min for CA in the presence of acetazolamide. There is absence of cytoplasmic and basolateral membraneassociatedstaining. A small amount of precipitate (arrows) is associated with the apical cell membrane; this is probably nonspecific binding of CO" to the glycocalyx. Original magnification x 8000. Bar = 1 pm.
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CA was also detected biochemically in homogenates of IMCD cells purified from rat papilla. Mean CA activity was 92 2 11 EUlmg protein (mean f SE; n = 5). The IMCD contains cells with three distinct ultrastructuralmorphologies: a cell with major characteristicsof collecting duct intercalated cells, a second whose predominating features are those of the collecting duct principal cell, and a third presumptively identical to the “IMCD cell” described by Madsen and co-workers(18). The distribution of these cell types varied with the IMCD subsegment, and the intensity of histochemical CA staining within each cell type varied. CA staining ranged from moderate to intense in intercalated-like cells and from little or none to intense in the principal and “IMCD cell” types. A cell comprising 10-20% of cells observed in IMCDl exhibited ultrastructural features of moderate to dark granular CAstaining cytoplasm, abundant centrally located mitochondria, and a substantial number of other tubulovesicularstructures in a largely subapical location (Figures4 and 5 ) . Compared with the luxuriant microplicated apical borders of the prototype for the intercalated cell, the turtle bladder mitochondria-rich or carbonic anhydraserich cell, the apical membranes of this cell are rather simplified (33). However, the rat kidney intercalated cells of the inner medulla also appear to have less complex apical cell borders than both the prototype and similar cells in the rat outer medulla (6,20). Nevertheless, to reflect this deviation in a cell with otherwise more characteristic intercalated cell features, we refer to it as intercalated-like. The intercalated-like cells in the IMCDi were interspersed among a majority population that displayed the morphology of principal cells (Figures 4 and 6), although they sometimesexhibited intense CA staining in substantial portions of their cytoplasm. In contrast to intercalated-likecells, they contained few mitochondria and subapical vesicles (Figure 6). A more conventional IMCDi principal cell is shown in Figure 7 . It presents a rather flat profile with finely granular cytoplasm containing few vesicular structures, a relatively small number of basally or laterally located mitochondria, short stubby apical microvilli, and a single long central cilium projecting from the apical membrane. There are also prominent basal infoldings, but less extensive lateral interlacing of cells than among the “IMCD cells” described below. The putative principal cell predominates in the IMCDl (80-90% of cells), becomes. less numerous and loses its central cilium in IMCD2, and disappears altogether in IMCD3. CA staining of widely varying intensity was observed among putative principal cells in both IMCDi and IMCD2. For example, the cell shown in the center of Figures 8 and 9 had the morphology described above, including the central cilium, and demonstrated
rather intense staining for CA. The immediately neighboring cells were morphologically indistinguishable, although central cilia were not present in the plane of this section. These neighboring cells showed a similar staining intensity in one case and, little, if any, staining in the other. The cell described above and shown in Fig. 7 , however, demonstrated identical morphology but much less staining, except for moderate reactivity of the basolateral infoldings. In IMCDl, at least half of cells with similar morphology exhibited moderate to intense CA staining. Moreover, this staining varied within the cytoplasm of such cells. Figure 10 shows gradients of staining for CA activity in the apical to basal direction within neighboring cells. In our hands, such gradients have been demonstrated not to be artifactual (9). In less intensely stained cells or areas of cells, punctate reaction product was observed in the cytoplasm and associated with apical microvilli (Figure 10) but little membrane associated staining was observed between cells, as in IMCD3 (Figure 13). The putative “IMCD cell” was low columnar to cubical, with a large basally located nucleus (Figure 11). As shown in Figures 12 and 13, mitochondria and subapical vesicular structureswere sparse to moderate in number. The apical plasma membrane had stubby microvilli with infrequent branching. The lateral and basal cell membranes, however, were highly branched and interlaced, with widely patent intercellular and subcellular spaces filled with amorphous material. The CA-reactive cytoplasm was moderately stained and granular in appearance. This cell type appeared in IMCDz and was the sole cell type present in IMCD3. In our hands, and in contrast to previous reports, the “IMCD cells” demonstrated histochemical staining for CA. In fact, a variable intensity of such staining was seen within both IMCD2 and IMCD3 subsegments. A low-power view (Figure 11)showed an intensely stained cell, several cells with moderate reaction product, and a few cells with little if any staining. The intensely stained cell in Figure 12 was not structurally distinguishable from its neighbors on the basis of mitochondria, subapical vesicles, and branched microvilli. Cells with less intense CA staining contained some punctate reaction product in their cytoplasm, along their apical processes (Figure ll), and prominently along their lateral cell membranes. Staining of the lateral membranes was sometimesheavy (Figure 13).
Discussion Histochemical CA activity in the rat IMCD was first demonstrated by Rosen (25). At the light microscopic level, the CA staining observed in the current study is similar to what was reported in the earlier report, especially if one considers a region such as shown
4
Figure 4. Low-power electron micrograph of an IMCQ containing two CA-reactive cells. The cell on the right has intercalated-likecell morphology and the cell on the left exhibits principal-like cell structure. Original magnification x 4200. Bar = 1 pn. Figure 5. A higher magnification image of the intercalated-likecell in Figure 4, showing moderate to dark CA staining. The cell contains many subapical vesicles and large mitochondria characteristic of cartical and outer medullary intercalated cells. Reaction product is cytoplasmic, most pronounced near the cell margins. Mitochondria lack CA staining. CA staining associated with the apical membrane is seen in the cell with CA-negative cytoplasm at left. Original magnification x 14,800. Bar = 1 pn. Figure 6. This principal-like cell, seen also in Figure 4, has short apical processes, a paucity of mitochondria, and few subapical vesicles. There is intense CA staining throughout the cytoplasm except for the central portion of the cell. CA staining associated with the apical membrane is seen in the cell with CA-negative cytoplasm at right. Original magnification x 11,600. Bar = 1 pm.
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Figure 9. Higher magnification of the CA-reactive principal cell in Figure 8. The cell has a central cilium, short microvilli, small numbers of mitochondria, and a low incidence of subapical vesicles. Original magnification x 14.800. Bar = 1 pm. Figure 10. A portion of an IMCD, just inside the inner strip of the outer medulla. The leftmost principal-like cell (small arrow) exhibits marked subapical CA staining and low basal staining, whereas the rightmost principal-like cell (large arrow) shpws the opposite staining pattern. Original magnification x 5700.Bar = 1 pm.
Figure 7. A low-power view of an IMCD, showing a principal cell with a central cilium and expressing a low level of CA staining except for moderate activity in the basal processes. Some of the surrounding cells with principal cell morphology, although lacking cilia in this plane of section, are more heavily CA reactive. Original magnification x 7300.Bar = 1 pm. Figure 8. Portion of an IMCD, with a highly CA-reactive principal cell. The central cilium (arrow), with marked CA staining, was confirmed in serial section to originate from this cell. Original magnification x 7300. Bar = 1 pm.
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Figure 13. Papillary tip "IMCD"cells exhibit heavy CA reactivity (arrows) associated with the lateral cell membranes. Original magnification x 8000. Bar = 1 pm.
in the right-hand portion of Figure 1 which appears to be most comparable to the early to mid-IMCD shown by Rosen. Even in his study, which differs from ours in a number of technical details, however, there is a suggestion of some heterogeneity of CA activity among cells (see ref. 25, Figures 4c and 4d). More recent studies, including some employing ultrastructural techniques, report that most CA activity in the distal portions of the nephron is contained within intercalated cells (17,22). The reactive cells reportedly comprise approximately 10% of IMCDi and are absent in IMCD2-3 (4.6). In the present study, cells partially resembling intercalated cells were observed in IMCDi. These contained many mitochondria and subapical vesicles but lacked the characteristic apical plasma membrane complexity. In addition, the cells showed some range of CA staining intensity. If these cells are indeed variants of the intercalated cell type, they may be less typical because of having been less activated by the acid-base status
of the animal. Activation would be expressed morphologically by expansion and multiplication of the apical cell border (20) and, at least in turtle bladder, by extension of intense staining for CA activity throughout the entire cytoplasm (9). The physiologicalcorrelate of these changes in morphology would be an increased rate of H+secretion. In any event, since the acid-base status of the animals usually has not been specified beyond reporting that the rats ate standard chow and drank tapwater, it is not clear whether acid-base status accounts for the differences in cell morphology and staining among different studies. Many of the cells comprising the majority population in the IMCD, and which also demonstrate heterogeneous CA staining, are presumably principal cells. This presumption is based on the paucity of mitochondria, absence of apical vesicles, simple microvillus apical border, and, most tellingly, the presence of a single long central cilium (19). A central cilium was observed both in cells
Figure 11. IMCD, cells at the center of a papillarytip show a diversity of CA staining patterns. There is a highly reactive cell (large arrow), a number of moderately stained cells (small arrows), and lateral cell membrane staining (arrowheads). Original magnification x 4000. Bar = 1 pm. Figure 12. Higher-magnificationview of the intensely stained cell in Figure 11. This cell possesses apical microvillisimilar to those of the surrounding less reactive cells. Subapical vesicles are present (white arrows). The basolateral cell processesare interdigitated and branched and surround amorphous extracellular material. The less reactive bordering cells possess fine punctate reaction product (black arrows) in the cytoplasm and apical membranes. Original magnification x 10,800. Bar = 1 urn.
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intensely stained for CA and in structurallysimilar cells with little staining. Staining of principal cells has been reported in the rat papilla but has been stated to become weaker and disappear altogether in IMCD2-3 (17). Unexpectedly, the present investigation revealed many CApositive cells in IMCD2-3. These cells lacked typical intercalated cell features and were morphologically indistinguishable from other cells lacking CA enzymatic activity. Presumably, the reactive and unreactive cells all belong to the distinct “IMCD cell” type of the terminal IMCD (18). It is doubtful that the failure to find CA activity in previous studies of this nephron segment is related to differences in rats. In the present study, rats were the same strain, although somewhat younger by weight, as those utilized by Clapp, Madsen, and co-workers(5,6,18-20). In addition, sections were not taken from peripheral regions of papilla in which a few intercalated cells have been observed as deep as in IMCDz ( 5 ) . Our observation of CA staining in cells out to the papillary tip may reflect preservation of CA by glutaraldehydeperfusion-fixation, the somewhat longer incubation times (10 min) employed to detect low levels of activity, and the use of non-embedded cryostat (10 pm) and vibratome (20 pm) sections. Previous work localizing antibodies to CA in rat kidney IMCD has found diminished to rare immunostaining in the terminal two thirds (3) The authors themselves point out, however, that although the anti-CA antibody raised against rat erythrocyte CA-C (CA-11) enzyme is specific for the soluble, cytoplasmic isoenzyme, there are discrepancies between weak immunohistochemicalstaining and pronounced CA histochemical staining in the proximal tubules and thick ascending limbs of the loop of Henle. They offer possible explanations, the most plausible of which is that certain tubules possess a CA isoform that is antigenically distinct from erythrocyte CA-C. This is suggested by the direct demonstration of positive histochemical and negative immunochemicalstaining in the same tissue (29). Uniqueness of CA isoenzymes in different regions of the kidney was also suggested by others (28,41). In addition to CA-11, the kidney is also known to contain a membrane-associatedType IV CA, believed to reside primarily in the apical membranes of proximal tubules. Functional evidence for luminal CA activity in inner stripe of OMCD and initial IMCD has been presented (32,36). Apical membrane CA activity is difficult to discern in cells with heavy cytoplasmic CA staining. However, reaction product distributed along the apical membrane of CA-negative cells is evident in Figures 5 and 6. The prominent membrane-associated CA staining in the terminal IMCD (Figure 13) does not appear to be in contact with the tubular lumen. The kidney also contains a Type V CA primarily associated with cortical, presumably proximal tubular, mitochondria (7). In the present studies, however, histochemical CA activity was not observed in mitochondria of any cell type in the IMCD. Finally, despite initially negative findings of biochemical CA activity in homogenates of papillae, a micromethod with increased sensitivity has detected CA activity in rat papilla, as reported in a previous study (1) and in disaggregated IMCD cells from this laboratory. These observations support the results of the present histochemical studies. IMCD acidification has been generally considered to be mediated by an electrogenicH’ pump with characteristics of an H’-ATPase (16,34,35,42). Cells morphologically similar to those proposed to
contain this transporter in cortical and outer medullary CD, however, have only been found in small numbers in the initial portion of the IMCD (6,18,19,22). Furthermore, only 10% of cells at the beginning of the initial third of the IMCD show immunolabeling for an H’-ATPase, and this labeling vanishes over the course of this subsegment (2). Although it seems likely that these were intercalated cells, no morphological confirmation was reported. The absence of staining for H+-ATPasemore distally in the IMCD is curious in view of the present results. The present differences from prior reports may be due to a technical problem, such as loss of soluble CA from less well-fixed sections utilized for antibody staining, a lack of H+secretion in intercalated cells and CA-positive cells of IMCD2-3, or the existence of an alternative mechanism(s) for H’ transport. In view of the finding of CA activity in a variety of cells throughout the IMCD and the relative contribution of this segment to overall renal acidification, at least under some circumstances, it is reasonable to postulate that cells other than the 10% of intercalated cells observed in the IMCDi possess an H’ transport capability. Although assigningproperties to cells in intact tissues or organs by extrapolation from cultured cells is hazardous, the demonstration of H+pump activity in IMCD cultures not considered to contain intercalated cells provides suggestive evidence for this notion (13,30). Moreover, there is evidence for a gastrictype H+,K+-ATPasein outer medullary CD (39,40), and we have reported preliminary evidence for this form of proton secretion in IMCD cultures (14). It is reasonable to speculate that some CApositive cells in situ may secrete acid by this mechanism. In summary, the present investigation demonstrates the occurrence of histochemical CA activity in cells distributed throughout the rat IMCD. The wide distribution of CA in a variety of cells with divergent morphological characteristicscasts doubt on the specificity of CA histochemical staining for marking intercalated cells. In addition, the results are consistent with the notion that cell types other than intercalated cells participate in IMCD acid secretion. The methodologies employed exclusively in the present study will have to be combined with future physiological studies of transport to fully examine this issue.
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