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作者：Wan-Young Kim, Joon-Ho Jung, Eun-Young Park, Chul-Woo Yang, Hyang Kim, Søren Nielsen, Kirsten M. Madsen, and Jin Kim作者单位：1 Department of Anatomy and Medical Research Center for Cell Death Disease Research Center, 3 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, 2 Department of Urology, Bundang Jesang Hospital, and 4 Department of Internal Medicine, Sungkyunkwan University, Kang
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& z7 [) A1 h, u1 F" f 【摘要】
$ d5 k4 M! Q* l1 t% n5 D/ o Recent studies of the distribution of PKC isoenzymes in the mouse kidney demonstrated that PKC-, - I, and - are expressed in intercalated cells. The purpose of this study was to identify the intercalated cell subtypes that express the different PKC isoenzymes and determine the location of the PKC isoenzymes within these cells. Adult C57BL/6 mice kidney tissues were processed for multiple-labeling immunohistochemistry. Antibodies against the vacuolar H -ATPase and pendrin were used to identify intercalated cell subtypes, whereas antibodies against calbindin D 28K and aquaporin-2 (AQP2) were used to identify connecting tubule cells and principal cells of the collecting duct, respectively. Within type A intercalated cells, PKC- was highly expressed in the apical part of the cells, whereas immunoreactivity for both PKC- and PKC- I was weak. Type B intercalated cells exhibited strong expression of PKC-, - I, and -. PKC- and - I were localized throughout the cytoplasm, whereas PKC- was restricted to the basal domain. Within non-A-non-B cells, immunoreactivity for both PKC- and PKC- I was high in intensity and localized diffusely in the cytoplasm, whereas PKC- was localized in the apical part of the cells. None of the PKC isoenzymes (PKC-, - I, or - ) were expressed in the calbindin D 28K -positive connecting tubule cells. Within AQP2-positive principal cells of the collecting duct, PKC- was expressed on the basolateral plasma membrane, but no significant staining was detected for PKC- I and -. In summary, this study demonstrates distinct and differential expression patterns of PKC-, - I, and - in the three subtypes of intercalated cells in the mouse kidney.
! d% d8 ?* d1 B 【关键词】 PKC I and immunohistochemistry* d: G0 I6 e# Z) j" F3 H
PKC IS A FAMILY OF PROTEIN KINASES that specifically phosphorylate serine/threonine. PKC plays a central role in intracellular signal transduction pathways of hormones, neurotransmitters, and growth factors and contributes significantly to the control of renal function such as cellular proliferation, differentiation, exocytosis, and ion and water transport ( 22, 23 ).1 {2 E# d( S8 U% ?, T/ R5 Q
) [) h) f- X f: _ N, xIn the kidney, PKC is involved in the regulation of blood flow in the glomerulus ( 31 ) and transport mechanisms of the uriniferous tubule ( 4, 5, 24, 32 ) and the collecting duct ( 3, 6, 10, 15 ). PKC-, -, -, -, -, -, and - have been detected in the rat kidney ( 11, 14, 17, 21, 26, 27 ), and PKC-, -, -, -, -, and - in the human kidney ( 14 ). Recently, Redling and colleagues ( 28 ) reported that PKC-, - I, and - are expressed in the intercalated cells of the mouse kidney. Although PKC was shown to be expressed in intercalated cells, these studies did not distinguish between the various subtypes of intercalated cells, which have distinctly different functions.1 W5 V9 ~. ~& }0 }1 z8 r
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Intercalated cells in the renal connecting tubule (CNT) and collecting duct assist with the regulation of acid-base balance by secreting or reabsorbing of H and HCO 3 -. They are divided into three groups according to immunocytochemical characteristics: type A, type B, and non-A-non-B cells ( 18, 19, 20, 34 ). There are a few functional reports about the involvement of PKC in acid-base balance ( 8, 29, 30, 37 ), but none of these reports described the role of PKC in intercalated cells. Because intercalated cells express type-specific acid-base transporters, such as H -ATPase, anion-exchanger 1 (AE1), and pendrin, it is critical to identify type-specific expression of PKC isozymes to understand the specific role of PKC in the subtypes of intercalated cells. Therefore, we studied the expression of PKC isoenzymes, I, and in the subtypes of mouse renal intercalated cells using multiple-labeling immunohistochemistry.
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7 G$ ]6 R7 s( ^5 N8 N9 h! AMATERIALS AND METHODS8 K, v- B c1 @) z: P
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Animals and Preservation of Kidneys) T. B9 a) V# [' {0 [
/ u. X: ~; X1 G' Y6 i) a: y$ R+ ^Male C57BL/6 mice, weighing 20-25 g, were used in all experiments. Animal care and experimental procedures were performed under approval from the Animal Care Committees of College of Medicine, the Catholic University of Korea. The animals were anesthetized with an intraperitoneal injection of urethane (16.5%), perfused with phosphate buffer (PBS; pH 7.4), and then fixed with 2% paraformaldehyde-lysine-periodate (PLP) solution, which was administered through the heart for 10 min. The kidneys were removed, cut into slices (2-mm thickness) that included the renal papilla, and then immersed in PLP solution for 12-16 h at 4°C.. V" s! C) @9 T; o+ ], ?1 V9 G
$ V5 ]; R. a. v, VAntibodies
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4 ^( g! m; @$ R8 ]! RAntibodies against PKC isoenzymes, I, and (Santa Cruz Biotechnology) were used. Intercalated cells were identified using an antibody against vacuolar-type H -ATPase B1/2 (Santa Cruz Biotechnology). An antibody against pendrin was used to label the apical plasma membranes of type B and non-A-non-B intercalated cells ( 20 ). Principal cells in the collecting duct were identified using an antibody against aquaporin-2 (AQP2; Chemicon, Temecula, CA) that labels the apical plasma membrane of principal cells. CNT cells were identified using an antibody against calbindin D 28K (Chemicon) that labels the cytoplasm of CNT cells ( 39 ).
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Light Microscopic Immunohistochemistry* q4 }/ b) f2 M. F7 A; l! _
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Single-labeling immunohistochemistry using preembedding methods. Fifty-micrometer-thick Vibratome sections were processed for immunohistochemistry using an indirect preembedding immunoperoxidase method. The sections were washed three times for 15 min each in PBS containing 50 mM NH 4 Cl. They were then incubated for 4 h in PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin ( solution A ). The tissue sections were incubated overnight at 4°C with PKC-, - I, and - antibodies diluted 1:2,000 in PBS containing 1% BSA ( solution B ). After several washes with PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin ( solution C ), the tissue sections were incubated for 2 h in a 1:100 dilution of a peroxidase-conjugated donkey anti-rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) in solution B. The tissues were then rinsed, first in solution C and subsequently in Tris buffer (50 mM Tris·HCl, pH 7.6). For the detection of horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine in the Tris buffer for 5 min. Then, colorization of the reaction was added with 0.01% H 2 O 2 for an 10-min incubation. After several washings in Tris buffer, the sections were dehydrated in a graded series of ethanol and embedded in Epon 812 (Polysciences, Warrington, CA).
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Multiple-labeling immunohistochemistry using postembedding methods. To identify the subtypes of intercalated cells that express PKC immunoreactivity, double-labeled immunohistochemistry was performed with antibodies against H -ATPase, pendrin, calbindin D 28K, and AQP2 ( Table 1 ). From the flat-embedded Vibratome sections processed for single immunolabeling of PKC-, - I, or -, sections from the cortex to the inner medulla were excised and glued onto empty blocks of Epon 812. Three successive 1.5-µm-thick sections were cut and treated for 10-15 min with a saturated solution of sodium hydroxide, diluted 1:1 in absolute ethanol, to remove the resin. After three brief rinses in absolute ethanol, the sections were hydrated with graded ethanol. After a rinse in tap water for 10 min, the tissue sections were incubated for 30 min with 1.4% H 2 O 2 in methanol, rinsed in tap water for 10 min, and incubated with 0.5% Triton X-100 in PBS for 15 min. The sections were rinsed in PBS three times for 10 min and incubated with 10% normal donkey serum for 1 h. Tissue sections were incubated with H -ATPase (1:400), pendrin (1:100), AQP2 (1:200), or calbindin D 28K antibody (1:400) overnight at 4°C. After several washes in PBS, the sections were incubated for 2 h with a DAKO Envision kit (DAKO, Glostrup, Denmark). For detection of peroxidase, Vector SG (Vector Laboratories, Burlingame, CA) was used as the chromogen to produce a grayish blue color, which is easily distinguished from the brown staining produced by 3,3'-diaminobenzidine in the preembedding procedure used for detection of PKC isoenzymes. The sections were washed with water, dehydrated, and mounted in Canada balsam.$ u- }! @+ b% T c1 ?
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Table 1. Multiple labeling by immunohistochemistry
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Localization of PKC-, - I, and - Immunoreactivity! z4 i/ n6 N" r( H
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Figure 1 shows low-magnification views of PKC-, - I, and - staining in longitudinal sections of mouse kidneys. The PKC-, - I, and - isoenzymes were strongly expressed in some tubular profiles, and their distribution patterns differed ( Fig. 1, A-C ).
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9 j, `: h" ]$ G, O$ U6 ~Fig. 1. Light micrographs of 50-µm-thick Vibratome sections illustrating immunostaining for PKC- ( A ), PKC- I ( B ), and PKC- ( C ) in mouse kidneys. Arrow, open arrows, and arrowheads indicate collecting ducts in the cortex (CO), outer (OSOM) and inner stripes (ISOM) of the outer medulla, respectively. Scale bars = 100 µm.* C# t D2 S2 D. }. e. z
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Figure 2 shows high-magnification views of PKC-, - I, and - staining in renal tubules. In the CNT, PKC-, - I and - immunoreactivity was observed in the apical domain or diffusely in the cytoplasm of intercalated cells. There was no significant labeling detectable in the CNT cells ( Fig. 2, A - C ). In the cortical collecting duct (CCD), PKC- and - I immunoreactivity was also observed in the apical domain or diffusely in the cytoplasm of intercalated cells ( Fig. 2, D and E ). Unlike PKC- and - I, distinct basal or apical PKC- immunolabeling of intercalated cells was observed in the CCD ( Fig. 2 F ). In the principal cells of the CCD, there was no PKC- I or - immunoreactivity, whereas weak PKC- immunoreactivity was observed on the basolateral plasma membrane ( Fig. 2 D ). In the outer stripe of the outer medulla, strong PKC- immunoreactivity was mainly observed on the basolateral plasma membrane of principal cells of the collecting duct and S3 segment of the proximal straight tubule ( Fig. 2 G ). In contrast, there was weak PKC- and - I immunoreactivity in the intercalated cells in this region ( Fig. 2, G and H ). Unlike PKC- and - I, PKC - was strongly expressed in the apical domain of most intercalated cells and in the basal domain of a few intercalated cells ( Fig. 2 I ). In the inner stripe part of the outer medullary collecting duct (OMCDi) and initial part of inner medullary collecting duct (IMCDi), the intensity of immunostaining with antibodies against PKC- and - I in the intercalated cells was much weaker compared with the CNT and CCD ( Fig. 2, J, K, M, and N ). In contrast, immunostaining for PKC- was strong in the intercalated cells of these regions ( Fig. 2, L and O ). In addition, PKC- was expressed strongly on the basolateral plasma membrane in the principal cells ( Fig. 2, J and M ), whereas no significant staining could be detected for PKC- I and - in the principal cells of these regions ( Fig. 2, K, L, N, and O ).1 f- w* |) [: J5 T- H* b
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Fig. 2. Light micrographs of 50-µm-thick Vibratome sections illustrating immunostaining for PKC- ( A, D, G, J, and M ), PKC- I ( B, E, H, K, and N ), and PKC- ( C, F, I, L, and O ) in the connecting tubule (CNT), cortical (CCD), outer stripe of outer medullary (OMCDo), inner stripe of outer medullary (OMCDi), and initial part of inner medullary (IMCDi) collecting duct of mouse kidneys. Arrows indicate intercalated cells with faint immunolabeling for PKC-. Asterisks indicate S3 segment of proximal tubules with basolateral immunolabeling for PKC-. Scale bar = 20 µm.
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; \% X9 n) D! m: T, s" R' vCellular Localization of PKC-, - I, and - in Subtypes of Intercalated Cells Determined by Multiple-Labeling Immunohistochemistry" }9 s7 b: a8 o. x+ p9 ?8 m
/ L4 K* i9 b$ k* \ t* p1 j8 NSubtypes of intercalated cells were easily identified by their pattern of immunostaining for H -ATPase and pendrin using multiple-labeling procedures on consecutive sections. Intercalated cells exhibiting apical staining for H -ATPase and no staining for pendrin were identified as type A cells, and they constituted a major portion of the intercalated cells in the CNT, CCD, OMCD, and IMCDi. The pendrin-positive intercalated cells could be subdivided into two distinct cell populations, type B and non-A-non-B cells, based on the pattern of H -ATPase immunostaining. The type B cells, characterized by basolateral and/or diffuse H -ATPase, were frequently observed in the CCD but were much less common in the CNT. Pendrin-positive intercalated cells exhibiting apical staining for H -ATPase were considered to be non-A-non-B cells. These non-A-non-B cells, which were the main form of intercalated cells in the CNT, were large cells that often protruded into the tubule lumen. Calbindin D 28K and AQP2 were used as a marker to identify the CNT and collecting duct, respectively. Calbindin D 28K immunostaining was observed in the cytoplasm of the CNT cells. AQP2 immunolabeling was present on the apical plasma membrane of the principal cells of the collecting duct.) R! h2 p, ~6 p0 \1 c
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PKC-. In the CNT, most cells with strong PKC- labeling exhibited apical H -ATPase and pendrin immunoreactivity, which is characteristic of non-A-non-B cells ( Fig. 3, A - C ). In the CCD, in contrast, most cells with strong PKC- labeling exhibited basolateral H -ATPase and apical pendrin immunoreactivity, which is characteristic of type B cells ( Fig. 3, D - F ). Intercalated cells with weak PKC- immunoreactivity in both the CNT and CCD were identified as type A cells with apical H -ATPase labeling and no pendrin immunoreactivity ( Fig. 3, D - F ). PKC- was also expressed on the basolateral plasma membrane of some AQP2-positive principal cells in the CCD ( Fig. 3, D - F ). However, there was no PKC- immunoreactivity in calbindin D 28K -positive CNT cells ( Fig. 3, A - C ). In the outer stripe part of the OMCD ( Fig. 4, A - C ), weak PKC- immunoreactivity was observed diffusely in both type A and type B intercalated cells, whereas strong PKC- immunoreactivity was observed on the basolateral plasma membrane of AQP2-positive principal cells and S3 segment of proximal tubules. In the inner stripe part of the OMCD ( Fig. 4, D - F ) and the IMCD ( Fig. 4, G - I ), all apical H -ATPase-positive type A cells exhibited very weak PKC- immunoreactivity, whereas there was strong PKC- immunoreactivity on the basolateral plasma membrane in AQP2-positive principal cells and IMCD cells." f+ B/ I; N$ P. U' _5 T5 e
' V7 r8 f1 `5 H$ g2 ~9 n8 aFig. 3. Light micrographs of 1.5-µm-thick consecutive sections of the CNT ( A - C ) and CCD ( D - F ) in mouse kidneys illustrating double immunostaining for PKC- (brown) and H -ATPase (blue; A and D ), PKC- (brown) and pendrin (blue; B and E ), PKC- (brown) and calbindin D 28K (blue; C ), or PKC- (brown) and AQP2 (blue; F ). PKC- is expressed in the cytoplasm of apical H -ATPase/apical pendrin-positive non-A-non-B intercalated cells (arrows) and of basolateral H -ATPase/apical pendrin-positive type B intercalated cells (double arrows), and in the apical domain of apical H -ATPase-positive/pendrin-negative type A intercalated cells (open arrows). There is no PKC- immunoreactivity in the calbindin D 28K -positive CNT cells, whereas AQP2-positive principal cells (arrowheads) have distinct basolateral PKC- immunoreactivity. Scale bar = 10 µm.) d. r$ |# T5 H7 d
6 j" m" l; D" H4 W# i5 RFig. 4. Light micrographs of 1.5-µm-thick consecutive sections of the OMCDo ( A - C ), OMCDi ( D - F ), and IMCDi ( G - I ) in mouse kidneys illustrating double immunostaining for PKC- (brown) and H -ATPase (blue; A, D, and G ), PKC- (brown) and pendrin (blue; B, E, and H ), and PKC- (brown) and AQP2 (blue; C, F, and I ). Immunoreactivity for PKC- in type A (open arrows) and type B intercalated cells (double arrows) is strong in the outer medullary collecting duct, whereas very weak PKC- immunoreactivity is seen in the type A intercalated cells (open arrows) of OMCDi and IMCDi. Note basolateral PKC- immunolabeling in AQP2-positive principal cells and S3 segment (asterisks) of proximal tubules. Scale bar = 10 µm.& e& E! X, e! D2 y3 r8 S' k
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PKC- I.. Non-A-non-B cells in the CNT exhibited moderate density of PKC- I immunoreactivity ( Fig. 5, A - C ). In contrast, type B cells in the CCD exhibited intense PKC- I immunoreactivity throughout the cytoplasm ( Fig. 5, D - F ). In type A cells of CNT and throughout the collecting duct, however, very faint PKC- I immunoreactivity was observed in the cytoplasm ( Fig. 5, D - I ). There was no PKC- I immunoreactivity in either calbindin D 28K -positive CNT cells ( Fig. 5, A - C ) or AQP2-positive principal cells of the collecting duct ( Fig. 5, D - I ).
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. d- \ W7 }3 zFig. 5. Light micrographs of 1.5-µm-thick consecutive sections of the CNT ( A - C ), CCD ( D - F ), and OMCDi ( G - I ) in mouse kidneys illustrating double immunostaining for PKC- I (brown) and H -ATPase (blue; A, D, and G ), PKC- I (brown) and pendrin (blue; B, E, and H ), PKC- I (brown) and calbindin D 28K (blue; C ), and PKC- I (brown) and AQP2 (blue; F and I ). Cells with strong PKC- I immunoreactivity in the cytoplasm are non-A-non-B cells (arrows) with apical H -ATPase/apical pendrin in the CNT and the type B cells with basolateral and/or diffuse H -ATPase/apical pendrin (double arrows). Cells with faint PKC- I immunoreactivity are type A cells with apical H -ATPase-positive/pendrin-negative (open arrows). No PKC- I immunoreactivity is seen in calbindin D 28K -positive CNT cells and AQP2-positive principal cells. Scale bar = 10 µm., \ l/ b; W \& t: Z f4 i
0 L; }2 _5 w; f0 j! P4 [5 u6 FPKC-. In the CNT and the collecting duct, immunoreactivity for PKC- was observed in the apical region of non-A-non-B ( Fig. 6, A - C ) and type A intercalated cells ( Fig. 6, D - I ). In contrast, PKC- was expressed on the basolateral plasma membrane of type B intercalated cells ( Fig. 6, D - F ). No PKC- immunoreactivity was observed in the calbindin D 28K -positive CNT cells ( Fig. 6, A - C ) or AQP2-positive principal cells ( Fig. 6, D - I ).
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Fig. 6. Light micrographs of 1.5-µm-thick consecutive sections of CNT ( A - C ), CCD ( D - F ), and OMCDi ( G - I ) in mouse kidneys illustrating double immunostaining for PKC- (brown) and H -ATPase (blue; A, D, and G), PKC- (brown) and pendrin (blue; B, E, and H ), and PKC- I (brown) and AQP2 (blue; I ), or single immunostaining for PKC- (brown; C and F ). Cells with strong PKC- immunoreactivity in the apical domain are non-A-non-B intercalated cells with apical H -ATPase/pendrin immunoreactivity (arrows) and type A cells with pendrin-negative/apical H -ATPase immunoreactivity (open arrows). Type B cells with apical pendrin/basolateral and/or diffuse H -ATPase, in contrast, exhibit distinct immunoreactivity for PKC- on the basolateral plasma membrane (double arrows). Insets in C and F show double immunostaining for PKC- and calbindin D 28K, and PKC- and AQP2, respectively. Note that there is no PKC- immunoreactivity in both calbindin D 28K -positive CNT cells and AQP2-positive principal cells of the collecting duct. Scale bar = 10 µm.( ]+ f% |* G! v- d
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The expression of PKC isoenzyme in subtypes of intercalated cells is summarized in Table 2.# A) b* p2 k8 }6 J8 X8 N, u
8 `5 A* w; {3 H$ R, U" xTable 2. Immunoreactivity of PKC isoforms in the intercalated cells of mouse kidneys' ]9 a% H) g* N9 R8 t
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I9 C d5 E1 l5 C% H% G9 IThe results of this study demonstrate that PKC-, - I, and - are expressed in the intercalated cells of the mouse kidney, which is consistent with previous observations ( 28 ). In addition, our results revealed a differential expression pattern of the three PKC-, - I, and - in the three subtypes of intercalated cells. PKC- and - I were highly expressed in the cytoplasm in both pendrin-positive type B and non-A-non-B intercalated cells but weakly expressed in the apical part of type A intercalated cells. In contrast, PKC- was highly expressed in the apical part of both type A and non-A-non-B intercalated cells but was restricted to the basal domain in type B intercalated cells. There was no immunoreactivity for PKC-, - I, and - in the calbindin D 28K -positive CNT cells. Within AQP2-positive principal cells of the collecting duct, PKC- was expressed on the basolateral plasma membrane, but no significant staining could be detected for PKC- I and -.
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Intercalated cells are subclassified into type A, type B, and non-A-non-B cells based on the presence or absence of the AE1 and pendrin, and the subcellular distribution of the H -ATPase ( 18, 33 ). The subcellular localization of the H -ATPase within an intercalated cell subtype correlates with whether the cell secretes or absorbs net H equivalents ( 7, 12 ).
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Acid-secreting type A intercalated cells express vacuolar H -ATPase on the apical side and extrude protons into the lumen, which functions in series with the basolateral band 3/AE1 Cl - /HCO 3 - exchanger ( 1, 13, 33, 36 ). HCO 3 - -secreting type B intercalated cells have an apical Cl - /HCO 3 - exchanger, pendrin, which functions in series with a basolateral H -ATPase ( 1, 12, 18, 36 ). Non-A-non-B intercalated cells appear to express both H -ATPase and pendrin on the apical side ( 18, 33, 34 ). Thus non-A-non-B intercalated cells would be capable of either HCO 3 - or acid secretion. Even through great progress has been made in the past decade in elucidating molecular mechanisms underlying acid-base transport in the intercalated cells, and the important role of H -ATPase, the regulation mechanisms of polarized expression and trafficking of H -ATPase are not fully understood. Winter et al. ( 38 ) recently suggested that PKC-dependent phosphorylation may be an important element of regulation of vacuolar H -ATPase in isolated OMCD. Welbourne et al. ( 37 ) also showed that troglitazone-induced acid-base responses appear to be mediated by a pathway involving PKC/ERK in LLC-PK 1 -F cell lines. The distribution patterns of PKC-, unlike PKC- and - I, are similar to those of H -ATPase in the three subtypes of intercalated cells. Both PKC- and H -ATPase were localized in the apical domain of both type A and non-A-non-B intercalated cells and in the basal part of type B intercalated cells. These findings indicate that in the modulation of polarized expression of H -ATPase in the intercalated cells of the mouse kidney may be mediated by PKC-.
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Besides PKC-, PKC- and - I were also intensely expressed in pendrin-positive type B and non-A-non-B intercalated cells but faintly in the type A intercalated cells. Orsenigo et al. ( 25 ) and Alvarez et al. ( 2 ) demonstrated that overall activation of PKC causes a reduction of transepithelial HCO 3 - transport rate. Taken together, our data support that PKC- and - I may play an important role in regulation of pendrin in mouse intercalated cells.' a8 h. |5 E7 n! m7 L" w8 L
% w% T4 w+ Q# `4 ^4 l; e8 U! XPKC has been shown to mediate the inhibitory influence of various hormones on water channels and sodium transport as well as inhibition of the secretory K channel in the CCD ( 3, 9, 16, 35 ). Studies in PKC- knockout mice ( 40 ) have provided evidence that PKC- contributes to urinary concentration. In the present study, PKC-, but not PKC- I and -, was detected on the basolateral membrane of AQP2-positive principal cells, and the intensity of immunoreactivity increased from the cortex to the medulla. These results are consistent with previous observations in rat kidney ( 27 ) and point to a potential role of PKC- in water and sodium transport in the collecting duct. However, Redling et al. ( 28 ) did not observe immunoreactivity for PKC- in AQP2-positive principal cells. This discrepancy may be due to the different sensitivity of the immunostaining methods.7 H+ l( k" x+ V0 N1 P/ ^, m
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In summary, our study demonstrates that PKC-, - I, and - are expressed in subtypes of intercalated cells of the mouse kidney. Within type A intercalated cells, PKC- was strongly expressed in the apical part of the cells, whereas immunoreactivity for both PKC- and - I was weak. Type B intercalated cells exhibited strong expression of PKC-, - I, and -, and PKC- and - I were localized throughout the cytoplasm, whereas PKC- was restricted to the basal domain. Within non-A-non-B cells, immunoreactivity for both PKC- and PKC- I was high in intensity and localized diffusely in the cytoplasm, whereas PKC- localized in the apical part of the cells. Thus a distinct and differential expression pattern of PKC-, - I, and - was observed in the three subtypes of intercalated cells of the mouse kidney, which may contribute to a better understanding of the distinct function of these isoenzymes within a given subcellular compartment of the intercalated cells. F7 X" u# z. b9 X9 H- R8 I6 C. G0 s
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This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the MRC for Cell Death Disease Research Center for MRC at the Catholic University of Korea (R13-2002-005-01001-0).: p5 d* c. E% m+ |. I- O' r$ s
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ACKNOWLEDGMENTS
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We are grateful for the technical assistance of Hong-Lim Kim.0 m! j1 p# F% G# T, [1 `, l6 ~
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Part of this work has been published in abstract form ( J Am Soc Nephrol 16: 341A, 2005).
9 U6 T4 ?3 k$ W( h 【参考文献】
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1 x3 L+ g, Y1 }8 \( JAlvarez BV, Vilas G, and Casey J. Metabolon disruption: a mechanism that regulates bicarbonate transport. EMBO J 24: 2499-2511, 2005.
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