Localization of the Epithelial Ca²⁺ Channel in Rabbit Kidney and Intestine

Isolation of Nephron Segments and Total RNA Extraction

Rabbit nephron segments were obtained by microdissection as described previously (6). Briefly, microdissection medium was prepared from Hanks' sterile solution (MEM 2126 ; Eurobio, Les Ulis, Cedex, France ; 137 mM NaCl, 5 mM KCl, 0.8 mM MgSO₄, 0.33 mM Na₂HPO4, 0.44 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂) supplemented with 1 mM lactate, 1 mM pyruvate, 1 mM glutamine, 1 mg/ml protease-free bovine serum albumin, and 20 mM Hepes, and pH was adjusted to 7.4. After anesthesia (Nembutal, 5 mg/100 g body wt), the left kidney was perfused with collagenase dissolved in microdissection medium (1.6 mg/ml). Thin pyramids, cut along the corticomedullary axis, were incubated for 25 min at 30°C in microdissection solution containing collagenase (1.2 mg/ml). The following structures were isolated according to morphologic and topographic criteria : glomeruli, proximal convoluted and straight tubules (PCT and PST), medullary and cortical thick ascending limbs of Henle's loop (mTAL, cTAL), distal convoluted tubules (DCT), cortical connecting tubules (CNT), and cortical collecting ducts (CCD). After elimination of cells and debris, pools of 10 to 50 tubules were transferred with 5 ml of dissection medium and processed for RNA extraction. Total RNA was extracted from isolated nephron segments and total kidney cortex according to Chomczynski and Sacchi and downscaled for small amounts (6, 7).

Reverse Transcription-PCR of Rabbit ECaC RNA

ECaC sense primer (5'-TGAACCTGGTGCGCGCACTGC-3') and ECaC antisense primer (5'-CCCAGGGAGTCCTGGGCCCGG-3') used in reverse transcription-PCR experiments were designed according to the rabbit ECaC cDNA sequence (GenBank accession no. AJ133128) and cover nucleotide positions 488 to 507 and 665 to 685 of the cDNA, respectively (1). The β-actin mRNA level was determined as a control to demonstrate the integrity of the isolated RNA from the nephron structures by using primers corresponding to the coding region of the human β-actin cDNA. The β-actin sense and antisense primers were : 5'-GCTACGAGCTGCCTGACGG-3' and 5'-GAGGCCAGGATGGAGCC-3', respectively, and bracket the sequence from 757 to 1084 bp of the human β-actin (8). Briefly, in a final volume of 34 μ1 the following compounds were added and maintained on ice : total RNA (approximately 20 ng) of isolated nephron structures and antisense primer (10 pmol). For denaturation, the tubes were heated at 70°C for 5 min and then equilibrated at 37°C. Each sample was separated into two incubations of 17 μ1 and supplemented with 12 μ1 of reverse transcriptase mix containing 6 μ1 of 5× first-strand buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl₂), 2 μ1 of 10 mM dNTP, 3 μ1 of 0.1 M dithiothreitol, and 1 μ1 of RNasin (40 U). The samples were incubated for 15 min at 37°C, and subsequently one reaction was supplemented with 200 U of Moloney murine leukemia virus-reverse transcriptase (Life Technologies, Breda, The Netherlands), whereas another reaction was used as a control for DNA contamination. The reverse transcription (RT) reaction was performed at 37°C for 1 h, followed by an increase to 90°C for 2 min. The amplification reaction was initiated by adding to each sample 50 μ1 of PCR mix containing 2 mM MgCl₂, 10 pmol of the sense primer, 0.01 mg/ml gelatin, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1.25 U Taq polymerase. The PCR was based on 40 cycles of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C, with an elongation time of 10 min being used after the last cycle. After the RT-PCR amplification step, an aliquot of 20 μ1 from each sample was run on a 2% agarose gel containing 2.5 μg/ml ethidium bromide, and bands were visualized using ultraviolet illumination.

Generation and Characterization of Antisera

Antiserum against ECaC was obtained by immunization of guinea pigs with ECaC C-tail protein and affinity-purified as described previously (1). To demonstrate the specificity of the ECaC antibody, immunoblots using a membrane fraction of ECaC-expressing oocytes and rabbit kidney cortex, and isolated rabbit duodenal brush-border membranes were performed. Unfortunately, we were unable to demonstrate a specific positive band of the appropriate molecular mass. Subsequently, we have generated two series of antibodies against a 15 AA synthetic peptide representing the C-tail of ECaC and against a fusion protein of the extracellular loop between transmembrane segment 1 and 2, respectively. Immunohistochemistry performed with these new antibodies demonstrated identical localization of ECaC in rabbit kidney cortex and duodenum as obtained in the present study. As with our first antibody, these new antibodies did not produce a specific signal on immunoblots, suggesting that the expression level of ECaC might be too low to detect the protein by immunoblots. Thus, three antibodies directed against different parts of the ECaC protein independently illustrated identical localization of ECaC. This convincingly demonstrates the specificity of the observed immunohistochemistry. Furthermore, Xenopus laevis oocytes were injected with 2 ng of ECaC cRNA. After 2 d, oocytes were fixed and stained for ECaC. Immunopositive staining was exclusively present in the plasma membrane of injected oocytes, whereas noninjected oocytes were devoid of any positive staining as shown previously (3). As an additional control, COS cells were transfected with rabbit ECaC cDNA and after 1 d cells were fixed and stained for ECaC. ECaC protein was observed in the plasma membrane of transfected COS cells, whereas nontransfected cells were negative (data not shown). Rabbit antisera against rabbit calbindin-D_28K and bovine calbindin-D_9K were characterized previously (1, 9). Mouse anti-sarcolemmal Na⁺-Ca²⁺-exchanger monoclonal antibody (mAb) (C2C12) was a generous gift from Dr. K. D. Philipson (Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, University of California, Los Angeles, School of Medicine, Los Angeles, CA), mouse anti-plasma membrane Ca²⁺-ATPase mAb (5F10) was kindly provided by Dr. J. T. Penniston (Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN), and the mouse monoclonal antibody against the thiazide-sensitive NaCl cotransporter (TSC) (JM5) was a gift from Dr. D. H. Ellison (Department of Medicine, University of Colorado School of Medicine, Denver, CO). The characterization of these mAb has been described elsewhere (10,11,12,13).

Immunohistochemistry

Kidneys and intestine were obtained from New Zealand White rabbits (approximately 0.5 kg). The organs were cut into pieces, placed in 1% (wt/vol) periodate-lysine-paraformaldehyde fixative (9) for 2 h, and overnight incubated in phosphate-buffered saline (PBS) containing 15% (wt/vol) sucrose. Subsequently, samples were frozen in liquid nitrogen and 7-μm sections were used for different staining procedures. Duodenum sections were boiled for 2 min, cooled to room temperature in citrate buffer (pH 6) containing 0.01 M C₆H₅Na₃O₇ and 0.01 M C₆H₈O₇, and thoroughly rinsed with PBS.

For immunoperoxidase staining, kidney and intestine sections were incubated for 30 min at room temperature in PBS containing 0.3% (vol/vol) H₂O₂ and washed three times with TN buffer (0.15 M NaCl, 0.1 M Tris/HCl, pH 7.5). Subsequently, sections were incubated for 30 min in TNB buffer (TN buffer containing 0.5% [wt/vol] blocking reagent from NEN Life Science Products, Zaventem, Belgium). Sections were incubated for 16 h at 4°C in TNB buffer containing the guinea pig anti-ECaC antiserum (1 : 50). Sections were washed three times with TNT buffer (TN buffer containing 0.05% [vol/vol] Tween 20) and incubated for 30 min at room temperature with peroxidase-conjugated goat anti-guinea pig antibody (1 : 50) (Sigma, St. Louis, MO). The sections were counterstained with hematoxylin Gill 2 and embedded in glycerol/gelatin as described previously (9).

For double immunofluorescence staining, periodate-lysine-paraformaldehyde-fixed frozen sections were preincubated in TNB buffer for 30 min and incubated for 16 h at 4°C with antiserum against ECaC (1 : 200) and with one of the antibodies against the Ca²⁺ transport proteins (calbindin-D_28K 1 : 200, calbindin-D_9K 1 : 100, NCX 1 : 100, or PMCA 1 : 400). Sections double-stained for ECaC and TSC were preincubated in TNB buffer for 30 min and incubated for 16 h at 4°C with antiserum against ECaC (1 : 200), subsequently washed with TNT buffer, and incubated for 1 h at room temperature with antiserum against TSC (1 : 3200).

After thorough washing with TNT buffer, tissue sections stained for ECaC together with NCX, PMCA, or TSC were incubated at room temperature for 1 h with goat anti-guinea pig-Alexa 594-conjugated anti-IgG (1 : 300) (Molecular Probes, Eugene, OR) for the detection of ECaC and with goat anti-mouse-Alexa 488-conjugated anti-IgG (1 : 300) (Molecular Probes) for the detection of the other transporters. Double staining of ECaC and calbindins was performed by incubation of the tissue sections for 1 h at room temperature with goat anti-guinea pig-FITC-conjugated anti-IgG (1 : 300) (Sigma) for the detection of ECaC and with goat anti-rabbit tetramethylrhodamine isothiocyanate-conjugated anti-IgG (1 : 300) (Sigma) for the detection of calbindin-D_28k and calbindin-D_9k, respectively. Kidney sections were incubated for 16 h at 4°C with FITC-conjugated peanut lectin (1 : 200) to visualize intercalated cells.

All sections were rinsed twice with TNB buffer and mounted in mowiol (Hoechst, Frankfurt, Germany) containing 2.5% (wt/vol) NaN₃ as described previously (1). All negative controls, including sections incubated with either preimmune serum, preabsorbed antiserum for 1 h with 10 μg/ml ECaC-glutathione S-transferase fusion protein, or solely with conjugated secondary antibodies, were devoid of any staining. Photographs were taken with a Zeiss Axioskop microscope equipped for epifluorescence illumination using Kodak EPH P1600X film or with a Bio-Rad MRC 1000 confocal laser scanning microscope.

Segmental Distribution of ECaC mRNA

RT-PCR on total RNA isolated from rabbit kidney cortex yielded a single amplified fragment of expected size of 197 bp (Figure 1). This band was absent when the RT step was omitted, which excludes amplification of contaminating genomic DNA (not shown). Sequence analysis confirmed that the amplified fragment encoded the part of the amino-terminal domain of rabbit ECaC between nucleotide positions 488 and 685. Subsequently, duplicate RT-PCR assays were performed on each of two batches of segmental RNA isolated from two rabbits. The acquired results demonstrate that the ECaC transcript is exclusively present in distal tubular segments. The expression was highest in CNT and decreased in the flanking DCT and CCD. No signal was observed in glomeruli, proximal tubules, thick ascending limb of Henle, or the outer medullary collecting duct. The integrity of isolated RNA was demonstrated by the presence of β-actin-specific fragments of the correct size (Figure 1). PCR water controls were consistently negative.

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Figure 1.

Reverse transcription-PCR analysis of epithelial Ca²⁺ channel (ECaC) mRNA expression in renal tubules. Total RNA was isolated from the indicated nephron structures microdissected from rabbit kidney, including glomeruli (GM), proximal convoluted and straight tubules (PCT and PST), medullary and cortical thick ascending limbs of Henle's loop (mTAL and cTAL), distal convoluted tubules (DCT), connecting tubules (CNT), and cortical and outer medullary collecting ducts (CCD and OMCD). Rabbit kidney cortex total RNA was used as a positive control. The size of the expected amplified rabbit ECaC fragment (197 bp) is indicated. β-actin mRNA was used as a control to demonstrate the intactness of isolated RNA. As a negative control, water was added instead of cDNA.

Immunohistochemical Localization of ECaC

The localization of ECaC was further investigated using an affinity-purified antibody raised against the carboxy terminus of ECaC as described previously (1, 3). The distribution of ECaC-specific immunoperoxidase staining in rabbit kidney cortex and duodenum is shown in Figure 2, A through F. In kidney, ECaC-positive staining was found in the superficial cortex, whereas outer and inner medulla were negative. Importantly, the exclusive cortical staining for ECaC was predominantly found along the apical membrane of the distal tubular segments (Figure 2, A through C). Staining was not observed in glomeruli, proximal tubular segments, or the loop of Henle. In intestine, ECaC was abundantly present in brush-border membranes of duodenum (Figure 2, D and E). Absorptive epithelial cells in the villi stained intensely for ECaC (Figure 2E), whereas crypt cells were negative (Figure 2F). Immunopositive staining for ECaC was not observed in ileum.

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Figure 2.

Immunoperoxidase staining for ECaC of periodate-lysine-paraformaldehyde-fixed sections of rabbit kidney cortex and duodenum. In kidney, apical localization of ECaC was observed in distal tubular segments (A through C). Sections of outer and inner medulla were negative (not shown). In duodenum, intense immunoperoxidase staining was observed in absorptive cells lining the villus tip (D and E), whereas crypt cells showed little or no staining (F). Ileum was negative (not shown). Bars : 25 μm in A through C ; 100 μm in D ; and 50 μm in E and F.

To determine more precisely the distribution of the ECaC protein along the distal nephron, several criteria were used. First, tubules were identified by their morphologic and anatomical features (14). Second, colocalization studies were performed with antisera against TSC, as a marker for DCT (13), and the Ca²⁺ transport proteins. Third, CNT and CCD were recognized by the appearance of intermingled intercalated cells. Discrimination between these segments was based on a different position in the cortex and superficial T branches, indicating transitions between these latter segments (15). Figure 3 shows double labeling of rabbit kidney cortex section for the presence of ECaC (Panel A) and TSC (Panel B) depicting immunopositive distal tubular segments. Some distal segments stained intensely for TSC, but were negative for ECaC. However, other tubules stained lightly for TSC and were also positive for ECaC. Because the former TSC-positive segments were previously identified as DCT and the latter as CNT (13), these findings suggest that ECaC is present in CNT, but not in DCT. The staining of ECaC did extend into CCD, as superficial T-branched tubules positive for ECaC were identified (Figure 3C). Immunopositive staining was not present in that part of the CCD located in medullary rays. A minority of the cells within the ECaC-positive distal tubules did not express ECaC, indicating that they are likely intercalated cells. Indeed, cells lacking ECaC were identified as intercalated cells using double fluorescence staining with peanut lectin (Figure 3D). The kidney section incubated with preabsorbed antiserum for 1 h with 10 μg/ml ECaC-glutathione S-transferase fusion protein was devoid of any staining (Figure 3E). Thus, ECaC is predominantly expressed in CNT, which is in agreement with the fact that this nephron segment is in rabbit the primary segment of hormone-regulated Ca²⁺ reabsorption (2, 5).

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Figure 3.

Distribution of ECaC along the distal nephron. Double immunofluorescence staining of rabbit kidney cortex sections showing staining for ECaC (A) and the thiazide-sensitive NaCl cotransporter (TSC) (B). The asterisks indicate DCT cells intensely stained for TSC but negative for ECaC, while the arrowheads mark CNT cells lightly positive for TSC and strongly stained for ECaC. G, glomerulus. (C) A superficial T-branched tubule positive for ECaC. (D) A superimposed picture of a CNT with immunopositive staining for ECaC (green) and peanut lectin (red). (E) The absence of immunopositive staining with ECaC antiserum preabsorbed for 1 h with 10 μg/ml ECaC-glutathione S-transferase fusion protein. G, glomerulus. Bars : 30 μm in A and B ; 15 μm in C and E ; and 8 μm in D.

ECaC Colocalizes with the Ca²⁺ Transport Proteins in Kidney and Intestine

Double immunofluorescence labeling experiments were performed to investigate the localization of ECaC with respect to the other Ca²⁺ transport proteins involved in transcellular Ca²⁺ transport. The antibodies used were raised against the canine sarcolemmal NCX, human erythrocyte PMCA, rabbit kidney calbindin-D_28k, and bovine intestinal calbindin-D_9k. Double staining of kidney sections revealed consistent colocalization of ECaC with calbindin-D_28k, NCX, and PMCA in the distal nephron (Figure 4). PMCA was also found in TAL (not shown). Immunopositive staining for NCX was restricted to the basolateral membrane of distal tubular cells, whereas staining for both calbindin-D_28k and PMCA was observed throughout the whole cell. In line with the kidney, a complete colocalization between ECaC, calbindin-D_9k, and PMCA was observed in duodenum (Figure 5). PMCA was present along the lateral membrane, whereas calbindin-D_9k was found throughout the whole cell. NCX could not be detected in duodenum.

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Figure 4.

Double immunofluorescence staining of rabbit kidney cortex sections showing staining for ECaC in distal tubular segments (A, C, and E) and calbindin-D_28K (B), Na⁺-Ca²⁺ exchanger (D), and PMCA (F). Bar, 30 μm.

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Figure 5.

Immunofluorescence staining of serial sections of rabbit duodenum showing staining for ECaC (A), calbindin-D_9K (B), and PMCA (C). Bar, 40 μm.