Disruption of Cadherin/Catenin Expression, Localization, and Interactions During HgCl2-Induced Nephrotoxicity

Jing Jiang*, Dana Dean{dagger}, Robert C. Burghardt{dagger} and Alan R. Parrish*,1

* Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center; and {dagger} Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University

Received March 2, 2004; accepted April 1, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cadherin/catenin complex is an essential regulator of intercellular adhesion and is critical for the establishment of epithelial cell polarity. The purpose of this study was to (1) determine the spatial pattern of cadherin and catenin expression, colocalization, and interaction along the mouse nephron, and (2) investigate the expression, localization, and interaction of proximal tubular cadherins and catenins during mercuric chloride-induced nephrotoxicity. Using a combination of Western blot analysis, colocalization studies, and coimmunoprecipitation, we conclude that two distinct cadherin/catenin complexes exist in adult mouse kidney proximal tubules: N-cadherin/ß-catenin/{alpha}-catenin and E-cadherin/ß-catenin/{alpha}-catenin/p120-catenin. In the distal tubule, E-cadherin/ß-catenin/{alpha}-catenin and E-cadherin/{gamma}-catenin/{alpha}-catenin complexes are present. Male C3H mice were challenged with 0–25 µmol/kg mercuric chloride ip (6–48 h) to assess the impact of nephrotoxicity on cadherin/catenin complexes. Plasma creatinine and blood urea nitrogen were increased between 6 and 48 h, indicating the onset of renal failure. In addition, histological evaluation demonstrated alterations in the proximal tubules. At 24 h, we observed decreases in Ksp- and N-cadherin, but not in E-cadherin. Additionally, {alpha}-catenin expression was decreased, in the absence of changes in ß-, {gamma}-, and p120-catenin. The early stages (6 h) of mercuric chloride-induced nephrotoxicity were associated with disruption of complex integrity. N-cadherin and {alpha}-catenin localizations were disrupted at 6 h. These changes in cadherin and catenin localization corresponded with a decrease in the coimmunoprecipitation of {alpha}-catenin with both ß-catenin and N-cadherin. Interestingly, these changes occurred at the same time that aberrant staining of Na+/K+-ATPase staining was seen. Taken together, these data suggest that alterations in cadherin and catenin expression, localization, and interaction are associated with nephrotoxicity.

Key Words: cadherin; catenin; mercury; nephrotoxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tubular epithelial cells, despite several unique segment-specific functions, share a common need for establishing and maintaining cell polarity (reviewed in Wagner and Molitoris, 1999Go). Unique apical and basolateral domains separate the glomerular filtrate from the blood and allow for the movement of ions, water, and macromolecules to and from each compartment. The cadherin/catenin complex is the predominant regulator of intercellular adhesion and subsequent establishment of cell polarity (reviewed in Takeichi, 1991Go), although tight junctions and desmosomes are also important regulators of intercellular adhesion. While disruption of intercellular adhesion has been implicated in several human nephropathies, including acute renal failure (reviewed in Molitoris and Marrs, 1999Go), a direct link between the disruption of cadherin/catenin complexes and functional renal deficits has not been established. However, a back leak of the glomerular filtrate into the interstitium in animal models of renal injury (Donohoe et al., 1978Go) and in human patients with renal failure (Moran and Myers, 1985Go; Myers et al., 1979Go) is compelling evidence for decreased intercellular adhesion as a critical component of renal injury.

The cadherin gene superfamily encodes for transmembrane proteins that regulate Ca++-dependent, homotypic intercellular adhesion. Type-I (classical) cadherins comprise a large family of highly homologous proteins, which includes E-(epithelial [L-CAM, uvomorulin]) N-(neuronal [A-CAM]), and P-(placental) cadherin (Kemler, 1992Go). The extracellular domain contains four highly conserved Ca++-binding "cadherin repeats" (EC1–4) and one membrane-proximal extracellular domain (EC5). Type-II (atypical) cadherins have a similar structure, but lack the conserved His-Ala-Val (HAV) sequence in the EC1 domain. Catenins, intracellular proteins that bind cadherins, are required for a functional intercellular adhesion complex. {alpha}-Catenin is linked to the cytoplasmic domain of cadherins via ß- or {gamma}-catenin; it does not directly bind to cadherin. Due to homology with vinculin, it is suggested that {alpha}-catenin links the cadherin/catenin complex to the cytoskeleton (Nagafuchi et al., 1991Go). p120-Catenin binds to the cadherin cytoplasmic domain and shares sequence homology with ß- and {gamma}-catenin but does not bind {alpha}-catenin (Reynolds et al., 1994Go).

The renal expression of cadherin molecules is complex, with reports of at least nine cadherins present in the kidney (E-, K-, Ksp-, N-, P-, R-, and T-cadherin; µ- and LKC protocadherins) (Cho et al., 1998Go; Dahl et al., 2002Go; Goldberg et al., 2002Go; Nouwen et al., 1993Go; Okazaki et al., 2002Go; Piepenhagen et al., 1995Go; Thomson et al., 1995Go). In addition to the developmental regulation of cadherin expression, evidence is accumulating that cadherin/catenin proteins are differentially expressed along the nephron. In the developing human kidney, N-cadherin is expressed in the proximal tubule and thin limb and E-cadherin is expressed in the thin limb, distal tubule and collecting tubule (Nouwen et al, 1993Go). In the mouse embryonic kidney, the proximal tubule progenitors express K-cadherin (a type-II cadherin), while the distal tubule cells express E-cadherin and the glomeruli express P-cadherin. Ultimately, K-cadherin is downregulated during maturation whereas E-cadherin expression remains in most, if not all, of the tubular epithelium (Cho et al., 1998Go). K-cadherin exhibits low homology to N-(38%), E-(35%), and P-cadherin (32%). It is expressed at high levels in fetal human kidney, and at low levels in adult kidney. In adult mouse kidney, E-cadherin is detected everywhere but the initial segment where the proximal tubule joins Bowman's capsule (Piepenhagen et al., 1995Go). This pattern of expression contrasts with adult human kidney, where E-cadherin is not detected in the proximal tubule (Nouwen et al, 1993Go). {alpha}- and ß-Catenin are expressed in all nephron segments, while {gamma}-catenin is only detected in distal tubules (Piepenhagen and Nelson, 1995Go). Recently, a novel 130-kDa, kidney-specific member of the cadherin superfamily (Ksp-cadherin) was identified. Although Ksp-cadherin lacks the HAV adhesion recognition sequence and possesses a truncated cytoplasmic domain that does not interact with catenins, it is capable of mediating intercellular adhesion (Thomson et al., 1995Go).

A rationale for studying the cadherin/catenin complex in mercuric chloride-induced acute renal failure (ARF) is the finding that a decrease in glomerular-filtration rate, as assessed by 3H-inulin clearance, was seen at the late proximal tubule but not at the Bowman's space in mercuric chloride-induced nephrotoxicity (Conger and Falk, 1986Go). This observation leds the authors to conclude "at 24 h in low-dose HgCl2-induced acute renal failure, tubular fluid back-leak is the major pathogenic factor." This suggests that disruption of intercellular adhesion, as evidenced by glomerular filtrate back-leak, is an important component of mercuric chloride-induced ARF in animal models, as well as human patients with ARF (Moran and Myers, 1985Go; Myers et al., 1979Go). The present study was designed to examine the expression and integrity of renal cadherin/catenin complexes in normal mouse kidney and during mercuric chloride-induced nephrotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. BUN and creatinine kits were purchased from Sigma Chemical Company (St. Louis, MO). Mouse monoclonal antibodies against N- and E-cadherin, and {alpha}-, ß-, {gamma}-, and p120-catenin were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against {alpha}-, ß-, {gamma}-catenin, and K-cadherin were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). An anti-Ksp-cadherin monoclonal antibody was the gift of Dr. Peter S. Aronson (Yale) or purchased from Zymed (South San Francisco, CA). A monoclonal anti-Na+/K+ ATPase {alpha}-1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). ECL detection reagents and nitrocellulose membranes were obtained from Amersham (Arlington Heights, IL). An immunohistochemistry kit (HistoMouse-SP) was purchased from Zymed (San Francisco, CA).

Animals. All animal protocols were approved by the Texas A&M University ULAC Committee (AUP 2001–268). Male C3H mice (20–25 g; Charles River) were maintained in the College of Medicine animal-care facility; this mouse strain was used based on the sensitivity of these animals to mercuric chloride (Tanaka-Kagawa et al., 1998Go). The animal room was temperature-controlled and on a 12/12 h light/dark cycle. Mice were given food and water ad libitum. Mice were injected with mercuric chloride (0–25 µmol/kg dissolved in PBS, total injection volume 250 µl, ip). Following anesthesia (isoflurane), the abdominal cavity was opened, and the kidneys were removed and weighed. The kidney capsule was removed and the kidneys sectioned (4 sagittal sections) and processed for analysis, as described below.

Western blots. Kidney samples were homogenized in 500 µl of lysis buffer (10 mM Tris/HCl, pH 7.6, 1% SDS; 1 mM PMSF; 1 mM leupeptin; 1 mM orthovandate) and boiled for 10 min. The homogenates were spun at 14,000 rpm for 10 min, and the supernatant was collected. Proteins were quantified by the Bradford method and diluted to 1 µg/µl in 2X sample buffer (250 mM Tris/HCl, pH 6.8, 4% SDS, 10% glycerol, 2% ß-mercaptoethanol, 0.006% bromphenol blue). Samples were boiled for 5 min prior to electrophoresis and protein was separated by 6% SDS–PAGE. Separated proteins were transferred onto a Hybond-ECL nitrocellulose membrane (Amersham) in transfer buffer containing 25 mM Tris, 200 mM glycine, 20% methanol, and 1% SDS. Nonspecific binding was blocked by incubation with TBST blocking buffer (0.1% Tween-20, 10 mM Tris, pH 7.5, 100 mM NaCl) supplemented with 5% nonfat dry milk for 1 h at room temperature. Primary antibodies (Table 1) were diluted in the same buffer and incubated at 4°C overnight. After subsequent washes with TBST, membranes were incubated with secondary antibody (1:20,000 TBST:5% nonfat dry milk) against the appropriate species for 1 h at room temperature. The blots were washed 3X in TBST and proteins detected with the Amersham ECL system and exposure to X-ray film (Kodak, Rochester, NY) prior to scanning and analysis via the UN-SCAN-IT program (Silk Scientific, Orem, UT).


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TABLE 1 Antibodies

 
Immunohistochemistry. Kidneys were sliced with a razor blade into 4 sagittal sections and placed in 4% paraformaldehyde for 24 h. At this time the sections were rinsed repeatedly with PBS, and placed in 70% ethanol for embedding. Five-µm sections from the paraffin-embedded tissues were used for histological evaluation following hematoxylin/eosin staining. Immunohistochemical localization of cadherins and catenins was performed by peroxidase/DAB staining with a commercially available system (HistoMouse-SP, Zymed). Sections were deparaffinized by xylene incubation for 12 min and rehydrated in a graded series of ethanol (95%, 80%, 70%, 50% ethanol) for 5 min each, then washed with PBS for 10 min. Peroxidase quenching was performed by incubation for 12 min with 9:1 dilution of methanol: 30% H2O2 to block endogenous peroxidase activity. After washing with PBS three times, sections were blocked with solutions A and B (Histomouse Kit). The primary antibodies (Table 1) were applied in a humidified chamber, at a dilution of 1:100 in room temperature, for 1 h. After rinsing in TBS, the sections were incubated for 30 min at room temperature with biotinylated secondary antibody. The streptavidin-peroxidase enzyme conjugate was added to each section for 15 min and peroxidase activity was visualized with substrate-chromogen mixture. Slides were counterstained with hematoxylin and mounted for light microscope study with mounting solution. Negative controls were incubated with blocking solution B in place of the primary antibody.

Immunofluorescence. Kidney sections were immediately frozen in Tissue-Tek OCT compound (Miles Scientific) in a liquid nitrogen bath. All tissues were stored at –80°C until use. Samples were cut on a cryostat and 8 µm sections collected on slides (Permafrost Plus), fixed in –20°C methanol for 10 min, and then air dried. Sections were treated with 1:20 blocking solutions (serum related to species in which the secondary antibody was generated) at room temperature for 1 h. Primary antibodies were added at appropriate dilutions (Table 1) overnight at 4°C. After washing (0.3% Tween in 0.02 M PBS; PBST), FITC-conjugated secondary antibodies (1:200) were added and sections incubated in the dark at room temperature for 1 h. Sections were mounted with anti-fade media (Molecular Probes) following several washes. Immunostained sections were visualized with a Zeiss Axioplan 2 microscope (Carl Zeiss) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu Corporation, Bridgewater, NJ) and images captured with Adobe Photoshop 5.0 software (Adobe Systems, Seattle, WA). Negative controls included both substituting appropriate species IgG for primary antibodies and substituting appropriate species serum for secondary antibodies in the staining reactions. On the basis of previously published studies (Piepenhagen and Nelson, 1995Go), we identified proximal tubules as {gamma}-catenin-negative.

Coimmunofluorescence. Following a 1-hr room temperature incubation in serum (1:20) related to species in which the secondary antibody was generated, 100 µl of primary antibody (1:100) was placed on each slide at 4°C, overnight. The slides were washed with PBST prior to 1-hr room temperature incubation with a secondary antibody coupled to FITC (1:200). The slides were again washed with PBST, and then blocked (serum related to species in which both the first and second secondary antibodies were generated) at room temperature for 1 hr. One hundred µl of primary antibody (1:100) was placed on each slide at room temperature for 1 h. Following washing, 100 µl of secondary antibody coupled to Texas Red (1:200) was incubated with each section at room temperature for 1 h. Sections were mounted with anti-fade media (Molecular Probes) following several washes. Immunostained sections were visualized using a Zeiss Axioplan 2 microscope, as described previously. Adobe Photoshop software was used to capture and merge individual FITC and Texas Red images.

Dual staining: Serial sections. For cases in which two antibodies were derived from the same source (i.e., two monoclonal antibodies; E- and N-cadherin), serial sections were used. The first and second sections were incubated with single monoclonal antibodies, and the third section was incubated with the monoclonal antibody used on the first section, each for 1 h. After washing with PBST, FITC-conjugated sheep antimouse IgG secondary antibody was incubated with first and third sections, while Texas red goat anti-mouse IgG secondary antibody was incubated with the second section. Following a second block, the third section was also incubated with a second primary antibody, followed by a Texas red goat antimouse IgG secondary antibody. Sections were mounted with antifade media (Molecular Probes) following several washes. Immunostained sections were visualized and images captured as described previously.

Immunoprecipitation. Kidney tissues were homogenized in 500 µl of lysis buffer (2% NP-40 in PBS; 1 mM PMSF, 1 mM leupeptin; 1 mM orthovandate). The samples were centrifuged for 15 min at 12,000 rpm (4°C). The supernatant was collected and incubated on a rocker platform for 30 min at 4°C with 50 µl of GammaBindTM Plus SepharoseTM; at 4°C, on a rocker platform. The samples were spun for 10 min at 12,000 rpm (4°C) and protein concentration determined using the Bradford assay. Five hundred µg of protein was incubated with 5 µg of antibody (TABLE 1) at 4°C for 1.5 h. GammaBindTM Plus SepharoseTM (25 µl) was added and the samples incubated at 4°C overnight. The proteins were collected by centrifugation for 10 min at 12,000 rpm at 4°C. The pellet was washed three times with lysis buffer prior to Western-blot analysis.

Renal function. Blood urea nitrogen (BUN) was assessed in collected blood samples using a commercially available kit (Sigma). Ten µl serum was added to a tube and incubated in 37°C water bath for 10 min. At this time, 1 µl phenol nitroprusside solution, 1 ml alkaline hypochlorite solution, and 5 ml H2O were added to each tube. The reaction was allowed to develop at room temperature for 30 min prior to spectrophotometric analysis.

Creatinine was also assessed with commercially available kits (Sigma). Three hundred µl of sample were added to a cuvette. To all cuvettes, 3 ml of alkaline picrate solution was added and incubated at room temperature for 10 min. Absorbance was read at 500 nm. After measurement, 0.1 ml acid reagent was added and the sample was incubated for 5 min at room temperature. Absorbance was then read again at 500 nm; the difference in absorbance before and after acidification correlates with the amount of creatinine.

Statistics. Data are expressed as mean ± SEM. An analysis of variance (ANOVA) was performed and a Dunnett's test was used to assess significant differences from the control group (p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Localization of Cadherins and Catenins in Normal Mouse Kidney
Initial experiments were designed to examine the protein expression of cadherins and catenins in the adult male C3H mouse kidney. E-, Ksp-, N-cadherin and {alpha}-, ß-, {gamma}- and p120-catenin were expressed in total cell lysates harvested from the kidney; however, P-cadherin and cadherin-11 (OB-cadherin) were not detected by Western blot analysis (data not shown).

In the next series of experiments, the spatial expression of cadherins and catenins was examined by immunohistochemistry in paraformaldehyde-fixed, paraffin-embedded sections. N-cadherin and p120-catenin were detected only in proximal tubules, while Ksp-cadherin was detected in both proximal and distal tubules (data not shown). E- and K-cadherin and {alpha}-, ß, and {gamma}-catenin were not detected with this strategy. Although these data demonstrated a distinct pattern of cadherin and catenin expression along the nephron, the inability to detect all of the cadherins and catenins in fixed, paraffin-embedded tissue necessitated development of an alternative strategy.

A coimmunofluorescence strategy using frozen tissue sections was employed to optimize antigen preservation and simultaneously detect two proteins. The colocalization of E- and N-cadherin is shown in Figure 1. The staining of N-cadherin was confined to the proximal tubules (Fig. 1A), while E-cadherin was expressed in both proximal and distal tubules; however, the intensity of the staining was greater in the distal segments (Fig. 1B). When the two images are merged, the colocalization of N- and E-cadherin in proximal tubules is evident, but not in the distal tubules (Fig. 1C). The specificity of the staining was demonstrated by the lack of a signal in the presence of sheep (Fig. 1D) or goat serum (Figs. 1E merged in 1F), or mouse IgG (Figs. 1G and 1H, merged in 1I) as controls for secondary and primary antibodies, respectively.



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FIG. 1. Colocalization of N- and E-cadherin in C3H mouse kidney: N-cadherin was confined to proximal tubules (A). E-cadherin staining was seen in all tubular segments; however, the staining was more intense in distal tubules (B). Colocalization of N- and E-cadherin was seen in proximal, but not distal tubules (C). D–I demonstrate the specificity of the staining; sheep serum (D) or goat serum (E) was substituted as controls for the secondary antibody (merged image in F). Mouse IgG (G and H) was substituted in place of the primary antibodies (merged image in I). The width of each field is 240 µm.

 
The colocalization of {alpha}-catenin with N- and E-cadherin was also examined. {alpha}-Catenin was expressed in both proximal and distal tubules (Fig. 2). Similar to previous results, N-cadherin was detected in proximal tubules by immunofluorescence (Fig. 2A). After merging, the colocalization of {alpha}-catenin (Fig. 2B) and N-cadherin in proximal tubules is depicted by the yellow staining (Fig. 2C). The distal tubules only express {alpha}-catenin, which remains as green staining in the merged image. Both E-cadherin (Fig. 2D) and {alpha}-catenin (Fig. 2E) were detected in proximal and distal tubules and colocalized as demonstrated by dual immunofluorescence staining (Fig. 2F). The specificity of the staining is demonstrated by the finding that there was no signal when the slides were incubated with rabbit or goat serum (control for secondary antibodies) (Figs. 2G–I), or goat or mouse IgG (control for the primary antibodies) (Figs. 2 J–L).



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FIG. 2. Colocalization of {alpha}-catenin and N-/E-cadherin in C3H mouse kidney: N-cadherin is localized in the proximal tubules (A), while {alpha}-catenin is found in all tubular segments (B). N-cadherin and {alpha}-catenin colocalize in proximal tubules, but not distal tubules (C). Higher magnification was used to demonstrate the precise colocalization of E-cadherin (D) and {alpha}-catenin (E) along the lateral borders of tubular epithelial cells; the merged image is shown in F. G–L demonstrate the specificity of the staining; rabbit serum (G) or goat serum (H) was substituted as controls for the secondary antibody (merged image in I). Goat IgG (J) or mouse IgG (K) was substituted in place of the primary antibodies (merged image in L). The width of each field is 240 µm with the exception of D–F (150 µm).

 
The colocalization of ß- and {gamma}-catenin with E- and N-cadherin is shown in Figure 3. Similar to previous results, N-cadherin was detected only in proximal tubules (Figs. 3A and 3D) and the expression of E-cadherin was higher in distal tubules than in proximal tubules (Figs. 3G and 3J). ß-catenin was expressed in both proximal and distal tubules (Figs. 3B and 3H), while the staining of {gamma}-catenin was confined to the distal tubules (Figs. 3E and 3K). ß-Catenin and N-cadherin colocalized only in the proximal tubule (yellow staining in Fig. 3C), while ß-catenin-positive/N-cadherin-negative distal tubules were also seen (green staining in Fig. 3C). ß-catenin and E-cadherin were colocalized in all tubules (Fig. 3I), suggesting that E-cadherin and ß-catenin are always colocalized, but that N-cadherin and ß-catenin are colocalized only in the proximal tubules. As expected, no colocalization between N-cadherin and {gamma}-catenin was seen (Fig. 3F). However, E-cadherin and {gamma}-catenin colocalize in distal tubular segments (Fig. 3L). Results of colocalization studies involving p120-catenin and N- or E-cadherin were equivocal (data not shown).



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FIG. 3. Colocalization of ß-/{gamma}-catenin and E-/N-cadherin in C3H mouse kidney: The staining of N-cadherin (A and D) is confined to the proximal tubules, while E-cadherin (G and J) is localized in both proximal and distal tubules, although the staining is more intense in the distal tubules. ß-Catenin is seen in both proximal and distal tubules (B and H) and co-localizes with N-cadherin only in proximal tubules (C), but with E-cadherin in all tubular segments (localize with N-cadherin (F), but with E-cadherin in distal tubules (L). The width of each field is 240 µm.

 
Interaction of Cadherin/Catenins in Normal Mouse Kidney
Although the immunofluorescence studies demonstrated co-localization of specific cadherin/catenin combinations, it remained to be determined if colocalized proteins immunoprecipitated as a cadherin/catenin complex. Cadherin/catenin interactions were investigated by immunoprecipitation/immnoblot analyses using antibodies previously demonstrated to have high specificity. Whole kidney extracts were prepared and immunoprecipitated with rabbit polyclonal antibodies against {alpha}-, ß-, and {gamma}-catenin, or a monoclonal antibody against p120. The immunoprecipitated complexes were separated by SDS-PAGE and probed for {alpha}-, ß-, {gamma}-, and p120-catenin and E- and N-cadherin (Fig. 4). An anti-{alpha} catenin antibody immunoprecipitated ß- and {gamma}-catenin, but not p120-catenin, and both E- and N-cadherin, although the band was very faint for {gamma}-catenin and N-cadherin. The ß-catenin antibody immunoprecipitated {alpha}-catenin and E- and N-cadherin, but not {gamma}- or p120-catenin. {gamma}-Catenin coimmunoprecipitated with {alpha}-catenin and E-cadherin, and to a very small extent, N-cadherin. Interestingly, p120-catenin only coimmunoprecipitated ß-catenin and E-cadherin, but not N-cadherin. This suggests that the p120-catenin in proximal tubules may only associate with E-cadherin, but not N-cadherin, consistent with the colocalization results. As a control, all of the catenins also coimmunoprecipitated themselves. In addition, anti-mouse or anti-rabbit IgG, or the GammaBindä Plus SepharoseTM beads alone did not precipitate any cadherins or catenins. The pattern of colocalization determined by immunofluorescence corresponds very well with the ability of catenins to coimmunoprecipitate other catenins or cadherins.



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FIG. 4. Coimmunoprecipitation of cadherins and catenins in C3H mouse kidney: Kidney extracts were immunoprecipitated with rabbit polyclonal antibodies against {alpha}-, ß-, and {gamma}-catenin or a monoclonal antibody against p120-catenin. The immunoprecipitates were then probed with monoclonal antibodies against {alpha}-, {gamma}-, ß-, p120-catenin, E- and N-cadherin (labeled at the bottom of the blot). The specificity of the immunoprecipitation was demonstrated by the finding that rabbit IgG (R), mouse IgG (M), or GammaBindTM Plus SepharoseTM (Gb) did not immunoprecipitate {alpha}-catenin or N-cadherin (right column); a positive control (PC) was also used on these blots. {alpha}-Catenin coimmunoprecipitated ß-catenin and E-cadherin, with faint bands seen with {gamma}-catenin and N-cadherin. ß-Catenin coimmunoprecipitated {alpha}-catenin, E- and N-cadherin, and {gamma}-catenin coimmunoprecipitated E-cadherin. p120-catenin coimmunoprecipitated only ß-catenin and E-cadherin. The cadherin/catenin band is indicated by an arrowhead ({blacktriangleright}) on the left side of each figure, while the heavy chain IgG band is denoted by a bracket on the right side (}).

 
Mercuric Chloride-Induced Acute Renal Failure
Blood urea nitrogen (BUN) and plasma creatinine are commonly used as clinical indicators of ARF. Following a single ip injection of mercuric chloride (6.25–25 µmol/kg), both were significantly elevated in a time- and dose-dependent manner (Fig. 5). BUN and creatinine were significantly increased following challenge with 25-µmol/kg mercuric chloride as early as 12 h after injection. There was no increase in BUN or creatinine at any time point following 6.25-µmol/kg mercuric chloride treatment. However, both 12.5 and 25-µmol/kg mercuric chloride doses significantly affected renal function as assessed by BUN and creatinine levels. Histological evaluation was also used to examine the kidney following mercuric chloride challenge. In H&E-stained sections of control tissue (Fig. 6), normal glomeruli and proximal/distal tubules are seen. Nuclear staining was well defined and the cells display normal morphology. Following 6 or 12 h challenges with either 12.5 or 25-mmol/kg mercuric chloride, organ architecture and cell structure were well maintained; however, evidence of damage was seen at 24 h. As expected, there was damage to, and a loss of, proximal tubule cells, and loss of nuclear staining (Figs. 6H and 6I, arrows). The injury was more pronounced with 25-mmol/kg mercuric chloride than with 12.5-mmol/kg dose. Whereas the glomeruli still appeared normal, the proximal tubules were specifically damaged by mercuric chloride challenge. Taken together, the biochemical and histological data suggest that necrotic damage induced by mercuric chloride is time-and dose-dependent. Cell death begins in a window between 6 and 24 h after injection with 12.5 or 25-µmol/kg mercuric chloride. Thus, we have identified an early stage (0–6 h) that may be associated with proximal tubular alterations prior to cell death, and a late stage (6–24 h) that is associated with cell death.



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FIG. 5. The impact of mercuric chloride on indexes of renal function: C3H mice were challenged with mercuric chloride (0–25 µmol/kg) injected ip. Mice were sacrificed at 6–48 h and the blood was collected for measurement of blood urea nitrogen (top) and plasma creatinine (bottom) using commercially available kits. In the left column, mice were challenged with 25-µmol/kg mercuric chloride for 6–48 h, while in the right-column mice were challenged with 12.5 and 25 µmol/kg mercuric chloride for 24 and 48 h. Each data point represents the mean ± SD of 12 mice (3 separate experiments, 4 mice per experiment); *significant difference from control (p < 0.05).

 


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FIG. 6. The impact of mercuric chloride on C3H mouse kidneys: C3H mice were challenged with 12.5 or 25-µmol/kg mercuric chloride, injected ip. Mice were sacrificed at 6, 12, and 24 h and the kidneys were fixed in 4% paraformaldehyde. Paraffin-embedded sections were stained with H&E for histological evaluation. Normal glomerular and tubular structure is seen in control samples (left column). Evidence of cell damage (loss of nuclear staining; arrow in H and I) is seen at 24 h with both concentrations of mercuric chloride, but not at 6 or 12 h. The width of the field is 150 µm.

 
Cell Adhesion Protein Expression: Late Stages of Mercuric Chloride-Induced ARF
The expression of adhesion molecules during the late stages of mercuric chloride-induced ARF was examined by Western-blot analysis. Interestingly, at 24-h postinjection, Ksp- and N-cadherin expressions were significantly decreased by either 12.5 (Ksp = 63.4 ± 17.4% control; n = 49.4 ± 6.6% control) or 25 µmol/kg (Ksp = 40.6 ± 12.7% control; n = 51.8 ± 11.0% control) mercuric chloride challenge, but not E-cadherin (88.1 ± 1.9% control; 92.0 ± 0.1% control for 12.5 and 25 µmol/kg, respectively) (Fig. 7). There was no significant difference between 6.25 µmol/kg mercuric chloride and control. In addition, there was no decrease of K-cadherin expression at any dose (data not shown). The same strategy was used to examine catenin expression. {alpha}-Catenin was significantly decreased by 25 µmol/kg mercuric chloride challenge (71.6 ± 14.6% control) (Fig. 7). However, there were no changes in the protein expression of ß- (103.9 ± 0.1% control], {gamma}- (122.3 ± 13.6 %control) or p120-catenin (107.6 ± 4.2 % control) following mercuric chloride challenge. These results suggest that the late stages of mercuric chloride-induced ARF are associated with a decrease in the expression of Ksp- and N-cadherin, and {alpha}-catenin; however, the concentration of mercuric chloride required to decrease cadherin expression is less than that for decreased {alpha}-catenin expression. Interestingly, each of these proteins is expressed in the proximal tubules, the site of mercuric chloride-induced damage. However, the expression pattern is not the sole factor in this selective response, as E-cadherin, ß- and p120-catenin are also expressed in the proximal tubules.



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FIG. 7. The impact of mercuric chloride on renal cadherin and protein expression: C3H mice were challenged with mercuric chloride (6.25–25 µmol/kg), injected ip. Mice were sacrificed at 24 h and cadherin and catenin expression determined via Western blot. Each lane represents extracts from 2 mice; similar results were seen in 6 animals.

 
Cadherin/Catenin Localization & Interaction: Early Stages of Mercuric Chloride-Induced ARF
Having established that the late stages of mercuric chloride-induced ARF were associated with specific changes in cadherin and catenin expression, the localization of these proteins during the early stages was next examined. Following 6 h of 25-µmol/kg mercuric chloride challenge, N-cadherin expression was significantly decreased on cell-cell border in certain, but not all, proximal tubules (Fig. 8). The effect of mercuric chloride appeared to be limited to cell-cell contact in certain cells, although, when using this method there was a diffuse staining in the cytoplasm, most likely due to the indirect detection method (peroxidase) that requires signal amplification. In addition, the staining pattern and distribution of {alpha}-catenin was significantly changed at the same time (Fig. 9), especially in proximal tubules.



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FIG. 8. The impact of mercuric chloride on localization of N-cadherin: C3H mice were challenged with 25 µmol/kg mercuric chloride injected ip. Mice were sacrificed at 6 h and the kidneys were fixed in 4% paraformaldehyde. Paraffin-embedded sections were processed for immunohistochemical localization of N-cadherin with a commercially available system (Zymed). Arrows indicate a loss of N-cadherin staining at cell-cell borders in mercuric chloride-treated kidneys (right panel). Similar results were seen in duplicate experiments. The width of the field is 150 µm.

 


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FIG. 9. Impact of mercuric chloride on expression and distribution of {alpha}-catenin: {alpha}-catenin staining was significantly decreased on cell-cell borders following a 6-h challenge with 25 µmol/kg mercuric chloride challenge in C3H mouse kidney proximal tubules. Control sections are shown in the left panel; mercuric chloride challenged sections are shown on the right. Arrows indicate a loss of {alpha}-catenin staining at cell-cell borders in mercuric chloride-treated kidneys (right panel). Similar results were seen in duplicate experiments. The width of the field is 240 µm.

 
In the proximal tubule, the N-cadherin/ß-catenin/{alpha}-catenin complex is proposed to be a major complex mediating cadherin-based intercellular adhesion based on our colocalization and coimmunoprecipitation studies. As the early stages of mercuric chloride-induced ARF are associated with alterations in N-cadherin and {alpha}-catenin localization, the ability of {alpha}-catenin to coimmunoprecipitate ß-catenin and N-cadherin was examined. A rabbit polyclonal antibody against {alpha}-catenin co-immunoprecipitated both proteins, although the N-cadherin band was quite faint. Following 6 h of challenge with mercuric chloride, {alpha}-catenin coimmunoprecipitated approximately 50% less ß-catenin (Fig. 10) and 60% less N-cadherin (data not shown), suggesting that the localization differences in {alpha}-catenin are also associated with functional differences (interaction). These data suggest that changes in the cadherin/catenin complexes are associated with early events in ARF, and occur prior to cell death.



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FIG. 10. Impact of mercuric chloride on the coimmunoprecipitation of {alpha}-catenin and ß-catenin: Following a 25-µmol/kg mercuric chloride challenge (6 h), kidney extracts were immunoprecipitated with a rabbit polyclonal {alpha}-catenin antibody, and the immunoprecipitates were probed with a monoclonal ß-catenin. Each lane represents an extract from a separate animal; the numbers at the bottom of each lane represent the mean pixel density as determined by the UN-SCAN-IT program. The ß-catenin band is indicated on the figure, as well as the heavy chain IgG band (HC). Similar results were seen in two separate experiments.

 
Disruption of Cadherin/Catenin Complexes: Association with Alterations in Na+K+-ATPase Localization
Seminal studies by Nelson and coworkers over the last decade have investigated the role of cadherins in the generation of epithelial cell polarity (Marrs et al., 1993Go; Mays et al., 1995Go). The Na+/K+-ATPase is specifically localized to the basolateral membrane of proximal tubular cells and is a common marker of cell polarity in the kidney (reviewed in Wagner and Molitoris, 1999Go). Therefore, we examined localization of Na+K+-ATPase to determine if the alterations in cadherin/catenin localization and co-immunoprecipitation were associated with alterations in Na+/K+-ATPase. As expected, in control sections the Na+/K+-ATPase was confined to the basal membranes (Figs. 11A and 11C). At the same time that the integrity of cadherin/catenin complexes was disrupted (6 h, 25 µmol/kg), a redistribution of staining to the lateral membrane was seen (Fig. 11B), as well as clear loss of staining on basal membranes of neighboring proximal tubules (Fig. 11D). These data indicate that disruption of the integrity of cadherin/catenin complexes is accompanied by alterations in the localization of Na+/K+-ATPase.



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FIG. 11. Impact of mercuric chloride on localization of Na+/K+ATPase: In control sections (left), staining is confined to the basolateral membrane. Mercuric chloride challenge (25 µmol/kg; 6 h) results in increased staining on the lateral borders, and a disruption of staining on the basal borders of neighboring tubules (right panels). Similar results were seen in duplicate experiments. The width of the field is 240 µm (top) and 150 µm (bottom).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our results demonstrate that E-, K-, Ksp-, and N-cadherin and {alpha}, ß-, {gamma}-, and p120-catenin are expressed in adult C3H mouse kidney. In the proximal tubule, E- (weak), Ksp-, and N-cadherin were expressed, while the distal tubules expressed only E- and Ksp-cadherin and ß-catenin were expressed in both proximal and distal tubules, while p120-catenin was confined to the proximal tubules, and {gamma}-catenin to the distal tubules. These data are in agreement with a number of previous studies that examined the localization of individual cadherins and catenins in the kidney. Specifically, previous work has shown Ksp-cadherin to be expressed in all tubular segments (Thomson and Aronson, 1999Go; Thomson et al., 1995Go). In addition, our data is similar to other reports in which N-cadherin was only detected in proximal tubules and E-cadherin was detected in both proximal and distal tubules (Nouwen et al., 1993Go; Piepenhagen et al., 1995Go). A consistent finding of our studies, however, was the fact that E-cadherin staining in the distal tubule is much more intense than that in the proximal tubule. E-cadherin is not detected in the proximal tubules of human (Nouwen et al., 1993Go) or rat kidneys (Jung et al., submitted), suggesting some species differences between cadherin expressions along nephron. {alpha}- and ß-Catenin are expressed in all nephron segments, while {gamma}-catenin is only detected in distal tubules (Piepenhagen and Nelson, 1995Go). Our data is the first to demonstrate the specific localization of p120-catenin in the proximal, but not distal, tubules. The species difference in E-cadherin expression suggests that disruption of N-cadherin complexes is critical in the rat and human, while in the mouse one must consider both E- and N-cadherin complexes during injury to the proximal tubule.

We also extend the understanding of cadherin/catenin complexes by examining the colocalization and interaction of cadherins and catenins in the mouse kidney. The colocalization and coimmunoprecipitation studies yielded very similar results. N- and E-cadherin colocalized in proximal, but not distal tubules. Both {alpha}- and ß-catenin colocalized with N-cadherin in proximal tubules, while no colocalization was seen between {gamma}-catenin and N-cadherin. E-cadherin was shown to colocalize with all of the catenins; {alpha}-, ß- and p120-catenin in proximal tubules and {alpha}-, ß-, and {gamma}-catenin in distal tubules. N-cadherin/ß-catenin/{alpha}-catenin co-immunoprecipitated, while {gamma}- and p120-catenin did not co-immunoprecipitate N-cadherin. As expected, based on colocalization, all catenins co-immunoprecipitated with E-cadherin. We conclude that in the proximal tubule, two distinct cadherin/catenin complexes exist: N-cadherin/ß-catenin/{alpha}-catenin and E-cadherin/ß-catenin/{alpha}-catenin/p120-catenin. In the distal tubule, E-cadherin/ß-catenin/{alpha}-catenin and E-cadherin/{gamma}-catenin/{alpha}-catenin complexes are present. These data are the first to define the spatial colocalization and co-immunoprecipitation patterns of renal cadherin and catenin complexes along the nephron.

K-cadherin is expressed in renal tubules (Cho et al., 1998Go; Shimazui et al., 2000Go), but was not detected in C3H mouse kidney, either by immuno-histochemistry or by immunofluorescence using antirabbit (from Dr. Gregory Dressler), and antigoat (Santa Cruz Biotechnology) polyclonal antibodies. One explanation is that K-cadherin is highly expressed during renal development and is then downregulated, thereby accounting for its low expression level in adult mouse kidney. As K-cadherin expression is downregulated, E-cadherin becomes more prominent in the mature tubules (Cho et al., 1998Go). However, faint bands were detected by Western blot. The function of p120-catenin is unclear and, most likely, cell-specific (Golenhofen and Drenckhahn, 2000). It is hypothesized that the cadherin/p120 complex is required for initial homotypic recognition, while ß- and {alpha}-catenin stabilize the cluster and strengthen cell–cell adhesion via interaction with the actin cytoskeleton. The finding that cadherin proteins lacking the p120-catenin-binding site do not mediate receptor clustering in vitro (Thoreson et al., 2000Go) supports this hypothesis. However, recent studies suggest that p120-catenin is not required for intercellular adhesion (Myster et al., 2003Go; Pacquelet et al., 2003Go) and that p120-catenin may inhibit cadherin-mediated adhesion in certain cell types (Aono et al., 1999Go). In the present study we demonstrated that p120 was localized in proximal tubules by immunohistochemistry and immunofluorescence but results of immunofluorescent colocalization were equivocal. However, immunoprecipitation studies demonstrated that p120-catenin complexes with ß-catenin and E-cadherin, but not with {gamma}-catenin and N-cadherin. The most plausible explanation for this is that p120-catenin in proximal tubules complexes only with E-cadherin. These data raise the intriguing question of the function of p120-catenin in the proximal tubule.

We demonstrate that mercuric chloride-induced nephrotoxicty is (1) associated with a selective loss of expression of adhesion proteins in the kidney during the late stages of injury (12–24 h), and (2) discrete alterations in cadherin/catenin intracellular localization and association occur prior to cell death (6 h). Based on the pattern of localization and coimmunoprecipitation, it is suggested that the selective protein loss cannot be accounted for solely by proximal tubular epithelial cell death. Prior to cell death (6 h) delocalization, loss of protein expression, and decreased protein:protein interactions in the N-cadherin/ß-catenin/{alpha}-catenin complex was observed. These data suggest that the early stages of renal injury may be associated with disruption of intercellular adhesion. The changes in the cadherin/catenin complex are not specific for mercuric chloride. Similar changes in protein expression and localization were seen in mice challenged with 20 mg/kg cisplatin (data not shown), a nephrotoxic compound (Megeyesi et al., 1998Go; Ramesh and Reeves, 2002Go). The mechanism(s) underlying the loss of complex integrity during the early stages of ARF, and protein loss during the late stages of ARF are currently under investigation. An important area of future investigation will be to understand the relationship between disruption of the cadherin/catenin complex and tight junctions. Although no impact of mercuric chloride on the protein expression of occludin and ZO-1 was seen at 24 h, a loss of ZO-2 was seen (data not shown). Importantly, the relationship between both the delocalization and loss of {alpha}-catenin and tight junction integrity is under investigation, as a clear relationship between {alpha}-catenin and tight junctions has been demonstrated (Itoh et al., 1997Go, 1999Go).

An important aspect of these studies was the association between disruption of cadherin/catenin complexes and alterations in the localization of the {alpha}1 subunit of Na+/K+-ATPase. At the same time, the integrity of cadherin/catenin complexes was disrupted; a redistribution of {alpha}1 staining to the lateral membrane was seen, as well as a decreased intensity of staining on basal membranes. While these data suggest that cell polarity may be altered, a number of important questions remain including whether the disruption of the cadherin/catenin complex by nephrotoxicants is a direct effect, or is indirect as a result of alterations in other important structural components, particularly the actin cytoskeleton. In addition, mercuric chloride has been shown to bind (Bhattacharya et al., 1997Go) Na+/K+-ATPase and disrupt membrane anchoring of this complex (Imesch et al., 1992Go). Given that recent data suggests that Na+/K+-ATPase activity is necessary to establish polarity (Rajasekaran et al., 2001Go), the alterations in cadherin/catenin complexes could be downstream from changes in the Na+/K+-ATPase.

In vivo evidence for disruption of cell–cell adhesion during renal injury in animal models is lacking, although some evidence suggests that it may be associated with renal injury. Bush et al. (2000)Go have shown that ischemia caused a loss of cell-surface E-cadherin, and degradation of E-cadherin to an approximate 80 kDa fragment, suggesting that disruption of the cadherin/catenin complex in the kidney may represent a critical event in ischemia-induced renal injury. In addition, bismuth, a nephrotoxic metal, was shown to specifically alter the cellular localization of N-, but not E-, cadherin in the mouse proximal tubular epithelium (Leussink et al., 2001Go). No changes in tight junction constituents were seen. These changes in the N-cadherin/catenin complex, in the absence of changes in E-cadherin and tight junctions are similar to our data using mercuric chloride-induced renal injury. Prozialeck and coworkers (2003)Go have recently extended their in vitro studies (reviewed in Prozialeck, 2000Go) investigating cadmium-induced disruption of cadherin/catenin complexes in the kidney. Specific changes in the localization of E- and N-cadherin and ß-catenin were observed in the proximal tubules of rats following subchronic challenge with cadmium chloride.

While evidence suggests that disruption of cell–cell adhesion is associated with renal failure in patients; understanding of the cause and effect of this relationship is limited by the lack of in vivo studies. It is clear that a number of toxic insults (metals, ischemia, toxins, etc.) disrupt cell–cell contacts in vitro, however, the in vivo relevance of these findings is uncertain. Many of the in vitro studies have focused on E-cadherin, despite the fact that expression of this cadherin is mostly limited to distal tubules. The large gap between our understanding of cell adhesion complexes and the role that disruption of homotypic cell adhesion may play in renal injury requires further investigation. However, the current studies demonstrate that ARF is associated with disruption of cadherin/catenin complexes at multiple levels: expression, localization, and interaction. These findings provide a basis for future investigations of the mechanism(s) involved in complex disruption during ARF, as well as the potential causal role of cadherin/catenin complexes in functional changes associated with renal pathophysiology.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Peter S. Aronson (Yale University) for providing the anti-Ksp cadherin monoclonal antibody and Dr. Gregory R. Dressler (University of Michigan) for providing an anti-K-cadherin antibody. This work was supported by the Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center and the Center for Environmental and Rural Health, Texas A&M University (ES09106).


    NOTES
 

1 To whom correspondence should be addressed at the Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114. Fax: (979) 845-0699. E-mail: parrish{at}medicine.tamu.edu.


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