Peter C. Lamar

Cadmium alters the localization of N-cadherin, E-cadherin, and β-catenin in the proximal tubule epithelium

Recent studies on proximal tubule-derived cells in culture have shown that Cd has relatively specific damaging effects on the cadherin-dependent junctions between the cells. The objective of the present study was to determine whether Cd can affect cadherin-dependent junctions in the proximal tubule epithelium in vivo. Male Sprague-Dawley rats received subcutaneous injections of Cd (0.6 mg/kg in isotonic saline, 5 days per week for up to 6 weeks). One day each week, 24-h urine samples were collected and analyzed for protein and creatinine. After 5-6 weeks, the Cd-treated animals developed significant proteinuria, with no change in creatinine excretion. Visualization of pan-cadherin immunoreactive materials by immunoperoxidase labeling showed that Cd caused a marked reduction in the intensity of cadherin labeling associated with the apical and the basolateral surfaces of the epithelial cells of the proximal tubule, but no change in the pattern of cadherin labeling in other segments of the nephron. Results of studies utilizing specific antibodies against N-cadherin, E-cadherin, and beta-catenin showed changes in the localization of all three molecules in the proximal tubule. Assessment of cell membrane integrity with trypan blue and ethidium homodimer showed no overt evidence of death in the proximal tubule epithelial cells. Additional results showed that Cd caused only a slight increase in the total levels of glutathione and no significant peroxidation of membrane lipids, indicating only a modest level of oxidative stress. These results indicate that Cd can disrupt cadherin-dependent cell-cell junctions in the proximal tubule, and they raise the possibility that a loss of cadherin-mediated adhesion may contribute to the nephrotoxic effects of Cd.

Differential expression of E-cadherin, N-cadherin and beta-catenin in proximal and distal segments of the rat nephron.

BackgroundThe classical cadherins such as E- and N-cadherin are Ca2+-dependent cell adhesion molecules that play important roles in the development and maintenance of renal epithelial polarity. Recent studies have shown that a variety of cadherins are present in the kidney and are differentially expressed in various segments of the nephron. However, the interpretation of these findings has been complicated by the fact that the various studies focused on different panels of cadherins and utilized different species. Moreover, since only a few of the previous studies focused on the rat, information regarding the expression and localization of renal cadherins in this important species is lacking. In the present study, we have employed dual immunofluorescent labeling procedures that utilized specific antibodies against either E- or N-cadherin, along with antibodies that target markers for specific nephron segments, to characterize the patterns of cadherin expression in frozen sections of adult rat kidney.ResultsThe results showed that N-cadherin is the predominant cadherin in the proximal tubule, but is essentially absent in other nephron segments. By contrast, E-cadherin is abundant in the distal tubule, collecting duct and most medullary segments, but is present only at very low levels in the proximal tubule. Additional results revealed different patterns of N-cadherin labeling along various segments of the proximal tubule. The S1 and S2 segments exhibit a fine threadlike pattern of labeling at the apical cell surface, whereas the S3 segment show intense labeling at the lateral cell-cell contacts.ConclusionsThese results indicate that E- and N-cadherin are differentially expressed in the proximal and distal tubules of rat kidney and they raise the possibility that differences in cadherin expression and localization may contribute to the differences in the susceptibility of various nephron segments to renal pathology or nephrotoxic injury.

Expression of kidney injury molecule-1 (Kim-1) in relation to necrosis and apoptosis during the early stages of Cd-induced proximal tubule injury

Cadmium (Cd) is a nephrotoxic industrial and environmental pollutant that causes a generalized dysfunction of the proximal tubule. Kim-1 is a transmembrane glycoprotein that is normally not detectable in non-injured kidney, but is up-regulated and shed into the urine during the early stages of Cd-induced proximal tubule injury. The objective of the present study was to examine the relationship between the Cd-induced increase in Kim-1 expression and the onset of necrotic and apoptotic cell death in the proximal tubule. Adult male Sprague-Dawley rats were treated with 0.6 mg (5.36 micromol) Cd/kg, subcutaneously, 5 days per week for up to 12 weeks. Urine samples were analyzed for levels of Kim-1 and the enzymatic markers of cell death, lactate dehydrogenase (LDH) and alpha-glutathione-S-transferase (alpha-GST). In addition, necrotic cells were specifically labeled by perfusing the kidneys in situ with ethidium homodimer using a procedure that has been recently developed and validated in the Prozialeck laboratory. Cryosections of the kidneys were also processed for the immunofluorescent visualization of Kim-1 and the identification of apoptotic cells by TUNEL labeling. Results showed that significant levels of Kim-1 began to appear in the urine after 6 weeks of Cd treatment, whereas the levels of total protein, alpha-GST and LDH were not increased until 8-12 weeks. Results of immunofluorescence labeling studies showed that after 6 weeks and 12 weeks, Kim-1 was expressed in the epithelial cells of the proximal tubule, but that there was no increase in the number of necrotic cells, and only a modest increase in the number of apoptotic cells at 12 weeks. These results indicate that the Cd-induced increase in Kim-1 expression occurs before the onset of necrosis and at a point where there is only a modest level of apoptosis in the proximal tubule.

INTERACTION OF CADMIUM (CD2+) WITH A 13-RESIDUE POLYPEPTIDE ANALOG OF A PUTATIVE CALCIUM-BINDING MOTIF OF E-CADHERIN

Previous studies from our laboratory have shown that Cd(2+) can selectively damage the tight junctions between epithelial cells in culture. Recently, we have obtained evidence suggesting that this effect may involve the interaction of Cd(2+) with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To begin to determine whether or not Cd(2+) might interact directly with the E-cadherin molecule, we studied the binding of Cd(2+) to peptide B, a synthetic, 13-residue polypeptide that corresponds to one of the extracellular Ca(2+) binding regions of mouse E-cadherin (also known as uvomorulin). The binding of Cd(2+) to peptide B was evaluated by using an equilibrium microdialysis technique and the radioactive isotope (109)Cd(2+). The effects of the binding on the conformation of the peptide were evaluated by circular dichroism (CD) spectroscopy. The results showed that Cd(2+) bound to peptide B, with a maximum of one Cd(2+) binding site per molecule and an apparent dissociation constant (K(d)) of 640 microM. The binding of Cd(2+) was reduced in the presence of excess Ca(2+), an effect that was overcome by raising the concentration of Cd(2+). Both Cd(2+) and Ca(2+) caused a shift in the CD spectrum of the peptide. However, the shift produced by Cd(2+) was about 3 times the magnitude of that produced by Ca(2+). These results indicate that Cd(2+) can interact with the Ca(2+) binding site on the peptide B molecule and distort the secondary structure of the peptide. These findings are consistent with the hypothesis that E-cadherin may be a direct molecular target for Cd(2+) toxicity.

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports

Recent studies have shown that Cd2+ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK1. The objective of the present studies was to examine the surface binding and uptake of Cd2+ by LLC-PK1 cells in relation to the disruption of cell-cell junctions. LLC-PK1 cells on Falcon Cell Culture Inserts were exposed to CdCl2 containing trace amounts of109Cd2+ from either the apical or the basolateral compartments, and the accumulation of109Cd2+ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3–4 times more Cd2+ from the basolateral compartment than from the apical compartment. The accumulation of Cd2+ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1–2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6–8 h. The Cd2+ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd2+ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd2+ accumulation was enhanced when Ca2+ was present at low concentrations (0.1 mM), and was greatly reduced when Ca2+ was present at high concentrations (10 mM). These results suggest that Cd2+ damages the junctions between LLC-PK1 cells by interacting with Ca2+-sensitive sites on the basolateral cell surface.

Chlamydia trachomatis Disrupts N-Cadherin-Dependent Cell-Cell Junctions and Sequesters β-Catenin in Human Cervical Epithelial Cells

ABSTRACT The cadherin/catenin complex serves as an important structural component of adherens junctions in epithelial cells. Under certain conditions, β-catenin can be released from this complex and interact with transcription factors in the nucleus to stimulate the expression of genes that regulate apoptosis and cell cycle control. While studying the effects of the bacterial pathogen Chlamydia trachomatis on human cervical epithelial cells in culture, we observed that C. trachomatis caused the epithelial cells to separate from each other without detaching from their growing surface. The objective of the present study was to determine if this effect might involve the disruption of the cadherin/catenin complex. Primary cultures of human cervical epithelial cells or HeLa cells were infected with C. trachomatis serovar E. Cadherin-like immunoreactive materials and β-catenin were visualized by immunofluorescence. Preliminary studies showed that N-cadherin was the primary cadherin expressed in both the primary cultures and the HeLa cells. In noninfected cells, N-cadherin and β-catenin were colocalized at the intercellular junctional complexes. By contrast, the infected cells showed a marked loss of both N-cadherin and β-catenin labeling from the junctional complexes and the concomitant appearance of intense β-catenin labeling associated with the chlamydial inclusion. The results of Western blot analyses of extracts of C. trachomatis showed no evidence of cross-reactivity with the β-catenin antibody. These results indicate that C. trachomatis causes the breakdown of the N-cadherin/β-catenin complex and that the organism can sequester β-catenin within the chlamydial inclusion. This could represent an important mechanism by which C. trachomatis alters epithelial cell function.

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells

SummaryPrevious studies from our laboratory have shown that Cd2+ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK1. The objective of the present studies was to determine whether or not Cd2+ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd2+ by adding CdCl2 to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10–40 µM Cd2+ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd2+ was added basolaterally than when it was added apically. Moreover, the effects of Cd2+ were qualitatively similar to those observed when the cells were incubated in Ca2+-free medium. These results show that Cd2+ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.

Direct Antiangiogenic Actions of Cadmium on Human Vascular Endothelial Cells

The vascular endothelium is a primary target of cadmium (Cd) toxicity, but little is known regarding a potential mechanism whereby Cd may inhibit angiogenesis. Recent findings showing that Cd can disrupt cadherin-mediated cell-cell adhesion suggested that Cd might inhibit angiogenesis by altering the function of VE-cadherin, a molecule that is essential for angiogenesis. To address this issue, endothelial cells (ECs) were exposed to Cd in the presence of serum and subjected to angiogenesis-related cell migration and tube formation assays. Initial examination of cytotoxicity showed that ECs are rather resistant to the acute cytotoxic effects of Cd even at concentrations up to 1 mM. However, 10 microM Cd decreased migration of ECs. Cd concentrations of 500 nM and greater significantly reduced organization of microvascular ECs into tubes. These antiangiogenic effects were evident even when ECs were preincubated with Cd and then washed to remove free Cd, indicating that Cd acted directly on the cells rather than on the extracellular matrix. Immunolocalization studies showed that Cd caused the redistribution of VE-cadherin from cell to cell contacts. These findings indicate that Cd acts in an angiostatic manner on ECs, and that this effect may involve alterations in the localization and function of VE-cadherin.

Effects of cadmium on E-cadherin and VE-cadherin in mouse lung

Exposure to Cd(2+) via inhalation or intratracheal instillation results in pulmonary edema, which is followed by the influx of leukocytes, the proliferation of type II pneumocytes and eventual scarring and fibrotic changes. While the general toxic effects of Cd(2+) in the lung have been well characterized, the specific molecular mechanisms underlying these effects have yet to be elucidated. Previously we have shown that Cd(2+) can disrupt the adhering junctions between various types of epithelial and endothelial cells in culture, most likely by perturbing the function of the Ca(2+) dependent cell adhesion molecules E-cadherin and VE-cadherin respectively. The objectives of this study were to determine whether respiratory exposure to Cd(2+) can alter the localization of E-cadherin and VE-cadherin in the lung, and to determine whether this effect may play a role in the acute pneumotoxic response to Cd(2+). Male CF-1 mice were exposed to CdCl(2) (0, 16.25, 32.5, 65 or 130 nmoles in 50 microl saline) via intratracheal instillation. After 24 hours, the lungs were removed and either subjected to bronchoalveolar lavage or analyzed for histopathologic changes. The results showed that Cd(2+) caused an increase in lung weight and in the protein content of the lavage fluid. These effects were accompanied by a pronounced decrease in the amount of E-cadherin in epithelial cells of the alveoli and small bronchioles and of VE-cadherin in vascular endothelial cells. Assessment of cell membrane integrity with ethidium homodimer-1 showed no evidence of severe injury or death in alveolar epithelial cells. These findings suggest that E-cadherin and VE-cadherin may be important early targets of Cd(2+) toxicity in the lung.

Comparison of the cytotoxic effects of cadmium chloride and cadmium-metallothionein in LLC-PK1 cells

Recent studies have shown that ionic cadmium (Cd2+) can selectively damage the tight junctions between LLC-PK1 cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd2+ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd2+ for 1-4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24-48 hours of exposure. Additional results showed that the cells accumulated Cd2+ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK1 cells, but that it can kill the cells after prolonged exposure.