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The environmental toxicant cadmium (Cd) enters the food chain. A substantial proportion of Cd in nutrients of plant origin is present as Cd-metallothionein (CdMT) and Cd-phytochelatin (CdPC) complexes, which may be absorbed and transcytosed intact by colonic enterocytes following bacterial fermentation and contribute to systemic Cd toxicity, e.g. in liver and kidneys. We have recently demonstrated that the receptor for human neutrophil gelatinase-associated lipocalin (hNGAL) is expressed in human colon and colon-like Caco-2 BBE cells where it mediates transcytosis of MT and PC. Here we show in colon-like Caco-2 BBE cells that hNGAL receptor (hNGAL-R) dependent toxicity is significantly higher with CdMT than with CdPC3 (2.5-50μM Cd(2+) complexed to MT or PC3 for ≤24h), using MTT assay. Fluorescence-labelled A546-MT, but not A488-PC3 (both 700nM), co-localizes with the lysosomal marker cathepsin-B, as determined by confocal microscopy. In transwell experiments with confluent monolayers, transcytosis efficiency (i.e. the ratio of basal delivery to apical decrease) of A546-MT is decreased compared to A488-PC3 (both 700nM). The tubulin polymerization disruptor nocodazole (16.7μM) almost abolished CdMT and CdPC3 toxicity, reduced apical uptake of both A546-MT and A488-PC3, but increased transcytosis efficiency of A546-MT compared to that of A488-PC3 by preventing trafficking of A546-MT to lysosomes. Hence, following hNGAL-R dependent endocytosis of CdMT/CdPC3 in colonic epithelia, a nocodazole-sensitive trafficking pathway may preferentially target CdMT, but not CdPC3, to lysosomes, causing increased colonic epithelial toxicity but reduced systemic toxicity., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Nickel and nickel compounds are carcinogens that target the lungs and kidneys causing cell death or cell survival adaptation. The multidrug resistance P-glycoprotein ABCB1 protects cells against toxic metabolites and xenobiotics and is upregulated in many cancer cell types. Here, we investigated the role of ABCB1 in nickel-induced stress signaling mediated by reactive oxygen species (ROS) and ceramides. In renal proximal tubule cells, nickel chloride (0.1-0.25 mM) increased both ROS formation, detected by 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and cellular ceramides, which were determined by lipid dot blot and surface immunostaining, culminating in decreased cell viability, increased DNA fragmentation, augmented PARP-1 cleavage, and increased ABCB1 mRNA and protein. Inhibitors of the de novo ceramide synthesis pathway (fumonisin B1, L-cycloserine) and an antioxidant (α-tocopherol) attenuated nickel-induced toxicity as well as induction of ABCB1. ABCB1 protects against nickel toxicity as PSC833, an ABCB1 blocker, augmented the decrease in cell viability by nickel. Moreover, nickel toxicity was attenuated in renal MDCK cells stably overexpressing ABCB1. In agreement with previous data that demonstrated extrusion of (glucosyl)ceramides by ABCB1 (Lee et al. in Toxicol Sci 121:343, 2011), PSC833 increased total cellular ceramides by >2-fold after nickel treatment. Further, glucosylceramide synthase (GCS) mRNA is upregulated by nickel at 3 h by ~1.5-fold but declined with prolonged exposures (6-24 h). Inhibition of GCS with C9DGJ or knockdown of GCS with siRNA significantly attenuated nickel toxicity. In conclusion, nickel induces a ROS-ceramide pathway to cause apoptotic cell death as well as activate adaptive survival responses, including upregulation of ABCB1, which improves cell survival by extruding proapoptotic (glucosyl)ceramides.

Cellular stress elicited by the toxic metal Cd(2+) does not coerce the cell into committing to die from the onset. Rather, detoxification and adaptive processes are triggered concurrently, allowing survival until normal function is restored. With high Cd(2+), death pathways predominate. However, if sublethal stress levels affect cells for prolonged periods, as in chronic low Cd(2+) exposure, adaptive and survival mechanisms may deregulate, such that tumorigenesis ensues. Hence, death and malignancy are the two ends of a continuum of cellular responses to Cd(2+), determined by magnitude and duration of Cd(2+) stress. Signaling cascades are the key factors affecting cellular reactions to Cd(2+). This review critically surveys recent literature to outline major features of death and survival signaling pathways as well as their activation, interactions and cross talk in cells exposed to Cd(2+). Under physiological conditions, receptor activation generates 2nd messengers, which are short-lived and act specifically on effectors through their spatial and temporal dynamics to transiently alter effector activity. Cd(2+) recruits physiological 2nd messenger systems, in particular Ca(2+) and reactive oxygen species (ROS), which control key Ca(2+)- and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca(2+) signals activate cell death effectors (ceramides, ASK1-JNK/p38, calpains, caspases) and/or cause irreversible damage to vital organelles, such as mitochondria and endoplasmic reticulum (ER), whereas low localized ROS/Ca(2+) levels act as 2nd messengers promoting cellular adaptation and survival through signal transduction (ERK1/2, PI3K/Akt-PKB) and transcriptional regulators (Ref1-Nrf2, NF-κB, Wnt, AP-1, bestrophin-3). Other cellular proteins and processes targeted by ROS/Ca(2+) (metallothioneins, Bcl-2 proteins, ubiquitin-proteasome system, ER stress-associated unfolded protein response, autophagy, cell cycle) can evoke death or survival. Hence, temporary or permanent disruptions of ROS/Ca(2+) induced by Cd(2+) play a crucial role in eliciting, modulating and linking downstream cell death and adaptive and survival signaling cascades.

The multidrug resistance (MDR) P-glycoprotein ABCB1 plays a major role in MDR of malignant cells and is regulated by various transcription factors, including Wnt/β-catenin/TCF4. The transcription factor PITX2 (Pituitary homeobox-2) is essential for embryonic development. PITX2 operates by recruiting and interacting with β-catenin to increase the expression of growth-regulating genes, such as cyclin D1/2 and c-Myc. The importance of PITX2 in malignancy is not yet known. Here we demonstrate that in the renal cancer cell lines ACHN and A498, the level of ABCB1 expression and function correlate with nuclear PITX2 localization and PITX2-luciferase reporter gene activity (A498 > ACHN). In A498 cells, doxorubicin toxicity is augmented by the ABCB1 inhibitor, PSC833. PITX2 overexpression increases ABCB1 expression and cell survival in ACHN cells. Silencing of PITX2 by siRNA downregulates ABCB1 and induces a greater chemotherapeutic response to doxorubicin in A498 cells, as determined by MTT cell viability and clonogenic survival assays. Two PITX2 binding sequences were identified in the ABCB1 promoter sequence. PITX2 binding was confirmed by chromatin immunoprecipitation. β-Catenin is not required for PITX2 upregulation of ABCB1 because ABCB1 mRNA increased and doxorubicin toxicity decreased upon PITX2 overexpression in β-catenin(-/-) cells. The data show for the first time that ABCB1 is a target gene of PITX2 transcriptional activity, promoting MDR and cell survival of cancer cells., (Copyright © 2013 UICC.)

The detrimental health effects of cadmium (Cd) were first described in the mid 19th century. As part of industrial developments, increasing usage of Cd has led to widespread contamination of the environment that threatens human health, particularly today. Rather than acute, lethal exposures, the real challenge in the 21st century in a global setting seems to be chronic low Cd exposure (CLCE), mainly from dietary sources. Ubiquity of Cd makes it a serious environmental health problem that needs to be thoroughly assessed because it already affects or will affect large proportions of the world's population. CLCE is a health problem that affects increasingly organ toxicity, especially nephrotoxicity, without a known threshold, implying that there is currently no safe limit for CLCE. In this chapter, we summarize current knowledge on the sources of Cd in the environment, describe the entry pathways for Cd into mammalian organisms, sum up the major organs targeted by acute or chronic Cd exposure and review the impact of Cd on organ function and human health. We also aim to put early pioneering studies on Cd poisoning into perspective in the context of recent ground-breaking prospective long-term population studies, which link CLCE to leading causes of diseases in modern societies - cancer, diabetes, and cardiovascular diseases, and of state-of-the-art studies detailing cellular and molecular mechanisms of acute and chronic Cd toxicity.

Cd²⁺ is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²⁺ on permeation and gating of Ca(v)3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd²⁺ rapidly blocked currents with 2 mM Ca²⁺ in a voltage-dependent manner. The block caused by Cd²⁺ was relieved at more-hyperpolarized potentials, which suggests that Cd²⁺ can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²⁺ and Na⁺ replaced by N-methyl-d-glucamine), Cd²⁺ carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²⁺). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²⁺ entry into cells during Cd²⁺ exposure. Incubation with radiolabeled ¹⁰⁹Cd²⁺ confirmed uptake of Cd²⁺ into cells with Ca(v)3.1 channels.

Cadmium (Cd) is nephrotoxic. Circulating Cd-metallothionein complexes (CdMT) are filtered by the kidney, reabsorbed by proximal tubule cells (PTC) via receptor-mediated endocytosis, and trafficked to lysosomes which results in apoptosis. ADP-ribosylation factors (Arfs) regulate vesicular trafficking. Arf1 is traditionally associated with the secretory pathway, but has been recently found involved in endocytotic trafficking in PTC. Hence, the role of Arf1 was investigated in MT-1 and transferrin (Tf) endocytosis, and in CdMT-1-induced cell death in a PTC line by overexpressing Arf1-wildtype (WT) or dominant-negative mutant Arf1-T31N. Endogenous Arf1 distribution in PTC was punctate throughout the cytosol, but was not detected in the plasma membrane. Arf1 colocalized with markers for sorting to late endosomes (Rab7, CLC6). Arf1 weakly overlapped with the late endosomal/lysosomal marker CLC7, but not with markers for early (Rab5, CLC5) and recycling endosomes (Rab11). Arf1-T31N significantly attenuated CdMT-1 toxicity by ∼60% when compared to Arf1-WT. However, overexpression of Arf1-T31N did not prevent internalization of Alexa Fluor 546-coupled Tf or MT-1 which accumulated in an EEA1-positive early endocytotic compartment, but not in Arf1-WT overexpressing cells. We conclude that Arf1 is involved in trafficking of protein-metal complexes, including CdMT, to late endosomes/lysosomes in renal PTC., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Cadmium (Cd(2+)) damages the kidney proximal tubule (PT) by ceramide-dependent apoptosis and is also a class I carcinogen. Multidrug resistance P-glycoprotein (MDR1, ABCB1) confers resistance to Cd(2+) apoptosis, and it has been hypothesized that ABCB1 can directly transport Cd(2+) as a mode of cellular protection. Our aim was to investigate the role of ABCB1 in Cd(2+) transport and ceramide apoptosis. In rat PT or Madin-Darby canine kidney (MDCK) cells overexpressing ABCB1, ABCB1-dependent efflux of rhodamine 123(+) (Rh123(+)) or (109)Cd(2+) were determined, and cell death was assayed with MTT, H-33342 nuclear staining, and monolayer integrity by impedance sensing (Electric cell-substrate impedance sensing [ECIS]). ABCB1 inhibitors (PSC833, UIC-2 antibody) did not affect (109)Cd(2+) efflux in PT cells though Rh123(+) transport was blocked. Furthermore, increased ABCB1 expression did not augment (109)Cd(2+) efflux but attenuated apoptosis by 10-50μM Cd(2+) or 5-25μM C(6)-ceramide, which was abolished by PSC833 (1μM). ECIS measurements of ABCB1-MDCK monolayers exhibited similar effects. Moreover, in ABCB1-MDCK cells, Cd(2+)-induced ceramide formation, determined by a diacylglycerol kinase assay, was abolished and increased extrusion of nitro-2-1,3-benzoxadiazol-4-yl (NBD)-C(6)-ceramide, and NBD-C(6)-glucosylceramide was observed compared with MDCK cells. Whereas pharmacological block of sphingomyelin synthase (0.1mM D609) or sphingosine kinase (1μM dimethylsphingosine), which increase the levels of ceramide and its metabolites, augmented Cd(2+)-induced apoptosis, Cd(2+) apoptosis was significantly decreased not only by prevention of de novo ceramide synthesis (0.1μM fumonisin B(1)) but also by inhibition of glucosylceramide synthase (2μM C(9)DGJ). We therefore conclude that Cd(2+) efflux is not the mechanism behind ABCB1-mediated protection from Cd(2+) apoptosis. Rather, the sphingolipid glucosylceramide may be the proapoptotic substrate extruded by ABCB1.

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush-border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Cadmium (Cd(2+)) is a nonessential divalent metal ion that causes toxicity in multiple organs in humans. In order for toxicity to occur Cd(2+) must first enter cells by utilizing transport pathways for essential metals. This review focuses on studies in which Cd(2+) transport was directly demonstrated by electrophysiological, radiotracer or Cd(2+)-sensitive fluorescent dye techniques. The chemistry of Cd(2+) and metal ions in general is addressed in the context of properties relevant for transport through membrane proteins, such as hydration energy. Apart from transport by the ZIP transporters SLC39A8 and SLC39A14, which is not topic of the review, uptake of free Cd(2+) has been demonstrated for the Fe(2+)/H(+) cotransporter divalent metal transporter 1. Moreover, the multiligand endocytic receptors megalin and cubilin take up cadmium-metallothionein complexes via receptor-mediated endocytosis. The role of ATP binding cassette transporters in Cd(2+) efflux from cells is also discussed. Both the multidrug resistance-associated protein 1 and cystic fibrosis transmembrane conductance regulator are likely to transport cadmium-glutathione complexes out of cells, whereas transport of free Cd(2+) by the multidrug resistance P-glycoprotein remains controversial. Finally, arguments for and against Cd(2+) transport by Ca(2+) channels are presented. Most N- and L-type Ca(2+) channels are closed at resting membrane potential (with the exception of CaV1.3 channels) and therefore unlikely to allow significant Cd(2+) influx under physiological conditions. CaV3.1 and CaV3.2 T-type calcium channels are permeated by divalent metal ions, such as Fe(2+) and Mn(2+) because of considerable "window" currents close to resting membrane potential and could be responsible for tonic Cd(2+) entry. TRPM7 and the mitochondrial Ca(2+) uniporter are other likely candidates for Cd(2+) transporters, whereas the role of Orai proteins, the store-operated calcium channels carrying Ca(2+) release-activated Ca(2+) current, in Cd(2+) influx remains to be investigated.

The transition metal cadmium (Cd) is an environmental pollutant which damages the kidneys. Chronic Cd exposure may induce renal fibrosis and/or cancer, but the signaling pathways involved are not understood. The Wnt pathway is a key signaling cascade responsible for renal development, fibrosis and cancer. Hence the effect of chronic in vivo Cd exposure (100 mg/l drinking water for 12 weeks) on transcriptional activation of the Wnt pathway and markers of epithelial-to-mesenchymal transition (EMT) was investigated in mouse kidneys. Cd exposure increased kidney Cd content from 0.023+/-0.001 microg/g to 61+/-7 microg/g wet weight (means+/-S.D. of 6-7 animals). This was accompanied by increased expression of Wnt ligands (Wnt3a/6/7a/7b/9a/9b/10a/11), as determined by RT-PCR. The Wnt receptors Frizzled (Fz1/2/4,5,7-10) were also upregulated, as were the co-receptors low-density lipoprotein receptor-related proteins 5/6. Immunoblots with Wnt10a and Fz7 antibodies also revealed increased protein expression induced by Cd exposure. In contrast, Wnt antagonists were largely unaffected. Upregulation of Wnt signaling components induced by Cd was corroborated by increased expression of Wnt target genes, i.e. cell proliferation and survival genes c-Myc, cyclin D1 and the multidrug transporter P-glycoprotein Abcb1b, which promote malignancy. Lastly the EMT markers Twist, fibronectin and collagen I, but not alpha-smooth muscle actin, were also upregulated, suggesting that Cd-induced changes of renal epithelial tissue characteristics towards fibrosis and cancer may be mediated by Wnt signaling., (2010 Elsevier Ireland Ltd. All rights reserved.)

Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd(2+)) causes renal cancer and is known to inactivate p53. G(2)/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd(2+)-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd(2+) (50-100 microM) induced DNA damage within 1-6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1-3 h, measured with dihydrorhodamine 123, and G(2)/M cell cycle arrest at 6 h, which were abolished by the antioxidant alpha-tocopherol (100 microM). Cd(2+)-induced G(2)/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G(2)/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 microM), which relieves G(2)/M transition block, abolished Cd(2+)-induced G(2) arrest and increased apoptosis. This was accompanied by prevention of Cd(2+)-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells Cd(2+)-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G(2)/M arrest. This may promote survival of premalignant PT cells and Cd(2+) carcinogenesis.

The cellular effects of the toxic metal cadmium (Cd) are manifold. A large proportion of the cellular reactions affected by ionic Cd(2+) are mediated by cellular signaling cascades. The aim of this review is to provide a principal understanding of the known physiological signaling cascades, which are recruited by Cd(2+), and to highlight the fact that Cd(2+), similarly to other toxic metals, disrupts physiological signal transduction. In principle, second messengers are generated at the time of receptor activation, are short-lived, and act specifically in space and time through non-covalent binding on effectors to transiently alter their activity. Signaling dysregulation induced by Cd(2+) is reflected by a permanent disruption of transducing modules, resulting in low and/or elevated and constant levels of second messengers, which overwhelm the control mechanisms of signaling. This disturbs physiological cellular functions, gene transcription and regulation and may result in cell death and/or stress-induced adaptation and survival as well as carcinogenesis. The impact of Cd(2+) on Ca(2+)-, cAMP-, NO-, ROS-, MAP-kinase-, PKB/Akt-, nuclear factor-kappa B-, and developmental signaling is critically discussed. The hierarchical as well as cooperative and integrative character of signaling cascades activated by Cd(2+) is illustrated in the kidney proximal tubule, a major target of Cd(2+) toxicity. This review also aspires to pinpoint new avenues of research that may contribute to a more differentiated view of the complex mechanisms underlying Cd(2+) toxicity in target tissues and eventually lead to rationales and strategies for prevention and therapy of Cd(2+) toxicity.

The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3) mediate facilitated and bidirectional diffusion of small (< or = 500Da) organic cations with broad specificities for endogenous substrates such as choline, acetylcholine and monoamine neurotransmitters, as well as a variety of xenobiotics. Importantly, besides a wide range of clinically used drugs, these also include several toxins like the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) and herbicide paraquat. OCT2-OCT-3 display differential tissue distribution: OCT1 is predominantly found in liver of humans, and liver and kidney in rodents; OCT2 is most strongly expressed in both human and rodent kidney, whereas is OCT3 primarily expressed in placenta, but also more widely detected in various tissues, including brain and lung. The physiological roles of OCTs as transporters for biogenic amines or acetylcholine in these tissues are still debated, in contrast to their involvement in providing access pathways for harmful/toxic cationic substrates into the body and particular tissues. This review highlights a novel role of human and rodent OCTs as carriers of the toxic fluorescent dye ethidium, as opposed to the less harmful related phenanthridine compound propidium, which is not transported. Additional uptake and efflux pathways for ethidium in pro- and eukaryotes are discussed. OCT-mediated pathways may determine major entry routes for ethidium into the body where toxicity via specific mechanisms may develop in tissues expressing OCTs. Considering the high affinity of OCTs for ethidium (K(m) = 1-2 microM) and their strong expression in various organs, strict safety guidelines for the handling of ethidium should be reinforced.

Apoptosis is a tightly regulated physiological process, which can be initiated by toxic stimuli, such as cadmium (Cd2+). Cd2+ (10-50 microM) induces a rapid increase in reactive oxygen species (ROS) (> or = 30 min) in a cell line derived from the S1 segment of rat kidney proximal tubule, without any apparent mitochondrial dysfunction. The sphingolipid ceramide is an important second messenger in apoptosis. Short exposure to Cd2+ (3h) causes an increase in ceramides, which occurs downstream of ROS formation, and may interact with cellular components, such as endoplasmic reticulum and mitochondria. Following apoptosis initiation, execution must take place. The classical executioners of apoptosis are caspases, a family of cysteine proteases. However, increasing studies report caspase-independent apoptosis, which questions the essentiality of caspases for apoptosis implementation. With low micromolar Cd2+ concentrations (< 10 microM), caspases are only activated after 24h and not at earlier time points, which supports the notion of caspase-independent apoptosis. Due to increased cytosolic Ca(2+) under pathological conditions, a role for the Ca2+-dependent proteases, calpains, has emerged. Calpain activation by Cd2+ (3-6h) seems to be regulated by ceramide levels, in order to induce apoptosis. Calpain and caspase substrates overlap but yield different fragments, which may explain their diverse downstream targets. Furthermore, calpains and caspases may interact with one another to enhance, as seen by Cd2+, or diminish apoptosis. In this review, we discuss novel roles for ceramides, calpains and caspases as part of Cd2+-induced apoptotic signalling pathways in the kidney proximal tubule and their in vivo relevance to Cd2+-induced nephrotoxicity.

In the present study we have employed an in vitro organ challenge model to study the post-challenge responses in parasite naïve and immune gastric tissue of sheep, in an attempt to identify the host derived factors involved in immune exclusion of Teladorsagia circumcincta larvae. Proteins present in the epithelial cells and mucus from ovine abomasa following parasite challenge in previously naïve and immune animals were analysed through Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof)-MS and shotgun proteomics. MALDI-ToF analysis of epithelial cell lysates revealed that a number of proteins identified were differentially expressed in naïve and immune cells. These included intelectin and lysozymes, which were present at higher levels in epithelial cell lysates derived from immune samples. A large number of proteins were identified in the mucosal wash from immune tissue which were not present in the mucosal wash of the naïve tissue. Some of these proteins were present in washes of immune tissue prior to the parasite challenge including immunoglobulin A, galectin 14 and 15 and sheep mast cell protease 1. However, other proteins, such as calcium activated chloride channel and intelectin were only detected in the washings from the challenged tissue. The latter may be related to an enhanced mucus release, which may result in entrapment of infective larvae and thus reduced establishment in tissue that has been previously challenged with the parasite. In conclusion, several proteins have been identified which may be involved, either directly or indirectly, in the exclusion and immune elimination of incoming infective larvae. In the present study, the usefulness of the in vitro model has been confirmed, and the global proteomic approach has identified proteins that had not previously been associated with parasite exclusion from abomasal mucosa, such as the calcium activated chloride channel.

Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 microM for 24 h) toxicity was significantly attenuated from 27.3+/-3.9% in ARF6-WT to 11.1+/-4.0% in ARF6-DN cells (n=6, P<0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0+/-4.6% versus 3.9+/-3.9% of ARF6-WT cells, n=3, P<0.01) and/or remained near the PM (89.3+/-5. 6% versus 45.2+/-14.3% of ARF6-WT cells, n=3, P<0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.

Secretion of enzymes and fluid induced by Ca(2+) in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K(+) conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K(+) permeability (IC(50) of approximately 10 microM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E(rev)) of -20.9 +/- 0.9 mV, and a single-channel K(+) conductance (g(K)) of 265.8 +/- 44.0 pS (n = 39). Replacement of cis 500 mM K(+) by 500 mM Na(+) shifted E(rev) to -2.4 +/- 3.6 mV (n = 3), indicating K(+) selectivity. Single-channel analysis identified several K(+) channel groups with distinct channel behaviors. K(+) channels with a g(K) of 651.8 +/- 88.0 pS, E(rev) of -22.9 +/- 2.2 mV, and open probability (P(open)) of 0.43 +/- 0.06 at 0 mV (n = 6) and channels with a g(K) of 155.0 +/- 11.4 pS, E(rev) of -18.3 +/- 1.8 mV, and P(open) of 0.80 +/- 0.03 at 0 mV (n = 3) were inhibited by 100 microM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca(2+)-activated K(+) channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC(50) of approximately 10 microM). Thus KCNQ1 may account for ZG K(+) conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.

The cellular distribution of the NH2-terminal electrogenic Na+-HCO3(-) cotransporter (NBCe1) variants NBCe1-A and NBCe1-B has been investigated in rat kidney and submandibular gland (SMG) under physiological conditions and after systemic acid-base perturbations. Moreover, the in vivo data were complemented in vitro by using an immortalized cell line derived from the S1 segment of the proximal tubule (PT) of normotensive Wistar-Kyoto rats (WKPT-0293 Cl.2). NBCe1-A was basolaterally localized in PT cells, whereas NBCe1-B exhibited intracellular and basolateral distribution. SMG showed transcript and protein expression for NBCe1-A and NBCe1-B. NBCe1-B was basolaterally localized in duct cells; NBCe1-A was found intracellularly in salivary striated ducts and apically in main duct cells. Acute metabolic acidosis significantly increased cells that showed basolateral NBCe1-A in the PT, indicating increased HCO3(-) reabsorption, and significantly decreased cells that exhibited basolateral NBCe1-B in the salivary ducts, suggesting decreased HCO3(-) secretion. Chronic acidosis had no effect on NBCe1 distribution in PT but significantly increased the percentage of cells with basolateral NBCe1-A in salivary striated duct cells, suggesting increased HCO3(-) reabsorption. In contrast, chronic alkalosis caused adaptive redistribution of NBCe1-A and NBCe1-B in renal PT, favoring decreased HCO3(-) reabsorption. In vitro, WKPT-0293 Cl.2 cells expressed key acid-base transporters. Extracellular alkalosis downregulated NBCe1-A protein. WKPT-0293 Cl.2 cells are therefore a useful model to study renal acid-base regulation in vitro. The results propose redistribution of the transporters as a potential posttranslational regulation modus during acid-base disturbances. Moreover, the data demonstrate that renal PT and salivary duct epithelia respond to acid-base disturbances by an opposite redistribution pattern for NBCe1-A and NBCe1-B, reflecting specialized functions as the HCO3(-)-reabsorbing and HCO3(-)-secreting epithelium, respectively.

Cadmium (Cd2+) induces renal proximal tubular (PT) damage, including disruption of the E-cadherin/beta-catenin complex of adherens junctions (AJs) and apoptosis. Yet, chronic Cd2+ exposure causes malignant transformation of renal cells. Previously, we have demonstrated that Cd(2+)-mediated up-regulation of the multidrug transporter Abcb1 causes apoptosis resistance in PT cells. We hypothesized that Cd2+ activates adaptive signaling mechanisms mediated by beta-catenin to evade apoptosis and increase proliferation. Here we show that 50 microM Cd2+, which induces cell death via apoptosis and necrosis, also causes a decrease of the trans-epithelial resistance of confluent WKPT-0293 Cl.2 cells, a rat renal PT cell model, within 45 min of Cd2+ exposure, as measured by electric cell-substrate impedance sensing. Immunofluorescence microscopy demonstrates Cd(2+)-induced decrease of E-cadherin at AJs and redistribution of beta-catenin from the E-cadherin/beta-catenin complex of AJs to cytosol and nuclei after 3 h. Immunoblotting confirms Cd(2+)-induced decrease of E-cadherin expression and translocation of beta-catenin to cytosol and nuclei of PT cells. RT-PCR shows Cd(2+)-induced increase of expression of c-myc and of the isoform Abcb1a at 3 h. The data prove for the first time that Cd2+ induces nuclear translocation of beta-catenin in PT cells. We speculate that Cd2+ activates beta-catenin/T-cell factor signaling to trans-activate proliferation and apoptosis resistance genes and promote carcinogenesis of PT cells.

A major target of cadmium (Cd(2+)) toxicity is the kidney proximal tubule (PT) cell. Cd(2+)-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3-6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by Cd(2+). Upstream processes underlying Cd(2+)-induced calpain activation remain unclear. We describe for the first time that 10-50 microM Cd(2+) causes a significant increase in ceramide formation by approximately 22% (3 h) and approximately 72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B(1) (3 microM) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3-6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that Cd(2+) enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C(6)-ceramide to PT cells increases cytosolic Ca(2+) and activates calpains. Apoptosis mediated by C(6)-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that Cd(2+)-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca(2+)-dependent calpain activation, which propagates kidney damage by Cd(2+).

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3-6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10-50 microM cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10-50 microM) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by 10 microM cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 microg/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 microM cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 microM) blocked apoptosis by 10 microM cadmium at 3-6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure.

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO (3)(-) into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO (3)(-) concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO(3) concentration. We hypothesized that BDL might affect the expression of cholangiocyte H(+) transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H(+)-ATPase (V-ATPase) subunit and of Na(+)/H(+) exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow approximately threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in approximately 60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na(+) and HCO (3)(-) reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO (3)(-) salvage to compensate for NHE3 down-regulation in BDL.

The nephrotoxic metal Cd(2+) causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K(+) cycle involving a K(+) uniporter and a K(+)/H(+) exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd(2+) on K(+) cycling in rat kidney cortex mitochondria. Cd(2+) (EC(50) approximately 19 microM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl >> choline chloride. Cd(2+)-induced swelling of energized mitochondria had a similar EC(50) value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca(2+) uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd(2+) influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (DeltaPsi(m)) induced by K(+) influx did not prevent contraction, but addition of the K(+)/H(+) exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 microM), abolished contraction, suggesting the mitochondrial pH gradient (DeltapH(m)) driving contraction. Accordingly, a quinine-sensitive partial dissipation of DeltapH(m) was coincident with the swelling-contraction phase. The data indicate that Cd(2+) enters the matrix through the MCU to activate a K(+) cycle. Initial K(+) load via a Cd(2+)-activated K(+) uniporter in the IMM causes osmotic swelling and breakdown of DeltaPsi(m) and triggers quinine-sensitive K(+)/H(+) exchange and contraction. Thus Cd(2+)-induced activation of a K(+) cycle contributes to the dissipation of the mitochondrial protonmotive force.

Cd(2+) induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound-induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd(2+)-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 muM Cd(2+) induced approximately 5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosis-inducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd(2+) concentrations at 24 h, suggesting that elevated Cd(2+) may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd(2+) caused swelling of energized isolated kidney cortex mitochondria (EC(50) approximately 9 muM) and cytochrome c release, which were independent of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd(2+) inhibited swelling and cytochrome c release induced by PTP openers (PO(4)(3-) or H(2)O(2)+Ca(2+)). The mitochondrial Ca(2+) uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La(3+), ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd(2+) inhibition of PO(4)(3-)-induced swelling. Using the Cd(2+)-sensitive fluorescent indicator FluoZin-1, Cd(2+) was also taken up by mitoplasts. The aquaporin inhibitor AgNO(3) abolished Cd(2+)-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd(2+) concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane.

The expression of K+-Cl- cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in alpha-cells, but not pancreatic beta-cells nor delta-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both alpha-cell and beta-cells. Exposure of isolated alpha-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 microM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in beta-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of alpha-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on beta-cell volume. In conclusion, KCCs are expressed in pancreatic alpha-cells and beta-cells. However, they make a significant contribution to volume homeostasis only in alpha-cells.

The mechanisms of cadmium-metallothionein (CdMT) uptake and toxicity in proximal tubule (PT) cells are not well understood. The effects of 10 microM CdCl2 or Cd7MT-1 (MT-1 saturated with 10 microM CdCl2) on 109Cd2+ uptake, viability, and MT levels of cultured rat PT cells were investigated. Apical 109Cd2+ uptake was measured in confluent monolayers, apoptosis was assessed with Hoechst 33342, and intracellular MT levels were monitored by immunofluorescence and quantitative morphometry. 109Cd2+ uptake into PTC increased over time and plateaued at 24 h. 109Cd7MT-1 uptake was delayed but reached a similar magnitude after 40 h. With Cd2+, apoptosis occurred within 4 h, peaked at 24 h, and declined at 48-72 h. Cd7MT-1 induced apoptosis after 24-36 h, reaching similar levels as with Cd2+ after 48 h. Cd2+ and Cd7MT-1 significantly increased intracellular MT immunoreactivity after 20 and 4 h, respectively. The weak base chloroquine and the inhibitor of phosphatidylinositol 3-kinases, LY-294002, selectively inhibited the effects of Cd7MT-1 on MT immunoreactivity and apoptosis. PT cells accumulated 109Cd7MT-1 in membrane vesicles associated with the late endo/lysosomal marker LAMP1 but less with the early endosomal marker Rab5a, which was abolished by chloroquine or LY-294002. Thus development of apoptosis followed the uptake kinetics of Cd2+ and Cd7MT-1. Endo/lysosomal inhibitors prevented uptake of Cd7MT-1 into endo/lysosomes and apoptosis but had no effect on these parameters with Cd2+, suggesting that apoptosis of PT cells is triggered by free cytosolic Cd2+, either by direct apical transport or by translocation of free Cd2+ from endo/lysosomes after endocytosis of Cd7MT-1.

Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca(2+)-dependent and -independent "docking" and "fusion" proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl(-) channel, a HCO-permeable member of the CLCA Ca(2+)-dependent anion channel family, and a KCNQ1 K(+) channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during "kiss and run" recycling. In pancreatic beta-cells, a granular ClC-3 Cl(-) channel provides a shunt pathway for a vacuolar-type H(+)-ATPase. Acidification "primes" the granules for Ca(2+)-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus.

Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at ca 135 kDa and ca 150 kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl(-) transport.

Several reports indicate that hypoglycemic sulfonylureas augment Ca(2+)-dependent insulin secretion via mechanisms other than inhibition of the ATP-sensitive K(+) channel. The effect involves a 65-kd protein in the granule membrane and culminates in intragranular acidification. Lowering of granule pH is necessary for the insulin granule to gain release competence. Proton pumping into the granule is driven by a v-type H(+)-ATPase, but requires simultaneous Cl(-) uptake into the granule via metabolically regulated ClC-3 Cl(-) channels to maintain electroneutrality. Here we discuss the possibility that modulation of granule ClC-3 channels represents the mechanism whereby sulfonylureas directly potentiate the beta-cell exocytotic machinery.