57 results on '"Lee, Wing-Kee"'
Search Results
2. A novel transgenic mouse model highlights molecular disruptions involved in the pathogenesis of Dent disease 1.
- Author
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Sakhi IB, De Combiens E, Frachon N, Durussel F, Brideau G, Nemazanyy I, Frère P, Thévenod F, Lee WK, Zeng Q, Klein C, Lourdel S, and Bignon Y
- Subjects
- Animals, Mice, Dent Disease genetics, Kidney Tubules, Proximal metabolism, Kidney Tubules, Proximal pathology, Mutation, Missense, Humans, Lipocalin-2 genetics, Lipocalin-2 metabolism, Autophagy genetics, Apoptosis genetics, Genetic Diseases, X-Linked, Nephrolithiasis, Chloride Channels genetics, Chloride Channels metabolism, Disease Models, Animal, Mice, Transgenic
- Abstract
Dent disease (DD) is a hereditary renal disorder characterized by low molecular weight (LMW) proteinuria and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl
- /H+ exchanger ClC-5 have been identified in patients with DD type 1. ClC-5 is essentially expressed in proximal tubules (PT) where it is thought to play a role in maintaining an efficient endocytosis of LMW proteins. However, the exact pathological roles of ClC-5 in progressive dysfunctions observed in DD type 1 are still unclear. To address this issue, we designed a mouse model carrying the most representative type of ClC-5 missense mutations found in DD patients. These mice showed a characteristic DD type 1 phenotype accompanied by altered endo-lysosomal system and autophagy functions. With ageing, KI mice showed increased renal fibrosis, apoptosis and major changes in cell metabolic functions as already suggested in previous DD models. Furthermore, we made the interesting new discovery that the Lipocalin-2-24p3R pathway might be involved in the progression of the disease. These results suggest a crosstalk between the proximal and distal nephron in the pathogenesis mechanisms involved in DD with an initial PT impairment followed by the Lipocalin-2 internalisation and 24p3R overexpression in more distal segments of the nephron. This first animal model of DD carrying a pathogenic mutation of Clcn5 and our findings pave the way aimed at exploring therapeutic strategies to limit the consequences of ClC-5 disruption in patients with DD type 1 developing chronic kidney disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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3. Cadmium transport by mammalian ATP-binding cassette transporters.
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Thévenod F and Lee WK
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- Humans, Animals, Biological Transport, Cadmium metabolism, ATP-Binding Cassette Transporters metabolism, ATP-Binding Cassette Transporters genetics
- Abstract
Cellular responses to toxic metals depend on metal accessibility to intracellular targets, reaching interaction sites, and the intracellular metal concentration, which is mainly determined by uptake pathways, binding/sequestration and efflux pathways. ATP-binding cassette (ABC) transporters are ubiquitous in the human body-usually in epithelia-and are responsible for the transfer of indispensable physiological substrates (e.g. lipids and heme), protection against potentially toxic substances, maintenance of fluid composition, and excretion of metabolic waste products. Derailed regulation and gene variants of ABC transporters culminate in a wide array of pathophysiological disease states, such as oncogenic multidrug resistance or cystic fibrosis. Cadmium (Cd) has no known physiological role in mammalians and poses a health risk due to its release into the environment as a result of industrial activities, and eventually passes into the food chain. Epithelial cells, especially within the liver, lungs, gastrointestinal tract and kidneys, are particularly susceptible to the multifaceted effects of Cd because of the plethora of uptake pathways available. Pertinent to their broad substrate spectra, ABC transporters represent a major cellular efflux pathway for Cd and Cd complexes. In this review, we summarize current knowledge concerning transport of Cd and its complexes (mainly Cd bound to glutathione) by the ABC transporters ABCB1 (P-glycoprotein, MDR1), ABCB6, ABCC1 (multidrug resistance related protein 1, MRP1), ABCC7 (cystic fibrosis transmembrane regulator, CFTR), and ABCG2 (breast cancer related protein, BCRP). Potential detoxification strategies underlying ABC transporter-mediated efflux of Cd and Cd complexes are discussed., (© 2024. The Author(s).)
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- 2024
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4. Global threat posed by metals and metalloids in the changing environment: a One Health approach to mechanisms of toxicity.
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Lee WK, Thévenod F, and Prenner EJ
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- Humans, One Health, Environmental Pollutants toxicity, Animals, Metalloids toxicity, Metals chemistry
- Published
- 2024
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5. Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line.
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Lee WK, Probst S, Scharner B, Deba T, Dahdouh F, and Thévenod F
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- Rats, Animals, Reactive Oxygen Species metabolism, Catalase metabolism, Catalase pharmacology, Hydrogen Peroxide metabolism, alpha-Tocopherol metabolism, alpha-Tocopherol pharmacology, Superoxide Dismutase-1 metabolism, Superoxide Dismutase-1 pharmacology, Oxidative Stress, Antioxidants pharmacology, Antioxidants metabolism, Kidney, Superoxide Dismutase metabolism, Cell Line, Cadmium toxicity, Superoxides metabolism, Cyclic N-Oxides, Metalloporphyrins, Spin Labels
- Abstract
Levels and chemical species of reactive oxygen/nitrogen species (ROS/RNS) determine oxidative eustress and distress. Abundance of uptake pathways and high oxygen consumption for ATP-dependent transport makes the renal proximal tubule particularly susceptible to cadmium (Cd
2+ )-induced oxidative stress by targeting ROS/RNS generation or antioxidant defence mechanisms, such as superoxide dismutase (SOD) or H2 O2 -metabolizing catalase (CAT). Though ROS/RNS are well-evidenced, the role of distinct ROS profiles in Cd2+ concentration-dependent toxicity is not clear. In renal cells, Cd2+ (10-50 µM) oxidized dihydrorhodamine 123, reaching a maximum at 2-3 h. Increases (up to fourfold) in lipid peroxidation by TBARS assay and H2 O2 by Amplex Red were evident within 30 min. ROS and loss in cell viability by MTT assay with 50 µM Cd2+ could not be fully reversed by SOD mimetics Tempol and MnTBAP nor by SOD1 overexpression, whereas CAT expression and α-tocopherol were effective. SOD and CAT activities were attenuated below controls only with >6 h 50 µM Cd2+ , yet augmented by up to 1.5- and 1.2-fold, respectively, by 10 µM Cd2+ . Moreover, 10 µM, but not 25-50 µM Cd2+ , caused 1.7-fold increase in superoxide anion (O2 •- ), detected by dihydroethidium, paralled by loss in cell viability, that was abolished by Tempol, MnTBAP, α-tocopherol and SOD1 or CAT overexpression. H2 O2 -generating NADPH oxidase 4 (NOX4) was attenuated by ~50% with 10 µM Cd2+ at 3 h compared to upregulation by 50 µM Cd2+ (~1.4-fold, 30 min), which was sustained for 24 h. In summary, O2 •- predominates with low-moderate Cd2+ , driving an adaptive response, whereas oxidative stress by elevated H2 O2 at high Cd2+ triggers cell death signaling pathways.Highlights Different levels of reactive oxygen species are generated, depending on cadmium concentration. Superoxide anion predominates and H2 O2 is suppressed with low cadmium representing oxidative eustress. High cadmium fosters H2 O2 by inhibiting catalase and increasing NOX4 leading to oxidative distress. Superoxide dismutase mimetics and overexpression were less effective with high versus low cadmium. Oxidative stress profile could dictate downstream signalling pathways., (© 2024. The Author(s).)- Published
- 2024
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6. Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.
- Author
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Thévenod F, Herbrechter R, Schlabs C, Pethe A, Lee WK, Wolff NA, and Roussa E
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- Humans, Lipocalin-2 metabolism, Cadmium metabolism, Iron metabolism, Metallothionein metabolism, Kidney Tubules, Proximal metabolism, Proteinuria metabolism, Endocytosis, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Organic Cation Transport Proteins metabolism, Metalloproteins metabolism, Nephrosis metabolism
- Abstract
The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.
- Published
- 2023
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7. [Pneumology in the Model Degree Program of the Medical Faculty Ostwestfalen Lippe, Germany].
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Schönhofer B, Garhy M, Bittner AB, Frankewitsch TF, Lätzsch R, Lee WK, Mertzlufft F, Moser FM, and Hornberg C
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- Humans, Germany, Faculty, Medical, Pulmonary Medicine
- Abstract
The specialist field of "pneumology" is still underrepresented in university clinics in Germany, but this is not the case at the newly founded medical faculty Ostwestfalen-Lippe (OWL) in Bielefeld. This is linked to representing pneumology and internal intensive care medicine in patient care, teaching and research across the board and the opportunity to actively help shape the development of the human medicine faculty in an exciting environment.The early anchoring of the subject "Pneumology" in the model degree program of medical school in OWL (begin winter semester 2021/22) contributes to further visibility and a university medical orientation. In this overview various issues of Pneumology in the Model Degree Program are explored by basic scientists, clinical teachers, members of the medical faculty and a student.In today's Evangelisches Klinikum Bethel (EvKB), pulmonary medicine has a long tradition. The hospital's first lung and infection center was opened in 1927. The EvKB's department for internal medicine, pneumology and intensive care medicine, which has been independent since 2009, is becoming a university clinic for pneumology within the medical faculty OWL. Relevant translational and interdisciplinary research can be intensified.There are 30 "Pneumology" teaching units in the model degree program, which are divided into two study sections using different formats, such as lectures, seminars, hands on courses and skills lab. It is represented in particular in the module complex "Circulation and Respiration". The content of the first phase of teaching was carried out by a module commission, with members representing the subjects involved in the module.Knowledge of the basics from, for example, physiology, pathophysiology, anatomy and pathology are taught to the students in the run-up to the pneumology course. Using the example of physiology, the presentation of the learning content of a basic subject is elaborated in this article.Half of all teaching units on pneumology of the entire course took place in the 2nd semester (in March and April 2022), so that students experienced the clinical relevance of the content at an early stage. There was a particular focus on obstructive airway and restrictive lung diseases. After imparting the basic knowledge of the physical examination of the lungs in the Skills Lab, the most important pathological findings in the above-mentioned diseases on inspection, palpation, auscultation and percussion are demonstrated and practised in patients as part of bedside teaching under supervision.Communication training is also longitudinally integrated into the modular teaching, with a total of more than 200 teaching hours and is performed interdisciplinary. In the "Circulation and Breathing" module eight hours are devoted to this with simulated patients, the anamnesis and therapy advice on classic cardiopulmonary diseases. For the students, integrating the teaching of basic theory and its clinical application for each organ systems represents a challenge in the model degree program, the advantages outweigh from today's perspective., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)
- Published
- 2022
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8. Renal hypoxia-HIF-PHD-EPO signaling in transition metal nephrotoxicity: friend or foe?
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Thévenod F, Schreiber T, and Lee WK
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- Humans, Hypoxia metabolism, Hypoxia pathology, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Kidney metabolism, Oxygen metabolism, Transcription Factors metabolism, Erythropoietin metabolism, Kidney Diseases pathology
- Abstract
The kidney is the main organ that senses changes in systemic oxygen tension, but it is also the key detoxification, transit and excretion site of transition metals (TMs). Pivotal to oxygen sensing are prolyl-hydroxylases (PHDs), which hydroxylate specific residues in hypoxia-inducible factors (HIFs), key transcription factors that orchestrate responses to hypoxia, such as induction of erythropoietin (EPO). The essential TM ion Fe is a key component and regulator of the hypoxia-PHD-HIF-EPO (HPHE) signaling axis, which governs erythropoiesis, angiogenesis, anaerobic metabolism, adaptation, survival and proliferation, and hence cell and body homeostasis. However, inadequate concentrations of essential TMs or entry of non-essential TMs in organisms cause toxicity and disrupt health. Non-essential TMs are toxic because they enter cells and displace essential TMs by ionic and molecular mimicry, e. g. in metalloproteins. Here, we review the molecular mechanisms of HPHE interactions with TMs (Fe, Co, Ni, Cd, Cr, and Pt) as well as their implications in renal physiology, pathophysiology and toxicology. Some TMs, such as Fe and Co, may activate renal HPHE signaling, which may be beneficial under some circumstances, for example, by mitigating renal injuries from other causes, but may also promote pathologies, such as renal cancer development and metastasis. Yet some other TMs appear to disrupt renal HPHE signaling, contributing to the complex picture of TM (nephro-)toxicity. Strikingly, despite a wealth of literature on the topic, current knowledge lacks a deeper molecular understanding of TM interaction with HPHE signaling, in particular in the kidney. This precludes rationale preventive and therapeutic approaches to TM nephrotoxicity, although recently activators of HPHE signaling have become available for therapy., (© 2022. The Author(s).)
- Published
- 2022
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9. Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer.
- Author
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Lee WK, Maaß M, Quach A, Poscic N, Prangley H, Pallott EC, Kim JL, Pierce JS, Ogretmen B, Futerman AH, and Thévenod F
- Subjects
- Ceramides metabolism, Doxorubicin pharmacology, Drug Resistance, Neoplasm, Endoplasmic Reticulum-Associated Degradation, Female, Humans, Male, RNA, Messenger genetics, Tandem Mass Spectrometry, Tumor Microenvironment, ATP Binding Cassette Transporter, Subfamily B biosynthesis, ATP Binding Cassette Transporter, Subfamily B genetics, ATP Binding Cassette Transporter, Subfamily B metabolism, Kidney Neoplasms drug therapy, Kidney Neoplasms genetics, Kidney Neoplasms metabolism, Membrane Proteins metabolism, Sphingolipids metabolism, Sphingosine N-Acyltransferase genetics, Sphingosine N-Acyltransferase metabolism, Tumor Suppressor Proteins
- Abstract
Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1
+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1 ) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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10. Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium.
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Probst S, Fels J, Scharner B, Wolff NA, Roussa E, van Swelm RPL, Lee WK, and Thévenod F
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- Animals, Apoptosis drug effects, Binding Sites, Binding, Competitive, Cadmium administration & dosage, Cell Death drug effects, Cell Line, Cells, Cultured, Deferoxamine pharmacology, Female, Gene Silencing, Iron administration & dosage, Kidney Tubules, Collecting cytology, Kidney Tubules, Collecting drug effects, Male, Mice, Mice, Inbred C57BL, Reactive Oxygen Species metabolism, Cadmium toxicity, Hepcidins genetics, Iron toxicity, Oxidative Stress drug effects
- Abstract
The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe
2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3 ] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5-5 μmol/l) and/or Fe2+ (50-100 μmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+ -induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+ -induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+ , but not Cd2+ . Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate., (© 2021. The Author(s).)- Published
- 2021
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11. Increased Endocytosis of Cadmium-Metallothionein through the 24p3 Receptor in an In Vivo Model with Reduced Proximal Tubular Activity.
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Zavala-Guevara IP, Ortega-Romero MS, Narváez-Morales J, Jacobo-Estrada TL, Lee WK, Arreola-Mendoza L, Thévenod F, and Barbier OC
- Subjects
- Animals, Male, Mice, Mice, Inbred C57BL, Nephrons metabolism, Cadmium metabolism, Endocytosis physiology, Kidney Tubules, Proximal metabolism, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Metallothionein metabolism, Receptors, Cell Surface metabolism
- Abstract
Background: The proximal tubule (PT) is the major target of cadmium (Cd
2+ ) nephrotoxicity. Current dogma postulates that Cd2+ complexed to metallothionein (MT) (CdMT) is taken up through receptor-mediated endocytosis (RME) via the PT receptor megalin:cubilin, which is the predominant pathway for reuptake of filtered proteins in the kidney. Nevertheless, there is evidence that the distal parts of the nephron are also sensitive to damage induced by Cd2+ . In rodent kidneys, another receptor for protein endocytosis, the 24p3 receptor (24p3R), is exclusively expressed in the apical membranes of distal tubules (DT) and collecting ducts (CD). Cell culture studies have demonstrated that RME and toxicity of CdMT and other (metal ion)-protein complexes in DT and CD cells is mediated by 24p3R. In this study, we evaluated the uptake of labeled CdMT complex through 24p3R after acute kidney injury (AKI) induced by gentamicin (GM) administration that disrupts PT function. Subcutaneous administration of GM at 10 mg/kg/day for seven days did not alter the structural and functional integrity of the kidney's filtration barrier. However, because of PT injury, the concentration of the renal biomarker Kim-1 increased. When CdMT complex coupled to FITC was administered intravenously, both uptake of the CdMT complex and 24p3R expression in DT increased and also colocalized after PT injury induced by GM. Although megalin decreased in PT after GM administration, urinary protein excretion was not changed, which suggests that the increased levels of 24p3R in the distal nephron could be acting as a compensatory mechanism for protein uptake. Altogether, these results suggest that PT damage increases the uptake of the CdMT complex through 24p3R in DT (and possibly CD) and compensate for protein losses associated with AKI.- Published
- 2021
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12. Teaching an old dog new tricks: reactivated developmental signaling pathways regulate ABCB1 and chemoresistance in cancer.
- Author
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Lee WK and Frank T
- Abstract
Oncogenic multidrug resistance (MDR) is a multifactorial phenotype intimately linked to deregulated expression of detoxification transporters. Drug efflux transporters, particularly the MDR P-glycoprotein ABCB1, represent a central mechanism by which not only chemotherapeutic drugs are extruded or sequestered to prevent drug delivery to their intracellular targets, but also for inhibiting apoptotic cell death cues, such as removal of proapoptotic signals. Several cell populations exhibiting the MDR phenotype co-exist within a tumor, such as cells forming the bulk tumor cell mass, cancer stem cells, and cancer persister cells. The key to regulation of ABCB1 expression is the cellular transcriptional machinery. Developmental signaling pathways (e.g, Hedgehog, Notch, Wnt/β-catenin, TGFβ, PITX2) are pivotal in governing cell proliferation, survival, differentiation and guiding cell migration during embryogenesis, and their reactivation during carcinogenesis, which is of particular significance for tumor initiation, progression, and metastasis, also leads to the upregulation of ABCB1. These pathways also drive and maintain cancer cell stemness, for which ABCB1 is used as a marker. In this review, the contribution of canonical and non-canonical developmental signaling pathways in transcriptional regulation of ABCB1 to confer MDR in cancer is delineated., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2021.)
- Published
- 2021
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13. Corrigendum: Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.
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Thévenod F, Lee WK, and Garrick MD
- Abstract
[This corrects the article DOI: 10.3389/fcell.2020.00848.]., (Copyright © 2021 Thévenod, Lee and Garrick.)
- Published
- 2021
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14. Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications.
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Thévenod F, Lee WK, and Garrick MD
- Abstract
Regulation of body fluid homeostasis is a major renal function, occurring largely through epithelial solute transport in various nephron segments driven by Na
+ /K+ -ATPase activity. Energy demands are greatest in the proximal tubule and thick ascending limb where mitochondrial ATP production occurs through oxidative phosphorylation. Mitochondria contain 20-80% of the cell's iron, copper, and manganese that are imported for their redox properties, primarily for electron transport. Redox reactions, however, also lead to reactive, toxic compounds, hence careful control of redox-active metal import into mitochondria is necessary. Current dogma claims the outer mitochondrial membrane (OMM) is freely permeable to metal ions, while the inner mitochondrial membrane (IMM) is selectively permeable. Yet we recently showed iron and manganese import at the OMM involves divalent metal transporter 1 (DMT1), an H+ -coupled metal ion transporter. Thus, iron import is not only regulated by IMM mitoferrins, but also depends on the OMM to intermembrane space H+ gradient. We discuss how these mitochondrial transport processes contribute to renal injury in systemic (e.g., hemochromatosis) and local (e.g., hemoglobinuria) iron overload. Furthermore, the environmental toxicant cadmium selectively damages kidney mitochondria by "ionic mimicry" utilizing iron and calcium transporters, such as OMM DMT1 or IMM calcium uniporter, and by disrupting the electron transport chain. Consequently, unraveling mitochondrial metal ion transport may help develop new strategies to prevent kidney injury induced by metals., (Copyright © 2020 Thévenod, Lee and Garrick.)- Published
- 2020
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15. Cell organelles as targets of mammalian cadmium toxicity.
- Author
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Lee WK and Thévenod F
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- Animals, Humans, Mammals, Organelles physiology, Plastics, Signal Transduction, Cadmium toxicity, Environmental Pollutants toxicity, Organelles drug effects
- Abstract
Ever increasing environmental presence of cadmium as a consequence of industrial activities is considered a health hazard and is closely linked to deteriorating global health status. General animal and human cadmium exposure ranges from ingestion of foodstuffs sourced from heavily polluted hotspots and cigarette smoke to widespread contamination of air and water, including cadmium-containing microplastics found in household water. Cadmium is promiscuous in its effects and exerts numerous cellular perturbations based on direct interactions with macromolecules and its capacity to mimic or displace essential physiological ions, such as iron and zinc. Cell organelles use lipid membranes to form complex tightly-regulated, compartmentalized networks with specialized functions, which are fundamental to life. Interorganellar communication is crucial for orchestrating correct cell behavior, such as adaptive stress responses, and can be mediated by the release of signaling molecules, exchange of organelle contents, mechanical force generated through organelle shape changes or direct membrane contact sites. In this review, cadmium effects on organellar structure and function will be critically discussed with particular consideration to disruption of organelle physiology in vertebrates.
- Published
- 2020
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16. Inverse Regulation of Lipocalin-2/24p3 Receptor/SLC22A17 and Lipocalin-2 Expression by Tonicity, NFAT5/TonEBP and Arginine Vasopressin in Mouse Cortical Collecting Duct Cells mCCD(cl.1): Implications for Osmotolerance.
- Author
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Probst S, Scharner B, McErlean R, Lee WK, and Thévenod F
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- Animals, Cell Line, Gene Expression Regulation drug effects, Kidney Cortex drug effects, Kidney Cortex metabolism, Kidney Tubules, Collecting drug effects, Kidney Tubules, Collecting metabolism, Ligands, Mice, Osmolar Concentration, Rats, Signal Transduction drug effects, Toll-Like Receptor 4 metabolism, Up-Regulation, Arginine Vasopressin pharmacology, Kidney Cortex cytology, Kidney Tubules, Collecting cytology, Lipocalin-2 metabolism, Organic Cation Transport Proteins metabolism, Transcription Factors metabolism
- Abstract
The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) apically, possibly to mediate high-affinity reabsorption of filtered proteins by endocytosis, although its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates SLC22A17 in cultured mouse inner-medullary CD cells, whereas activation of toll-like receptor 4 (TLR4), via bacterial lipopolysaccharides (LPS), downregulates SLC22A17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP), and downregulation by TLR4 signaling, which occur via the transcription factors NFAT5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity and AVP, and their associated signaling pathways, on the expression of SLC22A17 and its ligand, LCN2, in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity corresponded to 300 mosmol/L, whereas the addition of 50-100 mmol/L NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/L). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17 /SLC22A17 and Lcn2 /LCN2 expression. RNAi silenced Nfat5 , and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 was induced with LPS. Similar to Aqp2 , hyperosmotic/-tonic media and AVP upregulated Slc22a17 /SLC22A17, via activation of NFAT5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17 /SLC22A17. Conversely, though NFAT5 mediated the hyperosmolarity/-tonicity induced downregulation of Lcn2 /LCN2 expression, AVP reduced Lcn2 /LCN2 expression and predominantly apical LCN2 secretion, evoked by LPS, through a posttranslational mode of action that was independent of CREB signaling. In conclusion, the hyperosmotic/-tonic upregulation of SLC22A17 in mCCD(cl.1) cells, via NFAT5, and by AVP, via CREB, suggests that SLC22A17 contributes to adaptive osmotolerance, whereas LCN2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.
- Published
- 2019
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17. Channels, transporters and receptors for cadmium and cadmium complexes in eukaryotic cells: myths and facts.
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Thévenod F, Fels J, Lee WK, and Zarbock R
- Subjects
- Cadmium pharmacology, Cell Membrane drug effects, Cell Membrane metabolism, Coordination Complexes pharmacology, Eukaryotic Cells drug effects, Humans, Amino Acid Transport Systems metabolism, Cadmium metabolism, Coordination Complexes metabolism, Eukaryotic Cells metabolism, Ion Channels metabolism, Receptors, Cell Surface metabolism
- Abstract
Cadmium (Cd
2+ ) is a toxic and non-essential divalent metal ion in eukaryotic cells. Cells can only be targeted by Cd2+ if it hijacks physiological high-affinity entry pathways, which transport essential divalent metal ions in a process termed "ionic and molecular mimicry". Hence, "free" Cd2+ ions and Cd2+ complexed with small organic molecules are transported across cellular membranes via ion channels, carriers and ATP hydrolyzing pumps, whereas receptor-mediated endocytosis (RME) internalizes Cd2+ -protein complexes. Only Cd2+ transport pathways validated by stringent methodology, namely electrophysiology,109 Cd2+ tracer studies, inductively coupled plasma mass spectrometry, atomic absorption spectroscopy, Cd2+ -sensitive fluorescent dyes, or specific ligand binding and internalization assays for RME are reviewed whereas indirect correlative studies are excluded. At toxicologically relevant concentrations in the submicromolar range, Cd2+ permeates voltage-dependent Ca2+ channels ("T-type" CaV 3.1, CatSper), transient receptor potential (TRP) channels (TRPA1, TRPV5/6, TRPML1), solute carriers (SLCs) (DMT1/SLC11A2, ZIP8/SLC39A8, ZIP14/SLC39A14), amino acid/cystine transporters (SLC7A9/SLC3A1, SLC7A9/SLC7A13), and Cd2+ -protein complexes are endocytosed by the lipocalin-2/NGAL receptor SLC22A17. Cd2+ transport via the mitochondrial Ca2+ uniporter, ATPases ABCC1/2/5 and transferrin receptor 1 is likely but requires further evidence. Cd2+ flux occurs through the influx carrier OCT2/SLC22A2, efflux MATE proteins SLC47A1/A2, the efflux ATPase ABCB1, and RME of Cd2+ -metallothionein by the receptor megalin (low density lipoprotein receptor-related protein 2, LRP2):cubilin albeit at high concentrations thus questioning their relevance in Cd2+ loading. Which Cd2+ -protein complexes are internalized by megalin:cubilin in vivo still needs to be determined. A stringent conservative and reductionist approach is mandatory to verify relevance of transport pathways for Cd2+ toxicity and to overcome dissemination of unsubstantiated conjectures.- Published
- 2019
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18. Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule.
- Author
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Fels J, Scharner B, Zarbock R, Zavala Guevara IP, Lee WK, Barbier OC, and Thévenod F
- Subjects
- Animals, Cadmium pharmacology, Cadmium Poisoning, Cell Line, Kidney Tubules, Proximal cytology, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Metallothionein metabolism, Protein Binding, Rats, Receptors, Cell Surface metabolism, Albumins metabolism, Cadmium toxicity, Kidney Tubules, Proximal drug effects, Lipocalin-2 metabolism, beta 2-Microglobulin metabolism
- Abstract
Cadmium (Cd
2+ ) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+ -metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+ -metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105 -times higher than (Cd2+ )-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+ -binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+ -protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+ -protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+ -metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+ -β2-microglobulin, Cd2+ -albumin and Cd2+ -lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+ -protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.- Published
- 2019
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19. Oncogenic PITX2 facilitates tumor cell drug resistance by inverse regulation of hOCT3/SLC22A3 and ABC drug transporters in colon and kidney cancers.
- Author
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Lee WK and Thévenod F
- Subjects
- ATP Binding Cassette Transporter 1 genetics, ATP Binding Cassette Transporter, Subfamily G, Member 2 genetics, Antineoplastic Agents pharmacology, Caco-2 Cells, Cell Proliferation drug effects, Colonic Neoplasms drug therapy, Colonic Neoplasms genetics, Doxorubicin pharmacology, Gene Expression Regulation, Neoplastic, Glycogen Synthase Kinase 3 metabolism, Homeodomain Proteins genetics, Humans, Kidney Neoplasms drug therapy, Kidney Neoplasms genetics, Neoplasm Proteins genetics, Organic Cation Transport Proteins genetics, Phosphorylation, Signal Transduction, Transcription Factors genetics, Vincristine pharmacology, Homeobox Protein PITX2, ATP Binding Cassette Transporter 1 metabolism, ATP Binding Cassette Transporter, Subfamily G, Member 2 metabolism, Colonic Neoplasms metabolism, Drug Resistance, Multiple genetics, Drug Resistance, Neoplasm genetics, Homeodomain Proteins metabolism, Kidney Neoplasms metabolism, Neoplasm Proteins metabolism, Organic Cation Transport Proteins metabolism, Transcription Factors metabolism
- Abstract
Oncogenic pituitary homeobox 2 (PITX2), a de facto master regulator of developmental organ asymmetry, previously upregulated multidrug resistance (MDR) P-glycoprotein ABCB1 in A498 renal cell carcinoma (RCC) cells. The role of PITX2 isoforms in MDR cancers was investigated. Data mining correlated elevated PITX2 in >30% of cancers analyzed, maximally in colon (4.4-fold), confirmed in co-immunostaining of colon and renal cancer microarrays wherein ABCB1 concomitantly increased in RCC. Drug-resistant colorectal adenocarcinoma Colo320DM cells exhibited increased nuclear PITX2 (40-fold), PITX2 promoter activity (27-fold) and ABCB1 (8000-fold) compared to drug-sensitive Colo205. ABCB1 inhibitor PSC833/valspodar or PITX2 siRNA reversed doxorubicin resistance. Nuclei from Colo320DM and A498 cells harbored PITX2A/B1 and PITX2A/B1/B2/Cα/Cβ, respectively. ChIP-qPCR evidenced PITX2 promoter binding in drug exporters ABCB1, ABCC1, ABCG2 and importer hOCT3/SLC22A3. In A498, 786-O, Caki-1, Colo320DM, and Caco2 cells, PITX2 siRNA diminished exporters, increased hOCT3/SLC22A3 expression and activity, and reverted vincristine resistance. Heterologous PITX2 expression induced ABCB1, repressed hOCT3/SLC22A3, enhanced vincristine resistance and diminished proliferation inhibition wherein PITX2A and PITX2C were most effective. Furthermore, PITX2 activity and MDR depended on phosphorylation by GSK3 in A498 cells. Conclusively, oncogenic PITX2 limits sensitizing drug uptake and potentiates cytoprotective drug efflux, contributing to MDR phenotype., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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20. Proximal tubule transferrin uptake is modulated by cellular iron and mediated by apical membrane megalin-cubilin complex and transferrin receptor 1.
- Author
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Smith CP, Lee WK, Haley M, Poulsen SB, Thévenod F, and Fenton RA
- Subjects
- Animals, Cell Line, Transformed, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Inbred WKY, Iron metabolism, Kidney Tubules, Proximal metabolism, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Receptors, Cell Surface metabolism, Receptors, Transferrin metabolism, Transferrin metabolism
- Abstract
Receptor-mediated endocytosis is responsible for reabsorption of transferrin (Tf) in renal proximal tubules (PTs). Although the role of the megalin-cubilin receptor complex (MCRC) in this process is unequivocal, modalities independent of this complex are evident but as yet undefined. Here, using immunostaining and Tf-flux assays, FACS analysis, and fluorescence imaging, we report localization of Tf receptor 1 (TfR1), the cognate Tf receptor mediating cellular holo-Tf (hTf) acquisition, to the apical brush border of the PT, with expression gradually declining along the PT in mouse and rat kidneys. In functional studies, hTf uptake across the apical membrane of cultured PT epithelial cell (PTEC) monolayers increased in response to decreased cellular iron after desferrioxamine (DFO) treatment. We also found that apical hTf uptake under basal conditions is receptor-associated protein (RAP)-sensitive and therefore mediated by the MCRC but becomes RAP-insensitive under DFO treatment, with concomitantly decreased megalin and cubilin expression levels and increased TfR1 expression. Thus, as well as the MCRC, TfR1 mediates hTf uptake across the PT apical brush border, but in conditions of decreased cellular iron, hTf uptake is predominated by augmented apical TfR1. In conclusion, both the MCRC and TfR1 mediate hTf uptake across apical brush border membranes of PTECs and reciprocally respond to decreased cellular iron. Our findings have implications for renal health, whole-body iron homeostasis, and pathologies arising from disrupted iron balance., (© 2019 Smith et al.)
- Published
- 2019
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21. Sphingolipid abnormalities in cancer multidrug resistance: Chicken or egg?
- Author
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Lee WK and Kolesnick RN
- Subjects
- Humans, Membrane Microdomains metabolism, Phenotype, Sphingolipids biosynthesis, Drug Resistance, Multiple, Drug Resistance, Neoplasm, Neoplasms metabolism, Sphingolipids metabolism
- Abstract
The cancer multidrug resistance (MDR) phenotype encompasses a myriad of molecular, genetic and cellular alterations resulting from progressive oncogenic transformation and selection. Drug efflux transporters, in particular the MDR P-glycoprotein ABCB1, play an important role in MDR but cannot confer the complete phenotype alone indicating parallel alterations are prerequisite. Sphingolipids are essential constituents of lipid raft domains and directly participate in functionalization of transmembrane proteins, including providing an optimal lipid microenvironment for multidrug transporters, and are also perturbed in cancer. Here we postulate that increased sphingomyelin content, developing early in some cancers, recruits and functionalizes plasma membrane ABCB1 conferring a state of partial MDR, which is completed by glycosphingolipid disturbance and the appearance of intracellular vesicular ABCB1. In this review, the independent and interdependent roles of sphingolipid alterations and ABCB1 upregulation during the transformation process and resultant conferment of partial and complete MDR phenotypes are discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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22. Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17.
- Author
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Cabedo Martinez AI, Weinhäupl K, Lee WK, Wolff NA, Storch B, Żerko S, Konrat R, Koźmiński W, Breuker K, Thévenod F, and Coudevylle N
- Subjects
- Acute-Phase Proteins genetics, Acute-Phase Proteins metabolism, Animals, CHO Cells, Cricetinae, Cricetulus, Humans, Lipocalin-2, Lipocalins genetics, Lipocalins metabolism, Mice, Nuclear Magnetic Resonance, Biomolecular, Organic Cation Transport Proteins genetics, Organic Cation Transport Proteins metabolism, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Acute-Phase Proteins chemistry, Lipocalins chemistry, Organic Cation Transport Proteins chemistry, Proto-Oncogene Proteins chemistry
- Abstract
The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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23. Glutathione and mitochondria determine acute defense responses and adaptive processes in cadmium-induced oxidative stress and toxicity of the kidney.
- Author
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Nair AR, Lee WK, Smeets K, Swennen Q, Sanchez A, Thévenod F, and Cuypers A
- Subjects
- Animals, Apoptosis drug effects, Cadmium Chloride administration & dosage, Caspase 3 metabolism, Cell Line, Dose-Response Relationship, Drug, Female, Kidney drug effects, Kidney pathology, Kidney Tubules, Proximal drug effects, Kidney Tubules, Proximal pathology, Male, Mitochondria metabolism, Rats, Rats, Inbred F344, Rats, Inbred WKY, Reactive Oxygen Species metabolism, Cadmium Chloride toxicity, Glutathione metabolism, Mitochondria drug effects, Oxidative Stress drug effects
- Abstract
Cadmium (Cd(2+)) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd(2+) toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd(2+)-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd(2+) toxicity (1-30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd(2+) toxicity (1 mg/kg CdCl2 subcutaneously, 13 days). Whereas 30 µM Cd(2+) caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd(2+) in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd(2+) (1-10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd(2+)-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd(2+) exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd(2+) in renal PTCs as well as in adaptive processes associated with chronic renal Cd(2+) toxicity.
- Published
- 2015
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24. Live and Let Die: Roles of Autophagy in Cadmium Nephrotoxicity.
- Author
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Thévenod F and Lee WK
- Abstract
The transition metal ion cadmium (Cd
2+ ) is a significant environmental contaminant. With a biological half-life of ~20 years, Cd2+ accumulates in the kidney cortex, where it particularly damages proximal tubule (PT) cells and can result in renal fibrosis, failure, or cancer. Because death represents a powerful means by which cells avoid malignant transformation, it is crucial to clearly identify and understand the pathways that determine cell fate in chronic Cd2+ nephrotoxicity. When cells are subjected to stress, they make a decision to adapt and survive, or-depending on the magnitude and duration of stress-to die by several modes of death (programmed cell death), including autophagic cell death (ACD). Autophagy is part of a larger system of intracellular protein degradation and represents the channel by which organelles and long-lived proteins are delivered to the lysosome for degradation. Basal autophagy levels in all eukaryotic cells serve as a dynamic physiological recycling system, but they can also be induced by intra- or extracellular stress and pathological processes, such as endoplasmic reticulum (ER) stress. In a context-dependent manner, autophagy can either be protective and hence contribute to survival, or promote death by non-apoptotic or apoptotic pathways. So far, the role of autophagy in Cd2+ -induced nephrotoxicity has remained unsettled due to contradictory results. In this review, we critically survey the current literature on autophagy in Cd2+ -induced nephrotoxicity in light of our own ongoing studies. Data obtained in kidney cells illustrate a dual and complex function of autophagy in a stimulus- and time-dependent manner that possibly reflects distinct outcomes in vitro and in vivo . A better understanding of the context-specific regulation of cell fate by autophagy may ultimately contribute to the development of preventive and novel therapeutic strategies for acute and chronic Cd2+ nephrotoxicity.- Published
- 2015
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25. Rapamycin: a therapy of choice for endoplasmic reticulum stress-induced renal proximal tubule toxicity?
- Author
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Thévenod F, Lee WK, and Wolff NA
- Subjects
- Animals, Cell Survival drug effects, Cell Survival physiology, Endoplasmic Reticulum Stress physiology, Fanconi Syndrome chemically induced, Fanconi Syndrome immunology, Humans, Immunosuppressive Agents pharmacology, Kidney Tubules, Proximal drug effects, Kidney Tubules, Proximal immunology, Sirolimus pharmacology, Endoplasmic Reticulum Stress drug effects, Fanconi Syndrome drug therapy, Immunosuppressive Agents therapeutic use, Sirolimus therapeutic use
- Published
- 2015
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26. Cytosolic calcium measurements in renal epithelial cells by flow cytometry.
- Author
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Lee WK and Dittmar T
- Subjects
- Animals, Calcium analysis, Calcium Ionophores pharmacology, Cell Adhesion physiology, Coloring Agents chemistry, Cytosol chemistry, Cytosol drug effects, Epithelial Cells chemistry, Epithelial Cells metabolism, Ionomycin pharmacology, Kidney Tubules, Proximal chemistry, Kidney Tubules, Proximal cytology, Kidney Tubules, Proximal drug effects, Kidney Tubules, Proximal metabolism, Lactones pharmacology, Rats, Sesquiterpenes pharmacology, Signal Transduction drug effects, Tunicamycin pharmacology, Calcium metabolism, Cytosol metabolism, Flow Cytometry methods
- Abstract
A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.
- Published
- 2014
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27. Dynamic changes in saliva after acute mental stress.
- Author
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Naumova EA, Sandulescu T, Bochnig C, Al Khatib P, Lee WK, Zimmer S, and Arnold WH
- Subjects
- Adult, Biological Availability, Cross-Over Studies, Fluorides metabolism, Humans, Hydrocortisone metabolism, Hydrogen-Ion Concentration, Male, Young Adult, Saliva metabolism, Stress, Psychological metabolism
- Abstract
Stress-related variations of fluoride concentration in supernatant saliva and salivary sediment, salivary cortisol, total protein and pH after acute mental stress were assessed. The hypothesis was that stress reactions have no influence on these parameters. Thirty-four male students were distributed into two groups: first received the stress exposure followed by the same protocol two weeks later but without stress exposure, second underwent the protocol without stress exposure followed by the stress exposure two weeks later. The stressor was a public speech followed by tooth brushing. Saliva was collected before, immediately after stress induction and immediately, at 10, 30 and 120 min. after tooth brushing. Cortisol concentrations, total protein, intraoral pH, and fluoride content in saliva were measured. The data were analyzed statistically. Salivary sediment was ca 4.33% by weight of whole unstimulated saliva. Fluoride bioavailability was higher in salivary sediment than in supernatant saliva. The weight and fluoride concentration was not altered during 2 hours after stress exposure. After a public speech, the salivary cortisol concentration significantly increased after 20 minutes compared to the baseline. The salivary protein concentration and pH also increased. Public speaking influences protein concentration and salivary pH but does not alter the fluoride concentration of saliva.
- Published
- 2014
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28. Differential transcytosis and toxicity of the hNGAL receptor ligands cadmium-metallothionein and cadmium-phytochelatin in colon-like Caco-2 cells: implications for in vivo cadmium toxicity.
- Author
-
Langelueddecke C, Lee WK, and Thévenod F
- Subjects
- Acute-Phase Proteins drug effects, Caco-2 Cells, Cadmium toxicity, Cathepsin B metabolism, Cell Survival drug effects, Dose-Response Relationship, Drug, Humans, Intestinal Mucosa drug effects, Intestinal Mucosa pathology, Kinetics, Ligands, Lipocalin-2, Lipocalins drug effects, Lysosomes metabolism, Metallothionein toxicity, Phytochelatins toxicity, Protein Transport, Proto-Oncogene Proteins drug effects, Tubulin Modulators pharmacology, Acute-Phase Proteins metabolism, Cadmium metabolism, Intestinal Mucosa metabolism, Lipocalins metabolism, Metallothionein metabolism, Phytochelatins metabolism, Proto-Oncogene Proteins metabolism, Transcytosis
- Abstract
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.)
- Published
- 2014
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29. Nickel-induced cell death and survival pathways in cultured renal proximal tubule cells: roles of reactive oxygen species, ceramide and ABCB1.
- Author
-
Dahdouh F, Raane M, Thévenod F, and Lee WK
- Subjects
- ATP Binding Cassette Transporter, Subfamily B genetics, ATP Binding Cassette Transporter, Subfamily B metabolism, Animals, Apoptosis drug effects, Cell Survival drug effects, Dogs, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Glucosyltransferases antagonists & inhibitors, Glucosyltransferases genetics, Glucosyltransferases metabolism, Humans, Kidney Tubules, Proximal metabolism, Kidney Tubules, Proximal pathology, Madin Darby Canine Kidney Cells, RNA Interference, RNA, Messenger metabolism, Rats, Signal Transduction drug effects, Time Factors, Transfection, Up-Regulation, ATP Binding Cassette Transporter, Subfamily B drug effects, Glucosylceramides metabolism, Kidney Tubules, Proximal drug effects, Nickel toxicity, Oxidative Stress drug effects, Reactive Oxygen Species metabolism
- Abstract
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.
- Published
- 2014
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30. Cadmium and cellular signaling cascades: interactions between cell death and survival pathways.
- Author
-
Thévenod F and Lee WK
- Subjects
- Animals, Cell Survival drug effects, Environmental Exposure adverse effects, Humans, Oxidation-Reduction, Reactive Oxygen Species metabolism, Cadmium toxicity, Cell Death drug effects, Signal Transduction drug effects
- Abstract
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.
- Published
- 2013
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31. Substrate- and cell contact-dependent inhibitor affinity of human organic cation transporter 2: studies with two classical organic cation substrates and the novel substrate cd2+.
- Author
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Thévenod F, Ciarimboli G, Leistner M, Wolff NA, Lee WK, Schatz I, Keller T, Al-Monajjed R, Gorboulev V, and Koepsell H
- Subjects
- Animals, Apoptosis drug effects, Cadmium pharmacology, Catecholamine Plasma Membrane Transport Proteins metabolism, Cell Line, Cimetidine pharmacology, Corticosterone pharmacology, Humans, Kidney Tubules, Proximal metabolism, Organic Cation Transporter 2, Rats, Tetraethylammonium pharmacology, Cadmium metabolism, Organic Cation Transport Proteins antagonists & inhibitors, Organic Cation Transport Proteins metabolism
- Abstract
Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.
- Published
- 2013
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32. Pituitary homeobox 2 (PITX2) protects renal cancer cell lines against doxorubicin toxicity by transcriptional activation of the multidrug transporter ABCB1.
- Author
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Lee WK, Chakraborty PK, and Thévenod F
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors, ATP Binding Cassette Transporter, Subfamily B, Member 1 biosynthesis, Cell Line, Tumor, Cell Survival, Chromatin Immunoprecipitation, Cyclosporins pharmacology, Gene Expression Regulation, Neoplastic, Humans, Promoter Regions, Genetic, RNA Interference, RNA, Messenger biosynthesis, RNA, Small Interfering, Signal Transduction genetics, Transcription Factors genetics, Transcriptional Activation, beta Catenin genetics, Homeobox Protein PITX2, ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Antibiotics, Antineoplastic pharmacology, Carcinoma, Renal Cell drug therapy, Doxorubicin pharmacology, Homeodomain Proteins metabolism, Kidney Neoplasms drug therapy, Transcription Factors metabolism
- Abstract
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.)
- Published
- 2013
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33. Toxicology of cadmium and its damage to mammalian organs.
- Author
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Thévenod F and Lee WK
- Subjects
- Acute Disease, Animals, Cardiovascular Diseases chemically induced, Cardiovascular Diseases epidemiology, Cardiovascular Diseases metabolism, Cardiovascular Diseases pathology, Chronic Disease, Diabetes Mellitus chemically induced, Diabetes Mellitus epidemiology, Diabetes Mellitus metabolism, Diabetes Mellitus pathology, Heavy Metal Poisoning, Humans, Kidney Diseases chemically induced, Kidney Diseases epidemiology, Kidney Diseases metabolism, Metals, Heavy metabolism, Neoplasms chemically induced, Neoplasms epidemiology, Neoplasms metabolism, Neoplasms pathology, Poisoning epidemiology, Poisoning pathology, Cadmium adverse effects, Environmental Exposure adverse effects, Environmental Pollutants adverse effects, Poisoning metabolism
- Abstract
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.
- Published
- 2013
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34. ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.
- Author
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Lee WK, Chakraborty PK, Roussa E, Wolff NA, and Thévenod F
- Subjects
- Animals, Bestrophins, Cadmium toxicity, Calcium metabolism, Cell Death drug effects, Cell Lineage drug effects, Cell Membrane drug effects, Cell Membrane metabolism, Cell Nucleus drug effects, Cell Nucleus metabolism, Chloride Channels genetics, Cytoprotection drug effects, Enzyme Activation drug effects, Epithelial Cells drug effects, Kidney, Kinetics, Models, Biological, Phosphorylation drug effects, Rats, Reactive Oxygen Species metabolism, Signal Transduction drug effects, Thapsigargin pharmacology, Tunicamycin pharmacology, Unfolded Protein Response drug effects, Up-Regulation drug effects, Chloride Channels metabolism, Endoplasmic Reticulum Stress drug effects, Epithelial Cells enzymology, Epithelial Cells pathology, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Transcription Factor CHOP metabolism
- Abstract
Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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35. Cyclosporin a, but not FK506, induces osmotic lysis of pancreas zymogen granules, intra-acinar enzyme release, and lysosome instability by activating K+ channel.
- Author
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Lee WK, Braun M, Langelüddecke C, and Thévenod F
- Subjects
- Acinar Cells drug effects, Acinar Cells metabolism, Amylases metabolism, Animals, Biomarkers metabolism, Cathepsin B metabolism, Immunosuppressive Agents adverse effects, Lysosomes metabolism, Male, Osmosis drug effects, Pancreas blood supply, Pancreas Transplantation, Pancreatitis, Graft etiology, Permeability drug effects, Potassium Channels metabolism, Rats, Rats, Wistar, Reperfusion Injury etiology, Secretory Vesicles metabolism, Cyclosporine adverse effects, Immunosuppressive Agents pharmacology, Lysosomes drug effects, Pancreas drug effects, Potassium Channels drug effects, Secretory Vesicles drug effects, Tacrolimus pharmacology
- Abstract
Objectives: The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in zymogen granule (ZG) membranes, including a KCNQ1 K channel, promote hormone-stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities and enzyme secretion by CsA/FK506 contributes to pancreatitis., Methods: Rat ZGs and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single-channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting., Results: CsA (1-10 μM), but not FK506, enhanced ZGs osmotic lysis by selectively increasing K permeability up to 5-fold. Zymogen granule membrane K channels showed ∼2-fold increased single-channel open probability with CsA only. Cyclosporin A selectively increased basal (∼2-fold), but not cholecystokinin-octapeptide (1 nM)-induced amylase secretion in K medium only. Cyclosporin A (5 μM), but not FK506, increased cathepsin B release from lysosomes., Conclusions: Cyclosporin A selectively opens the ZG K channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZGs and may aggravate or fuel acute allograft pancreatitis following hypoxia-reperfusion injury.
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- 2012
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36. Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis.
- Author
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Langelueddecke C, Roussa E, Fenton RA, Wolff NA, Lee WK, and Thévenod F
- Subjects
- Albumins metabolism, Animals, CHO Cells, Cricetinae, Cricetulus, Kidney Medulla cytology, Kidney Medulla drug effects, Kidney Medulla metabolism, Kidney Tubules, Distal cytology, Kidney Tubules, Distal drug effects, Lipocalin-2, Male, Metallothionein toxicity, Quinolinium Compounds metabolism, Rats, Transferrin metabolism, Acute-Phase Proteins metabolism, Endocytosis drug effects, Gene Expression Regulation drug effects, Kidney Tubules, Distal metabolism, Lipocalins metabolism, Oncogene Proteins metabolism
- Abstract
In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.
- Published
- 2012
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37. Acute short-term mental stress does not influence salivary flow rate dynamics.
- Author
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Naumova EA, Sandulescu T, Al Khatib P, Thie M, Lee WK, Zimmer S, and Arnold WH
- Subjects
- Adult, Humans, Male, Models, Statistical, Psychometrics, Salivary Proteins and Peptides metabolism, Speech, Stress, Psychological psychology, Surveys and Questionnaires, Time Factors, Young Adult, Hydrocortisone metabolism, Saliva metabolism, Stress, Psychological metabolism
- Abstract
Background: Results of studies that address the influence of stress on salivary flow rate and composition are controversial. The aim of this study was to reveal the influence of stress vulnerability and different phases of stress reactivity on the unstimulated and stimulated salivary flow rate. We examined that acute mental stress does not change the salivary flow rate. In addition, we also examined the salivary cortisol and protein level in relation to acute mental stress stimuli., Methods: Saliva of male subjects was collected for five minutes before, immediately, 10, 30 and 120 min after toothbrushing. Before toothbrushing, the subjects were exposed to acute stress in the form of a 2 min public speech. Salivary flow rate and total protein was measured. The physiological stress marker cortisol was analyzed using enzyme-linked immunosorbent assay. To determine the subjects' psychological stress reaction, the State-Trait-Anxiety Inventory State questionnaire (STAI) data were obtained. The subjects were divided into stress subgroup (S1) (psychological reactivity), stress subgroup (S2) (psychological and physiological reactivity) and a control group. The area under the curve for salivarycortisol concentration and STAI-State scores were calculated. All data underwent statistical analysis using one-way analysis of variance., Results: Immediately after stress exposure, all participants exhibited a psychological stress reaction. Stress exposure did not change the salivary flow rate. Only 69% of the subjects continued to display a physiological stress reaction 20 minutes after the public talk. There was no significant change in the salivary flow rate during the psychological and the physiological stress reaction phases relative to the baseline., Conclusions: Acute stress has no impact on the salivary flow rate; however, there may be other responses through salivary proteins that are increased with the acute stress stimuli. Future studies are needed to examine specific proteins and their possible roles in acute stress responses.
- Published
- 2012
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38. Role of Arf1 in endosomal trafficking of protein-metal complexes and cadmium-metallothionein-1 toxicity in kidney proximal tubule cells.
- Author
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Wolff NA, Lee WK, and Thévenod F
- Subjects
- ADP-Ribosylation Factor 1 analysis, Animals, Biological Transport, Cells, Cultured, Endocytosis, Metallothionein metabolism, Protein Transport, Rats, Transferrin metabolism, Vesicular Transport Proteins analysis, rab GTP-Binding Proteins analysis, rab7 GTP-Binding Proteins, ADP-Ribosylation Factor 1 physiology, Endosomes metabolism, Kidney Tubules, Proximal drug effects, Metallothionein toxicity
- Abstract
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.)
- Published
- 2011
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39. ABCB1 protects kidney proximal tubule cells against cadmium-induced apoptosis: roles of cadmium and ceramide transport.
- Author
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Lee WK, Torchalski B, Kohistani N, and Thévenod F
- Subjects
- 4-Chloro-7-nitrobenzofurazan analogs & derivatives, 4-Chloro-7-nitrobenzofurazan metabolism, ATP Binding Cassette Transporter, Subfamily B metabolism, Animals, Benzimidazoles, Biological Transport, Cell Line, Ceramides metabolism, Cyclosporins metabolism, Dogs, Electrophoresis, Polyacrylamide Gel methods, Fluorescent Dyes, Glucosylceramides metabolism, Glucosyltransferases antagonists & inhibitors, Humans, Kidney Tubules, Proximal pathology, Oxadiazoles metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism, Plasmids, Rats, Sphingolipids metabolism, Transfection methods, Up-Regulation, ATP Binding Cassette Transporter, Subfamily B genetics, Apoptosis, Cadmium toxicity, Kidney Tubules, Proximal cytology
- Abstract
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.
- Published
- 2011
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40. Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking.
- Author
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Wolff NA, Liu W, Fenton RA, Lee WK, Thévenod F, and Smith CP
- Subjects
- Animals, Cation Transport Proteins biosynthesis, Cation Transport Proteins genetics, Cell Line, Cell Membrane metabolism, Male, Protein Transport, RNA, Messenger biosynthesis, RNA, Messenger genetics, Rats, Rats, Sprague-Dawley, Cation Transport Proteins metabolism, Iron metabolism, Iron Overload metabolism, Kidney Tubules, Proximal metabolism
- Abstract
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.)
- Published
- 2011
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41. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells.
- Author
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Chakraborty PK, Lee WK, Molitor M, Wolff NA, and Thévenod F
- Subjects
- Animals, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors drug effects, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Cadherins drug effects, Cadherins genetics, Cadherins metabolism, Cell Proliferation drug effects, Cell Transformation, Neoplastic genetics, Cells, Cultured, Electrophoretic Mobility Shift Assay, Humans, Immunoblotting, Immunoprecipitation, Kidney Tubules, Proximal metabolism, Microscopy, Confocal, Microscopy, Fluorescence, Rats, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction physiology, Transcription Factor 4, Transcription Factors drug effects, Transcription Factors genetics, Transcription Factors metabolism, Transfection, Up-Regulation, Wnt Proteins genetics, Wnt Proteins metabolism, beta Catenin drug effects, beta Catenin genetics, beta Catenin metabolism, Cadmium toxicity, Carcinogens toxicity, Cell Transformation, Neoplastic metabolism, Kidney Tubules, Proximal drug effects, Signal Transduction drug effects, Wnt Proteins drug effects
- Abstract
Background: The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/beta-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/beta-catenin signaling are known to contribute to carcinogenesis., Results: We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 microM, 3-9 h) caused nuclear translocation of beta-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of beta-catenin with AJ components (E-cadherin, alpha-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability., Conclusions: Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.
- Published
- 2010
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42. Cadmium-induced DNA damage triggers G(2)/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells.
- Author
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Bork U, Lee WK, Kuchler A, Dittmar T, and Thévenod F
- Subjects
- Animals, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins metabolism, Cell Death, Cell Division drug effects, Cells, Cultured, Checkpoint Kinase 1, Checkpoint Kinase 2, DNA-Binding Proteins metabolism, G2 Phase drug effects, Kidney Tubules, Proximal drug effects, Kidney Tubules, Proximal physiology, Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Protein Kinases drug effects, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, Rats, Reactive Oxygen Species metabolism, Signal Transduction, Staurosporine analogs & derivatives, Staurosporine pharmacology, Tumor Suppressor Proteins metabolism, Cadmium pharmacology, Cell Cycle drug effects, Cyclin B metabolism, DNA Damage, Kidney Tubules, Proximal cytology, Protein Kinases metabolism, Tumor Suppressor Protein p53 deficiency
- Abstract
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.
- Published
- 2010
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43. Organic cation transporters: physiology, toxicology and special focus on ethidium as a novel substrate.
- Author
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Lee WK, Wolff NA, and Thévenod F
- Subjects
- Animals, Humans, Ethidium metabolism, Organic Cation Transport Proteins metabolism, Trypanocidal Agents metabolism
- Abstract
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.
- Published
- 2009
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44. Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule.
- Author
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Lee WK, Reichold M, Edemir B, Ciarimboli G, Warth R, Koepsell H, and Thévenod F
- Subjects
- Animals, Biological Transport, Active physiology, CHO Cells, Cricetinae, Cricetulus, Gene Expression Regulation physiology, Humans, Inhibitory Concentration 50, Kidney Tubules, Proximal cytology, Mice, Mice, Knockout, Paraquat chemistry, Paraquat pharmacology, Propidium chemistry, Propidium pharmacology, Protein Isoforms, Substrate Specificity, Coloring Agents pharmacokinetics, Ethidium pharmacokinetics, Kidney Tubules, Proximal metabolism, Organic Cation Transport Proteins genetics, Organic Cation Transport Proteins metabolism
- Abstract
The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.
- Published
- 2009
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45. Novel roles for ceramides, calpains and caspases in kidney proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies.
- Author
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Lee WK and Thévenod F
- Subjects
- Humans, In Vitro Techniques, Kidney Tubules, Proximal drug effects, Apoptosis physiology, Cadmium toxicity, Calpain physiology, Caspases physiology, Ceramides physiology, Kidney Tubules, Proximal cytology
- Abstract
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.
- Published
- 2008
- Full Text
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46. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells.
- Author
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Wolff NA, Lee WK, Abouhamed M, and Thévenod F
- Subjects
- ADP-Ribosylation Factor 6, Animals, Endocytosis physiology, Kidney Tubules, Proximal drug effects, Kidney Tubules, Proximal metabolism, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Metallothionein toxicity, Rats, ADP-Ribosylation Factors physiology, Endocytosis drug effects, Kidney Tubules, Proximal cytology, Metallothionein metabolism, Transferrin metabolism
- Abstract
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.
- Published
- 2008
- Full Text
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47. Evidence for KCNQ1 K+ channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion.
- Author
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Lee WK, Torchalski B, Roussa E, and Thévenod F
- Subjects
- Amylases metabolism, Animals, Cholagogues and Choleretics pharmacology, Chromans pharmacology, Enzyme Precursors, Gene Expression Regulation physiology, Ion Channel Gating drug effects, KCNQ1 Potassium Channel antagonists & inhibitors, KCNQ1 Potassium Channel genetics, Male, Pancreas enzymology, Rats, Rats, Wistar, Secretory Vesicles enzymology, Sulfonamides pharmacology, Cell Membrane metabolism, Cholecystokinin pharmacology, KCNQ1 Potassium Channel metabolism, Pancreas metabolism, Secretory Vesicles metabolism
- Abstract
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.
- Published
- 2008
- Full Text
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48. Cadmium induces nuclear translocation of beta-catenin and increases expression of c-myc and Abcb1a in kidney proximal tubule cells.
- Author
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Thévenod F, Wolff NA, Bork U, Lee WK, and Abouhamed M
- Subjects
- ATP Binding Cassette Transporter 1, Active Transport, Cell Nucleus physiology, Animals, Cadherins biosynthesis, Cadherins genetics, Cell Line, Transformed, Kidney Tubules, Proximal cytology, Kidney Tubules, Proximal drug effects, Proto-Oncogene Proteins c-myc biosynthesis, Rats, Up-Regulation genetics, beta Catenin biosynthesis, beta Catenin genetics, ATP-Binding Cassette Transporters biosynthesis, ATP-Binding Cassette Transporters genetics, Cadmium physiology, Cell Nucleus metabolism, Kidney Tubules, Proximal metabolism, Proto-Oncogene Proteins c-myc genetics, Up-Regulation drug effects, beta Catenin metabolism
- Abstract
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.
- Published
- 2007
- Full Text
- View/download PDF
49. Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells.
- Author
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Lee WK, Torchalski B, and Thévenod F
- Subjects
- Animals, Apoptosis drug effects, Caspase 3 metabolism, Cell Line, Transformed, Cytosol metabolism, Enzyme Inhibitors pharmacology, Fumonisins pharmacology, Oxidoreductases antagonists & inhibitors, Oxidoreductases metabolism, Rats, Rats, Inbred WKY, Signal Transduction drug effects, Signal Transduction physiology, Apoptosis physiology, Cadmium toxicity, Calpain metabolism, Ceramides metabolism, Kidney Tubules, Proximal cytology
- Abstract
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+).
- Published
- 2007
- Full Text
- View/download PDF
50. Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells.
- Author
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Abouhamed M, Wolff NA, Lee WK, Smith CP, and Thévenod F
- Subjects
- Animals, Apoptosis drug effects, Apoptosis physiology, Cation Transport Proteins genetics, Cell Line, Endocytosis, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Cation Transport Proteins deficiency, Cation Transport Proteins metabolism, Kidney Tubules, Proximal cytology, Kidney Tubules, Proximal drug effects, Metallothionein toxicity, RNA Interference
- Abstract
Chronic exposure to Cd2+ causes renal proximal tubular (PT) damage. Cd2+ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd2+ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd2+ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 +/- 9.6 and 36.9 +/- 14.4% (n = 5-6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 microM CdMT-1 by 35.6 +/- 4.2% (n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd2+ toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd2+-induced cell death signaling are not affected by DMT1 knockdown. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.
- Published
- 2007
- Full Text
- View/download PDF
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