Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons... more
Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons control blood filtration, as well as resorption and recycling of inorganic and organic ions, sugars, amino acids, peptides, vitamins, water and further metabolites of endogenous and exogenous origin. All nephron segments consist of polarized cells and express high levels of Pals1. Mice that are functionally haploid for Pals1 develop a lethal phenotype, accompanied by heavy proteinuria and the formation of renal cysts. However, on a cellular level, it is still unclear if reduced cell polarization, incomplete cell-cell contact formation, or an altered Pals1-dependent gene expression accounts for the renal phenotype. To address this, we analyzed the transcriptomes of Pals1-haploinsufficient kidneys and the littermate controls by gene set enrichment anal...
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In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global... more
In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SAR...
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This editorial summarizes the 12 scientific papers published in the Special Issue “Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations 2 [...]
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The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of... more
The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cell...
Research Interests: Biology, Virology, Medicine, In Vitro, Virus, and 3 moreViruses, Immunoprecipitation, and Hepatitis B virus
Einleitung Cisplatin ist ein wirksames Chemotherapeutikum für die Behandlung von Tumoren im Kindesalter, limitiert durch seine Oto- und Nephrotoxizität. Die individuell unterschiedliche Cisplatin-Toleranz spricht für das Vorhandensein... more
Einleitung Cisplatin ist ein wirksames Chemotherapeutikum für die Behandlung von Tumoren im Kindesalter, limitiert durch seine Oto- und Nephrotoxizität. Die individuell unterschiedliche Cisplatin-Toleranz spricht für das Vorhandensein genetischer Prädispositionsfaktoren. Unterschiede[for full text, please go to the a.m. URL]
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Background Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling... more
Background Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. Methods Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. Results Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)–associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signali...
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The discovery of the anticancer properties of platinum derivatives by Rosenberg represents a milestone in the development of chemotherapeutic protocols for tumor treatment [...]
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A new zebrafish study identifies compounds that shield ears and kidneys against an anticancer drug.
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The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many... more
The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many transport systems, which are specifically expressed in the different parts of the nephron, the functional unit of the kidneys. These transport systems are involved, for example, in the reabsorption of sodium, glucose, and other important solutes and peptides from the primary urine. They are also important in the reabsorption of water and thereby production of a concentrated urine. However, several studies have shown the importance of transport systems for different tumor entities. Transport systems, for example, contributed to the proliferation and migration of cancer cells and thereby on tumor progression. They could also serve as drug transporters that could enable drug resistance by outward transport of, for example, chemotherapeutic agents and other drugs. Although many renal transporters have been characterized in detail with respect to the significance for proper kidney function, their role in renal cancer progression is less known. Here, we describe the types of renal cancer and review the studies that analyzed the role of organic cation transporters of the SLC22-family and of the aquaporin water channel family in kidney tumors.
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The human organic cation transporter 2 (hOCT2) is highly expressed in proximal tubules of the kidneys, where it plays an important role in the secretion of organic cations. Since many drugs are organic cations, hOCT2 has relevant... more
The human organic cation transporter 2 (hOCT2) is highly expressed in proximal tubules of the kidneys, where it plays an important role in the secretion of organic cations. Since many drugs are organic cations, hOCT2 has relevant pharmacological implications. The hOCT2 gene is polymorphic, and the nonsynonymous single nucleotide polymorphism (SNP) causing the substitution of alanine at position 270 of the protein sequence with serine (Ala270Ser) is present with high frequency in the human population. Therefore, Ala270Ser has potentially important pharmacologic consequences. Here, we analyzed the transport properties and rapid regulation of hOCT2 wildtype and hOCT2 Ala270Ser expressed in human embryonic kidney cells using real-time uptake measurements. Moreover, we compared the expression of hOCT2 in the plasma membrane determined by biotinylation experiments and the cellular transport and toxicity of cisplatin measured by inductively coupled plasma mass spectrometry and a viability ...
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Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in... more
Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles—observed in many histologic analyses of renal diseases—is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the ...
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In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs... more
In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This “one-segment-one-proteome-approach” defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module...
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In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a... more
In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinur...
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In the article “Immunohistochemical localization of OCT2 in the cochlea of various species,” which appeared in the September 2015 Issue of Laryngoscope, Hellberg et al. investigated the expression of the organic cation transporter 2... more
In the article “Immunohistochemical localization of OCT2 in the cochlea of various species,” which appeared in the September 2015 Issue of Laryngoscope, Hellberg et al. investigated the expression of the organic cation transporter 2 (Oct2) in the cochlea of different animals by immunohistochemical analysis. The issue is of importance because of the role of Oct2 as an uptake transporter for ototoxic and nephrotoxic drugs such as cisplatin, and such an analysis has the potential to predict which cells are particularly sensitive to cisplatin toxicity. However, immunohistochemical studies are difficult to perform because the availability of specific antibodies against these transporters is limited. Using a commercial available antibody, Hellberg et al. observed an Oct2 staining in the rat cochlea in the supporting cells of the organ of Corti and in type I spiral ganglion cells. The specificity of the staining was tested using rat kidney sections. Here the authors showed a staining of renal tubules and concluded that, because of the known expression of Oct2 in the kidneys, the antibody that they used specifically detects Oct2. However, by a closer examination of the renal labeling, it is evident that the antibody stains the apical membrane of tubular epithelial cells. It is known that Oct2 in the rat kidneys is heavily expressed on the basolateral membrane of proximal tubules cells. For this reason, the antibody used by the authors seems not to be specific, at least for what concerns the renal transporters. The results of the study on the localization of Oct2 in the rat cochlea should be therefore interpreted with caution.
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This work presents a quantitative bioimaging method for platinum based on laser ablation-inductively coupled plasma-mass spectrometry and its application for a biomedical study concerning toxic side effects of cisplatin. To trace the... more
This work presents a quantitative bioimaging method for platinum based on laser ablation-inductively coupled plasma-mass spectrometry and its application for a biomedical study concerning toxic side effects of cisplatin. To trace the histopathology back to cisplatin, platinum was localized and quantified in major functional units of testicle, cochlea, kidney, nerve and brain sections from cisplatin treated mice. The direct consideration of the histology enables precise interpretation of the Pt images and the novel quantitative evaluation approach allows significantly more precise investigations than the pure image. For the first time, platinum was detected and quantified in all major injured structures including organ of Corti of cochlea and seminiferous tubule of testicle. In this way, proximal tubule in kidney, Leydig cells in testicle, stria vascularis and organ of Corti in cochlea and nerve fibers in sciatic nerves are confirmed as targets of cisplatin in these organs. However, the accumulation of platinum in almost all investigated structures also raises questions about more complex pathogenesis including direct and indirect interruption of several biological processes.
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One of the most common setups for elemental bioimaging, the hyphenation of a laser ablation (LA) system and an inductively coupled plasma mass spectrometer (ICP-MS), was expanded by adding full scan mass spectrometric information as... more
One of the most common setups for elemental bioimaging, the hyphenation of a laser ablation (LA) system and an inductively coupled plasma mass spectrometer (ICP-MS), was expanded by adding full scan mass spectrometric information as another dimension of information. While most studies deal with the analysis of typically not more than up to 10 isotopes per scan cycle, a fast scanning quadrupole mass analyzer was utilized to record the full mass spectrum of interest in this work. Mass-to-charge ratios from 6 to 250 were observed within one cycle. Besides the x- and y-position on the ablated sample and the intensity, the m/z-ratio served as fourth variable for each pixel of the obtained data, closing thereby the gap between "inorganic" and "organic" mass spectrometric imaging techniques. The benefits of this approach include an improved control of interferences, the discovery of unexpected elemental distributions, the possibility to plot isotopic ratios, and to inte...
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The processes of cellular proliferation and progressive acquisition of a specialized phenotype show a remarkable degree of coordination that involves both intracellular programming and intercellular communication. One of the major... more
The processes of cellular proliferation and progressive acquisition of a specialized phenotype show a remarkable degree of coordination that involves both intracellular programming and intercellular communication. One of the major incentives for studying factors that regulate the processes of cellular proliferation and differentiation is the recognition of their potential contribution to tumorigenesis. In normal cells, stimulatory and inhibitory events are believed to be under the control of growth factors and growth inhibitory factors, which are known to be protooncogene products. Growth regulatory mechanisms usually involve the binding of a growth factor to a specific receptor on the cell surface, which then through an intracellular biochemical cascade leads to cell division. The cell regulation pathways initiated by growth factors may be subverted at several distinct levels in cancer cells. Studies of oncogenes have shown that they may function as abnormal growth factors or abnor...
Research Interests: Biology, Cell Biology, Medicine, Cell Division, Signal Transduction, and 15 moreGrowth, Humans, Pubmed, Phenotype, Minerva Medica, Epidermal Growth Factor, Oncogenes, Carcinogenesis, Neoplasms, Amino Acid Sequence, Growth Factor, Cell Growth, Intracellular, Molecular Sequence Data, and Neoplasm metastasis
1. Organic cation transporters (OCTs) translocate endogenous (e.g. dopamine) and exogenous (e.g. drugs) substances of cationic nature and, therefore, play an important role in the detoxification of exogenous compounds. This review aims to... more
1. Organic cation transporters (OCTs) translocate endogenous (e.g. dopamine) and exogenous (e.g. drugs) substances of cationic nature and, therefore, play an important role in the detoxification of exogenous compounds. This review aims to furnish essential information on OCTs, with an emphasis on pharmacological aspects. 2. Analysis of the literature on OCTs makes clear that there is a species- and organ-specific distribution of the different isoforms, which can also be differentially regulated. OCTs are responsible for the excretion and/or distribution of many drugs and also for serious tissue-specific side-effects such as cisplatin-induced nephrotoxicity. The presence of single nucleotide polymorphisms in these transporters significantly influences the response of patients to medication, as demonstrated for the antidiabetic drug metformin. 3. A substantial amount of research has to be undertaken to clarify further the OCT structure-function relationships specifically to define the role of oligomerization on their activity and regulation, to identify intracellular interaction partners of OCTs, and to characterize their pharmacogenetic aspects.
Research Interests: Physiology, Chemistry, Regulation, Drug, Humans, and 14 moreToxicity, Metformin, Pharmacogenetics, Cisplatin, Single Nucleotide Polymorphism, Oral Hypoglycemic Agents, Transporters, Transporter, Protein isoforms, Ion Transport, Structure activity Relationship, Antineoplastic Agents, Biochemistry and cell biology, and Pharmacology and pharmaceutical sciences
Research Interests: Endocrinology, Medicine, Internal Medicine, Animals, Renal failure, and 15 moreEnd Stage Renal Failure, Male, Animal Model, Renal Function, Behavioral Animal Models, Clinical Sciences, Rats, cystatin C, Rat Model, Chronic Kidney Failure, Nephrectomy, Predictive value of tests, Creatinine, Cysteine Proteinase Inhibitors, and Glomerular Filtration Rate
RATIONALEBiological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved... more
RATIONALEBiological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement‐containing species in biological samples such as tissue sections.METHODSA UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)‐MS, whereas atmospheric pressure chemical ionization (APCI)‐MS with an orbitrap mass analyzer was utilized for molecular MS.RESULTSSimultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for ...
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The lungs are a site for the uptake, accumulation, and storage of exogenous basic amines. The compound N-N-N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3 propanediamine (HIPDM) is a basic amine that can be labelled with... more
The lungs are a site for the uptake, accumulation, and storage of exogenous basic amines. The compound N-N-N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3 propanediamine (HIPDM) is a basic amine that can be labelled with radioactive iodine and detected by external counting. Intravenously injected 123I-HIPDM is extracted by the human lung, where it is retained in a slowly effluxable pool. In the present study, we measured HIPDM lung kinetics and subcellular distribution in rabbits given i.v. 125I-HIPDM. Rabbits were killed from 2 min to 5 h after injection, and the radioactivity retained in their lungs was measured. Subcellular lung fractions (nuclear, mitochondrial, lysosomal, microsomal and postmicrosomal supernatant) were assayed for HIPDM radioactivity, protein content, and distribution of specific marker enzymes. HIPDM lung clearance in rabbits was nearly identical to that of humans. Virtually all the HIPDM radioactivity in lungs (98+/-1%) was associated with subce...
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This editorial summarizes the 22 scientific papers published in the Special Issue “Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment” of the International Journal of... more
This editorial summarizes the 22 scientific papers published in the Special Issue “Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment” of the International Journal of Molecular Sciences [...]
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Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into... more
Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins—MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performa...
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Organic cation transporters (OCT) play an important role in mediating cellular uptake of several pharmaceuticals, such as the antidiabetic drug metformin and the platinum-derived chemotherapeutics. Since these drugs can also affect the... more
Organic cation transporters (OCT) play an important role in mediating cellular uptake of several pharmaceuticals, such as the antidiabetic drug metformin and the platinum-derived chemotherapeutics. Since these drugs can also affect the pancreas, here it was investigated whether these transporters are expressed in this organ. An interaction between OCT2 and the glucose transporter 2 (GLUT2), which is expressed with important functional consequences in the kidneys and in the pancreas, has already been demonstrated elsewhere. Therefore, here it was further investigated whether the two proteins have a functional relationship. It was demonstrated that OCT2 is expressed in pancreas, probably in β cells of Langerhans islets, together with GLUT2. However, a co-localization was only evident in a cell-line model of rat pancreatic β cells under incubation with high glucose concentration. High glucose stimulated OCT2 expression and activity. On the other side, studies conducted in human embryon...
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The human organic cation transporter 2 (hOCT2) mediates renal and neuronal cellular cisplatin and oxaliplatin uptake, and therefore plays a significant role in the development of side effects associated with these chemotherapeutic drugs.... more
The human organic cation transporter 2 (hOCT2) mediates renal and neuronal cellular cisplatin and oxaliplatin uptake, and therefore plays a significant role in the development of side effects associated with these chemotherapeutic drugs. Autophagy is induced by cisplatin and oxaliplatin treatment and is believed to promote cell survival under stressful conditions. We examined in vitro the role of hOCT2 on autophagy induced by cisplatin and oxaliplatin. We also explored the effect of autophagy on toxicities of these platinum derivatives. Our results indicate that autophagy, measured as LC3 II accumulation and reduction in p62 expression level, is induced in response to cisplatin and oxaliplatin in HEK293-hOCT2 but not in wild-type HEK293 cells. Furthermore, inhibition of autophagy is associated with higher toxicity of platinum derivatives, and starvation was found to offer protection against cisplatin-associated toxicity. In conclusion, activation of autophagy could be a potential st...
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The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here,... more
The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to w...
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Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SF) play a key role. Baricitinib and Tofacitinib both act intracellularly, blocking the ATP-binding side of JAK proteins and thereby the... more
Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SF) play a key role. Baricitinib and Tofacitinib both act intracellularly, blocking the ATP-binding side of JAK proteins and thereby the downstream signalling pathway via STAT-3. Therefore, we investigated the role of organic cation transporters (OCTs) in Baricitinib and Tofacitinib cellular transport. Methods: OCT expression was analysed in SF isolated from RA and osteoarthritis (OA) patients, as well as peripheral blood mononuclear cells. The interaction of Baricitinib and Tofacitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of both drugs was quantified using LC/MS. Target inhibition for both drugs was tested using Western blot for phosphorylated JAK1 and STAT3 upon stimulation with IL-6. Results: MATE-1 expression increased in OASF compared to RASF. The other OCTs were not differentially expressed. The transport of Baricitinib was not...
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Vectorial transport of organic cations (OCs) in renal proximal tubules is mediated by sequential action of human OC transporter 2 (hOCT2) and human multidrug and toxic extrusion protein 1 and 2K (hMATE1 and hMATE2K), expressed in the... more
Vectorial transport of organic cations (OCs) in renal proximal tubules is mediated by sequential action of human OC transporter 2 (hOCT2) and human multidrug and toxic extrusion protein 1 and 2K (hMATE1 and hMATE2K), expressed in the basolateral (hOCT2) and luminal (hMATE1 and hMATE2K) plasma membranes, respectively. It is well known that hOCT2 activity is subjected to rapid regulation by several signaling pathways, suggesting that renal OC secretion may be acutely adapted to physiological requirements. Therefore, in this work, the acute regulation of hMATEs stably expressed in human embryonic kidney cells was characterized using the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) as a marker. A specific regulation of ASP+ transport by hMATE1 and hMATE2K measured in uptake and efflux configurations was observed. In the example of hMATE1 efflux reduction by inhibition of casein kinase II, it was also shown that this regulation is able to modify transcellul...
Research Interests: Genetics, Chemistry, Cell Biology, Medicine, Kidney, and 4 moreMolecular sciences, Efflux, Transporter, and Secretion
Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 (... more
Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 ( SLC22A1/hOCT1) and hOCT2 ( SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63...
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Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular... more
Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular processing of platinum. In this study, we addressed the establishment of cisplatin resistance in the absence of copper transporter ATP7B that has been previously found to be overexpressed in various resistant cells. Cisplatin sensitivity, induction of apoptosis, drug accumulation, and transporter gene expression were determined in hepatoma cell lines. Knockout or overexpression of copper transporter ATP7B did not affect cisplatin sensitivity. Cisplatin resistant cells showed a stably reduced cisplatin accumulation and a downregulation of organic cation transporter 3 (OCT3). In contrast, OCT3 overexpression could reverse resistance. Reduced MT1 expression was detected in the resistant cell line, however transient and highly dependent on the presence of...
Research Interests: Biology, Cancer Research, Apoptosis, Medicine, MT, and 4 moreCisplatin, OCT, Efflux, and Transporter
Recent therapeutic approaches of rheumatoid arthritis (RA) address the use of small molecules such as tyrosine kinase inhibitors (TKIs). However, the TKIs developed to date have important side effects and/or scarce efficacy in... more
Recent therapeutic approaches of rheumatoid arthritis (RA) address the use of small molecules such as tyrosine kinase inhibitors (TKIs). However, the TKIs developed to date have important side effects and/or scarce efficacy in inflammatory diseases such as RA. Since intracellular effective TKIs must enter the cell to reach their intracellular targets, here we investigated the interaction of the TKI saracatinib, a dual inhibitor of c-Src and c-Abl signaling, with transporters for organic cations as well as the role of these transporters for the biological effect of saracatinib in human RA-synovial fibroblasts (hRASF). Saracatinib significantly reduced proliferation of hRASF. The cellular saracatinib uptake was mainly dependent on the human novel organic cation transporter 1 (hOCTN1), which showed the highest apparent affinity for saracatinib among all other transporters for organic cations analyzed here. In hRASF, saracatinib biologic function was dependent on hOCTN1. Further analysi...
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Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug... more
Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters. We initially found that organic cation transporters (OCTs), uptake carriers of metformin and oxaliplatin, were inhibited by several clinically used TKIs. Mechanistic studies showed that these TKIs inhibit the Src family kinase Yes1, which was found to be essential for OCT2 tyrosine phosphorylation and function. Yes1 inhibition in vivo diminished OCT2 activity, significantly mitigating oxaliplatin-induced acute sensory neuropathy. Along with OCT2, other SLC-family drug transporters are potentially part of an extensive 'transporter-phosphoproteome' with unique susceptibility to TKIs. On the basis of these fi...
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Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates... more
Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulte...
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Assuming that genetic variants of the SLC22A2 and SLC31A1 transporter affect patients' susceptibility to cisplatin-induced ototoxicity, we compared the distribution of 11 SLC22A2 variants and the SLC31A1 variant rs10981694 between... more
Assuming that genetic variants of the SLC22A2 and SLC31A1 transporter affect patients' susceptibility to cisplatin-induced ototoxicity, we compared the distribution of 11 SLC22A2 variants and the SLC31A1 variant rs10981694 between patients with and without cisplatin-induced ototoxicity. Genotyping was performed in 64 pediatric patients and significant findings were re-evaluated in 66 adults. The SLC22A2 polymorphism rs316019 (c.808G>T; Ser270Ala) was significantly associated with protection from cisplatin-induced ototoxicity in the pediatric (p = 0.022) and the adult cohort (p = 0.048; both: Fisher's exact test). This result was confirmed by multiple logistic regression analysis accounting for age which was identified as a relevant factor for ototoxicity as well (rs316019: OR [G/T vs G/G] = 0.12, p = 0.009; age: OR [per year]: 0.84, p = 0.02). These results identified rs316019 as potential pharmacogenomic marker for cisplatin-induced ototoxicity and point to a critical ro...
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Purpose: Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2−/−mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis... more
Purpose: Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2−/−mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2−/−mice, we hypothesized that alternate pathways are involved in the observed injury. Experimental Design: Studies were done in wild-type, Oct1/2−/−, or p53-deficient animals, all on an FVB background, receiving cisplatin intraperitoneally at 15 mg/kg. Cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays. Results: KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that the most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wild-type (P = 2.40 × 10−11) and Oct1/2−/−mice (P = 1.92 × 10−8). This was confirmed by demonstrating that homozygosity for...
Research Interests: Pharmacology, Biology, Kidney diseases, Medicine, Kidney, and 13 moreMice, Female, Animals, Male, Cisplatin, Biological markers, Nephrotoxicity, Toluene, Knockout Mice, Antineoplastic Agents, Gene expression profiling, real time polymerase chain reaction, and reverse transcriptase polymerase chain reaction
Research Interests: Physiology, Chemistry, Fluorescence Microscopy, Biology, Medicine, and 14 moreFluorescence Resonance Energy Transfer, Western blotting, Humans, Mutation, Medical Physiology, HeLa cells, Transporter, Cysteine, Transfection, Protein Binding, Extracellular, Biochemistry and cell biology, Cell Membrane, and Tissue culture techniques
Research Interests:
To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after... more
To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP+ uptake, whereas regulation by p56 lck tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA+, TPA+, and quinine were changed differently in each mutant (EC50 in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in μmol: TEA+: 105, 153, 56, 1135, 484, 498, 518; TPA+: 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, ...
Research Interests:
Purpose: Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving... more
Purpose: Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving chemotherapeutic agents. Experimental Design: Creatinine transport was studied in transfected HEK293 cells in vitro and in wild-type mice and age-matched organic cation transporter 1 and 2–deficient [Oct1/2(−/−)] mice ex vivo and in vivo. Clinical pharmacogenetic and transport inhibition studies were done in two separate cohorts of cancer patients. Results: Compared with wild-type mice, creatinine clearance was significantly impaired in Oct1/2(−/−) mice. Furthermore, creatinine inhibited organic cation transport in freshly isolated proximal tubules from wild-type mice and humans, but not in those from Oct1/2(−/−) mice. In a genetic association analysis (n = 590), several polymorphisms around the OCT2/SLC22A2 gene locus, including rs2504954 (P = 0.000873), were s...