RESUMO
Endogenous positively charged organic substances, including neurotransmitters and cationic uremic toxins, as well as exogenous organic cations such as the anti-diabetic medication metformin, serve as substrates for organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). These proteins facilitate their transport across cell membranes. Vectorial transport through the OCT/MATE axis mediates the hepatic and renal excretion of organic cations, regulating their systemic and local concentrations. Organic cation transporters are part of the remote sensing and signaling system, whose activity can be regulated to cope with changes in the composition of extra- and intracellular fluids. Glucose, as a source of energy, can also function as a crucial signaling molecule, regulating gene expression in various organs and tissues. Its concentration in the blood may fluctuate in specific physiological and pathophysiological conditions. In this work, the regulation of the activity of organic cation transporters was measured by incubating human embryonic kidney cells stably expressing human OCT1 (hOCT1), hOCT2, or hMATE1 with high glucose concentrations (16.7 mM). Incubation with this high glucose concentration for 48 h significantly stimulated the activity of hOCT1, hOCT2, and hMATE1 by increasing their maximal velocity (Vmax), but without significantly changing their affinity for the substrates. These effects were independent of changes in osmolarity, as the addition of equimolar concentrations of mannitol did not alter transporter activity. The stimulation of transporter activity was associated with a significant increase in transporter mRNA expression. Inhibition of the mechanistic target of rapamycin (mTOR) kinase with Torin-1 suppressed the transporter stimulation induced by incubation with 16.7 mM glucose. Focusing on hOCT2, it was shown that incubation with 16.7 mM glucose increased hOCT2 protein expression in the plasma membrane. Interestingly, an apparent trend towards higher hOCT2 mRNA expression was observed in kidneys from diabetic patients, a pathology characterized by high serum glucose levels. Due to the small number of samples from diabetic patients (three), this observation must be interpreted with caution. In conclusion, incubation for 48 h with a high glucose concentration of 16.7 mM stimulated the activity and expression of organic cation transporters compared to those measured in the presence of 5.6 mM glucose. This stimulation by a diabetic environment could increase cellular uptake of the anti-diabetic drug metformin and increase renal tubular secretion of organic cations in an early stage of diabetes.
Assuntos
Metformina , Proteínas de Transporte de Cátions Orgânicos , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/genética , Metformina/farmacologia , Metformina/metabolismo , Cátions/metabolismo , RNA MensageiroRESUMO
Aquaporin-2-4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2-4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2-3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.
Assuntos
Aquaporina 3/metabolismo , Aquaporina 4/metabolismo , Movimento Celular/fisiologia , Medula Renal/citologia , Túbulos Renais Coletores/metabolismo , Animais , Aquaporina 3/genética , Aquaporina 4/genética , Bucladesina/farmacologia , Movimento Celular/efeitos dos fármacos , Forma Celular , Células Cultivadas , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Concentração Osmolar , Cultura Primária de Células , Ratos , Trocador 1 de Sódio-Hidrogênio/metabolismo , beta Catenina/metabolismoRESUMO
Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4 weeks) low-dose (4 mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2-/-). Functional and molecular analysis showed that OCT1/2-/- mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2-/- mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Fator 1 de Transcrição de Octâmero/genética , Transportador 2 de Cátion Orgânico/genética , Animais , Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Ototoxicidade/etiologia , Ototoxicidade/genética , Proteômica , Transdução de Sinais/efeitos dos fármacosRESUMO
Tyrosine kinase inhibitors have emerged as a therapeutic option for rheumatic diseases such as systemic sclerosis (SSc). Because tyrosine kinases like c-Abl kinase are important for fibroblast activation and fibrosis development in SSc, the c-Abl inhibitor imatinib was proposed for SSc treatment. Transporters for organic cations have become increasingly recognized as an important determinant for uptake and efficacy of tyrosine kinase inhibitors. Therefore, we investigated the role of organic cation transporters in the uptake of imatinib. Moreover, the influence of important SSc pathogenetic factors, like PDGF and Notch pathway activation on these uptake processes, has been studied. We showed that organic cation transporters OCT1-3, novel organic cation transporters OCTN1/2, and the multidrug and toxin extrusion protein MATE1 are expressed in healthy dermal and SSc fibroblasts. Decreased expression levels of MATE1 and decreased imatinib uptake were measured in SSc fibroblasts. In small interfering RNA experiments, MATE1 was identified as key transporter for imatinib uptake and biological effect in dermal fibroblasts. Furthermore, PDGF reduced imatinib uptake by decreasing MATE1 expression in SSc fibroblasts, but not in healthy fibroblasts. Blocking the Notch pathway in SSc fibroblasts increased MATE1 transporter expression and imatinib uptake. In conclusion, MATE1-mediated transport governs therapeutic efficacy of imatinib in SSc.
Assuntos
Mesilato de Imatinib/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Receptores Notch/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Biópsia , Células Cultivadas , Derme/citologia , Derme/metabolismo , Derme/patologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mesilato de Imatinib/uso terapêutico , Proteínas de Transporte de Cátions Orgânicos/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Escleroderma Sistêmico/patologia , Transdução de SinaisRESUMO
Acute cellular renal allograft rejection (AR) frequently occurs after kidney transplantations. It is a sterile T-cell mediated inflammation leading to increased local glucose metabolism. Here we demonstrate in an allogeneic model of Brown Norway rat kidneys transplanted into uninephrectomized Lewis rats the successful implementation of the recently developed glucose chemical exchange saturation transfer (glucoCEST) magnetic resonance imaging. This technique is a novel method to assess and differentiate AR. Renal allografts undergoing AR showed significantly increased glucoCEST contrast ratios of cortex to medulla of 1.61 compared to healthy controls (1.02), syngeneic Lewis kidney to Lewis rat transplants without rejection (0.92), kidneys with ischemia reperfusion injury (0.99) and kidneys affected by cyclosporine A toxicity (1.10). Receiver operating characteristic curve analysis showed an area under the curve value of 0.92, and the glucoCEST contrast ratio predicted AR with a sensitivity of 100% and a specificity of 69% at a threshold level over 1.08. In defined animal models of kidney injuries, the glucoCEST contrast ratios of cortex to medulla correlated positively with mRNA expression levels of T-cell markers (CD3, CD4, CD8a/b), but did not correlate to impaired renal perfusion. Thus, the glucoCEST parameter may be valuable for the assessment and follow up treatment of AR.
Assuntos
Aloenxertos/diagnóstico por imagem , Rejeição de Enxerto/diagnóstico por imagem , Transplante de Rim/efeitos adversos , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Traumatismo por Reperfusão/diagnóstico por imagem , Aloenxertos/imunologia , Aloenxertos/patologia , Animais , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8 , Meios de Contraste , Ciclosporina/toxicidade , Modelos Animais de Doenças , Glucose/administração & dosagem , Glucose/metabolismo , Rejeição de Enxerto/induzido quimicamente , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Rim/imunologia , Rim/patologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante Homólogo/efeitos adversosRESUMO
CD63 is a ubiquitously expressed member of the tetraspanin superfamily. Using a mating-based split-ubiquitin-yeast 2-hybrid system, pull-down experiments, total internal reflection fluorescence microscopy, Förster resonance energy transfer, and biotinylation assays, we found that CD63 interacts with human organic cation transporter 2 (hOCT2), which transports endogenous and exogenous substrates, such as neurotransmitters and drugs in several epithelial cells. CD63 overexpression affects cellular localization of hOCT2 expressed in human embryonic kidney (HEK)293 cells. Studies with CD63-knockout mice indicate that in renal proximal tubules, CD63 determines the insertion of the mouse ortholog of the transporter into the proper membrane domain and mediates transporter regulation by trafficking processes. In polarized Madin-Darby kidney canine kidney (MDCK) epithelial cells, CD63 and hOCT2 colocalize with the small GTPase Rab4, which controls the rapid recycling from sorting endosomes back to the cell surface. Suitable negative and positive control experiments were performed for each experimental approach. Empty vector transfected cells and wild-type mice were used as control. CD63 seems to play a role in the recycling of hOCT2 from endosomes to the basolateral membrane in polarized epithelia. These data indicate that CD63 has a previously uncharacterized function in regulating trafficking of specific membrane proteins in polarized cells.-Schulze, U., Brast, S., Grabner, A., Albiker, C., Snieder, B., Holle, S., Schlatter, E., Schröter, R., Pavenstädt, H., Herrmann, E., Lambert, C., Spoden, G. A., Florin, L., Saftig, P., Ciarimboli, G. Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.
Assuntos
Túbulos Renais Proximais/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Tetraspanina 30/metabolismo , Animais , Membrana Celular/metabolismo , Cães , Endossomos/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Transportador 2 de Cátion Orgânico , Ligação Proteica , Transporte Proteico , Tetraspanina 30/genética , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
With this study, we wanted to prove the hypothesis that the unique extracellular osmolality within the renal medulla modulates a specific gene expression pattern. The physiologic functions of the kidneys are mediated by the segment-specific expression of key proteins. So far, we have limited knowledge about the mechanisms that control this gene expression pattern. The hyperosmolality in the renal medullary interstitium is of major importance as a driving force for urine concentration. We made use of primarily cultured rat renal inner medullary collecting-duct cells and microarray analysis to identify genes affected by the environmental osmolality of the culture medium. We identified hundreds of genes that were either induced or repressed in expression by hyperosmolality in a time- and osmolality-dependent fashion. Further analysis demonstrated that many of them, physiologically, showed a kidney- and even collecting-duct-specific expression, including secreted proteins, kinases, and transcription factors. On the other hand, we identified factors, down-regulated in expression, that have a diuretic effect. In conclusion, the kidney is the only organ that has such a hyperosmotic environment, and study provides an excellent method for controlling tissue-specific gene expression.-Schulze Blasum, B., Schröter, R., Neugebauer, U., Hofschröer, V., Pavenstädt, H., Ciarimboli, G., Schlatter E., Edemir, B. The kidney-specific expression of genes can be modulated by the extracellular osmolality.
Assuntos
Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Concentração Osmolar , Cloreto de Sódio/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Espaço Extracelular/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismoRESUMO
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 findings we propose that TKIs, an important and rapidly expanding class of therapeutics, can functionally modulate pharmacologically important proteins by inhibiting protein kinases essential for their post-translational regulation.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/efeitos dos fármacos , Fosfotirosina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-yes/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Camundongos , Modelos Moleculares , Transportadores de Ânions Orgânicos/efeitos dos fármacos , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/efeitos dos fármacos , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismoRESUMO
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) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Injúria Renal Aguda/patologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cisplatino , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Camundongos , Proteínas de Transporte de Cátions Orgânicos/deficiência , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
AIM: 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. PATIENTS & METHODS: Genotyping was performed in 64 pediatric patients and significant findings were re-evaluated in 66 adults. RESULTS: 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). CONCLUSION: These results identified rs316019 as potential pharmacogenomic marker for cisplatin-induced ototoxicity and point to a critical role of SLC22A2 for cisplatin transport in humans and its contribution to the organ specific side effects of this drug. Original submitted 17 September 2014; Revision submitted 19 December 2014.
Assuntos
Proteínas de Transporte de Cátions/genética , Cisplatino/efeitos adversos , Neoplasias/tratamento farmacológico , Proteínas de Transporte de Cátions Orgânicos/genética , Adolescente , Adulto , Criança , Pré-Escolar , Cisplatino/administração & dosagem , Transportador de Cobre 1 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Feminino , Estudos de Associação Genética , Genótipo , Haplótipos , Humanos , Masculino , Neoplasias/genética , Neoplasias/patologia , Transportador 2 de Cátion Orgânico , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The kidney is a major target for drug-induced injury, primarily due the fact that it transports a wide variety of chemical entities into and out of the tubular lumen. Here, we investigated the expression of the main xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1 at an mRNA and/or protein level. RPTEC/TERT1 cells expressed OCT2, OCT3, OCTN2, MATE1, MATE2, OAT1, OAT3 and OAT4. The functionality of the OCTs was demonstrated by directional transport of the fluorescent dye 4-Di-1-ASP. In addition, P-glycoprotein activity in RPTEC/TERT1 cells was verified by fluorescent dye retention in presence of various P-glycoprotein inhibitors. In comparison to proliferating cells, contact inhibited RPTEC/TERT1 cells expressed increased mRNA levels of several ABC transporter family members and were less sensitive to cyclosporine A. We conclude that differentiated RPTEC/TERT1 cells are well suited for utilisation in xenobiotic transport and pharmacokinetic studies.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Túbulos Renais Proximais/citologia , Proteínas de Transporte/genética , Linhagem Celular , Humanos , Túbulos Renais Proximais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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 a p53-null allele partially reduced renal tubular damage, whereas loss of p53 in Oct1/2(-/-)mice (p53(-/-)/Oct1/2(-/-)) completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(-/-)/Oct1/2(-/-)mice. CONCLUSIONS: These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Fator 1 de Transcrição de Octâmero/fisiologia , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Antineoplásicos/metabolismo , Benzotiazóis/farmacologia , Biomarcadores/metabolismo , Cisplatino/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Fator 1 de Transcrição de Octâmero/antagonistas & inibidores , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Transportador 2 de Cátion Orgânico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
HISTORY AND ADMISSION FINDINGS: A 67-year-old male patient developed progressive renal failure following successful treatment of a soft tissue sarcoma that comprised surgical resection after neoadjuvant radiochemotherapy with the application of doxorubicin (cumulative dose 180 mg/m²) and ifosfamide (cumulative dose 33 g/m²). INVESTIGATIONS: Plasma creatinine concentration was elevated to 4.5 mg/dl. Upon detection of glucosuria and α1-microglobulinuria renal biopsy was performed. DIAGNOSIS, TREATMENT AND COURSE: Histologic analysis revealed massively injured tubules that could be explained by a toxic effect of ifosfamide. Glomeruli were not affected and appeared normal. After two months of conservative therapy, the patient developed an uremic syndrome requiring hemodialysis. Ever since kidney function did not recover albeit preserved diuresis. CONCLUSIONS: Ifosfamide can cause end-stage renal disease by a tubulotoxic effect that may be the result of a selective intracellular uptake into the proximal tubule via the human organic cation transporter 2 (OCT2).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Falência Renal Crônica/induzido quimicamente , Falência Renal Crônica/diagnóstico , Sarcoma/tratamento farmacológico , Neoplasias de Tecidos Moles/tratamento farmacológico , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Diagnóstico Diferencial , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Humanos , Ifosfamida/efeitos adversos , Ifosfamida/uso terapêutico , Masculino , Sarcoma/complicações , Neoplasias de Tecidos Moles/complicações , Resultado do TratamentoRESUMO
The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.
Assuntos
Canais de Cloreto/metabolismo , Túbulos Renais Proximais/metabolismo , Podócitos/metabolismo , Reabsorção Renal , Trifosfato de Adenosina/farmacologia , Aldosterona/sangue , Animais , Anoctamina-1 , Células Cultivadas , Canais de Cloreto/deficiência , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Feminino , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Podócitos/efeitos dos fármacos , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Reabsorção Renal/efeitos dos fármacos , Renina/sangue , Fatores de Tempo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 µM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.
Assuntos
Diazepam/farmacologia , Fluoxetina/farmacologia , Ketamina/farmacologia , Fator 3 de Transcrição de Octâmero/metabolismo , Psicotrópicos/farmacologia , Serotonina/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Células Cultivadas , Células HEK293 , Histamina/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Compostos de Amônio Quaternário/farmacologia , Especificidade da EspécieRESUMO
The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2−/− mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2−/−) mice. OCT-mediated transport in proximal tubules of mOCT2−/− mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2−/− mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation
Assuntos
Transporte Biológico Ativo/fisiologia , Transporte de Íons/fisiologia , Túbulos Renais Proximais/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por SubstratoRESUMO
The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function (1). At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR (2). We herein present in addition small animal positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation (3). Further, this method can be applied for follow-up to improve monitoring of transplant rejection (4).
Assuntos
Radioisótopos de Flúor , Fluordesoxiglucose F18 , Rejeição de Enxerto/diagnóstico por imagem , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Animais , Ratos , Ratos Endogâmicos Lew , Transplantes/diagnóstico por imagemRESUMO
UNLABELLED: We proposed small-animal PET with (18)F-FDG-labeled T lymphocytes as a new method for image-based diagnosis of acute allogeneic renal transplant rejection (AR) established in a rat model. METHODS: One and 2 h after tail vein injection of 30 × 10(6) ex vivo (18)F-FDG-labeled human T cells into male 10-wk-old uninephrectomized, allogeneically transplanted rats (aTX; Lewis-brown Norway [LBN] to Lewis), whole-body radioactivity distribution was assessed in vivo by small-animal PET (postoperative day 4), and percentage injected dose (%ID) as a parameter of T-cell infiltration was assessed and compared between graft and native kidney. In vivo results were confirmed by autoradiography and staining of human CD3 after postmortem dissection. Syngeneically transplanted rats (sTX) (LBN to LBN), rats with ischemia-reperfusion injury (IRI) (45-min warm ischemia), and rats subjected to acute cyclosporine A (CSA) toxicity (50 mg/kg for 2 d intraperitoneally) served as controls. RESULTS: The accumulation of labeled cells was significantly elevated in allografts with AR (1.07 ± 0.28 %ID), compared with native control kidneys (0.49 ± 0.18 %ID) (P < 0.0001). No differences were found among native controls, sTX, CSA toxicity, and kidneys with IRI. In vivo uptake of (18)F-FDG cells measured in the PET scanner correlated with results obtained by autoradiography, histologic evaluation, and polymerase chain reaction. CONCLUSION: We proposed graft PET imaging using (18)F-FDG-labeled T cells as a new option to detect rat renal AR with a low dose of (18)F-FDG in a noninvasive, fast, and specific manner in rats.
Assuntos
Fluordesoxiglucose F18 , Rejeição de Enxerto/diagnóstico por imagem , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Transplante de Rim/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Linfócitos T/patologia , Animais , Rastreamento de Células/métodos , Rejeição de Enxerto/patologia , Masculino , Compostos Radiofarmacêuticos , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Kidney transplanted patients are often treated with immunosuppressive, antihypertensive, and antibiotic drugs such as cyclosporine A (CsA), ß-blockers, and fluoroquinolones, respectively. Organic cation transporters (OCT) expressed in the basolateral membrane of proximal tubules represent an important drug excretion route. In this work, the renal expression of OCT after syngeneic and allogeneic kidney transplantation in rats with or without CsA immunosuppression was studied. Moreover, the interactions of CsA, ß-blockers (pindolol/atenolol), and fluoroquinolones (ofloxacin/norfloxacin) with rOCT1, rOCT2, hOCT1, and hOCT2 in stably transfected HEK293-cells were studied. Kidney transplantation was associated with reduced expression of rOCT1, while rOCT2 showed only reduced expression after allogeneic transplantation. All drugs interacted subtype- and species-dependently with OCT. However, only atenolol, pindolol, and ofloxacin were transported by hOCT2, the main OCT in human kidneys. While CsA is not an OCT substrate, it exerts a short-term effect on OCT activity, changing their affinity for some substrates. In conclusion, appropriate drug dosing in transplanted patients is difficult partly because OCT are down-regulated and because concomitant CsA treatment may influence the affinity of the transporters. Moreover, drug-drug competition at the transporter can also alter drug excretion rate.
Assuntos
Antagonistas Adrenérgicos beta/metabolismo , Fluoroquinolonas/metabolismo , Transplante de Rim/efeitos adversos , Túbulos Renais Proximais/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Animais , Western Blotting , Linhagem Celular , Ciclosporina/uso terapêutico , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This study characterizes the complex mechanisms of acute regulation of organic cation (OC) transport across the basolateral membrane of isolated mouse proximal tubules. The fluorescent substrate ASP(+), 4-(-4-(dimethylamino) styryl-N-methylpyridinium, was used to quantify OC transport using a microtiter plate based fluorescence reader method. Inhibition of phosphatidylinositol-3-kinase, of p56 tyrosine kinase, stimulation of PKC and inhibition of PKA reduced ASP(+)-uptake. ASP(+)-kinetic and Dixon plot analyses revealed effects on transporter trafficking as explanation for the inhibition of ASP(+)-uptake by these pathways. Angiotensin II (AII) via stimulation of Ca(2+)/calmodulin increased ASP(+)-uptake. This effect aroused from an altered substrate affinity. Bafilomycin, an inhibitor of the vacuolar H(+)-ATPase and thus endosomal and lysosomal function, reduced ASP(+)-uptake, but did not prevent the AII effect on ASP(+)-uptake. Bafilomycin seemed to diminish the recycling rate of OCTs and hence to reduce the amount of transporters in the membrane. AII via Ca(2+)/calmodulin increased the substrate affinity of the remaining OCTs. The involvement of the cytoskeleton in acute regulation of OCTs became obvious as colchicine induced inhibition of microtubule polymerisation reduced ASP(+)-uptake. Acute regulation of mouse OCTs mostly involves changes in trafficking from and to the plasma membrane and only in the case of AII/CaM changes in substrate affinity.