RESUMO
The natural compound curcumin has been shown to have therapeutic potential against a wide range of diseases such as cancer. Curcumin reduces cell viability of renal cell carcinoma (RCC) cells when combined with TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that specifically targets cancer cells, by helping overcome TRAIL resistance. However, the therapeutic effects of curcumin are limited by its low bioavailability. Similar compounds to curcumin with higher bioavailability, such as demethoxycurcumin (DMC) and 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), can potentially have similar anticancer effects and show a similar synergy with TRAIL, thus reducing RCC viability. This study aims to show the effects of DMC and EF24 in combination with TRAIL at reducing ACHN cell viability and ACHN cell migration. It also shows the changes in death receptor 4 (DR4) expression after treatment with these compounds individually and in combination with TRAIL, which can play a role in their mechanism of action.
Assuntos
Compostos de Benzilideno/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Diarileptanoides/farmacologia , Neoplasias Renais/tratamento farmacológico , Piperidonas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Apoptose , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Movimento Celular , Quimioterapia Combinada , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Células Tumorais CultivadasRESUMO
Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.
Assuntos
Apoptose/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Hipóxia Celular/fisiologia , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , CamundongosRESUMO
The pancreatic ß-cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end-stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi-organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN-BD11 rat clonal pancreatic ß-cell line using Ctns-targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient-stimulated insulin secretion were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox-sensitive transcription factor NF-κB were increased in cells with silenced Ctns. From this study, the cystinotic-like pancreatic ß-cell model demonstrated that the altered oxidative status of the cell, resulted in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient-stimulated insulin secretion.
Assuntos
Trifosfato de Adenosina/metabolismo , Cistina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Estresse Oxidativo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Apoptose , Linhagem Celular , Exocitose , NF-kappa B/metabolismo , RatosRESUMO
ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified ß-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.
Assuntos
Albuminas/metabolismo , Canais de Cloreto/metabolismo , Endocitose/fisiologia , Glutamil Aminopeptidase/metabolismo , Túbulos Renais Proximais/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Rim/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , RatosRESUMO
The incidence of Type 2 diabetes is increasing rapidly worldwide, and understanding the mechanisms of its complications including diabetic nephropathy (DN) is important in the discovery of early biomarkers, understanding the causative mechanisms of its complications and identifying therapeutic targets. DN is characterized by glomerulosclerosis, tubulointerstitial fibrosis and tubular atrophy. The tubular component of the disease is important in progression of disease. In vitro models are a valuable alternative to animal studies and an effective way to explore mechanisms of human disease. Several proximal tubular cell lines have been used in studying mechanisms of DN. Key extracellular conditions that contribute to damage to the proximal tubule in DN include hyperglycaemia, proteinuria, and hypoxia and inflammation. According to current knowledge, these exert their effects through changes in transforming growth factor beta signalling, the renin-angiotensin system, dysregulation of pathways such as the polyol pathway, hexosamine pathway and protein kinase C pathway and through formation of advanced glycation end products. Studies in cell culture models have been instrumental in the delineation of these processes. However, all of the existing cell culture models have limitations including dedifferentiation. To bring research forward along with technological advances, such as major advances in 'omics' methodologies, a more suitable model is necessary. The RPTEC/TERT1 cell line is a promising alternative to previous proximal tubular epithelial cell lines due to features that resemble the cell type in vivo, such as its epithelial characteristics, maintenance of functional capabilities, glucose handling, expression of the primary cilium and transport activity including albumin. This cell line will facilitate identification of mechanisms of DN with potential to identify new therapeutic targets.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Túbulos Renais Proximais/patologia , Animais , Diabetes Mellitus Tipo 2/complicações , Progressão da Doença , Humanos , Técnicas In VitroRESUMO
Chemical carcinogens are substances which induce malignant tumours, increase their incidence or decrease the time taken for tumour formation. Often, exposure to chemical carcinogens results in tissue specific patterns of tumorigenicity. The very same anatomical, biochemical and physiological specialisations which permit the kidney to perform its vital roles in maintaining tissue homeostasis may in fact increase the risk of carcinogen exposure and contribute to the organ specific carcinogenicity observed with numerous kidney carcinogens. This review will address the numerous mechanisms which play a role in the concentration, bioactivation, and uptake of substances from both the urine and blood which significantly increase the risk of cancer in the kidney.
Assuntos
Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Carcinógenos/metabolismo , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/patologia , Animais , Carcinogênese/metabolismo , Humanos , Neoplasias Renais/metabolismoRESUMO
The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO(3)) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO(3) resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO(3) exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO(3) cause significant deciliation in a model of the proximal tubule. With KBrO(3), this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO(3) exposure.
Assuntos
Bromatos/toxicidade , Carcinógenos/toxicidade , Ciclo Celular/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Ocratoxinas/toxicidade , Fatores de Ribosilação do ADP/análise , Linhagem Celular , Cílios/efeitos dos fármacos , Cílios/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Imunofluorescência , Humanos , Túbulos Renais Proximais/ultraestrutura , Nifedipino/toxicidade , Transcriptoma/efeitos dos fármacos , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown. METHODS: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin. RESULTS: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways. CONCLUSIONS: We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Albumina Sérica/metabolismo , Animais , Células Cultivadas , Endocitose , Proteínas Imediatamente Precoces/genética , Gambás , Proteínas Serina-Treonina Quinases/genética , Albumina Sérica/análise , Distribuição Tecidual , Xantenos/análise , Xantenos/farmacocinéticaRESUMO
Molecular O(2) and CO(2) are the primary substrate and product of aerobic metabolism, respectively. Levels of these physiologic gases in the cell microenvironment vary dramatically both in health and in diseases, such as chronic inflammation, ischemia, and cancer, in which metabolism is significantly altered. The identification of the hypoxia-inducible factor led to the discovery of an ancient and direct link between tissue O(2) and gene transcription. In this study, we demonstrate that mammalian cells (mouse embryonic fibroblasts and others) also sense changes in local CO(2) levels, leading to altered gene expression via the NF-κB pathway. IKKα, a central regulatory component of NF-κB, rapidly and reversibly translocates to the nucleus in response to elevated CO(2). This response is independent of hypoxia-inducible factor hydroxylases, extracellular and intracellular pH, and pathways that mediate acute CO(2)-sensing in nematodes and flies and leads to attenuation of bacterial LPS-induced gene expression. These results suggest the existence of a molecular CO(2) sensor in mammalian cells that is linked to the regulation of genes involved in innate immunity and inflammation.
Assuntos
Dióxido de Carbono/metabolismo , Regulação da Expressão Gênica/imunologia , Imunidade Inata/fisiologia , Inflamação/metabolismo , NF-kappa B/imunologia , Animais , Western Blotting , Células Cultivadas , Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Inflamação/imunologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologiaRESUMO
Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.
Assuntos
Carcinógenos/toxicidade , DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Micotoxinas/toxicidade , Ocratoxinas/toxicidade , Animais , Linhagem Celular , DNA/genética , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Especificidade da Espécie , Testes de ToxicidadeRESUMO
Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.
Assuntos
Bromatos/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Ratos , Junções Íntimas/metabolismo , Testes de ToxicidadeRESUMO
The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation, but this combination can also result in increased adverse effects. We previously showed that not only CsA treatment but also its combination with SRL decreased paracellular permeability in renal proximal tubular cells by modification of the tight junction proteins, claudins, through ERK1/2 signaling pathway. In this present study, evidence is presented that not only CsA but also the combination of CsA/SRL may have adverse effects on the barrier function of renal proximal cells, at least in part, through the expression of the cytokine transforming growth factor (TGF)-ß(1). CsA treatment upregulated TGF-ß(1) gene expression and this upregulation was enhanced when CsA and SRL were applied together. Addition of TGF-ß(1) (5 ng/ml) altered the barrier function with increased transepithelial electrical resistance (TER) and claudin-1 expression. Use of a TGF-ß(1)-blocking antibody or blockage of TGF-ß(1) receptor kinase activity with SD208 prevented the CsA- and CsA/SRL-induced increase in TER. No evidence was found in the present studies to indicate that CsA or CsA/SRL treatment activated the TGF-ß(1) Smad canonical signaling pathway, whereas addition of TGF-ß(1) (5 ng/ml) did activate the Smad pathway. Addition of the ERK1/2 signaling inhibitor U0126 was able to prevent the TGF-ß(1)-mediated increase in TER and claudin expression. It is most likely that the CsA- and CsA/SRL-induced increases in TGF-ß(1) expression may not be sufficient to trigger the Smad pathway but however may trigger other TGF-ß(1) receptor-mediated signaling including the ERK1/2 signaling pathway.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sirolimo/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Butadienos/farmacologia , Linhagem Celular , Claudina-1 , Impedância Elétrica , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Nitrilas/farmacologia , Pteridinas/farmacologia , Proteínas Smad/metabolismo , Suínos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Regulação para CimaRESUMO
The scavenger receptor megalin binds to albumin in the microvilli of the renal proximal tubule, and transports the ligand to the intravillar cleft for processing by endocytosis. Albumin endocytosis in the proximal tubule is regulated by protein complexes containing a number of transmembrane and accessory proteins including PDZ scaffolds such as NHERF1 and NHERF2. PDZ scaffold proteins bind to class I PDZ binding motifs (S/T-X-Φ) in the extreme C-terminus of targets. Megalin contains a functional PDZ binding motif (SDV) in its distal terminus, however a potential interaction with the NHERF proteins has not been investigated. As megalin associates with NHE3 in the microvilli and NHE3 is tethered to the intravillar cleft via its interaction with NHERF1, we investigated if there is a direct interaction between megalin and NHERF1 in renal proximal tubule cells. Using confocal microscopy we determined that megalin and NHERF1 co-localise in the apical region in proximal tubule cells. Immunoprecipitation experiments performed using rat kidney lysate indicated that megalin bound NHERF1 in vivo. Using fusion proteins and peptides, we determined that PDZ2 of NHERF1 bound to megalin and that this interaction was via the C-terminus of megalin directly and in the absence of any accessory protein. We next investigated which domain in megalin was regulating this interaction. Using GST fusion proteins we determined that the loss of the most distal C-terminus of megalin containing the PDZ binding motif (SDV) did not alter its ability to bind to NHERF1. Significantly, we then identified an internal NHERF binding domain in the C-terminus of megalin. Using peptide studies we were able to demonstrate that NHERF1 bound to an internal PDZ binding motif in megalin and that a loss of a single threonine residue abolished the interaction between megalin and NHERF1. Finally, in proximal tubule cells, silencing NHERF1 increased megalin expression. Therefore, we have identified a novel protein interaction in proximal tubule cells and specifically identified a new internal PDZ binding motif in the C-terminus of megalin.
Assuntos
Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosfoproteínas/genética , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/análise , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The calcineurin inhibitor cyclosporine A (CsA) is a widely used immunosuppressive agent. However, nephrotoxicity is a serious side effect observed in patients which limits clinical use of CsA. CsA nephrotoxicity is associated with tubulointerstitial injury progressing to nephropathy. This is typically diagnosed by invasive renal biopsy and is often only detected when the disease process is well advanced. Therefore identification of novel, early indicators of CsA nephrotoxicity could be clinically advantageous. This study aimed to establish a murine model of CsA nephrotoxicity and to identify urinary proteins that may indicate the onset of CsA-induced nephropathy using 2-D gel electrophoresis. CsA nephrotoxicity was induced in CD-1 mice by daily CsA administration for 4weeks. By week 4, elevated serum creatinine and proteinuria were observed after CsA treatment indicating significant renal dysfunction. Decreased cadherin-1, increased α-smooth muscle actin and fibroblast specific protein 1 in kidney tissue indicated disruption of normal tubular architecture. Alterations in podocin and uromodulin were also observed which may indicate damage to other segments of the nephron. Proteomic analysis of urine identified a number of differentially regulated proteins that may be involved in early CsA nephropathy including cadherin 1, superoxide dismutase and vinculin. These findings suggest novel mechanisms of CsA nephrotoxicity and identify novel potential markers of the disease.
Assuntos
Ciclosporina/toxicidade , Modelos Animais de Doenças , Nefrite/induzido quimicamente , Nefrite/patologia , Animais , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomerulonefrite Membranosa/induzido quimicamente , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Nefrite/metabolismoRESUMO
BACKGROUND: high-mobility group box protein 1 (HMGB-1) is a chromatin-binding protein that bends DNA, thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. METHODS: we established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMCs). The model was characterized using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. RESULTS: activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased α-smooth muscle actin expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products and through induction of transforming growth factor-ß(1) secretion from PTECs. CONCLUSIONS: these results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis.
Assuntos
Células Epiteliais/imunologia , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Proteína HMGB1/genética , Proteína HMGB1/imunologia , Actinas/genética , Actinas/metabolismo , Análise de Variância , Caderinas/metabolismo , Movimento Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Junções Intercelulares/metabolismo , Túbulos Renais Proximais/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Hibridização de Ácido Nucleico , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated. METHODS: Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort. RESULTS: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells. CONCLUSION: The accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.
Assuntos
Cílios/patologia , Células Epiteliais/patologia , Nefropatias/diagnóstico , Túbulos Renais/patologia , Lipocalina-2/análise , Fatores de Ribosilação do ADP/análise , Fatores de Ribosilação do ADP/urina , Biomarcadores/análise , Linhagem Celular , Cílios/química , Células Epiteliais/citologia , Humanos , Nefropatias/patologia , Nefropatias/urina , Túbulos Renais/citologia , Lipocalina-2/urinaRESUMO
The E2A gene encodes two distinct basic helix-loop-helix transcription factors, E12 and E47. E12 and E47 modulate expression of their target genes through formation of homodimers and heterodimers with other basic helix-loop-helix transcription factors. E2A proteins are thought to play critical roles in regulation of cell commitment, growth and differentiation in a range of cell types including lymphocytes, muscle cells and neurons. Emerging evidence suggests that E2A proteins also play key roles in the process of epithelial mesenchymal transition, a mechanism which contributes significantly to kidney disease progression and tumour metastasis. Further understanding of the diverse effects of E2A proteins may lead to novel therapeutic approaches to targeting important disease process.
Assuntos
Fenótipo , Fatores de Transcrição TCF/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Dimerização , Desenvolvimento Embrionário/efeitos dos fármacos , Expressão Gênica , Humanos , Linfócitos/fisiologia , Mesoderma/citologia , Dados de Sequência Molecular , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
Cyclosporine A (CsA) significantly improves the success of organ transplantation, however renal fibrosis, characterised by severe tubulointerstitial fibrosis is a complication of CsA therapy. Previously we have reported the involvement of PKC-beta isoforms in a model of CsA-induced tubulointerstitial fibrosis and we have now further elucidated this role. Treatment of human proximal tubular epithelial cells with CsA resulted in increased fibronectin production which coincided with increased PKC activity. To delineate the respective contributions of the two PKC-beta isoforms in fibrotic events, we overexpressed PKC-betaI, -betaII, or both in combination. Overexpression of the two PKC-beta isoforms induced morphological alterations, secretion of the profibrotic cytokine TGF-beta1, and fibronectin release from proximal tubular cells however PKC-betaII induced more significant effects in all parameters examined. Inhibition of PKC-beta completely abrogated the CsA-induced increase in fibronectin secretion demonstrating a direct antifibrotic effect of PKC-beta inhibition. Further studies also identified a role for the p44/42 mitogen activated kinase signalling pathway in CsA-induced fibrotic effects downstream of PKC-beta. Overall, these findings demonstrate a central role for PKC-beta, and particularly PKC-betaII in the development of tubulointerstitial fibrosis and suggest that PKC-beta may be a viable therapeutic target in CsA nephropathy.
Assuntos
Células Epiteliais/enzimologia , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Proteína Quinase C/metabolismo , Butadienos/farmacologia , Linhagem Celular , Forma Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Fibronectinas/biossíntese , Fibrose , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imidazóis/farmacologia , Isoenzimas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fenótipo , Proteína Quinase C beta , Piridinas/farmacologia , Pironas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Albuminuria is a key marker of renal injury and a major risk factor for cardiovascular disease. In vivo imaging techniques with fluorescent albumin have allowed visualization of its movement within the whole kidney but they could not distinguish between intact and degraded albumin. To visualize albumin degradation in proximal tubular cells in vivo we used an albumin conjugate (dye quenched (DQ)-albumin), which only fluoresces when it is degraded. In cultured proximal tubule cells, the fluorescent signal from DQ-albumin was dependent on endocytosis and lysosomal function and showed that at any time about 40% of endocytosed DQ-albumin was degraded. Significant accumulation of conventional Texas Red-labeled albumin and degraded DQ-albumin was found in rat proximal tubules 5 min after injection. Importantly, no hint of DQ-albumin was detected in the serum, suggesting that the fluorescent signal in the proximal tubules was derived from tubular degradation of intact albumin. Our study shows that DQ-albumin, together with conventional fluorescent conjugates of intact albumin, can be used to visualize albumin degradation by proximal tubules in vivo.
Assuntos
Albuminas/metabolismo , Diagnóstico por Imagem/métodos , Túbulos Renais Proximais/metabolismo , Animais , Diagnóstico por Imagem/normas , Endocitose , Corantes Fluorescentes , Túbulos Renais Proximais/ultraestrutura , Lisossomos/metabolismo , Masculino , Microscopia de Fluorescência , Ratos , Ratos WistarRESUMO
In renal pathologies tubulo-interstitial fibrosis results from an aberrant wound-healing ability where the normal epithelial tissue is substituted for scar tissue caused by accumulation of extracellular matrix proteins (ECM). During the wound-healing process, epithelial cells may undergo epithelial-mesenchymal transition (EMT) acquiring a mesenchymal-like phenotype that allows cells to migrate and re-epithelialize the wound site. It has been reported that chronic inflammation and uremic milieu are involved in wound-healing and enhanced kidney damage in chronic kidney disease (CKD) patients. In this study we evaluated reactive carbonyl compounds (RCC) effects on renal wound healing. The compounds resulting from carbonyl stress evaluated in this study were glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and 4-hydroxy-hexenal (HHE). Wound repair ability was evaluated by the wound healing assay using HK-2 cells. EMT was evaluated by morphological, protein and transcriptional changes using microscopy, western blot, zymography and RT-qPCR. Changes in the vimentin network and primary cilia were assessed by immunofluorescence. Our data demonstrated that MDA and GO delay wound closure mediated by vimentin disruption, which caused collagen I mRNA decrease, and deciliation. In contrast, HHE treatment (and MGO to a minor degree) induced morphological changes and increased mesenchymal marker expression and gelatinase activity in HK-2 cells. In this study, we have demonstrated for the first time that exposure to RCC differentially affects wound healing in proximal tubular epithelia. A better comprehension of effects of uremic toxins on wound healing and fibrosis and migration is necessary to seek mechanisms to slow down renal fibrosis.