RESUMO
Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-ß-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage.
Assuntos
Cisplatino , MAP Quinase Quinase Quinases , Camundongos Knockout , Transdução de Sinais , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Animais , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Camundongos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de SinalRESUMO
Acute kidney injury is a devasting clinical syndrome resulting from multiple causes, characterized by an abrupt deterioration of kidney function for which there is no pharmacologic treatment. Cilastatin has demonstrated direct nephroprotective effects in acute kidney injury and now is shown to be effective to specifically target therapeutically loaded nanoparticles to the proximal tubule to treat acute kidney injury.
Assuntos
Injúria Renal Aguda , Cilastatina , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Humanos , Cilastatina/uso terapêutico , Cilastatina/administração & dosagem , Animais , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , NanopartículasRESUMO
Oxidative stress plays a crucial role in the development and progression of various kidney diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is the primary transcription factor that protects cells from oxidative stress by regulating cytoprotective genes including those involved in the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and cell death. Antimycin A, an inhibitor of complex III of the electron transport chain, causes oxidative stress and reduces GSH levels. In this study, we induced mitochondrial damage in rat renal proximal tubular cells using antimycin A and investigated cellular viability and levels of NRF2 and GSH. Treatment with antimycin A altered the expression of antioxidant genes, including reduction in the transcription of glutathione-cysteine ligase subunits (Gclc and Gclm) and glutathione reductase (Gsr1), followed by a reduction in total GSH content with a concomitant decrease in NRF2 protein expression. AR-20007, previously described as an NRF2 activator, stabilizes and increases NRF2 protein expression in cells. By stimulating NRF2, AR-20007 increased the expression of antioxidant and detoxifying enzymes, thereby enhancing protection against oxidative stress induced by antimycin A. These data suggest that NRF2 activation effectively inhibits antimycin A-induced oxidative stress and that NRF2 may be a promising therapeutic target for preventing cell death during acute kidney injury.
Assuntos
Antimicina A , Células Epiteliais , Glutationa , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Antimicina A/farmacologia , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Glutationa/metabolismo , Ratos , Estresse Oxidativo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Morte Celular/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Antioxidantes/farmacologia , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismoRESUMO
Cisplatin-induced nephrotoxicity restricts its clinical use against solid tumors. The present study elucidated the pharmacological effects of Renogrit, a plant-derived prescription medicine, using cisplatin-induced human renal proximal tubular (HK-2) cells and Caenorhabditis elegans. Quantification of phytochemicals in Renogrit was performed on HPTLC and UHPLC platforms. Renogrit was assessed in vitro in HK-2 cells post-exposure to clinically relevant concentration of cisplatin. It was observed that renoprotective properties of Renogrit against cisplatin-induced injury stem from its ability to regulate renal injury markers (KIM-1, NAG levels; NGAL mRNA expression), redox imbalance (ROS generation; GST levels), and mitochondrial dysfunction (mitochondrial membrane potential; SKN-1, HSP-60 expression). Renogrit was also found to modulate apoptosis (EGL-1 mRNA expression; protein levels of p-ERK, p-JNK, p-p38, c-PARP1), necroptosis (intracellular calcium accumulation; RIPK1, RIPK3, MLKL mRNA expression), mitophagy (lysosome population; mRNA expression of PINK1, PDR1; protein levels of p-PINK1, LC3B), and inflammation (IL-1ß activity; protein levels of LXR-α). More importantly, Renogrit treatment did not hamper normal anti-proliferative effects of cisplatin as observed from cytotoxicity analysis on MCF-7, A549, SiHa, and T24 human cancer cells. Taken together, Renogrit could be a potential clinical candidate to mitigate cisplatin-induced nephrotoxicity without compromising the anti-neoplastic properties of cisplatin.
Assuntos
Apoptose , Caenorhabditis elegans , Cisplatino , Mitofagia , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Animais , Humanos , Mitofagia/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Extratos Vegetais/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Antineoplásicos/efeitos adversos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologiaRESUMO
Bisphenol P (BPP) has been detected in human biological samples; however studies on its nephrotoxicity are scarce. Given the susceptibility of kidneys to endocrine-disrupting chemicals, there is an urgent need to investigate the renal toxicity of BPP. This study aimed to evaluate the effects of different concentrations of BPPs on the kidneys of C57BL/6 mice and elucidate the underlying mechanisms of renal damage using a combination of mouse renal transcriptomic data and human renal proximal tubular epithelial cells (HK-2). Mice were exposed to BPP (0, 0.3, 30, 3000 µg/kg bw/d) via gavage for 5 weeks. Renal injury was assessed based on changes in body and kidney weights, serum renal function indices, and histopathological examination. Transcriptomic analysis identified differentially expressed genes and pathways, whereas cellular assays were used to measure cell viability, reactive oxygen species (ROS), apoptosis, and the expression of key genes and proteins. The results show that BPP exposure induces renal injury, as evidenced by increased body weight, abnormal renal function indices, and renal tissue damage. Transcriptomic analysis revealed alterations in genes and pathways related to oxidative stress, p53 signaling, autophagy, and apoptosis. Cellular experiments confirmed that BPP induces oxidative stress and apoptosis. Furthermore, BPP exposure significantly inhibits autophagy, potentially exacerbating apoptosis and contributing to kidney injury. Treatment with a ROS inhibitor (N-Acetylcysteine, NAC) mitigated BPP-induced autophagy inhibition and apoptosis, implicating oxidative stress as a key factor. BPP exposure may lead to renal injury through excessive ROS accumulation, oxidative stress, inflammatory responses, autophagy inhibition, and increased apoptosis. The effects of NAC highlight the role of oxidative stress in BPP-induced nephrotoxicity. These findings enhance our understanding of BPP-induced nephrotoxicity and underscore the need to control BPP exposure to prevent renal disease. This study emphasized the importance of evaluating the safety of new Bisphenol A analogs, including BPP, in environmental toxicology.
Assuntos
Células Epiteliais , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fenóis , Animais , Humanos , Camundongos , Apoptose/efeitos dos fármacos , Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Rim/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Estresse Oxidativo/efeitos dos fármacos , Fenóis/toxicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.
Assuntos
Metabolismo Energético , Inflamação , Estresse Oxidativo , Tramadol , Estresse Oxidativo/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Tramadol/efeitos adversos , Tramadol/farmacologia , Metabolismo Energético/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Humanos , Linhagem Celular , Animais , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/farmacologiaRESUMO
It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.
Assuntos
Albuminas , Angiotensina II , Endocitose , Células Epiteliais , Glucose , Túbulos Renais Proximais , Receptor Tipo 1 de Angiotensina , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Angiotensina II/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Albuminas/metabolismo , Suínos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Losartan/farmacologiaRESUMO
Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney.
Assuntos
Injúria Renal Aguda , Dexametasona , Células Epiteliais , Túbulos Renais Proximais , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Animais , Dexametasona/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , Humanos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Nanopartículas , Masculino , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismoAssuntos
Compostos Benzidrílicos , Biomarcadores , Glucosídeos , Insuficiência Cardíaca , Túbulos Renais Proximais , Inibidores do Transportador 2 de Sódio-Glicose , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Benzidrílicos/uso terapêutico , Biomarcadores/sangue , Glucosídeos/uso terapêutico , Glucosídeos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologiaRESUMO
This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson's staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1's 3'-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.
Assuntos
Apoptose , Proliferação de Células , Células Epiteliais , Glucose , Fator de Crescimento Insulin-Like I , Túbulos Renais , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Glucose/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular , Túbulos Renais/metabolismo , Túbulos Renais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 3/genéticaRESUMO
Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Humanos , Técnicas de Cultura de Células em Três Dimensões/métodos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Técnicas de Cultura de Células/métodos , Antineoplásicos/farmacologia , Automação , Automação Laboratorial/métodos , Neoplasias/patologia , Neoplasias/tratamento farmacológicoRESUMO
BACKGROUND: The protection of the diabetic kidney by Empagliflozin (EMPA) is attributed to its interaction with the sodium glucose cotransporter 2 located on proximal tubular epithelial cells (PTECs). Estrogen-related receptor α (ESRRA), known for its high expression in PTECs and association with mitochondrial biogenesis, plays a crucial role in this process. This study aimed to explore the impact of ESRRA on mitochondrial mass in diabetic tubular injury and elucidate the mechanism underlying the protective effects of EMPA. METHODS: Mitochondrial changes in PTECs of 16-week-old diabetic mice were assessed using transmission electron microscopy (TEM) and RNA-sequences. In vivo, EMPA administration was carried out in db/db mice for 8 weeks, while in vitro experiments involved modifying ESRRA expression in HK2 cells using pcDNA-ESRRA or EMPA. RESULTS: Evaluation through TEM revealed reduced mitochondrial mass and swollen mitochondria in PTECs, whereas no significant changes were observed under light microscopy. Analysis of RNA-sequences identified 110 downregulated genes, including Esrra, associated with mitochondrial function. Notably, ESRRA overexpression rescued the loss of mitochondrial mass induced by high glucose (HG) in HK2 cells. EMPA treatment ameliorated the ultrastructural alterations and mitigated the downregulation of ESRRA both in db/db mice and HG-treated HK2 cells. CONCLUSION: The diminished expression of ESRRA is implicated in the decline of mitochondrial mass in PTECs during the early stages of diabetes, highlighting it as a key target of EMPA for preventing the progression of diabetic kidney injury.
Assuntos
Compostos Benzidrílicos , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Glucosídeos , Mitocôndrias , Animais , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Compostos Benzidrílicos/farmacologia , Camundongos , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/metabolismo , Masculino , Receptor ERRalfa Relacionado ao Estrogênio , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/ultraestrutura , Camundongos Endogâmicos C57BL , Linhagem Celular , Receptores de Estrogênio/metabolismoRESUMO
Pharmacokinetic changes induced by radiation following radiotherapy ("RT-PK" phenomenon) are of great significance to the effectiveness and safety of chemotherapeutic agents in clinical settings. The aims of this study were to clarify the organic anion transporters (Oats) involved in the "RT-PK" phenomenon of bestatin in rats following X-ray irradiation and to elucidate its potential mechanism via vitamin D signalling. Pharmacokinetic studies, uptake assays using rat kidney slices and primary proximal tubule cells, and molecular biological studies were performed. Significantly increased plasma concentrations and systemic exposure to bestatin were observed at 24 and 48 h following abdominal X-ray irradiation, regardless of oral or intravenous administration of the drugs in rats. Reduced renal clearance and cumulative urinary excretion of bestatin were observed at 24 and 48 h post-irradiation in rats following intravenous administration. The uptake of the probe substrates p-aminohippuric acid and oestrone 3-sulfate sodium in vitro and the expression of Oat1 and Oat3 in vivo were reduced in the corresponding models following irradiation. Moreover, the upregulation of the vitamin D receptor (Vdr) in mRNA and protein levels negatively correlated with the expressions and functions of Oat1 and Oat3 following irradiation. Additionally, elevated plasma urea nitrogen levels and histopathological changes were observed in rats after exposure to irradiation. The "RT-PK" phenomenon of bestatin occurs in rats after exposure to irradiation, possibly resulting in the regulation of the expressions and activities of renal Oats via activation of the Vdr signalling pathway.
Assuntos
Regulação para Baixo , Rim , Receptores de Calcitriol , Animais , Ratos , Receptores de Calcitriol/metabolismo , Masculino , Rim/metabolismo , Rim/efeitos da radiação , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Ratos Sprague-Dawley , Raios X , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/efeitos da radiação , Túbulos Renais Proximais/efeitos dos fármacos , Leucina/análogos & derivadosRESUMO
Ochratoxin A (OTA), a secondary fungal metabolite known for its nephrotoxic effects, is prevalent in various feeds and food items. Our recent study suggests that OTA-induced nephrotoxicity is linked to the Sigma-1 receptor (Sig-1R)-mediated mitochondrial pathway apoptosis in human proximal tubule epithelial-originated kidney-2 (HK-2) cells. However, the contribution of Sig-1R to OTA-induced nephrotoxicity involving other forms of regulated cell death, such as ferroptosis, remains unexplored. In this investigation, cell viability, malondialdehyde (MDA) levels, glutathione (GSH) levels, and protein expressions in HK-2 cells treated with OTA and/or Ferrostatin-1/blarcamesine hydrochloride/BD1063 dihydrochloride were assessed. The results indicate that a 24 h-treatment with 1 µM OTA significantly induces ferroptosis by inhibiting Sig-1R, subsequently promoting nuclear receptor coactivator 4 (NCOA4), long-chain fatty acid-CoA ligase 4 (ACSL4), arachidonate 5-lipoxygenase (ALOX5), autophagy protein 5 (ATG5), and ATG7, inhibiting ferritin heavy chain (FTH1), solute carrier family 7 member 11 (SLC7A11/xCT), glutathione peroxidase 4 (GPX4), peroxiredoxin 6 (PRDX6), and ferroptosis suppressor protein 1 (FSP1), reducing GSH levels, and increasing MDA levels (P < 0.05). In conclusion, OTA induces ferroptosis by inhibiting Sig-1R, subsequently promoting ferritinophagy, inhibiting GPX4/FSP1 antioxidant systems, reducing GSH levels, and ultimately increasing lipid peroxidation levels in vitro.
Assuntos
Ferroptose , Ocratoxinas , Receptores sigma , Receptor Sigma-1 , Ocratoxinas/toxicidade , Ferroptose/efeitos dos fármacos , Receptores sigma/metabolismo , Humanos , Linhagem Celular , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismoRESUMO
Cisplatin is an effective chemotherapeutic agent widely used for the treatment of various solid tumors. However, cisplatin has an important limitation in its use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Thrombomodulin (TM) is well known not only for its role as a cofactor in the clinically important natural anticoagulation pathway but also for its anti-inflammatory properties. Here, we investigated the effects of TM in cisplatin-induced AKI. In mice intraperitoneally injected with 15 mg/kg cisplatin, TM (10 mg/kg) or PBS was administered intravenously at 24 h after cisplatin injection. TM significantly attenuated cisplatin-induced nephrotoxicity with the suppressed elevation of blood urea nitrogen and serum creatinine, and reduced histological damages. Actually, TM treatment significantly alleviated oxidative stress-induced apoptosis by reducing reactive oxygen species (ROS) levels in cisplatin-treated renal proximal tubular epithelial cells (RPTECs) in vitro. Furthermore, TM clarified cisplatin-induced apoptosis by reducing caspase-3 levels. In addition, TM attenuated the endoplasmic reticulum (ER) stress signaling pathway in both renal tissues and RPTECs to protect the kidneys from cisplatin-induced AKI. These findings suggest that TM is a potential protectant against cisplatin-induced nephrotoxicity through suppressing ROS generation and ER stress in response to cisplatin.
Assuntos
Injúria Renal Aguda , Apoptose , Cisplatino , Estresse do Retículo Endoplasmático , Estresse Oxidativo , Espécies Reativas de Oxigênio , Trombomodulina , Cisplatino/efeitos adversos , Animais , Trombomodulina/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Apoptose/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Antineoplásicos/efeitos adversos , Antineoplásicos/toxicidade , Camundongos Endogâmicos C57BL , Nitrogênio da Ureia Sanguínea , Transdução de Sinais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologiaRESUMO
Increased dietary phosphate consumption intensifies renal phosphate burden. Several mechanisms for phosphate-induced renal tubulointerstitial fibrosis have been reported. Considering the dual nature of phosphate as both a potential renal toxin and an essential nutrient for the body, kidneys may possess inherent protective mechanisms against phosphate overload, rather than succumbing solely to injury. However, there is limited understanding of such mechanisms. To identify these mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) analysis of the kidneys of control and dietary phosphate-loaded (Phos) mice at a time point when the Phos group had not yet developed tubulointerstitial fibrosis. scRNA-seq analysis identified the highest number of differentially expressed genes in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these differentially expressed genes, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid ß-oxidation (FAO) in the PTECs. This activation was further substantiated through various experiments, including the use of an FAO activity visualization probe. Compared with wild-type mice, Ppara knockout mice exhibited exacerbated tubulointerstitial fibrosis in response to phosphate overload. Experiments conducted with cultured PTECs demonstrated that activation of the PPAR-α/FAO pathway leads to improved cellular viability under high-phosphate conditions. The Phos group mice showed a decreased serum concentration of free fatty acids, which are endogenous PPAR-α agonists. Instead, experiments using cultured PTECs revealed that phosphate directly activates the PPAR-α/FAO pathway. These findings indicate that noncanonical metabolic reprogramming via endogenous activation of the PPAR-α/FAO pathway in PTECs is essential to counteract phosphate toxicity.NEW & NOTEWORTHY This study revealed the activation of peroxisome proliferator-activated receptor-α and fatty acid ß-oxidation in proximal tubular epithelial cells as an endogenous mechanism to protect the kidney from phosphate toxicity. These findings highlight noncanonical metabolic reprogramming as a potential target for suppressing phosphate toxicity in the kidneys.
Assuntos
Túbulos Renais Proximais , PPAR alfa , Fosfatos , Animais , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR alfa/genética , Fosfatos/metabolismo , Fosfatos/toxicidade , Fibrose , Camundongos Endogâmicos C57BL , Masculino , Camundongos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Ácidos Graxos/metabolismo , Camundongos Knockout , OxirreduçãoRESUMO
Cisplatin (CDDP) is administered as an anticancer drug across a broad spectrum of cancer treatments, but it causes severe renal damage. Several studies have attempted to elucidate the cause of CDDP-induced renal injury, but the detailed mechanism remains unclear. We previously found that S3 cells are more sensitive to CDDP than S1 and S2 cells by using immortalized cells derived from S1, S2, and S3 segments of proximal tubules. In this study, we investigated the potential contribution of reactive oxygen species (ROS) to the sensitivity of S3 cells to CDDP. The results showed that S3 cells have high sensitivity to CDDP, paraquat (PQ) and three ROS substances. To examine the mechanisms underlying the sensitivity to ROS in S3 cells, we compared the cellular responses of CDDP- and PQ-exposed S3 cells. The results indicated that the levels of intracellular ROS and lipid peroxides were increased in S3 cells after CDDP and PQ exposure. The intracellular levels of antioxidant proteins such as thioredoxin, thioredoxin reductase 1 and glutathione peroxidase 4 were also increased by exposure to PQ, but these proteins were decreased by CDDP exposure in S3 cells. Furthermore, the levels of intracellular free Fe2+ were increased by CDDP exposure only in S3 cells but not S1 or S2 cells, and cytotoxicity by exposure to CDDP in S3 cells was suppressed by ferroptosis inhibitors. These results suggested that the induction of ferroptosis due to the ROS production through attenuation of the antioxidant system and elevated free Fe2+ is partly responsible for the sensitivity of S3 cells to CDDP.
Assuntos
Antineoplásicos , Cisplatino , Ferroptose , Túbulos Renais Proximais , Espécies Reativas de Oxigênio , Cisplatino/toxicidade , Cisplatino/farmacologia , Ferroptose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/toxicidade , Antineoplásicos/farmacologia , Animais , Paraquat/toxicidade , Linhagem Celular , Linhagem Celular Transformada , Camundongos , Sobrevivência Celular/efeitos dos fármacosRESUMO
During the progression of diabetic kidney disease, proximal tubular epithelial cells respond to high glucose to induce hypertrophy and matrix expansion leading to renal fibrosis. Recently, a non-canonical PTEN has been shown to be translated from an upstream initiation codon CUG (leucine) to produce a longer protein called PTEN-Long (PTEN-L). Interestingly, the extended sequence present in PTEN-L contains cell secretion/penetration signal. Role of this non-canonical PTEN-L in diabetic renal tubular injury is not known. We show that high glucose decreases expression of PTEN-L. As a mechanism of its function, we find that reduced PTEN-L activates Akt-2, which phosphorylates and inactivate tuberin and PRAS40, resulting in activation of mTORC1 in tubular cells. Antibacterial agent acriflavine and antiviral agent ATA regulate translation from CUG codon. Acriflavine and ATA, respectively, decreased and increased expression of PTEN-L to altering Akt-2 and mTORC1 activation in the absence of change in expression of canonical PTEN. Consequently, acriflavine and ATA modulated high glucose-induced tubular cell hypertrophy and lamininγ1 expression. Importantly, expression of PTEN-L inhibited high glucose-stimulated Akt/mTORC1 activity to abrogate these processes. Since PTEN-L contains secretion/penetration signals, addition of conditioned medium containing PTEN-L blocked Akt-2/mTORC1 activity. Notably, in renal cortex of diabetic mice, we found reduced PTEN-L concomitant with Akt-2/mTORC1 activation, leading to renal hypertrophy and lamininγ1 expression. These results present first evidence for involvement of PTEN-L in diabetic kidney disease.
Assuntos
Nefropatias Diabéticas , Glucose , Túbulos Renais Proximais , Alvo Mecanístico do Complexo 1 de Rapamicina , PTEN Fosfo-Hidrolase , Animais , Humanos , Masculino , Camundongos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/genética , Regulação para Baixo/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Transdução de SinaisRESUMO
Diabetic nephropathy (DN) is one of the most serious and frequent complications among diabetes patients and presently constitutes vast the cases of end-stage renal disease worldwide. Tubulointerstitial fibrosis is a crucial factor related to the occurrence and progression of DN. Oridonin (Ori) is a diterpenoid derived from rubescens that has diverse pharmacological properties. Our previous study showed that Ori can protect against DN by decreasing the inflammatory response. However, whether Ori can alleviate renal fibrosis in DN remains unknown. Here, we investigated the mechanism through which Ori affects the Wnt/ß-catenin signaling pathway in diabetic rats and human proximal tubular epithelial cells (HK-2) exposed to high glucose (HG) levels. Our results revealed that Ori treatment markedly decreased urinary protein excretion levels, improved renal function and alleviated renal fibrosis in diabetic rats. In vitro, HG treatment increased the migration of HK-2 cells while reducing their viability and proliferation rate, and treatment with Ori reversed these changes. Additionally, the knockdown of ß-catenin arrested cell migration and reduced the expression levels of Wnt/ß-catenin signaling-related molecules (Wnt4, p-GSK3ß and ß-catenin) and fibrosis-related molecules (α-smooth muscle actin, collagen I and fibronectin), and Ori treatment exerted an effect similar to that observed after the knockdown of ß-catenin. Furthermore, the combination of Ori treatment and ß-catenin downregulation exerted more pronounced biological effects than treatment alone. These findings may provide the first line of evidence showing that Ori alleviates fibrosis in DN by inhibiting the Wnt/ß-catenin signaling pathway and thereby reveal a novel therapeutic avenue for treating tubulointerstitial fibrosis.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Diterpenos do Tipo Caurano , Fibrose , Via de Sinalização Wnt , Animais , Humanos , Masculino , Ratos , beta Catenina/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/etiologia , Diterpenos do Tipo Caurano/farmacologia , Diterpenos do Tipo Caurano/uso terapêutico , Fibrose/tratamento farmacológico , Rim/patologia , Rim/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Glomerular issues and affected podocytes are at the origin of 80% of chronic kidney disease cases. Thus, acquiring a deeper understanding in this domain is necessary to halt progressive kidney damage. In this study, the authors investigated the harmful impact of podocyte-cleaved soluble retinoic acid receptor responder protein-1 on podocytes and proximal tubular cells and identified matrix metalloprotease 23 as the enzyme responsible for cleaving retinoic acid receptor responder protein-1. These findings provide new insights into chronic kidney disease progression, suggesting innovative treatment avenues.