RESUMEN
Dyslipidemia is the most prevalent independent risk factor for patients with chronic kidney disease (CKD). Lipid-induced NLRP3 inflammasome activation in kidney-resident cells exacerbates renal injury by causing sterile inflammation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that modulates the cellular redox balance; however, the exact role of Nrf2 signaling and its regulation of the NLRP3 inflammasome in hyperlipidemia-induced kidney injury are poorly understood. In this study, we demonstrated that activation of the mtROS-NLRP3 inflammasome pathway is a critical contributor to renal tubular epithelial cell (RTEC) apoptosis under hyperlipidemia. In addition, the Nrf2/ARE signaling pathway is activated in renal tubular epithelial cells under hyperlipidemia conditions both in vivo and in vitro, and Nrf2 silencing accelerated palmitic acid (PA)-induced mtROS production, mitochondrial injury, and NLRP3 inflammasome activation. However, the activation of Nrf2 with tBHQ ameliorated mtROS production, mitochondrial injury, NLRP3 inflammasome activation, and cell apoptosis in PA-induced HK-2 cells and in the kidneys of HFD-induced obese rats. Furthermore, mechanistic studies showed that the potential mechanism of Nrf2-induced NLRP3 inflammasome inhibition involved reducing mtROS generation. Taken together, our results demonstrate that the Nrf2/ARE signaling pathway attenuates hyperlipidemia-induced renal injury through its antioxidative and anti-inflammatory effects through the downregulation of mtROS-mediated NLRP3 inflammasome activation.
Asunto(s)
Células Epiteliales , Hiperlipidemias , Inflamasomas , Túbulos Renales , Factor 2 Relacionado con NF-E2 , Proteína con Dominio Pirina 3 de la Familia NLR , Transducción de Señal , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/complicaciones , Hiperlipidemias/inmunología , Células Epiteliales/metabolismo , Ratas , Humanos , Túbulos Renales/patología , Túbulos Renales/metabolismo , Masculino , Línea Celular , Apoptosis , Elementos de Respuesta Antioxidante , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Ratas Sprague-DawleyRESUMEN
Background: Lipid metabolism disorders lead to lipotoxicity. The hyperlipidemia-induced early stage of renal injury mainly manifests as podocyte damage. CD36 mediates fatty acid uptake and the subsequent accumulation of toxic lipid metabolites, resulting in podocyte lipotoxicity. Methods: Male Sprague-Dawley rats were divided into two groups: the normal control group and the high-fat diet group (HFD). Podocytes were cultured and treated with palmitic acid (PA) and sulfo-N-succinimidyl oleate (SSO). Protein expression was measured by immunofluorescence and western blot analysis. Boron-dipyrromethene staining and Oil Red O staining was used to analyze fatty acid accumulation. Results: Podocyte foot process (FP) effacement and marked proteinuria occurred in the HFD group. CD36 protein expression was upregulated in the HFD group and in PA-treated podocytes. PA-treated podocytes showed increased fatty acid accumulation, reactive oxygen species (ROS) production, and actin cytoskeleton rearrangement. However, pretreatment with the CD36 inhibitor SSO decreased lipid accumulation and ROS production and alleviated actin cytoskeleton rearrangement in podocytes. The antioxidant N-acetylcysteine suppressed PA-induced podocyte FP effacement and ROS generation. Conclusions: CD36 participated in fatty acid-induced FP effacement in podocytes via oxidative stress, and CD36 inhibitors may be helpful for early treatment of kidney injury.
RESUMEN
Lysosomal dysfunction and impaired autophagic flux are involved in the pathogenesis of lipotoxicity in the kidney. Here, we investigated the role of transcription factor EB (TFEB), a master regulator of autophagy-lysosomal pathway, in palmitic acid induced renal tubular epithelial cells injury. We examined lipid accumulation, autophagic flux, expression of Ps211-TFEB, and nuclear translocation of TFEB in HK-2 cells overloaded with palmitic acid (PA). By utilizing immunohistochemistry, we detected TFEB expression in renal biopsy tissues from patients with diabetic nephropathy and normal renal tissue adjacent to surgically removed renal carcinoma (controls), as well as kidney tissues from rat fed with high-fat diet (HFD) and low-fat diet (LFD). We found significant lipid accumulation, increased apoptosis, accompanied with elevated Ps211-TFEB, decreased nuclear TFEB, reduced lysosome biogenesis and insufficient autophagy in HK-2 cells treated with PA. Kidney tissues from patients with diabetic nephropathy had lower nuclear and total levels of TFEB than that in control kidney tissues. Level of renal nuclear TFEB in HFD rats was also lower than that in LFD rats. Exogenous overexpression of TFEB increased the nuclear TFEB level in HK-2 cells treated with PA, promoted lysosomal biogenesis, improved autophagic flux, reduced lipid accumulation and apoptosis. Our results collectively indicate that PA is a strong inducer for TFEB phosphorylation modification at ser211 accompanied with lower nuclear translocation of TFEB. Impairment of TFEB-mediated lysosomal biogenesis and function by palmitic acid may lead to insufficient autophagy and promote HK-2 cells injury.
Asunto(s)
Nefropatías Diabéticas , Ácido Palmítico , Ratas , Humanos , Animales , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Nefropatías Diabéticas/metabolismo , Autofagia , Lisosomas/metabolismo , Células Epiteliales/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismoRESUMEN
Aims: Oxidative stress plays a crucial role in podocyte injury in diabetic nephropathy (DN). tert-Butylhydroquinone (tBHQ) is an activator of Nrf2 that exerts protective effects in diabetic mice, but the underlying mechanism of tBHQ in the podocytes of DN is not fully understood. Materials and methods: A high glucose (HG)-induced HK2 cell model and streptozotocin-induced rat model of DN were established and treated with tBHQ or apocynin. The expression levels of Nrf2, HO-1, NOX2 and NOX4 were determined by Western blot or immunohistochemical staining. The level of oxidative stress in podocytes or kidney tissues was assessed using DCFH-DA or dihydroethidium (DHE) staining. Cell injury was assessed by F-actin staining and flow cytometry analysis. Key findings: We showed that HG treatment increased the expressions of NOX2 and NOX4 and enhanced ROS production in podocytes. Inhibition of NADPH oxidase activity by apocynin dramatically attenuated HG-induced ROS production and further alleviated cell injury and apoptosis in podocytes. Moreover, we found that HG inhibited the Nrf2/HO-1 signalling pathway in podocytes; however, tBHQ treatment significantly activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity, and attenuated ROS production and cell injury in HG-treated podocytes. Furthermore, we observed that tBHQ treatment partially attenuated renal injury, activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity and reduced ROS generation in the kidneys of STZ-induced diabetic rats. Significance: These results suggest that tBHQ exerts a protective role in hyperglycaemia-induced podocyte injury, and that the potential protective mechanism of tBHQ involves inhibiting NADPH oxidase-derived ROS generation by activating the Nrf2/HO-1 signalling pathway.
RESUMEN
Chronic kidney disease (CKD) is often accompanied by dyslipidemia, and abnormal lipid metabolism in proximal tubule cells is considered closely related to the dysfunction of proximal tubule cells and eventually leads to accelerated kidney damage. Nuclear factor E2-related factor 2 (Nrf2), known as a redox-sensitive transcription factor, is responsible for regulating cellular redox homeostasis. However, the exact role of Nrf2 in dyslipidemia-induced dysfunction of proximal tubule cells is still not fully elucidated. In the present study, we showed that palmitic acid (PA) induced mitochondrial damage, excessive mitochondrial reactive oxygen species (ROS) (mtROS) generation, and cell injury in HK-2 cells. We further found that mtROS generation was involved in PA-induced mitochondrial dysfunction, cytoskeletal damage, and cell apoptosis in HK-2 cells. In addition, we demonstrated that the Nrf2/ARE signaling pathway was activated in PA-induced HK-2 cells and that silencing Nrf2 dramatically aggravated PA-induced mtROS production, mitochondrial damage, cytoskeletal damage and cell apoptosis in HK-2 cells. However, the mitochondrial antioxidant MitoTEMPOL effectively eliminated these negative effects of Nrf2 silencing in HK-2 cells under PA stimulation. Moreover, activation of the Nrf2/ARE signaling pathway with tBHQ attenuated renal injury, significantly reduced mtROS generation, and improved mitochondrial function in rats with HFD-induced obesity. Taken together, these results suggest that the Nrf2/ARE-mediated antioxidant response plays a protective role in hyperlipidemia-induced renal injury by ameliorating mtROS-mediated mitochondrial dysfunction and that enhancing Nrf2 antioxidant signaling provides a potential therapeutic strategy for kidney injury in CKD with hyperlipidemia.
RESUMEN
To investigate its effect and molecular regulatory mechanism on vascular calcification, EPO was added to vascular smooth muscle cells cultured in vitro and injected intraperitoneally into SD rats. The effect of EPO on VSMC calcification was determined by alizarin red staining and ALP activity. Differentially expressed genes were screened by transcriptome sequencing and the relationship and function were verified. We found EPO promotes VSMC calcification in vitro and blood calcification in vivo in a dose-dependent manner. A total of 88 upregulated genes and 59 downregulated genes were detected in transcriptome sequencing, among which the expression of genes associated with bone formation exhibited a marked increase, namely the GATA6 transcription factor, BMP2, RUNX2, OPN, and OCN. Dual luciferase assay has indicated that the binding of GATA6 to BMP2 promoter facilitates the transcription of BMP2. Taken together, findings indicate that EPO can enhance the calcification of VSMCs by activating the GATA6/BMP2 signal axis.
Asunto(s)
Eritropoyetina/toxicidad , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Calcificación Vascular/inducido químicamente , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Células Cultivadas , Eritropoyetina/administración & dosificación , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Regulación de la Expresión Génica , Inyecciones Intraperitoneales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Osteogénesis/genética , Ratas Sprague-Dawley , Transducción de Señal , Transcriptoma , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/patologíaRESUMEN
Lipid accumulation in podocytes can lead to the destruction of cellular morphology, in addition to cell dysfunction and apoptosis, which is a key factor in the progression of chronic kidney disease (CKD). Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Coptis chinensis, which has been reported to have a lipidlowering effect and prevent CKD progression. Therefore, the present study aimed to investigate the effect of BBR on palmitic acid (PA)induced podocyte apoptosis and its specific mechanism using an in vitro model. Cell death was measured using the Cell Counting Kit8 colorimetric assay. Cell apoptotic rate was assessed by flow cytometry. The expression of endoplasmic reticulum (ER) stress and apoptosisrelated proteins was detected by western blotting or immunofluorescence. Reactive oxygen species (ROS) were evaluated by 2',7'dichlorofluorescein diacetate fluorescence staining. The results of the present study revealed that BBR treatment decreased PAinduced podocyte apoptosis. In addition, 4phenylbutyric acid significantly reduced PAinduced cell apoptosis and the expression of ER stressrelated proteins, which indicated that ER stress was involved in PAinduced podocyte apoptosis. In addition, Nacetylcysteine inhibited PAinduced excessive ROS production, ER stress and cell apoptosis of podocytes. BBR also significantly reduced PAinduced ROS production and ER stress in podocytes. These results suggested that PA mediated podocyte apoptosis through enhancing ER stress and the production of ROS. In conclusion, BBR may protect against PAinduced podocyte apoptosis, and suppression of ROSdependent ER stress may be the key mechanism underlying the protective effects of BBR.
Asunto(s)
Berberina/farmacología , Ácido Palmítico/efectos adversos , Podocitos/citología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Ratones , Estrés Oxidativo/efectos de los fármacos , Podocitos/efectos de los fármacos , Podocitos/metabolismoRESUMEN
AIMS: Although autophagy impairment is a well-established cause of muscle atrophy and P300 has recently been identified as an important regulator of autophagy, the effects of P300 on autophagy and muscle atrophy in type 2 diabetes (T2D) remain unexplored. We aimed at characterizing the role of P300 in diabetic muscle and its underlying mechanism. MAIN METHODS: Protein levels of phosphorylated P300, total P300, acetylated histone H3, LC3, p62 and myosin heavy chain, and mRNA levels of Atrogin-1 and MuRF1 were analyzed in palmitic acid (PA)-treated myotubes and db/db mice. Autophagic flux was assessed using transmission electron microscopy, immunofluorescence and mRFP-GFP-LC3 lentivirus transfection in cells. Muscle weight, blood glucose and grip strength were measured in mice. Hematoxylin and eosin (H&E) staining was performed to determine changes in muscle fiber size. To investigate the effects of P300 on autophagy and myofiber remodeling, a P300 specific inhibitor, c646, was utilized. 3-Methyladenine (3-MA) was utilized to inhibit autophagosomes formation, and chloroquine (CQ) was used to block autophagic flux. KEY FINDINGS: Phosphorylation of P300 in response to PA enhanced its activity and subsequently suppressed autophagic flux, leading to atrophy-related morphological and molecular changes in myotubes. Inhibition of P300 reestablished autophagic flux and ameliorated PA-induced myotubes atrophy. However, this effect was largely abolished by co-treatment with the autophagy inhibitor CQ. In vivo results demonstrated that inhibition of P300 partially rescued muscle wasting in db/db mice, accompanied with autophagy reactivation. SIGNIFICANCE: The findings revealed that T2D-induced overactivation of P300 contributes to muscle atrophy by blocking autophagic flux.
Asunto(s)
Autofagia/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Atrofia Muscular/metabolismo , Animales , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Proteína p300 Asociada a E1A/genética , Fuerza de la Mano/fisiología , Masculino , Ratones , Ratones Transgénicos , Atrofia Muscular/genética , Atrofia Muscular/patología , Mioblastos/metabolismo , Mioblastos/patologíaRESUMEN
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD), and renal tubular cell dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH) is an enzyme that can hydrolyze epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs) into the less biologically active metabolites. Inhibition of sEH has multiple beneficial effects on renal function, however, the exact role of sEH in hyperglycemia-induced dysfunction of tubular cells is still not fully elucidated. In the present study, we showed that human proximal tubular epithelial (HK-2) cells revealed an upregulation of sEH expression accompanied by the impairment of autophagic flux, mitochondrial dysfunction, ubiquitinated protein accumulation and enhanced endoplasmic reticulum (ER) stress after high glucose (HG) treatment. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which resulted in excessive reactive oxygen species (ROS) generation, Bax translocation, cytochrome c release, and apoptosis. However, t-AUCB, an inhibitor of sEH, partially reversed these negative outcomes. Moreover, we also observed increased sEH expression, impaired autophagy flux, mitochondrial dysfunction and enhanced ER stress in the renal proximal tubular cells of db/db diabetic mice. Notably, inhibition of sEH by treatment with t-AUCB attenuated renal injury and partially restored autophagic flux, improved mitochondrial function, and reduced ROS generation and ER stress in the kidneys of db/db mice. Taken together, these results suggest that inhibition of sEH by t-AUCB plays a protective role in hyperglycemia-induced proximal tubular injury and that the potential mechanism of t-AUCB-mediated protective autophagy is involved in modulating mitochondrial function and ER stress. Thus, we provide new evidence linking sEH to the autophagic response during proximal tubular injury in the pathogenesis of DN and suggest that inhibition of sEH can be considered a potential therapeutic strategy for the amelioration of DN.
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Nefropatías Diabéticas/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Epóxido Hidrolasas/farmacología , Mitocondrias/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/tratamiento farmacológico , Estrés del Retículo Endoplásmico/fisiología , Epóxido Hidrolasas/genética , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/patología , Riñón/patología , Mitocondrias/metabolismoRESUMEN
Diabetic nephropathy (DN), the primary cause of end-stage renal disease (ESRD), is often accompanied by dyslipidemia, which is closely related to the occurrence and development of DN and even the progression to ESRD. Mitophagy, the selective degradation of damaged and dysfunctional mitochondria by autophagy, is a crucial mitochondrial quality control mechanism, and largely regulated by PINK1 (PTEN-induced putative kinase 1)/Parkin signaling pathway. In the present study, we demonstrated that PA induced mitochondrial damage and excessive mitoROS generation in podocytes. We also found PA treatment resulted in the activation of mitophagy by increasing co-localization of GFP-LC3 with mitochondria and enhancing the formation of mitophagosome, stabilization of PINK1 and mitochondrial translocation of Parkin, which indicated that PINK1/Parkin pathway was involved in PA-induced mitophagy in podocytes. Furthermore, inhibition of mitophagy by silencing Parkin dramatically aggravated PA-induced mitochondrial dysfunction, mitoROS production, and further enhanced PA-induced apoptosis of podocytes. Finally, we showed that PINK1/Parkin pathway were up-regulated in kidney of high fat diet (HFD)-induced obese rats. Taken together, our results suggest that PINK1/Parkin mediated mitophagy plays a protective role in PA-induced podocytes apoptosis through reducing mitochondrial ROS production and that enhancing mitophagy provides a potential therapeutic strategy for kidney diseases with hyperlipidemia, such as DN.
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Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitofagia/genética , Ácido Palmítico/farmacología , Podocitos/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Dieta Alta en Grasa , Silenciador del Gen , Riñón/metabolismo , Riñón/fisiopatología , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Obesidad/metabolismo , Podocitos/metabolismo , Podocitos/ultraestructura , Proteínas Quinasas/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Diabetes mellitus is a metabolic disorder that can lead to blood-brain barrier (BBB) disruption and cognitive decline. However, the mechanisms of BBB breakdown in diabetes are still unclear. Soluble epoxide hydrolase (sEH) is an enzyme that degrades epoxyeicosatrienoic acids (EETs), which have multiple protective effects on vascular structure and functions. In the current study, we showed increased vascular permeability of the BBB, which was accompanied by upregulation of sEH and downregulation of 14,15-EET. Moreover, the sEH inhibitor t-AUCB restored diabetic BBB integrity in vivo, and 14,15-EET prevented ROS accumulation and MEC injury in vitro. t-AUCB or 14,15-EET treatment provoked AMPK/HO-1 activation under diabetic conditions in vivo and in vitro. Thus, we suggest that decreased EET degradation by sEH inhibition might be a potential therapeutic approach to attenuate the progression of BBB injury in diabetic mice via AMPK/HO-1 pathway activation.
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Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Diabetes Mellitus Tipo 2/complicaciones , Epóxido Hidrolasas/antagonistas & inhibidores , Sustancias Protectoras/uso terapéutico , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Epóxido Hidrolasas/metabolismo , Hemo-Oxigenasa 1/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND Increased lipid accumulation in renal tubular epithelial cells (TECs) contributes to their injury and dysfunction and progression of tubulointerstitial fibrosis. Berberine (BBR), a natural plant alkaloid isolated from traditional medicine herbs, is effective in lowing serum lipid, and has a protective effect on chronic kidney disease (CKD) with dyslipidemia, including diabetic nephropathy. The aim of this study was to investigate the effect of BBR on palmitate (PA)-induced lipid accumulation and apoptosis in TECs. MATERIAL AND METHODS Human kidney proximal tubular epithelial cell line (HK-2) cells were treated with PA, BBR, and/or palmitoyltransferase 1A (CPT1A) inhibitor Etomoxir. Intracellular lipid content was assessed by Oil Red O and Nile Red staining. Cell apoptosis rate was evaluated by flow cytometry assay. The expression of apoptosis-related protein cleaved-caspase3 and fatty acid oxidation (FAO)-regulating proteins, including CPT1A, peroxisome proliferator-activated receptor α (PPARα), and PPARγ co-activator-1α (PGC1α), was measured by Western blot analysis and immunofluorescence. RESULTS In the present study, PA treatment increased intracellular lipid deposition accompanied by elevated apoptosis in TECs compared with control group, whereas the protein expression of CPT1A, PPARα, and PGC1α, did not correspondingly increase in TECs. BBR significantly up-regulated the protein expression of CPT1A, PPARα, and PGC1α in TECs treated with or without PA, and reversed PA-induced intracellular lipid accumulation and apoptosis. Moreover, the CPT1A inhibitor Etomoxir counteracted the protective effect of BBR in TECs. CONCLUSIONS These in vitro findings suggest that PA can induce intracellular lipid accumulation and apoptosis in TECs, and the mechanism may be associated with inducing defective FAO, whereas BBR can protect TECs against PA-induced intracellular lipid accumulation and apoptosis by promoting FAO.
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Apoptosis/efectos de los fármacos , Berberina/farmacología , Células Epiteliales/patología , Túbulos Renales/patología , Palmitatos/toxicidad , Sustancias Protectoras/farmacología , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Línea Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.
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Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Ácido Palmítico/farmacología , Podocitos/citología , Especies Reactivas de Oxígeno/metabolismo , Animales , Caspasas/metabolismo , Células Cultivadas , Ratones , Podocitos/efectos de los fármacosRESUMEN
Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis.