RESUMEN
Long non-coding RNAs (lncRNAs) are a group of epigenetic regulators that have been implicated in kidney diseases including acute kidney injury (AKI). However, very little is known about the specific lncRNAs involved in AKI and the mechanisms underlying their pathologic roles. Here, we report a new lncRNA derived from the pseudogene GSTM3P1, which mediates ischemic AKI by interacting with and promoting the degradation of mir-668, a kidney-protective microRNA. GSTM3P1 and its mouse orthologue Gstm2-ps1 were induced by hypoxia in cultured kidney proximal tubular cells. In mouse kidneys, Gstm2-ps1 was significantly upregulated in proximal tubules at an early stage of ischemic AKI. This transient induction of Gstm2-ps1 depends on G3BP1, a key component in stress granules. GSTM3P1 overexpression increased kidney proximal tubular apoptosis after ATP depletion, which was rescued by mir-668. Notably, kidney proximal tubule-specific knockout of Gstm2-ps1 protected mice from ischemic AKI, as evidenced by improved kidney function, diminished tubular damage and apoptosis, and reduced kidney injury biomarker (NGAL) induction. To test the therapeutic potential, Gstm2-ps1 siRNAs were introduced into cultured mouse proximal tubular cells or administered to mice. In cultured cells, Gstm2-ps1 knockdown suppressed ATP depletion-associated apoptosis. In mice, Gstm2-ps1 knockdown ameliorated ischemic AKI. Mechanistically, both GSTM3P1 and Gstm2-ps1 possessed mir-668 binding sites and downregulated the mature form of mir-668. Specifically, GSTM3P1 directly bound to mature mir-668 to induce its decay via target-directed microRNA degradation. Thus, our results identify GSTM3P1 as a novel lncRNA that promotes kidney tubular cell death in AKI by binding mir-668 to inducing its degradation.
RESUMEN
The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.
Asunto(s)
Enfermedades Renales , Obstrucción Ureteral , Ratones , Animales , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismo , Factor 1 Inducible por Hipoxia , Enfermedades Renales/patología , Hipoxia , Autofagia/genética , Fibrosis , Cloroquina/farmacologíaRESUMEN
Nephrotoxicity is a major side effect of cisplatin, a widely used cancer therapy drug. However, the mechanism of cisplatin nephrotoxicity remains unclear and no effective kidney protective strategies are available. Here, we report the induction of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in both in vitro cell culture and in vivo mouse models of cisplatin nephrotoxicity. Notably, PFKFB3 was mainly induced in the nucleus of kidney tubular cells, suggesting a novel function other than its canonical role in glycolysis. Both pharmacological inhibition and genetic silencing of PFKFB3 led to the suppression of cisplatin-induced apoptosis in cultured renal proximal tubular cells (RPTCs). Moreover, cisplatin-induced kidney injury or nephrotoxicity was ameliorated in renal proximal tubule-specific PFKFB3 knockout mice. Mechanistically, we demonstrated the interaction of PFKFB3 with cyclin-dependent kinase 4 (CDK4) during cisplatin treatment, resulting in CDK4 activation and consequent phosphorylation and inactivation of retinoblastoma tumor suppressor (Rb). Inhibition of CDK4 reduced cisplatin-induced apoptosis in RPTCs and kidney injury in mice. Collectively, this study unveils a novel pathological role of PFKFB3 in cisplatin nephrotoxicity through the activation of the CDK4/Rb pathway, suggesting a new kidney protective strategy for cancer patients by blocking PFKFB3.
Asunto(s)
Lesión Renal Aguda , Neoplasias , Ratones , Animales , Cisplatino/toxicidad , Quinasa 4 Dependiente de la Ciclina/farmacología , Quinasa 4 Dependiente de la Ciclina/uso terapéutico , Riñón/patología , Apoptosis , Lesión Renal Aguda/inducido químicamente , Neoplasias/patologíaRESUMEN
Kidney transplantation is a standard care for end stage renal disease, but it is also associated with a complex pathogenesis including ischemia-reperfusion injury, inflammation, and development of fibrosis. Over the past decade, accumulating evidence has suggested a role of epigenetic regulation in kidney transplantation, involving DNA methylation, histone modification, and various kinds of non-coding RNAs. Here, we analyze these recent studies supporting the role of epigenetic regulation in different pathological processes of kidney transplantation, i.e., ischemia-reperfusion injury, acute rejection, and chronic graft pathologies including renal interstitial fibrosis. Further investigation of epigenetic alterations, their pathological roles and underlying mechanisms in kidney transplantation may lead to new strategies for the discovery of novel diagnostic biomarkers and therapeutic interventions.
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Trasplante de Riñón , Daño por Reperfusión , Metilación de ADN , Epigénesis Genética , Femenino , Fibrosis , Humanos , Trasplante de Riñón/efectos adversos , Masculino , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
Cold storage/rewarming is an inevitable process for kidney transplantation from deceased donors, which correlates closely with renal ischemia-reperfusion injury (IRI) and the occurrence of delayed graft function. Histone deacetylases (HDAC) are important epigenetic regulators, but their involvement in cold storage/rewarming injury in kidney transplantation is unclear. In the present study, we showed a dynamic change of HDAC3 in a mouse model of kidney cold storage followed by transplantation. We then demonstrated that the selective HDAC3 inhibitor RGFP966 could reduce acute tubular injury and cell death after prolonged cold storage with transplantation. RGFP966 also improved renal function, kidney repair and tubular integrity when the transplanted kidney became the sole life-supporting graft in the recipient mouse. In vitro, cold storage of proximal tubular cells followed by rewarming induced remarkable cell death, which was suppressed by RGFP966 or knockdown of HDAC3 with shRNA. Inhibition of HDAC3 decreased the mitochondrial pathway of apoptosis and preserved mitochondrial membrane potential. Collectively, HDAC3 plays a pathogenic role in cold storage/rewarming injury in kidney transplantation, and its inhibition may be a therapeutic option.
Asunto(s)
Acrilamidas/uso terapéutico , Histona Desacetilasas/efectos de los fármacos , Trasplante de Riñón , Fenilendiaminas/uso terapéutico , Daño por Reperfusión/prevención & control , Aloinjertos , Animales , Apoptosis , Frío , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Túbulos Renales Proximales/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Preservación de Órganos/efectos adversos , ARN Interferente PequeñoRESUMEN
Cisplatin is a widely used chemotherapy drug with notorious nephrotoxicity. Na+-glucose cotransporter 2 inhibitors are a class of novel antidiabetic agents that may have other effects in the kidneys besides blood glucose control. In the present study, we demonstrated that canagliflozin significantly attenuates cisplatin-induced nephropathy in C57BL/6 mice and suppresses cisplatin induced renal proximal tubular cell apoptosis in vitro. The protective effect of canagliflozin was associated with inhibition of p53, p38 and JNK activation. Mechanistically, canagliflozin partially reduced cisplatin uptake by kidney tissues in mice and renal tubular cells in culture. In addition, canagliflozin enhanced the activation of Akt and inhibited the mitochondrial pathway of apoptosis during cisplatin treatment. The protective effect of canagliflozin was diminished by the phosphatidylinositol 3-kinase/Akt inhibitor LY294002. Notably, canagliflozin did not affect the chemotherapeutic efficacy of cisplatin in A549 and HCT116 cancer cell lines. These results suggest a new application of canagliflozin for renoprotection in cisplatin chemotherapy. Canagliflozin may protect kidneys by reducing cisplatin uptake and activating cell survival pathways.
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Apoptosis/efectos de los fármacos , Canagliflozina/farmacología , Cisplatino , Enfermedades Renales/prevención & control , Riñón/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células Cultivadas , Citocromos c/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Riñón/enzimología , Riñón/patología , Enfermedades Renales/inducido químicamente , Enfermedades Renales/enzimología , Enfermedades Renales/patología , Masculino , Ratones Endogámicos C57BL , Fosforilación , Ratas , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Renal interstitial fibrosis is a common pathological feature of chronic kidney disease that may involve changes of metabolism in kidney cells. In the present study, we first showed that blockade of glycolysis with either dichloroacetate (DCA) or shikonin to target different glycolytic enzymes reduced renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Both inhibitors evidently suppressed the induction of fibronectin and collagen type I in obstructed kidneys, with DCA also showing inhibitory effects on collagen type IV and α-smooth muscle actin (α-SMA). Histological examination also confirmed less collagen deposition in DCA-treated kidneys. Both DCA and shikonin significantly inhibited renal tubular apoptosis but not interstitial apoptosis in UUO. Macrophage infiltration after UUO injury was also suppressed. Shikonin, but not DCA, caused obvious animal weight loss during UUO. To determine whether shikonin and DCA worked on tubular cells and/or fibroblasts, we tested their effects on cultured renal proximal tubular BUMPT cells and renal NRK-49F fibroblasts during hypoxia or transforming growth factor-ß1 treatment. Although both inhibitors reduced fibronectin and α-SMA production in NRK-49F cells during hypoxia or transforming growth factor-ß1 treatment, they did not suppress fibronectin and α-SMA expression in BUMPT cells. Altogether, these results demonstrate the inhibitory effect of glycolysis inhibitors on renal interstitial fibrosis. In this regard, DCA is more potent for fibrosis inhibition and less toxic to animals than shikonin.
Asunto(s)
Ácido Dicloroacético/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Glucólisis/efectos de los fármacos , Enfermedades Renales/prevención & control , Túbulos Renales/efectos de los fármacos , Naftoquinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Obstrucción Ureteral/complicacionesRESUMEN
Acute kidney injury (AKI) is a major public health concern associated with high morbidity and mortality. Despite decades of research, the pathogenesis of AKI remains incompletely understood and effective therapies are lacking. An increasing body of evidence suggests a role for epigenetic regulation in the process of AKI and kidney repair, involving remarkable changes in histone modifications, DNA methylation and the expression of various non-coding RNAs. For instance, increases in levels of histone acetylation seem to protect kidneys from AKI and promote kidney repair. AKI is also associated with changes in genome-wide and gene-specific DNA methylation; however, the role and regulation of DNA methylation in kidney injury and repair remains largely elusive. MicroRNAs have been studied quite extensively in AKI, and a plethora of specific microRNAs have been implicated in the pathogenesis of AKI. Emerging research suggests potential for microRNAs as novel diagnostic biomarkers of AKI. Further investigation into these epigenetic mechanisms will not only generate novel insights into the mechanisms of AKI and kidney repair but also might lead to new strategies for the diagnosis and therapy of this disease.
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Lesión Renal Aguda/genética , Epigénesis Genética , Riñón/patología , MicroARNs/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Metilación de ADN , Humanos , Riñón/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Histone deacetylase inhibitors (HDACi) have therapeutic effects in models of various renal diseases including acute kidney injury (AKI); however, the underlying mechanism remains unclear. Here we demonstrate that two widely tested HDACi (suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)) protect the kidneys in cisplatin-induced AKI by enhancing autophagy. In cultured renal proximal tubular cells, SAHA and TSA enhanced autophagy during cisplatin treatment. We further verified the protective effect of TSA against cisplatin-induced apoptosis in these cells. Notably, inhibition of autophagy by chloroquine or by autophagy gene 7 (Atg7) ablation diminished the protective effect of TSA. In mice, TSA increased autophagy in renal proximal tubules and protected against cisplatin-induced AKI. The in vivo effect of TSA was also abolished by chloroquine and by Atg7 knockout specifically from renal proximal tubules. Mechanistically, TSA stimulated AMPK and inactivated mTOR during cisplatin treatment of proximal tubule cells and kidneys in mice. Together, these results suggest that HDACi may protect kidneys by activating autophagy in proximal tubular cells.
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Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Autofagia , Cisplatino/efectos adversos , Inhibidores de Histona Desacetilasas/uso terapéutico , Túbulos Renales Proximales/patología , Lesión Renal Aguda/patología , Adenilato Quinasa/metabolismo , Animales , Autofagia/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Cloroquina/farmacología , Citoprotección/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Túbulos Renales Proximales/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Vorinostat/farmacología , Vorinostat/uso terapéuticoRESUMEN
Diabetic kidney disease, a leading cause of end-stage renal disease, has become a serious public health problem worldwide and lacks effective therapies. Autophagy is a highly conserved lysosomal degradation pathway that removes protein aggregates and damaged organelles to maintain cellular homeostasis. As important stress-responsive machinery, autophagy is involved in the pathogenesis of various diseases. Emerging evidence has suggested that dysregulated autophagy may contribute to both glomerular and tubulointerstitial pathologies in kidneys under diabetic conditions. This review summarizes the recent findings regarding the role of autophagy in the pathogenesis of diabetic kidney disease and highlights the regulation of autophagy by the nutrient-sensing pathways and intracellular stress signaling in this disease. The advances in our understanding of autophagy in diabetic kidney disease will facilitate the discovery of a new therapeutic target for the prevention and treatment of this life-threatening diabetes complication.
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Autofagia/fisiología , Nefropatías Diabéticas/fisiopatología , Nefropatías Diabéticas/terapia , Terapias en Investigación , Animales , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Homeostasis/fisiología , Humanos , Riñón/metabolismo , Riñón/fisiología , Podocitos/metabolismo , Podocitos/fisiología , Transducción de Señal/fisiología , Terapias en Investigación/métodos , Terapias en Investigación/tendenciasRESUMEN
The pathogenesis of chronic kidney disease (CKD) is complex and apparently multifactorial. Hypoxia or decrease in oxygen supply in kidney tissues has been implicated in CKD. Hypoxia inducible factors (HIF) are a small family of transcription factors that are mainly responsive to hypoxia and mediate hypoxic response. HIF plays a critical role in renal fibrosis during CKD through the modulation of gene transcription, crosstalk with multiple signaling pathways, epithelial-mesenchymal transition, and epigenetic regulation. Moreover, HIF also contributes to the development of various pathological conditions associated with CKD, such as anemia, inflammation, aberrant angiogenesis, and vascular calcification. Treatments targeting HIF and related signaling pathways for CKD therapy are being developed with promising clinical benefits, especially for anemia. This review presents an updated analysis of hypoxia response, HIF, and their associated signaling network involved in the pathogenesis of CKD.
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Factor 1 Inducible por Hipoxia/metabolismo , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal , Animales , Hipoxia de la Célula , Humanos , Factor 1 Inducible por Hipoxia/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/terapiaRESUMEN
Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing.
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Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Exosomas/metabolismo , Túbulos Renales/metabolismo , Cicatrización de Heridas , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Receptores ErbB/antagonistas & inhibidores , Exosomas/patología , Gefitinib , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Ratones , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Quinazolinas/farmacología , Transducción de Señal , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.
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Apoptosis , Autofagia/fisiología , Enfermedades Renales/patología , Riñón/citología , Proteína Quinasa C-delta/fisiología , Animales , Células Cultivadas , Cisplatino/toxicidad , Enfermedades Renales/inducido químicamente , Masculino , RatonesRESUMEN
Ischemia-reperfusion injury contributes to tissue damage and organ failure in clinical settings, but the underlying mechanism remains elusive and effective therapies are still lacking. Here, we identified microRNA 687 (miR-687) as a key regulator and therapeutic target in renal ischemia-reperfusion injury. We show that miR-687 is markedly upregulated in the kidney during renal ischemia-reperfusion in mice and in cultured kidney cells during hypoxia. MiR-687 induction under these conditions was mediated by hypoxia-inducible factor-1 (HIF-1). Upon induction in vitro, miR-687 repressed the expression of phosphatase and tensin homolog (PTEN) and facilitated cell cycle progression and apoptosis. Blockade of miR-687 preserved PTEN expression and attenuated cell cycle activation and renal apoptosis, resulting in protection against kidney injury in mice. Collectively, these results unveil a novel HIF-1/miR-687/PTEN signaling pathway in ischemia-reperfusion injury that may be targeted for therapy.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Renales/fisiopatología , MicroARNs/genética , Proteínas de Microfilamentos/genética , Fosfohidrolasa PTEN/genética , Daño por Reperfusión/fisiopatología , Análisis de Varianza , Animales , Northern Blotting , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Proteínas de Microfilamentos/metabolismo , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transducción de Señal , Tensinas , Regulación hacia ArribaRESUMEN
ER stress has been implicated in the pathogenesis of both acute and chronic kidney diseases. However, the molecular regulation of ER stress in kidney cells and tissues remains poorly understood. In this study, we examined tunicamycin-induced ER stress in renal proximal tubular cells (RPTC). Tunicamycin induced the phosphorylation and activation of PERK and eIF2α within 2 h in RPTC, which was followed by the induction of GRP78 and CHOP. Consistently, tunicamycin also induced apoptosis in RPTC. Interestingly, mTOR was activated rapidly during tunicamycin treatment, as indicated by phosphorylation of both mTOR and p70S6K. Inhibition of mTOR with rapamycin partially suppressed the phosphorylation of PERK and eIF2a and the induction of CHOP and GRP78 induction during tunicamycin treatment. Rapamycin also inhibited apoptosis during tunicamycin treatment and increased cell survival. Collectively, the results suggest that mTOR plays a regulatory role in ER stress, and inhibition of mTOR may have potential therapeutic effects in ER stress-related renal diseases.
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Apoptosis , Estrés del Retículo Endoplásmico , Túbulos Renales Proximales/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Tunicamicina/farmacologíaRESUMEN
AKI is pathologically characterized by sublethal and lethal damage of renal tubules. Under these conditions, renal tubular cell death may occur by regulated necrosis (RN) or apoptosis. In the last two decades, tubular apoptosis has been shown in preclinical models and some clinical samples from patients with AKI. Mechanistically, apoptotic cell death in AKI may result from well described extrinsic and intrinsic pathways as well as ER stress. Central converging nodes of these pathways are mitochondria, which become fragmented and sensitized to membrane permeabilization in response to cellular stress, resulting in the release of cell death-inducing factors. Whereas apoptosis is known to be regulated, tubular necrosis was thought to occur by accident until recent work unveiled several RN subroutines, most prominently receptor-interacting protein kinase-dependent necroptosis and RN induced by mitochondrial permeability transition. Additionally, other cell death pathways, like pyroptosis and ferroptosis, may also be of pathophysiologic relevance in AKI. Combination therapy targeting multiple cell-death pathways may, therefore, provide maximal therapeutic benefits.
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Lesión Renal Aguda/fisiopatología , Muerte Celular , Riñón/patología , Animales , Apoptosis , Permeabilidad de la Membrana Celular , Estrés del Retículo Endoplásmico , Humanos , Etiquetado Corte-Fin in Situ , Hierro/química , Mitocondrias/metabolismo , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Necrosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de SeñalRESUMEN
Renal ischemia-reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia-reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or "reperfusion" of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia-reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.
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Lesión Renal Aguda/genética , Adenosina Trifosfato/metabolismo , Daño del ADN , Túbulos Renales/metabolismo , Riñón/irrigación sanguínea , Daño por Reperfusión/genética , Acetilcisteína/metabolismo , Lesión Renal Aguda/metabolismo , Animales , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Glucosa/metabolismo , Histonas/genética , Histonas/metabolismo , Riñón/metabolismo , Túbulos Renales/irrigación sanguínea , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reperfusión , Daño por Reperfusión/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In kidneys, each tubular epithelial cell contains a primary cilium that protrudes from the apical surface. Ciliary dysfunction was recently linked to acute kidney injury (AKI) following renal ischemia-reperfusion. Whether ciliary regulation is a general pathogenic mechanism in AKI remains unclear. Moreover, the ciliary change during AKI and its underlying mechanism are largely unknown. Here we examined the change of primary cilium and its role in tubular cell apoptosis and AKI induced by cisplatin, a chemotherapy agent with notable nephrotoxicity. In cultured human proximal tubular HK-2 epithelial cells, cilia became shorter during cisplatin treatment, followed by apoptosis. Knockdown of Kif3a or Polaris (cilia maintenance proteins) reduced cilia and increased apoptosis during cisplatin treatment. We further subcloned HK-2 cells and found that the clones with shorter cilia were more sensitive to cisplatin-induced apoptosis. Mechanistically, cilia-suppressed cells showed hyperphosphorylation or activation of ERK. Inhibition of ERK by U0126 preserved cilia during cisplatin treatment and protected against apoptosis in HK-2 cells. In C57BL/6 mice, U0126 prevented the loss of cilia from proximal tubules during cisplatin treatment and protected against AKI. U0126 up-regulated Polaris, but not Kif3a, in kidney tissues. It is suggested that ciliary regulation by ERK plays a role in cisplatin-induced tubular apoptosis and AKI.
Asunto(s)
Lesión Renal Aguda/patología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cilios/fisiología , Cisplatino/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Túbulos Renales/efectos de los fármacos , Lesión Renal Aguda/enzimología , Animales , Butadienos/farmacología , Caspasas/metabolismo , Línea Celular , Activación Enzimática , Humanos , Túbulos Renales/enzimología , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Nitrilos/farmacologíaRESUMEN
Bax and Bak, two pro-apoptotic Bcl-2 family proteins, have been implicated in acute kidney injury following renal ischemia/reperfusion; however, definitive evidence for a role of these genes in the disease process is lacking. Here we first examined two Bax-deficient mouse models and found that only conditional Bax deletion specifically from proximal tubules could ameliorate ischemic acute kidney injury. Global (whole mouse) knockout of Bax enhanced neutrophil infiltration without significant effect on kidney injury. In contrast, global knockout of Bak protected mice from ischemic acute kidney injury with improved renal function. Interestingly, in these models, Bax or Bak knockout attenuated renal tubular cell apoptosis without significantly affecting necrotic tubular damage. Cytochrome c release in ischemic acute kidney injury was also suppressed in conditional Bax- or global Bak-knockout mice. In addition, Bak deficiency prevented mitochondrial fragmentation in ischemic acute kidney injury. Thus, our gene-knockout studies support a critical role of Bax and Bak in tubular cell apoptosis in ischemic acute kidney. Furthermore, necrosis and apoptosis have distinguishable regulatory functions.
Asunto(s)
Lesión Renal Aguda/metabolismo , Túbulos Renales Proximales/metabolismo , Daño por Reperfusión/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Lesión Renal Aguda/prevención & control , Animales , Apoptosis , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Túbulos Renales Proximales/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Necrosis , Infiltración Neutrófila , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7-knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.