RESUMEN

Macroautophagy/autophagy activation in renal tubular epithelial cells protects against acute kidney injury (AKI). However, the role of immune cell autophagy, such as that involving macrophages, in AKI remains unclear. In this study, we discovered that macrophage autophagy was an adaptive response during AKI as mice with macrophage-specific autophagy deficiency (atg5-/-) exhibited higher serum creatinine, more severe renal tubule injury, increased infiltration of ADGRE1/F4/80+ macrophages, and elevated expression of inflammatory factors compared to WT mice during AKI induced by either LPS or unilateral ischemia-reperfusion. This was further supported by adoptive transfer of atg5-/- macrophages, but not WT macrophages, to cause more severe AKI in clodronate liposomes-induced macrophage depletion mice. Similar results were also obtained in vitro that bone marrow-derived macrophages (BMDMs) lacking Atg5 largely increased pro-inflammatory cytokine expression in response to LPS and IFNG. Mechanistically, we uncovered that atg5 deletion significantly upregulated the protein expression of TARM1 (T cell-interacting, activating receptor on myeloid cells 1), whereas inhibition of TARM1 suppressed LPS- and IFNG-induced inflammatory responses in atg5-/- RAW 264.7 macrophages. The E3 ubiquitin ligases MARCHF1 and MARCHF8 ubiquitinated TARM1 and promoted its degradation in an autophagy-dependent manner, whereas silencing or mutation of the functional domains of MARCHF1 and MARCHF8 abolished TARM1 degradation. Furthermore, we found that ubiquitinated TARM1 was internalized from plasma membrane into endosomes, and then recruited by the ubiquitin-binding autophagy receptors TAX1BP1 and SQSTM1 into the autophagy-lysosome pathway for degradation. In conclusion, macrophage autophagy protects against AKI by inhibiting renal inflammation through the MARCHF1- and MARCHF8-mediated degradation of TARM1.Abbreviations: AKI, acute kidney injury; ATG, autophagy related; Baf, bafilomycin A1; BMDMs, bone marrow-derived macrophages; CCL2/MCP-1, C-C motif chemokine ligand 2; CHX, cycloheximide; CQ, chloroquine; IFNG, interferon gamma; IL, interleukin; IR, ischemia-reperfusion; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; MARCHF, membrane associated ring-CH-type finger; NC, negative control; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; NLRP3, NLR family, pyrin domain containing 3; NOS2, nitric oxide synthase 2, inducible; Rap, rapamycin; Wort, wortmannin; RT-qPCR, real-time quantitative polymerase chain reaction; Scr, serum creatinine; SEM, standard error of mean; siRNA, small interfering RNA; SYK, spleen tyrosine kinase; TARM1, T cell-interacting, activating receptor on myeloid cells 1; TAX1BP1, Tax1 (human T cell leukemia virus type I) binding protein 1; TECs, tubule epithelial cells; TNF, tumor necrosis factor; WT, wild type.

RESUMEN

Transcription factors can affect autophagy activity by promoting or inhibiting the expression of autophagic and lysosomal genes. As a member of the zinc finger family DNA-binding proteins, ZKSCAN3 has been reported to function as a transcriptional repressor of autophagy, silencing of which can induce autophagy and promote lysosomal biogenesis in cancer cells. However, studies in Zkscan3 knockout mice showed that the deficiency of ZKSCAN3 did not induce autophagy or increase lysosomal biogenesis. In order to further explore the role of ZKSCAN3 in the transcriptional regulation of autophagic genes in human cancer and non-cancer cells, we generated ZKSCAN3 knockout HK-2 (non-cancer) and Hela (cancer) cells via the CRISPR/Cas9 system and analyzed the differences in gene expression between ZKSCAN3 deleted cells and non-deleted cells through fluorescence quantitative PCR, western blot and transcriptome sequencing, with special attention to the differences in expression of autophagic and lysosomal genes. We found that ZKSCAN3 may be a cancer-related gene involved in cancer progression, but not an essential transcriptional repressor of autophagic or lysosomal genes, as the lacking of ZKSCAN3 cannot significantly promote the expression of autophagic and lysosomal genes.

Asunto(s)

Autofagia , Regulación de la Expresión Génica , Animales , Ratones , Humanos , Autofagia/genética , Células HeLa , Lisosomas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Hyperuricemia can induce acute and chronic kidney damage, but the pathological mechanism remains unclear. The potential role of AMP-activated protein kinase (AMPK) α2 in hyperuricemia-induced renal injury was investigated in this study. Acute and chronic hyperuricemic nephropathy was induced by administering intraperitoneal injections of uric acid and oxonic acid to AMPK α2 knockout and wild-type mice. Changes in renal function, histopathology, inflammatory cell infiltration, renal interstitial fibrosis, and urate deposition were analyzed. In both acute and chronic hyperuricemic nephropathy mouse models, knockout of AMPK α2 significantly reduced serum creatinine levels and renal pathological changes. The tubular expression of kidney injury molecule-1 was also reduced in hyperuricemic nephropathy mice deficient in AMPK α2. In addition, knockout of AMPK α2 significantly suppressed the infiltration of renal macrophages and progression of renal interstitial fibrosis in mice with chronic hyperuricemic nephropathy. Knockout of AMPK α2 reduced renal urate crystal deposition, probably through increasing the expression of the uric acid transporter, multidrug resistance protein 4. In summary, AMPK α2 is involved in acute and chronic hyperuricemia-induced kidney injury and may be associated with increased urate crystal deposition in the kidney.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Hiperuricemia , Enfermedades Renales , Fallo Renal Crónico , Proteínas Quinasas Activadas por AMP/genética , Animales , Modelos Animales de Enfermedad , Fibrosis , Hiperuricemia/inducido químicamente , Hiperuricemia/genética , Riñón/patología , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Ratones , Ratones Noqueados , Ácido Úrico/efectos adversos , Ácido Úrico/metabolismo

RESUMEN

OBJECTIVE: Lupus nephritis (LN) is a major complication and cause of death among patients with SLE. This research used in vivo and in vitro experiments to explore the therapeutic potential of metformin in kidney injury from LN-induced inflammation. METHODS: In vivo study, 8-week-old MRL/MpJ-Faslpr/J (MRL/lpr) mice were randomly divided into two groups (n=12 each): daily administration of 0.3 mg/mL metformin in drinking water and control (water only). Body weight and urinary samples were measured biweekly. Mice were sacrificed after 8-week treatment to harvest serum, lymph nodes, spleen and kidneys. In vitro study, human kidney-2 (HK-2) cells were pretreated with 1 mM metformin for 1 hour and then stimulated with 20 µg/mL lipopolysaccharides (LPS) or 10 ng/mL tumour necrosis factor-α (TNF-α) for another 48 hours. Protein was collected for subsequent analysis. RESULTS: We found that metformin administration improved renal function in MRL/lpr lupus-prone mice, measured by decreased urea nitrogen and urinary proteins. Metformin reduced immunoglobulin G and complement C3 deposition in glomeruli. The treatment also downregulated systemic and renal inflammation, as seen in decreased renal infiltration of F4/80-positive macrophages and reduced splenic and renal MCP-1 (monocyte chemoattractant protein-1) and TNF-α, and renal IL-1ß (interleukin 1ß) expression. Metformin administration decreased renal expression of necroptosis markers p-RIPK1 (phosphorylated receptor-interacting protein kinase 1) and p-MLKL, along with tubular injury marker KIM-1 (kidney injury molecule-1) in lupus mice. In addition, metformin alleviated the necroptosis of HK-2 cells stimulated by LPS and TNF-α, evidencing by a decrease in the expression of necroptosis markers p-RIPK1, p-RIPK3 and p-MLKL, and the inflammasome-related markers NLRP3 (NLR family pyrin domain containing 3), ASC (apoptosis-associated speck-like protein containing a CARD), caspase-1. Mechanistically, metformin treatment upregulated p-AMPK (phosphorylated AMP-activated protein kinase) and downregulated p-STAT3 (phosphorylated signal transducer and activator of transcription 3) expression in the kidneys. Moreover, AMPKα2 knockdown abolished the protective effects of metformin in vitro. CONCLUSIONS: Metformin alleviated kidney injury in LN though suppressing renal necroptosis and inflammation via the AMPK/STAT3 pathway.

Asunto(s)

Lupus Eritematoso Sistémico , Nefritis Lúpica , Metformina , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/farmacología , Animales , Humanos , Inflamación , Riñón/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/uso terapéutico , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Nefritis Lúpica/complicaciones , Nefritis Lúpica/tratamiento farmacológico , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Ratones Endogámicos MRL lpr , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/farmacología , Factor de Transcripción STAT3/uso terapéutico , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/uso terapéutico

RESUMEN

BACKGROUND: Hydroxychloroquine (HCQ) has been recommended as a basic treatment for lupus nephritis (LN) during this decade based on its ability to improve LN-related renal immune-mediated inflammatory lesions. As a classical lysosomal inhibitor, HCQ may inhibit lysosomal degradation and disrupt protective autophagy in proximal tubular epithelial cells (PTECs). Therefore, the final renal effects of HCQ on LN need to be clarified. METHOD: HCQ was administered on spontaneous female MRL/lpr LN mice with severe proteinuria daily for 4 weeks. Moreover, the MRL/lpr mice with proteinuric LN were subjected to cisplatin-induced or unilateral ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) after 2 weeks of HCQ preadministration. RESULTS: As expected, HCQ treatment increased the survival ratio and downregulated the levels of serum creatinine in the mice with LN, ameliorated renal lesions, and inhibited renal interstitial inflammation. Unexpectedly, HCQ preadministration significantly increased susceptibility to and delayed the recovery of AKI complicated by LN, as demonstrated by an increase in PTEC apoptosis and expression of the tubular injury marker KIM-1 as well as the retardation of PTEC replenishment. HCQ preadministration suppressed the proliferation of PTECs by arresting cells in G1/S phase and upregulated the expression of cell cycle inhibitors. Furthermore, HCQ preadministration disrupted the PTEC autophagy-lysosomal pathway and accelerated PTEC senescence. CONCLUSION: HCQ treatment may increase susceptibility and delay the recovery of AKI complicated by LN despite its ability to improve LN-related renal immune-mediated inflammatory lesions. The probable mechanism involves accelerated apoptosis and inhibited proliferation of PTECs via autophagy-lysosomal pathway disruption and senescence promotion.

Asunto(s)

Lesión Renal Aguda , Nefritis Lúpica , Lesión Renal Aguda/inducido químicamente , Animales , Femenino , Hidroxicloroquina/farmacología , Riñón/patología , Ratones , Ratones Endogámicos MRL lpr

RESUMEN

Objective:To explore the protective effect and mechanism of scutellarin (Scu) on sepsis associated-acute kidney injury (SA-AKI).Methods:① In vivo experiment: 36 male C57BL/6 mice were divided into normal saline (NS) control group, lipopolysaccharide (LPS) induced SA-AKI model group (LPS group), 20 mg/kg Scu control group (Scu 20 control group), and 5, 10, 20 mg/kg Scu pretreatment groups by random number table with 6 mice in each group. The SA-AKI model was reproduced by intraperitoneal injection of 10 mg/kg LPS. The NS control group was injected with NS intraperitoneally. The Scu pretreatment groups were intraperitoneally injected with different doses of Scu every day before LPS injection for 1 week. Scu 20 control group was injected with 20 mg/kg Scu for 1 week. After 24 hours of LPS treatment, mice in each group were sacrificed, kidney tissues were collected, and kidney injury was detected by hematoxylin-eosin (HE) staining. Western blotting was used to detect the protein expression levels of nuclear factor-κB (NF-κB) signaling pathway related molecules, apoptosis-related proteins and cysteine-rich protein 61-connective tissue growth factor-nephroblastoma overexpressed gene 1 (CCN1). ② In vitro experiment: human renal tubular epithelial cell line HK-2 was cultured in vitro and used for experiment when the cells fused to 80%. In the cells without LPS treatment and after 100 g/L LPS treatment, pcDNA3.1-CCN1 and small interfering RNA (siRNA) CCN1 sequence were transfected to overexpress and inhibit CCN1 expression, respectively, to observe whether CCN1 was involved in NF-κB signaling pathway activation and apoptosis. In addition, 100g/L LPS and 20 μmol/L Scu were added into HK-2 cells transfected with and without CCN1 siRNA to investigate the mechanism of protective effect of Scu on LPS-induced HK-2 cells injury. Results:① The results of in vivo experiment: the renal function of SA-AKI mice induced by LPS was significantly decreased, and had kidney histological damage and severely damaged renal tubules. Scu could alleviate renal function and histological damage in a dose-dependent manner. Western blotting results showed Scu could reduce the protein expression of NF-κB signaling pathway related molecules and CCN1 in the renal tissue, and had a significant alleviating effect on apoptosis, indicating that CCN1 was involved in NF-κB signaling pathway activation and apoptosis. ② The results of in vitro experiment: in HK-2 cells not treated with LPS, CCN1 overexpression had no effect on apoptosis related protein and pro-inflammatory factors of NF-κB signaling pathway. In HK-2 cells treated with LPS, overexpression of CCN1 significantly inhibited the mRNA expressions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), with significant differences as compared with cells stimulated only by LPS [IL-1β mRNA (2 -ΔΔCT): 3.20±0.57 vs. 4.88±0.69, TNF-α mRNA (2 -ΔΔCT): 2.99±0.44 vs. 5.00±0.81, MCP-1 mRNA (2 -ΔΔCT): 2.81±0.50 vs. 5.41±0.75, all P < 0.05], and the apoptosis-related protein was significantly down-regulated. However, when siRNA was used to inhibit the expression of CCN1, the mRNA expressions of pro-inflammatory factors were significantly increased as compared with cells stimulated only by LPS [IL-1β mRNA (2 -ΔΔCT): 6.01±1.13 vs. 4.88±0.69, TNF-α mRNA (2 -ΔΔCT): 5.15±0.86 vs. 5.00±0.81, all P < 0.05], and apoptosis-related protein was significantly up-regulated. In the LPS-induced HK-2 cells, the mRNA expressions of pro-inflammatory factors were significantly down-regulated after Scu treatment as compared with cells stimulated only by LPS [IL-1β mRNA (2 -ΔΔCT) : 2.55±0.50 vs. 6.15±1.04, TNF-α mRNA (2 -ΔΔCT): 2.58±0.40 vs. 3.95±0.52, MCP-1 mRNA (2 -ΔΔCT): 2.64±0.44 vs. 6.21±0.96, all P < 0.05], and apoptosis-related protein was also significantly reduced. When the expression of CCN1 was inhibited by siRNA, the protective effect of Scu on cells was weakened, which showed that the mRNA expressions of pro-inflammatory factors in cells was significantly up-regulated compared with the cells without inhibition of CCN1 expression [IL-1β mRNA (2 -ΔΔCT): 5.34±0.76 vs. 2.55±0.50, TNF-α mRNA (2 -ΔΔCT): 3.66±0.54 vs. 2.58±0.40, MCP-1 mRNA (2 -ΔΔCT): 5.15±0.79 vs. 2.64±0.44, all P < 0.05], and the expression of apoptosis related protein was also significantly up-regulated. Conclusions:Scu could protect the renal function in SA-AKI mice, and the protective effect is associated with NF-κB signaling pathway and CCN1. Thus, Scu could alleviate LPS-induced kidney injury by regulating the NF-κB signaling pathway.

RESUMEN

Cyclosporine A (CsA) is an immunosuppressor widely used for the prevention of acute rejection during solid organ transplantation. However, severe nephrotoxicity has substantially limited its long-term usage. Recently, an impaired autophagy pathway was suggested to be involved in the pathogenesis of chronic CsA nephrotoxicity. However, the underlying mechanisms of CsA-induced autophagy blockade in tubular cells remain unclear. In the present study, we observed that CsA suppressed the activation and expression of transcription factor EB (TFEB) by increasing the activation of mTOR, in turn promoting lysosomal dysfunction and autophagy flux blockade in tubular epithelial cells (TECs) in vivo and in vitro. Restoration of TFEB activation by Torin1-mediated mTOR inhibition significantly improved lysosomal function and rescued autophagy pathway activity, suppressing TEC injury. In summary, targeting TFEB-mediated autophagy flux represents a potential therapeutic strategy for CsA-induced nephrotoxicity.

Asunto(s)

Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ciclosporina/toxicidad , Células Epiteliales/patología , Túbulos Renales/patología , Lisosomas/patología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Inmunosupresores/toxicidad , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Serina-Treonina Quinasas TOR/genética

RESUMEN

ERK, an extracellular signal-regulated protein kinase, is involved in various biological responses, such as cell proliferation and differentiation, cell morphology maintenance, cytoskeletal construction, apoptosis, and canceration of cells. In this study, we focused on ERK pathway on cellular injury and autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells and explored the potential mechanisms underlying it. By using antioxidants N-acetylcysteine and catalase, we found that ERK pathway was activated by a reactive oxygen species- (ROS-) dependent mechanism after exposure to urinary proteins. What is more, ERK inhibitor U0126 could decrease the release of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and the number of apoptotic cells induced by urinary proteins, indicating the damaging effects of ERK pathway in mediating cellular injury and apoptosis in HK-2 cells. Interestingly, we also found that the increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II (a key marker of autophagy) and the decreased expression of p62 (autophagic substrate) induced by urinary proteins were reversed by U0126, suggesting autophagy was activated by ERK pathway. Furthermore, rapamycin reduced urinary protein-induced NGAL and KIM-1 secretion and cell growth inhibition, while chloroquine played the opposite effect, indicating that autophagy activation by ERK pathway was an adaptive response in the exposure to urinary proteins. Taken together, our results indicate that activated ROS-ERK pathway can induce cellular injury and in the meantime provide an autophagy-associated adaptive response in urinary protein-irritated renal tubular epithelial cells.

Asunto(s)

Autofagia , Células Epiteliales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Túbulos Renales Proximales/enzimología , Nefrosis Lipoidea/enzimología , Estrés Oxidativo , Proteinuria/enzimología , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/farmacología , Apoptosis , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Nefrosis Lipoidea/patología , Nefrosis Lipoidea/orina , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteinuria/patología , Proteinuria/orina , Transducción de Señal

RESUMEN

Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro models were established. LIN or dissolvent was given, and histologic analysis, quantitative (q)RT-PCR, serum creatinine and blood urea nitrogen testing were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify the target protein of LIN, and small interfering RNA (siRNA) transfection was applied to explore the crucial role of identified protein. First, we found that LIN inhibited kidney injury in an in vivo IRI model and decreased the expression of interleukin-12 (IL-12) p40 in vivo and in vitro IRI models. To explore the mechanism of LIN, we collected raw data from a public microarray database and identified E26 oncogene homolog 2 (ETS2) as a crucial protein of LIN according to microarray analysis and PPI. Meanwhile, qRT-PCR indicated that IL-12 p40 showed no significant difference between ETS2 knock down group and LIN treated ETS2 knock down group after hypoxia reoxygenation treatment. In addition, according to molecular docking the contact area is highly conserved and located on a PPI domain of ETS2 which indicates that LIN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression. Our study demonstrated the anti-inflammatory effect of LIN during IRI-AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-AKI.

Asunto(s)

Lesión Renal Aguda/prevención & control , Glicósidos/farmacología , Interleucina-12/antagonistas & inhibidores , Proteína Proto-Oncogénica c-ets-2/antagonistas & inhibidores , Lesión Renal Aguda/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glicósidos/química , Humanos , Interleucina-12/química , Interleucina-12/metabolismo , Masculino , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-ets-2/química , Proteína Proto-Oncogénica c-ets-2/metabolismo , Ratas , Ratas Wistar

RESUMEN

Macroautophagy/autophagy dysregulation has been noted in diabetic nephropathy; however, the regulatory mechanisms controlling this process remain unclear. In this study, we showed that SMAD3 (SMAD family member 3), the key effector of TGFB (transforming growth factor beta)-SMAD signaling, induces lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis. The pharmacological inhibition or genetic deletion of SMAD3 restored lysosome biogenesis activity by alleviating the suppression of TFEB, thereby protecting lysosomes from depletion and improving autophagic flux in renal tubular epithelial cells in diabetic nephropathy. Mechanistically, we found that SMAD3 directly binds to the 3'-UTR of TFEB and inhibits its transcription. Silencing TFEB suppressed lysosome biogenesis and resulted in a loss of the protective effects of SMAD3 inactivation on lysosome depletion under diabetic conditions. In conclusion, SMAD3 promotes lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis; this may be an important mechanism underlying autophagy dysregulation in the progression of diabetic nephropathy.Abbreviations: AGEs: advanced glycation end products; ATP6V1H: ATPase H+ transporting V1 subunit H; CTSB: cathepsin B; ChIP: chromatin immunoprecipitation; Co-BSA: control bovine serum albumin; DN: diabetic nephropathy; ELISA: enzyme-linked immunosorbent assay; FN1: fibronectin 1; HAVCR1/TIM1/KIM-1: hepatitis A virus cellular receptor 1; LAMP1: lysosomal associated membrane protein 1; LMP: lysosome membrane permeabilization; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NC: negative control; SIS3: specific inhibitor of SMAD3; SMAD3: SMAD family member 3; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TECs: tubular epithelial cells; TFEB: transcription factor EB; TGFB1: transforming growth factor beta 1; TGFBR1: transforming growth factor beta receptor 1; UTR: untranslated region; VPS11: VPS11 core subunit of CORVET and HOPS complexes.

Asunto(s)

Autofagia , Diabetes Mellitus , Nefropatías Diabéticas , Proteína smad3 , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Diabetes Mellitus/metabolismo , Células Epiteliales/metabolismo , Humanos , Lisosomas/metabolismo , Transducción de Señal , Proteína smad3/metabolismo

RESUMEN

BACKGROUND Cell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-ß-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-ß has not been explored well. MATERIAL AND METHODS The effects of autophagy inhibition on TGF-ß-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-ß with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf). RESULTS Autophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-ß-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-ß treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-ß treated HK-2 cells. Western blot analysis showed that TGF-ß-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors. CONCLUSIONS Inhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-ß that contributes to the induction of tubulointerstitial fibrosis.

Asunto(s)

Autofagia/efectos de los fármacos , Cloroquina/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Macrólidos/farmacología , Insuficiencia Renal Crónica/patología , Factor de Crecimiento Transformador beta/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibronectinas/efectos de los fármacos , Fibronectinas/metabolismo , Fibrosis , Humanos , Técnicas In Vitro , Túbulos Renales/citología , Insuficiencia Renal Crónica/metabolismo , Proteína de Retinoblastoma/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo

RESUMEN

As one of the main gynecological cancers, ovarian cancer (OC) has an unfavourable outcomes owing to its high recurrence and metastasis rate. Our previous studies have revealed that LINC01296 functions as an oncogene in OC, but the underlying mechanism has not been explored. The aim of this paper was to further investigate that how LINC01296 plays a role in OC. Through online software prediction, miR-29c-3p has been discriminated as the target miRNA of LINC01296 for further research, and subsequent luciferase assay confirmed bioinformatics prediction. Then the data obtained from the two databases (GSE119055 and GSE83693) were analyzed by GEO2R for differential gene analysis. The results indicated that the miR-29c-3p was lowly expressed in OC tissues than that in normal ovarian tissues, and its expression in recurrent OC tissues was lower than that in primary OC tissues. Simultaneously, Kaplan-Meier survival analysis illustrated that the lower expression of miR-29c-3p was interrelated to unfavourable outcomes of OC. Further, the qRT-PCR data revealed that the miR-29c-3p expression in OC cell lines (SKOV-3 and OVCAR-3) was markedly declined than that in normal control cells (IOSE80). Subsequently, the functional experiments, such as CCK8, colony formation and Transwell assays, prompted that inhibition of miR-29c-3p can obviously increase the proliferation, invasion and migration of OVCAR3 and SKOV3 cells compared with control group, while downregulation of LINC01296 showed an opposite result. It is worth noting that downregulation of LINC01296 can reverse the effect of miR-29c-3p suppression on OC cells. Finally, we detected the changes of EMT-related proteins by western blot experiment, and reached a similar conclusion that knockdown of LINC01296 reversed the EMT caused by miR-29c-3p inhibition. In sum up, the cancer-promoting function of LINC01296 was achieved by regulating the expression of miR-29c-3p, and LINC01296/miR-29c-3p axis mediates the mechanical regulation of EMT in OC cells, hoping to provide the novel biomarkers and possibilities for OC therapy.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Interferencia de ARN , ARN Largo no Codificante/genética , Regiones no Traducidas 3' , Biomarcadores de Tumor , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/terapia

RESUMEN

Autophagy, the intracellular lysosomal degradation process plays a pivotal role in podocyte homeostasis in diabetic kidney disease (DKD). Lysosomal function, autophagic activity, and their actions were investigated in vitro and in vivo. We found that LC3-II- and p62-positive vacuoles accumulated in podocytes of patients with DKD. Moreover, we found that advanced glycation end products (AGEs) could increase the protein expression of LC3-II and p62 in a dose- and time-dependent manner in cultured podocytes. However, the mRNA expression of LC3B, Beclin-1 or ATG7, as well as the protein level of Beclin-1 or ATG7 did not change significantly in the AGE-treated cells compared with that in control groups, suggesting that AGEs did not induce autophagy. In addition, AGEs led to an increase in the number of autophagosomes but not autolysosomes, accompanied with a failure in lysosomal turnover of LC3-II or p62, indicating that the degradation of autophagic vacuoles was blocked. Furthermore, we observed a dramatic decrease in the enzymatic activities, and the degradation of DQ-ovalbumin was significantly suppressed after podocytes were treated with AGEs. Plasma-irregular lysosomal-associated membrane protein 1 granules accompanied with the diffusion of cathepsin D expression and acridine orange redistribution were observed in AGE-treated podocytes, indicating that the lysosomal membrane permeability was triggered. Interestingly, we also found that AGEs-induced autophagic inhibition and podocyte injury were mimicked by the specific lysosomotropic agent, L-leucyl-L-leucine methyl ester. The exacerbated apoptosis and Rac-1-dependent actin-cytoskeletal disorganization were alleviated by an improvement in the lysosomal-dependent autophagic pathway by resveratrol plus vitamin E treatment in AGE-treated podocytes. However, the rescued effects were reversed by the addition of leupeptin, a lysosomal inhibitor. It suggests that restoring lysosomal function to activate autophagy may contribute to the development of new therapeutic strategies for DKD.

Asunto(s)

Nefropatías Diabéticas/terapia , Lisosomas/metabolismo , Podocitos/metabolismo , Autofagia , Humanos

RESUMEN

Objective To investigate the predictive value of neutrophil to lymphocyte ratio (NLR) at admission for mortality risk during hospitalization in patients with acute ischemic stroke (AIS). Methods Patients with AIS admitted to the Department of Neurology, the First Affiliated Hospital of China Medical University from January 2017 to December 2018 were enrolled retrospectively. The demographic data and all baseline clinical data were collected, and compared between the in-hospital death group and survival group. Multivariate logistic regression analysis was used to determine independent influencing factors for in-hospital death in patients with AIS. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of NLR for death during hospitalization. Results A total of 266 patients with AIS were enrolled, with an average age of 65 years, 168 were males (63.2% ), 98 were females (36.8% ), 52 died in hospital (19.5% ), and 214 (80.5% ) survived. The NLR of the death group was significantly higher than that of the survival group (median [ interquartile range]: 7.6 [4.6-14.0] vs. 2.4 [1.8-4.0]; Z=7.727, P<0.001). Multivariate logistic regression analysis showed that advanced age (odds ratio [OR] 1.07, 95% confidence interval [CI] 1.01-1.14; P=0.009), previous history of stroke or transient ischemic attack (OR 9.06, 95% CI 2.06-39.88; P=0.004), high NIHSS score (OR 1.13, 95% CI 1.04-1.24; P=0.004), and high NLR (OR 1.23, 95% CI 1.02-1.48; P=0.024) were the independent risk factors for death during hospitalization in patients with AIS. ROC curve analysis showed that the area under the curve of NLR predicting death during hospitalization for patients with AIS was 0.846 (95% CI 0.786-0.905; P<0.001). When the cut-off value of NLR was 4.52, the sensitivity and specificity were 76.9% and 80.8% respectively. The positive predictive value was 49.4% , and the negative predictive value was 93.5% . Conclusions The increased NLR level at admission had certain predictive value for the death of patients with AIS during hospitalization.

RESUMEN

OBJECTIVE: To study the association between superior mesenteric artery hemodynamic indexes and scores of lower gastrointestinal symptoms rating scales(LGSRS) in patients with type 2 diabetic mellitus. METHODS: Totally 142 inpatients with type 2 diabetes with average age of 58.76±12.32 yrs were enrolled, who were treated from August 2016 to March 2018. The history, gender, age,course and BMI were recorded, and fasting blood glucose(FBG), glycosylated hemoglobin(HbA1c), 2-hour postprandial blood glucose(PBG), total cholesterol(TC), triglyceride(TG), urine ACR and LGSRS were determined. Ultrasonic scanning of mesenteric artery was performed for hemodynamic indexes, including artery inner diameter(ID), peak systolic velocity(PSV), end-diastolic velocity(EDV), and resistance index(RI)at starting part,first level branch, and second level branch from root of the superior mesenteric artery(SMA).Patients were divided into 2 groups according to their LGSRS, 74 patients with LGSRS≥6 were in positive group, and 68 patients with LGSRS0.05), but the age and DD were significantly higher in positive group than in control group(P0.05). 3. There were no significant difference between positive group and control group in ID at starting part and first level branch of SMA, while ID at second level branch was significantly increased in positive group compared with control group [(3.83±0.85)mm vs.(3.53±0.90)mm, P<0.05)].4. RI at first(0.816±0.059 vs 0.842±0.063,P<0.05) and second level branch(0.813±0.076 vs 0.845±0.073, P<0.05) and PSV at first level branch[(110.89±46.89)cm/s vs(95.72±36.59)cm/s,P<0.05] were significantly high in positive group; there were no difference in other hemodynamic indexes between the groups. 5.Adjusted by age,DD,glycemic and lipidemic profile,Logistic regression showed that ID at first(RR=2.092,95%CI 1.080-4.050,P=0.029) and second level branch(RR=0.491,95%CI 0.252-0.955,P=0.36) and EDV at second level branch(RR=0.897,95%CI 0.824-0.976,P=0.012) were independent factors influencing LGSRS(P<0.05). CONCLUSION: Ultrosonic hemodynamic abnormalities in the superior mesenteric artery might be important factor in development of lower gastrointestinal tract symptoms in patients with type 2 diabetes.

RESUMEN

BACKGROUND/AIMS: Massive proteinuria, a significant sign of nephrotic syndrome (NS), has the potential to injure tubular epithelial cells (TECs). Furosemide is widely used for the treatment of edema, a common manifestation of NS. However, whether furosemide treatment affects massive proteinuria-induced TEC injury in patients with NS is unknown. METHODS: The effect of furosemide on TEC damage was investigated in vitro. In addition, a clinical study was conducted to study whether the short-term treatment of nephrotic edema with furosemide could exacerbate TEC injury. RESULTS: The proliferation of in vitro human kidney-2 (HK-2) cells exposed to massive urinary protein (8 mg/mL) significantly decreased (P<0.05), while the levels of kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin (NGAL) in the supernatants significantly increased (P<0.05). Importantly, furosemide treatment did not further increase the expression of Kim-1 and NGAL in HK-2 cells upregulated by massive proteinuria. For the clinical study, 26 patients with NS, all prescribed the recommended dosage of prednisone (1 mg/kg/day), were randomly assigned to two groups. One group (n=13) received furosemide (60-120 mg/day, intravenously) for 1 week; the remaining participants (control group) did not receive furosemide or any other diuretics. The results showed that the 24-h urine volume in the furosemide-treated group was slightly, but not significantly, higher than that in the control group (P>0.05). In addition, serum levels of BUN, Scr, Cys C, and urinary Kim-1 and NGAL were not significantly different between the two groups (all P>0.05). Twenty-three patients underwent a renal biopsy. Of these, 22 patients exhibited vacuolar degeneration of the TECs; 8 patients showed brush border membrane shedding of the TECs; and 12 patients showed protein casts. However, there were no significant differences between the two groups (all P>0.05). CONCLUSION: In summary, massive proteinuria induced the injury of TECs in patients with NS, and furosemide treatment did not aggravate this injury.

Asunto(s)

Furosemida/uso terapéutico , Síndrome Nefrótico/prevención & control , Proteinuria/patología , Adolescente , Adulto , Biomarcadores/análisis , Biomarcadores/sangre , Estudios de Casos y Controles , Línea Celular , Supervivencia Celular/efectos de los fármacos , Niño , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Furosemida/farmacología , Humanos , Enfermedades Renales/complicaciones , Enfermedades Renales/patología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Lipocalina 2/análisis , Masculino , Persona de Mediana Edad , Síndrome Nefrótico/complicaciones , Prednisona/uso terapéutico , Proteinuria/complicaciones , Método Simple Ciego , Adulto Joven

RESUMEN

Transcription factor EB(TFEB)is a member of the MiTF/TFE family and plays an important role in cell stress,metabolism,cancer and so on.There are relatively few studies on the role of TFEB in renal diseases.TFEB was initially found to be highly expressed in TFEB-fusion renal cell carcinoma and plays a key role in the development of re-nal cell carcinoma.Blocking the downstream signaling pathway activated by TFEB would be a promising treatment for TFEB-fusion renal cell carcinoma.On the contrary,the expression of TFEB in renal intrinsic cells is decreased in diabetic kidney disease,leading to a blockage in the autophagy-lysosome pathway.TFEB enhances the ability of cell stress and self-repair,and then delays the progress of diabetic kidney disease.In cystine nephropathy,TFEB expression is reduced in re-nal tubular epithelial cells and compensatory activation is insufficient as well.TFEB over-expression effectively eliminates intracellular cystine and repairs damaged lysosome,which is expected to alleviate or cure the Fanconi syndrome.In summa-ry,TFEB plays a key role in different kidney diseases,and targeted regulation of TFEB provides new hope for the treatment of kidney diseases.

RESUMEN

Dysregulation of autophagy-mediated podocyte homeostasis is proposed to play a role in idiopathic membranous nephropathy (IMN). In the present study, autophagic activity and lysosomal alterations were investigated in podocytes of IMN patients and in cultured podocytes exposed to sublytic terminal complement complex, C5b-9. C5b-9 upregulated the number of LC3 positive puncta and the expression of p62 in patient podocytes and in C5b-9 injuried podocyte model. The lysosomal turnover of LC3-II was not influenced, although the BECN1 expression level was upregulated after exposure of podocytes to C5b-9. C5b-9 also caused a significant increase in the number of autophagosomes but not autolysosomes, suggesting that C5b-9 impairs the lysosomal degration of autophagosomes. Moreover, C5b-9 exacerbated the apoptosis of podocytes, which could be mimicked by chloroquine treatment, indicating that C5b-9 triggered podocyte injury, at least partially through inhibiting autophagy. Subsequent studies revealed that C5b-9 triggered lysosomal membrane permeabilization, which likely caused the decrease in enzymatic activity, defective acidification of lysosomes, and suppression of DQ-ovalbumin degradation. Taken together, our results suggest that the lysosomal-dependent autophagic pathway is blocked by C5b-9, which may play a key role in podocyte injury during the development of IMN.

Asunto(s)

Autofagia , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Glomerulonefritis Membranosa/metabolismo , Lisosomas/metabolismo , Podocitos/metabolismo , Transducción de Señal , Adulto , Autofagosomas/metabolismo , Autofagia/inmunología , Permeabilidad de la Membrana Celular , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Femenino , Glomerulonefritis Membranosa/tratamiento farmacológico , Glomerulonefritis Membranosa/inmunología , Glomerulonefritis Membranosa/patología , Humanos , Lisosomas/ultraestructura , Masculino , Persona de Mediana Edad , Podocitos/patología

RESUMEN

Acute and repeated exposures to ketamine mimic aspects of positive, negative, and cognitive symptoms of schizophrenia in humans. Recent studies by our group and others have shown that chronicity of ketamine use may be a key element for establishing a more valid model of cognitive symptoms of schizophrenia. However, current understanding on the long-term consequences of ketamine exposure on brain circuits has remained incomplete, particularly with regard to microstructural changes of white matter tracts that underpin the neuropathology of schizophrenia. Thus, the present study aimed to expand on previous investigations by examining causal effects of repeated ketamine exposure on white matter integrity in a non-human primate model. Ketamine or saline (control) was administered intravenously for 3 months to male adolescent cynomolgus monkeys (n = 5/group). Diffusion tensor imaging (DTI) experiments were performed and tract-based spatial statistics (TBSS) was used for data analysis. Fractional anisotropy (FA) was quantified across the whole brain. Profoundly reduced FA on the right side of sagittal striatum, posterior thalamic radiation (PTR), retrolenticular limb of the internal capsule (RLIC) and superior longitudinal fasciculus (SLF), and on the left side of PTR, middle temporal gyrus and inferior frontal gyrus were observed in the ketamine group compared to controls. Diminished white matter integrity found in either fronto-thalamo-temporal or striato-thalamic connections with tracts including the SLF, PTR, and RLIC lends support to similar findings from DTI studies on schizophrenia in humans. This study suggests that chronic ketamine exposure is a useful pharmacological paradigm that might provide translational insights into the pathophysiology and treatment of schizophrenia.

RESUMEN

Receptor tyrosine kinase-like orphan receptor 2 is an enzyme-linked receptor which specifically modulates WNT5A signaling and plays an important role in tumorigenesis, invasion, and metastasis; however, the precise role of receptor tyrosine kinase-like orphan receptor 2 in cancer is controversial. The purpose of this study was to investigate the expression and role of receptor tyrosine kinase-like orphan receptor 2 in ovarian carcinoma and clarify the biological functions and interactions of receptor tyrosine kinase-like orphan receptor 2 with non-canonical Wnt pathways in ovarian cancer. The result of the human ovary tissue microarray revealed that the receptor tyrosine kinase-like orphan receptor 2-positive rate increased in malignant epithelial ovarian cancers and was extremely higher in the metastatic tumor tissues, which was also higher than that in the malignant ovarian tumor tissues. In addition, high expression of receptor tyrosine kinase-like orphan receptor 2 was closely related with ovarian cancer grading. The expression of receptor tyrosine kinase-like orphan receptor 2 protein was higher in SKOV3 and A2780 cells than OVCAR3 and 3AO cells. Knockdown of receptor tyrosine kinase-like orphan receptor 2 inhibited ovarian cancer cell proliferation, migration, invasion, and induced morphologic as well as digestive state alterations in stably transfected SKOV3 cells. Detailed study further revealed that silencing of receptor tyrosine kinase-like orphan receptor 2 reversed the epithelial-mesenchymal transition and inhibited non-canonical Wnt signaling. Our findings suggest that receptor tyrosine kinase-like orphan receptor 2 may be an important regulator of epithelial-mesenchymal transition, primarily regulated the non-canonical Wnt signaling pathway in ovarian cancer cells, and may display a promising therapeutic target for ovarian cancer.

Asunto(s)

Carcinogénesis/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Ováricas/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/biosíntesis , Anciano , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/antagonistas & inhibidores , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/genética