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2.
Proc Natl Acad Sci U S A ; 121(35): e2403424121, 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39159367

RESUMEN

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.


Asunto(s)
Proteínas Portadoras , Comovirus , Nicotiana , Enfermedades de las Plantas , Proteínas de Plantas , Vigna , Comovirus/metabolismo , Comovirus/fisiología , Comovirus/genética , Vigna/virología , Vigna/metabolismo , Nicotiana/virología , Nicotiana/metabolismo , Nicotiana/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Enfermedades de las Plantas/virología , Proteasas de Cisteína/metabolismo , Proteasas de Cisteína/genética , Plantas Modificadas Genéticamente , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Potyvirus/fisiología , Potyvirus/metabolismo , Endopeptidasas
3.
Pharmacol Res ; 207: 107306, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39002871

RESUMEN

Many inflammatory disorders, including diabetic kidney disease (DKD), are associated with pyroptosis, a type of inflammation-regulated cell death. The purpose of this work was to ascertain the effects of apabetalone, which targets BRD4, a specific inhibitor of the bromodomain (BRD) and extra-terminal (BET) proteins that target bromodomain 2, on kidney injury in DKD. This study utilized pharmacological and genetic approaches to investigate the effects of apabetalone on pyroptosis in db/db mice and human tubular epithelial cells (HK-2). BRD4 levels were elevated in HK-2 cells exposed to high glucose and in db/db mice. Modulating BRD4 levels led to changes in the generation of inflammatory cytokines and cell pyroptosis linked to NLRP3 inflammasome in HK-2 cells and db/db mice. Likewise, these cellular processes were mitigated by apabetalone through inhibition BRD4. Apabetalone or BRD4 siRNA suppressed PLK1 expression in HK-2 cells under high glucose by P300-dependent H3K27 acetylation on the PLK1 gene promoter, as demonstrated through chromatin immunoprecipitation and immunoprecipitation assays. To summarize, apabetalone relieves renal proptosis and fibrosis in DKD. BRD4 regulates the P300/H3K27ac/PLK1 axis, leading to the activation of the NLRP3 inflammasome and subsequent cell pyroptosis, inflammation, and fibrosis. These results may provide new perspectives on DKD treatment.


Asunto(s)
Proteínas de Ciclo Celular , Nefropatías Diabéticas , Ratones Endogámicos C57BL , Quinasa Tipo Polo 1 , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Piroptosis , Factores de Transcripción , Animales , Piroptosis/efectos de los fármacos , Humanos , Proteínas de Ciclo Celular/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Línea Celular , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Masculino , Factores de Transcripción/metabolismo , Histonas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína p300 Asociada a E1A/metabolismo , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Riñón/efectos de los fármacos , Riñón/patología , Riñón/metabolismo , Transducción de Señal/efectos de los fármacos , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos , Proteínas que Contienen Bromodominio , Proteínas Nucleares
4.
Diabetes Res Clin Pract ; 209: 111594, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38403176

RESUMEN

BACKGROUND: The relationship between Bowman's capsule thickening and progression of diabetic kidney disease (DKD) remains uncertain. METHODS: Renal biopsy specimens from 145 DKD patients and 20 control subjects were evaluated for Bowman's capsule thickness. Immunohistochemical staining assessed col4α2, laminin ß1, and albumin expression. In a discovery cohort of 111 DKD patients with eGFR ≥ 30 ml/min/1.73 m2, thickening was classified as fibrotic or exudative. The composite endpoint included CKD stage 5, dialysis initiation, and renal disease-related death. Prognosis was analyzed using Kaplan-Meier and Cox regression analyses. Two validation cohorts were included. RESULTS: Three types of thickening were observed: fibrotic, exudative, and periglomerular fibrosis. Parietal epithelial cell matrix protein accumulation contributed to fibrotic thickening, while albumin was present in exudative thickening. Bowman's capsule was significantly thicker in DKD patients (5.74 ± 2.09 µm) compared to controls (3.38 ± 0.43 µm, P < 0.01). In discovery cohort, the group of exudative thickning had a poorer prognosis(median time 20 months vs 57 months, P = 0.000). Cox multivariate analysis revealed that exudative thickening of Bowman's capsule were associated with a poor prognosis. The validation cohorts confirmed the result. CONCLUSIONS: Various mechanisms contribute to Bowman's capsule thickening in DKD. The proportion of exudative thickening may serve as a valuable prognostic indicator for DKD patients.


Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Fallo Renal Crónico , Humanos , Cápsula Glomerular/metabolismo , Cápsula Glomerular/patología , Nefropatías Diabéticas/patología , Fallo Renal Crónico/patología , Diálisis Renal , Albúminas , Diabetes Mellitus/patología
5.
Acta Diabetol ; 61(6): 705-714, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38400938

RESUMEN

AIMS: Several studies have reported dietary microorganisms' beneficial effects on human health. We aimed to detect the potential association between dietary live microbe intake and diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus (T2DM) through a cross-sectional analysis of the National Health and Nutrition Examination Survey from 1999 to 2018. METHODS: According to the Sanders classification system of dietary live microbes, the study participants were divided into three groups: low, medium, and high live microbe groups. In patients with T2DM, DKD was assessed by glomerular filtration rate (< 60 mL/min/1.73 m2 using the Chronic Kidney Disease Epidemiology Collaboration algorithm), proteinuria (urinary albumin to creatinine ratio ≥ 30 mg/g), or both. Weighted univariate and multivariate logistic regression and subgroup analyses were conducted to investigate the independent association between dietary live microbe and DKD. RESULTS: The study included 3836 participants, of whom 1467 (38.24%) had DKD for the diagnosis. Our study demonstrated that participants in the high dietary live microbe group were more likely to be older, female, non-Hispanic White, have higher education levels, have a lower prevalence of smoking, have a high poverty-income ratio, have higher energy intake, lower haemoglobin (HbA1c) and serum creatinine levels, and lower risk of progression. After adjustment for covariates, patients in the high dietary live microbe group had a low prevalence of DKD, whereas no significant association with DKD was found between the medium and low dietary live microbe groups. No statistically significant interaction was observed in all subgroup analyses except for HbA1c (p for interaction < 0.05). CONCLUSIONS: Our results indicate that high dietary live microbe intake was associated with a low DKD prevalence.


Asunto(s)
Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Encuestas Nutricionales , Humanos , Femenino , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Masculino , Estudios Transversales , Persona de Mediana Edad , Nefropatías Diabéticas/epidemiología , Nefropatías Diabéticas/etiología , Anciano , Adulto , Dieta/estadística & datos numéricos , Estados Unidos/epidemiología , Tasa de Filtración Glomerular
6.
Diabetol Metab Syndr ; 15(1): 197, 2023 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-37821982

RESUMEN

BACKGROUND: To investigate the diagnostic value of serum lncRNA growth arrest-specific transcript 5 (lncRNA GAS5) and microRNA-21 (miR-21) in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), and elucidate their roles in the pathogenesis. METHODS: A microarray technology was used asses lncRNA GAS5 and miR-21 expression profiles in non-anticoagulant blood from 44 patients including T2DM without DN group (DM), T2DM with DN group (DN), and healthy controls group (N), followed by real-time PCR validation. Logistic regression and receiver operating characteristic (ROC) curves were applied to evaluate the clinical indicators among normal, T2DM, and DN patients. RESULTS: The serum lncRNA GAS5 expression in T2DM and DN patients was significantly down-regulated compared with the N group, while the expression of miR-21 was significantly up-regulated (all P < 0.05). Fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) were negatively correlated with serum lncRNA GAS5, and FBG was independently correlated with serum lncRNA GAS5. Urinary microalbumin, total cholesterol (TC), creatinine (Cr), urea, and systolic blood pressure (SBP) were significantly positively correlated with serum miR-21. Glomerular filtration rate (GFR) and albuminuria (ALB) were negatively correlated with serum miR-21, and ALB was independently correlated with serum miR-21. Serum lncRNA GAS5, miR-21 and lncRNA GAS5/miR-21 showed good diagnostic efficiency as the "diagnostic signature" of T2DM and DN. CONCLUSION: The lncRNA GAS5/miR-21 diagnostic signature may be a more effective non-invasive biomarker for detecting T2DM. In addition, miR-21 alone may be a more accurate serum biomarker for the early screening of DN patients.

7.
Food Funct ; 14(20): 9279-9286, 2023 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-37772927

RESUMEN

Objectives: There is growing evidence that antioxidant-rich diets protect against chronic kidney disease (CKD). However, the relationship between the Composite Dietary Antioxidant Index (CDAI), an important measure of an antioxidant diet, and CKD has received little attention. Therefore, here we investigated the relationship between the CDAI and CKD through a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES) 2011-2018 data. Methods: The CDAI was calculated based on the intake of six dietary antioxidants. A survey-based multivariate linear regression analysis was performed to analyze the independent relationship between the CDAI and CKD. Weighted multivariate regression and subgroup analyses were conducted to explore the relationship between the CDAI and CKD. Results: A total of 6874 NHANES participants represented 181.9 million non-institutionalized US residents (mean age, 46.43 ± 0.38 years; 49.87% female; 40.62% non-Hispanic white; 20.24% non-Hispanic black; and 13.94% Mexican American). The weighted linear regression model with full adjustment for confounding variables was -0.0155 (-0.0417, 0.0107) for Q2 (P for trend <0.0001), -0.0052 (-0.0346, 0.0242) for Q3 (P for trend <0.0001), and -0.0305 (-0.0491, -0.0120) for Q4 (P for trend = 0.0094) upon comparison with the lowest quartile of the CDAI. None of the interactions in any subgroup analysis were statistically significant except for individuals with a history of diabetes or the aged population (≥60 years) (P for interaction <0.05). Conclusions: The CDAI was positively associated with a lower prevalence of CKD in adults in the United States. Further large-scale prospective studies are required to analyze the role of the CDAI in CKD.

8.
iScience ; 26(9): 107609, 2023 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-37664593

RESUMEN

Tubulointerstitial abnormalities contribute to the progression of diabetic kidney disease (DKD). However, the underlying mechanism of the pathobiology of tubulointerstitial disease is largely unknown. Here, we showed that MYCT1 expression was downregulated in in vitro and in vivo DKD models. Adeno-associated virus (AAV)-Myct1 significantly attenuated renal dysfunction and tubulointerstitial fibrosis in diabetic db/db mice and downregulated Sp1 transcription and TGF-ß1/SMAD3 pathway activation. In human proximal tubular epithelial cells, high glucose-induced high expression of SP1 and TGF-ß1/SMAD3 pathway activation as well as overaccumulation of extracellular matrix (ECM) were abrogated by MYCT1 overexpression. Mechanistically, the binding of VDR to the MYCT1 promoter was predicted and confirmed using dual-luciferase reporter and ChIP analysis. VDR transcriptionally upregulates MYCT1. Our data reveal MYCT1 as a new and potential therapeutic target in treating DKD.

9.
Front Pharmacol ; 14: 1069348, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36874012

RESUMEN

Objectives: Dimeric pyruvate kinase (PK) M2 (PKM2) plays an important role in promoting the accumulation of hypoxia-inducible factor (HIF)-1α, mediating aberrant glycolysis and inducing fibrosis in diabetic kidney disease (DKD). The aim of this work was to dissect a novel regulatory mechanism of Yin and Yang 1 (YY1) on lncRNA-ARAP1-AS2/ARAP1 to regulate EGFR/PKM2/HIF-1α pathway and glycolysis in DKD. Materials and methods: We used adeno-associated virus (AAV)-ARAP1 shRNA to knocked down ARAP1 in diabetic mice and overexpressed or knocked down YY1, ARAP1-AS2 and ARAP1 expression in human glomerular mesangial cells. Gene levels were assessed by Western blotting, RT-qPCR, immunofluorescence staining and immunohistochemistry. Molecular interactions were determined by RNA pull-down, co-immunoprecipitation, ubiquitination assay and dual-luciferase reporter analysis. Results: YY1, ARAP1-AS2, ARAP1, HIF-1α, glycolysis and fibrosis genes expressions were upregulated and ARAP1 knockdown could inhibit dimeric PKM2 expression and partly restore tetrameric PKM2 formation, while downregulate HIF-1α accumulation and aberrant glycolysis and fibrosis in in-vivo and in-vitro DKD models. ARAP1 knockdown attenuates renal injury and renal dysfunction in diabetic mice. ARAP1 maintains EGFR overactivation in-vivo and in-vitro DKD models. Mechanistically, YY1 transcriptionally upregulates ARAP1-AS2 and indirectly regulates ARAP1 and subsequently promotes EGFR activation, HIF-1α accumulation and aberrant glycolysis and fibrosis. Conclusion: Our results first highlight the role of the novel regulatory mechanism of YY1 on ARAP1-AS2 and ARAP1 in promoting aberrant glycolysis and fibrosis by EGFR/PKM2/HIF-1α pathway in DKD and provide potential therapeutic strategies for DKD treatments.

10.
Front Immunol ; 13: 835879, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35280997

RESUMEN

Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease (ESRD). Existing treatments cannot control the progression of diabetic nephropathy very well. In diabetic nephropathy, Many monocytes and macrophages infiltrate kidney tissue. However, the role of these cells in the pathogenesis of diabetic nephropathy has not been fully elucidated. In this study, we analyzed patient kidney biopsy specimens, diabetic nephropathy model animals. Meanwhile, we cocultured cells and found that in diabetic nephropathy, damaged intrinsic renal cells (glomerular mesangial cells and renal tubular epithelial cells) recruited monocytes/macrophages to the area of tissue damage to defend against and clear cell damage. This process often involved the activation of different types of macrophages. Interestingly, the infiltrating macrophages were mainly M1 (CD68+iNOS+) macrophages. In diabetic nephropathy, crosstalk between the Notch pathway and NF-κB signaling in macrophages contributed to the polarization of macrophages. Hyperpolarized macrophages secreted large amounts of inflammatory cytokines and exacerbated the inflammatory response, extracellular matrix secretion, fibrosis, and necroptosis of intrinsic kidney cells. Additionally, macrophage depletion therapy with clodronate liposomes and inhibition of the Notch pathway in macrophages alleviated the pathological changes in kidney cells. This study provides new information regarding diabetic nephropathy-related renal inflammation, the causes of macrophage polarization, and therapeutic targets for diabetic nephropathy.


Asunto(s)
Nefropatías Diabéticas , Nefritis , Animales , Nefropatías Diabéticas/patología , Femenino , Fibrosis , Humanos , Inflamación/metabolismo , Riñón/patología , Macrófagos/metabolismo , Masculino , Necroptosis , Nefritis/patología
11.
J Cell Mol Med ; 24(22): 12994-13009, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32969198

RESUMEN

The persistent transactivation of epidermal growth factor receptor (EGFR) causes subsequent activation of the TGF-ß/Smad3 pathway, which is closely associated with fibrosis and cell proliferation in diabetic nephropathy (DN), but the exact mechanism of persistent EGFR transactivation in DN remains unclear. ARAP1, a susceptibility gene for type 2 diabetes, can regulate the endocytosis and ubiquitination of membrane receptors, but the effect of ARAP1 and its natural antisense long non-coding RNA (lncRNA), ARAP1-AS2, on the ubiquitination of EGFR in DN is not clear. In this study, we verified that the expression of ARAP1 and ARAP1-AS2 was significantly up-regulated in high glucose-induced human proximal tubular epithelial cells (HK-2 cells). Moreover, we found that overexpression or knockdown of ARAP1-AS2 could regulate fibrosis and HK-2 cell proliferation through EGFR/TGF-ß/Smad3 signalling. RNA pulldown assays revealed that ARAP1-AS2 directly interacts with ARAP1. Coimmunoprecipitation, dual-immunofluorescence and ubiquitination assays showed that ARAP1 may maintain persistent EGFR activation by reducing EGFR ubiquitination through competing with Cbl for CIN85 binding. Taken together, our results suggest that the lncRNA ARAP1-AS2 may promote high glucose-induced proximal tubular cell injury via persistent EGFR/TGF-ß/Smad3 pathway activation by interacting with ARAP1.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Túbulos Renales Proximales/metabolismo , ARN Largo no Codificante , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proliferación Celular , Nefropatías Diabéticas/metabolismo , Receptores ErbB/metabolismo , Glucosa , Humanos , Hibridación Fluorescente in Situ , Túbulos Renales Proximales/patología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Oligonucleótidos Antisentido/farmacología , Unión Proteica , ARN Largo no Codificante/genética , RNA-Seq , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ubiquitina/metabolismo
12.
Kidney Int ; 98(5): 1225-1241, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32610050

RESUMEN

Polycystin-1 (PC1) and -2 (PC2), products of the PKD1 and PKD2 genes, are mutated in autosomal dominant polycystic kidney disease (ADPKD). They localize to the primary cilia; however, their ciliary function is in dispute. Loss of either the primary cilia or PC1 or PC2 causes cyst formation. However, loss of both cilia and PC1 or PC2 inhibits cyst growth via an unknown pathway. To help define a pathway, we studied cilium length in human and mouse kidneys. We found cilia are elongated in kidneys from patients with ADPKD and from both Pkd1 and Pkd2 knockout mice. Cilia elongate following polycystin inactivation. The role of intraflagellar transport proteins in Pkd1-deficient mice is also unknown. We found that inactivation of Ift88 (a gene expressing a core component of intraflagellar transport) in Pkd1 knockout mice, as well as in a new Pkd2 knockout mouse, shortened the elongated cilia, impeded kidney and liver cystogenesis, and reduced cell proliferation. Multi-stage in vivo analysis of signaling pathways revealed ß-catenin activation as a prominent, early, and sustained event in disease onset and progression in Pkd2 single knockout but not in Pkd2.Ift88 double knockout mouse kidneys. Additionally, AMPK, mTOR and ERK pathways were altered in Pkd2 single knockout mice but only AMPK and mTOR pathway alteration were rescued in Pkd2.Ift88 double knockout mice. Thus, our findings advocate an essential role of polycystins in the structure and function of the primary cilia and implicate ß-catenin as a key inducer of cystogenesis downstream of the primary cilia. Our data suggest that modulating cilium length and/or its associated signaling events may offer novel therapeutic approaches for ADPKD.


Asunto(s)
Quistes , Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Animales , Cilios , Quistes/genética , Humanos , Riñón , Hígado , Ratones , Ratones Noqueados , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/genética
13.
Biol Pharm Bull ; 43(3): 558-564, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32115515

RESUMEN

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease. Current therapies for DKD are insufficient. Therefore, there is an urgent need for identifying new therapies. An increasing number of micro RNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been demonstrated to modulate the progression of diabetic kidney disease. Nevertheless, until now, there have been few reports evaluating the relevance of circular RNAs (circRNAs) in DKD. circRNAs have been reported to regulate the occurrence and development of multiple diseases. In this study, we intended to explore the circRNA expression profiles and determine the role of circRNA in DKD. We identified a series of dysregulated circRNAs in glucose-stressed HK-2 cells using circRNA microarray analysis. Among the candidate circRNAs, we found that circACTR2 was upregulated and may be involved in inflammation and pyroptosis. Knockdown of circACTR2 significantly decreased pyroptosis, interleukin (IL)-1ß release and collagen IV and fibronectin production, indicating the effective regulation by circACTR2 of cell death and inflammation. Overall, our study identified a new circRNA, circACTR2, that regulates high glucose-induced pyroptosis, inflammation and fibrosis in proximal tubular cells. The present study preliminarily explores the role of circRNAs in pyroptosis of tubular cells, and provides novel insight into the pathogenesis of DKD and new therapeutic strategies.


Asunto(s)
Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Glucosa/efectos adversos , Piroptosis/genética , Piroptosis/fisiología , ARN Circular/deficiencia , ARN Circular/genética , Proteína 2 Relacionada con la Actina , Línea Celular , Colágeno/metabolismo , Células Epiteliales , Fibronectinas/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Expresión Génica , Humanos , Interleucina-1beta/metabolismo , Riñón/metabolismo
14.
Diabetes Metab Syndr Obes ; 13: 313-323, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32104028

RESUMEN

BACKGROUND: Apoptosis has been repeatedly linked with diabetic kidney disease (DKD), which is a programmed cell death mediated by effector caspases-3, 6 and 7, targeting >600 substrates. However, the pathophysiologic correlations of this process remain obscure. As a putative tumor suppressor, gasdermin E (GSDME) was recently reported to be cleaved by caspase-3 to produce a GSDME-N fragment which targets the plasma membrane to switch apoptosis to secondary necrosis. However, it remains elusive whether GSDME is involved in the regulation of DKD. METHODS: To evaluate the therapeutic potential of caspase-3 inhibition in DKD, we administered caspase-3 inhibitor Z-DEVD-FMK to STZ-induced diabetic mice for eight weeks. Albuminuria, renal function, pathological changes, and indicators of secondary necrosis and fibrosis were evaluated. In vitro, human tubule epithelial cells (HK-2 cells) were subjected to high-glucose treatment. Secondary necrosis was determined by LDH release, GSDME cleavage, and morphological feature under confocal microscopy. Z-DEVD-FMK and GSDME inhibition by shRNA were administered to suppress the cleavage and expression of GSDME. Flow cytometry, cytotoxicity assay and immunoblot were used to assess cell death and fibrogenesis. RESULTS: Caspase-3 inhibition by Z-DEVD-FMK ameliorated albuminuria, renal function, and tubulointerstitial fibrosis in diabetic mice. The nephroprotection mediated by Z-DEVD-FMK was potentially associated with inhibition of GSDME. In vitro, molecular and morphological features of secondary necrosis were observed in glucose-stressed HK-2 cells, evidenced by active GSDME cleavage, ballooning of the cell membrane, and release of cellular contents. Here we showed that caspase-3 inhibition prevented GSDME activation and cell death in glucose-treated tubular cells. Specifically, knocking down GSDME directly inhibited secondary necrosis and fibrogenesis. CONCLUSION: These data suggest GSDME-dependent secondary necrosis plays a crucial role in renal injury, and provides a new insight into the pathogenesis of DKD and a promising target for its treatment.

15.
J Cell Physiol ; 235(7-8): 5787-5795, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31975379

RESUMEN

The epithelial-mesenchymal transition (EMT) plays an important role in diabetic renal fibrosis. The ARAP1 gene is located near risk alleles for Type 2 diabetes, and its function has been linked to cytoskeleton rearrangement, Golgi apparatus remodeling, and endocytic trafficking of membrane receptors. The role of ARAP1 and its antisense RNA, ARAP1-AS2, in the pathogenesis of diabetes is unclear. To clarify the roles of ARAP1 and its antisense RNA in diabetes and related complications, we examined if the expression of these transcripts changed under high glucose (HG) conditions. To do this, we examined transcript levels in HK-2 cells, and explored the roles of ARAP1 and ARAP1-AS2 in the EMT process in HK-2 cells. We found increased expression of ARAP1-AS2 and ARAP1 in HK-2 cells under HG condition, and observed that the overexpression of ARAP1-AS2 significantly increased the EMT process. In addition, HG upregulated Cdc42-GTP levels in HK-2 cells, and increased cytoskeleton rearrangement, cell viability, and migration. After knockdown of ARAP1, the level of Cdc42-GTP was decreased; cytoskeleton reorganization, cell viability, and migration processes were decreased; and EMT and expression of fibrosis marker protein. Overall, our results indicated that ARAP1-AS2/ARAP1 may participate in cytoskeleton rearrangement and EMT processes in HK-2 cells through increased Cdc42-GTP levels.


Asunto(s)
Proteínas Portadoras/genética , Diabetes Mellitus Tipo 2/genética , Células Epiteliales/metabolismo , Proteínas Activadoras de GTPasa/genética , ARN Largo no Codificante/genética , Proteína de Unión al GTP cdc42/genética , Alelos , Movimiento Celular/genética , Citoesqueleto/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Glucosa/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Factores de Riesgo
16.
Med Sci Monit ; 24: 846-854, 2018 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-29428962

RESUMEN

BACKGROUND To investigate the protective effect of ursolic acid (UA) on high glucose (HG)-induced human glomerular mesangial cell injury and to determine whether UA inhibits cell proliferation and reactive oxygen species (ROS) production by suppressing PI3K/Akt/mTOR pathway activation. MATERIAL AND METHODS Human mesangial cells were cultured with normal glucose (NG group), high glucose (HG group), mannitol (mannitol hypertonic control group), or high glucose with different concentrations (0.5, 1.0, and 2.0 mmol/L) of UA (HG+UA groups). Cell proliferation and intracellular ROS levels were assessed by methyl thiazolyl tetrazolium (MTT) and dichloro-dihydro-fluorescein diacetate (DCFH-DA) flow cytometry assays, respectively. Western blotting was used to detect mesangial cell expression of PI3K/Akt/mTOR pathway components, including Akt, p-Akt, mTOR, and p-mTOR, and proteins related to cell injury, including TGF-ß1 and fibronectin (FN). mRNA expression of TGF-ß1 and FN were evaluated using real-time quantitative polymerase chain reaction (PCR). RESULTS Abnormal proliferation was observed in human glomerular mesangial cells at 48 h after treatment with HG, and UA suppressed the HG-induced proliferation of mesangial cells in a dose-dependent manner. UA inhibited ROS generation and oxidative stress in mesangial cells and mitigated mesangial cell injury. Treatment with UA reduced Akt and mTOR phosphorylation levels in mesangial cells exposed to HG (p<0.05 vs. HG) and downregulated protein and mRNA expression of TGF-ß1 and FN in these cells (p<0.05 vs. HG). CONCLUSIONS UA attenuated mesangial cell proliferation and ROS generation by inhibiting HG-mediated PI3K/Akt/mTOR pathway activation, thereby ameliorating mesangial cell damage.


Asunto(s)
Glucosa/toxicidad , Células Mesangiales/enzimología , Células Mesangiales/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Triterpenos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Mesangiales/efectos de los fármacos , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ácido Ursólico
17.
Clin Nephrol ; 87(6): 293-300, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28332473

RESUMEN

OBJECTIVE: To analyze the correlations of renal tissue elastography with clinical biochemical indicators and pathological changes in patients with chronic kidney disease (CKD) as well as to explore the potential for renal tissue elastography as a new, noninvasive method for the dynamic monitoring of renal disease progression, efficacy assessment, and prognosis evaluation. METHODS: Patients admitted to the Department of Nephrology of the First Affiliated Hospital of China Medical University from August 2014 to January 2015 who had undergone renal biopsies were selected. A total of 113 patients with CKD and 16 healthy controls were enrolled in this study, including 61 males and 52 females. In total, 23 cases of IgA nephropathy, 39 cases of membranous nephropathy, 15 cases of minimal-change nephropathy (MCN), and 7 cases of focal segmental glomerulosclerosis were included. The Young's moduli (YM) of the renal cortex and medulla were measured using an AixPlorer Doppler ultrasound with full digital color from Supersonic Imagine. The correlations between the YM of renal tissue and clinical biochemical indicators of blood and urine and the differences in Young's moduli among the different pathological changes in the patients with CKD were analyzed. RESULTS: The YM of the CKD patients was significantly higher than that of the control group (p < 0.05), and the YM of the renal cortex and medulla gradually increased with the progression of CKD. The YM of the renal cortex in the stage-G5 CKD patients was significantly higher than that of the CKD patients in stages G1 - G3 (p < 0.05). The YM of the renal medulla of the CKD patients in stages G3 - 5 was significantly higher than that of the CKD patients in stages G1 - G2. On univariate analysis, the YM of the renal cortex was correlated with systolic blood pressure, serum creatinine, cystatin C, serum albumin, serum phosphorus, calcium and phosphorus products, uric acid, iPTH, urinary N-acetyl-glucosaminidase (NAG), eGFR, and hemoglobin levels. And the YM of the renal medulla was correlated with systolic blood pressure, serum creatinine, serum albumin, uric acid, iPTH, urinary microalbumin (MA), urinary NAG, and hemoglobin levels. On multivariate analysis, serum cystatin C (ß = 0.485, p = 0.018) and uric acid (ß = 0.418, p = 0.039) levels were independently correlated with the YM of the renal cortex, while serum creatinine (ß = 0.380, p = 0.019) and uric acid (ß = 0.482, p = 0.004) levels, as well as smoking (ß = 0.337, p = 0.009), were independently correlated with the YM of the renal medulla. The YMs of the renal cortex in patients with membranous nephropathy and IgA nephropathy were significantly higher than those in the patients with CN (p < 0.05). The YM of the renal cortices of the patients in phases IV and V of IgA nephropathy based on the Lee grading system were significantly higher than those of the patients in phases II and III (p < 0.05). According to the Oxford classification for IgA nephropathy, the Young's moduli of the renal cortex and medulla in T1 and T2 patients were significantly higher than those in T0 patients (p < 0.05). The YM of the renal cortex and medulla showed no statistically-significant differences among the different stages of membranous nephropathy. CONCLUSIONS: The YM of the renal cortex and medulla are associated with the progression of renal insufficiency, and renal ultrasound elastography shows promise as a new means of assessing the stage of CKD. Renal ultrasound elastography is expected to become a new, noninvasive method for the early diagnosis of CKD and the dynamic monitoring of disease progression as well as the assessment of efficacy and prognosis.
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Asunto(s)
Diagnóstico por Imagen de Elasticidad , Riñón , Insuficiencia Renal Crónica , Presión Sanguínea , Femenino , Humanos , Riñón/diagnóstico por imagen , Riñón/fisiopatología , Masculino , Insuficiencia Renal Crónica/diagnóstico por imagen , Insuficiencia Renal Crónica/epidemiología , Insuficiencia Renal Crónica/fisiopatología
18.
Nephrol Dial Transplant ; 32(8): 1285-1293, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26567247

RESUMEN

BACKGROUND: Autophagy plays an important role in the maintenance of podocyte homeostasis. Reduced autophagy may result in limited renal cell function during exposure to high glucose conditions. In this study we investigated the effects of ursolic acid (UA) on autophagy and podocyte injury, which were induced by high glucose. METHODS: Conditionally immortalized murine podocytes were cultured in media supplemented with high glucose and the effects of the PI3K inhibitor LY294002 and UA on protein expression were determined. miR-21 expression was detected by real-time RT-PCR. Activation of the PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related proteins and expression of podocyte marker proteins were determined by western blot. Immunofluorescence was used to monitor the accumulation of LC3 puncta. Autophagosomes were also observed by transmission electron microscopy. RESULTS: During exposure to high glucose conditions, the normal level of autophagy was reduced in podocytes, and this defective autophagy induced podocyte injury. Increased miR-21 expression, decreased PTEN expression and abnormal activation of the PI3K/Akt/mTOR pathway were observed in cells that were cultured in high glucose conditions. UA and LY294002 reduced podocyte injury through the restoration of defective autophagy. Our data suggest that UA inhibits miR-21 expression and increases PTEN expression, which in turn inhibits Akt and mTOR and restores normal levels of autophagy. CONCLUSIONS: Our data suggest that podocyte injury is associated with reduced levels of autophagy during exposure to high glucose conditions, UA attenuated podocyte injury via an increase in autophagy through miR-21 inhibition and PTEN expression, which inhibit the abnormal activation of the PI3K/Akt/mTOR pathway.


Asunto(s)
Antiinfecciosos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/toxicidad , MicroARNs/genética , Podocitos/efectos de los fármacos , Triterpenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Células Cultivadas , Ratones , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Podocitos/metabolismo , Podocitos/patología , Transducción de Señal/efectos de los fármacos , Edulcorantes/toxicidad , Serina-Treonina Quinasas TOR/metabolismo , Ácido Ursólico
19.
Cell Death Dis ; 7(11): e2445, 2016 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-27809300

RESUMEN

The aim of our study was to investigate the role of autophagy, a homeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins, in regulating the survival of mesangial cells treated with advanced glycation end products (AGEs). In the present study, AGEs induced mitochondrial depolarization and led to mitochondrial-dependent apoptosis in mesangial cells, as shown by the loss of the mitochondrial membrane potential; increased Bax processing; increased Caspase-9, Caspase-3 and PARP cleavage; and decreased Bcl-2 expression. Meanwhile, AGEs also triggered autophagy flux in mesangial cells, as confirmed by the presence of autophagic vesicles, the conversion of LC3II/LC3I and the increase/decrease in Beclin-1/p62 expression. Interestingly, this study reported apparent apoptosis and autophagy that were dependent on reactive oxygen species (ROS) production. Scavenging ROS with N-acetyl-l-cysteine could prevent the appearance of the autophagic features and reverse AGE-induced apoptosis. Moreover, AGE-triggered mitophagy, which was confirmed by the colocalization of autophagosomes and mitochondria and Parkin translocation to mitochondria, played a potential role in reducing ROS production in mesangial cells. Additionally, inhibition of autophagy significantly enhanced AGE-induced cell apoptosis. Taken together, our data suggest that ROS were the mediators of AGE-induced mesangial cell apoptosis and that autophagy was likely to be the mechanism that was triggered to repair the ROS-induced damage in the AGE-treated cells and thereby promote cell survival. This study provides new insights into the molecular mechanism of autophagy involved in AGE-induced apoptosis in mesangial cells.


Asunto(s)
Apoptosis , Autofagia , Productos Finales de Glicación Avanzada/metabolismo , Células Mesangiales/citología , Células Mesangiales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Potencial de la Membrana Mitocondrial , Células Mesangiales/ultraestructura , Mitocondrias/metabolismo , Mitofagia , Modelos Biológicos , Ratas , Transducción de Señal
20.
PLoS One ; 10(2): e0117400, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25689721

RESUMEN

OBJECTIVE: To investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions. METHODS: Rat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy. RESULTS: Compared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression. CONCLUSIONS: Ursolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.


Asunto(s)
Autofagia/efectos de los fármacos , Diabetes Mellitus/patología , Células Mesangiales/patología , Transducción de Señal/efectos de los fármacos , Triterpenos/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Citoprotección/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Glucosa/farmacología , Hipertrofia , Riñón/efectos de los fármacos , Células Mesangiales/efectos de los fármacos , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fagosomas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Serina-Treonina Quinasas TOR/metabolismo , Ácido Ursólico
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