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Background: Nephronophthisis (NPHP) is a rare autosomal recessive inherited tubulointerstitial nephropathy, the most prevalent genetic cause of end-stage renal disease (ESRD) in children. Convincing evidence indicated that the overall prevalence of NPHP in adult-onset ESRD is very likely to be an underestimation. Therefore, understanding the genetic background and clinicopathologic features of adult-onset NPHP is warranted. Case presentation: we reported one intriguing case with concurrent NPHP3 c.2694-2_2694-1delAG (splicing) variant and c.1082C > G (p.S361C) variant. A 48-year-old male was admitted to our hospital, complained about renal dysfunction for 10 years, and found right renal space-occupying lesion for 1 week. One of the most interesting clinical features is adult-onset ESRD, which differs from previous cases. Another discovery of this study is that the NPHP harboring NPHP3 deletion may be associated with clear cell renal cell carcinoma. Conclusion: In conclusion, we report two mutations in the NPHP3 gene that cause NPHP with adult-onset ESRD and renal clear cell carcinoma in a Chinese family, enriching the clinical features of NPHP.
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OBJECTIVE: Investigating the impact of centromere protein N (CENP-N) on radiosensitivity of nasopharyngeal carcinoma (NPC) cells. METHODS: Using immunohistochemistry and immunofluorescence to detect CENP-N expression in tissues from 35 patients with radiosensitive or radioresistant NPC. Assessing the effect of combined CENP-N knockdown and radiotherapy on various cellular processes by CCK-8, colony formation, flow cytometry, and Western blotting. Establishing a NPC xenograft model. When the tumor volume reached 100 mm3, a irradiation dose of 6 Gy was given, and the effects of the combined treatment were evaluated in vivo using immunofluorescence and Western blotting techniques. RESULTS: The level of CENP-N was significantly reduced in radiosensitive tissues of NPC (p < 0.05). Knockdown of CENP-N enhanced NPC radiosensitivity, resulting in sensitizing enhancement ratios (SER) of 1.44 (5-8 F) and 1.16 (CNE-2Z). The combined treatment showed significantly higher levels of proliferation suppression, apoptosis, and G2/M phase arrest (p < 0.01) compared to either CENP-N knockdown alone or radiotherapy alone. The combined treatment group showed the highest increase in Bax and γH2AX protein levels, whereas the protein Cyclin D1 exhibited the greatest decrease (p < 0.01). However, the above changes were reversed after treatment with AKT activator SC79. In vivo, the mean volume and weight of tumors in the radiotherapy group were 182 ± 54 mm3 and 0.16 ± 0.03 g. The mean tumor volume and weight in the combined treatment group were 84 ± 42 mm3 and 0.04 ± 0.01 g. CONCLUSION: Knockdown of CENP-N can enhance NPC radiosensitivity by inhibiting AKT/mTOR.
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Neoplasias Nasofaríngeas , Proteínas Proto-Oncogênicas c-akt , Humanos , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/radioterapia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/radioterapia , Linhagem Celular Tumoral , Tolerância a Radiação/genética , Serina-Treonina Quinases TOR , Proliferação de Células/efeitos da radiação , Apoptose/genéticaRESUMO
AIMS: To investigate the long-term alterations in immune function and spontaneous inflammation in mice following specific knockout of Notch2 (Notch2KO) in Treg cells. MAIN METHODS: A Treg cell-specific Notch2 knockout mouse model was constructed, and the mice were named Notch2KO mice. The pathological changes and inflammatory cell infiltration in the lungs, skin, and liver of the mice at 2, 6, 9, and 12 months of age were evaluated by HE staining. The expression of Th1/Th2/Th17/Treg transcription factors was detected by Western blotting. The proportion of CD4 + T-cell subsets was determined by flow cytometry. The levels of Th1/Th2/Th17/Treg cytokines were measured by enzyme-linked immunosorbent assays (ELISAs). KEY FINDINGS: The expression level of Notch2 in Treg cells from the Notch2KO mice was significantly decreased compared with that in Treg cells from the control mice (P < 0.05). HE staining showed that compared with the control mice, the Notch2KO mice displayed spontaneous inflammation and had a large amount of inflammatory cell infiltration in the lungs and skin (P < 0.05). The number of Treg cells, the expression level of Foxp3, and the level of IL-10 were reduced in the Notch2KO mice compared with the control mice (P < 0.05), and these metrics further decreased with increasing age (P < 0.05). In contrast, the number of Th1/Th2 cells, the expression level of T-bet/GATA3, and the levels of Th1 cytokines (IFN-γ)/Th2 cytokines (IL-4, IL-5, and IL-13) were significantly increased in the Notch2KO mice (P < 0.05), and these metrics further increased with increasing age (P < 0.05). There was no significant change in the number of Th17 cells, the expression of RORγt, or the level of IL-17. Further analysis showed that the balance of Th1/Th2 and Treg/Th17 cells in the Notch2KO mice was shifted, and the ratio showed a downward trend over time (P < 0.05). SIGNIFICANCE: The number and function of Treg cells can be severely inhibited by a specific knockout of Notch2 in Treg cells, leading to immune disorders that gradually worsen over time.
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Subpopulações de Linfócitos T , Linfócitos T Reguladores , Animais , Camundongos , Citocinas/metabolismo , Homeostase , Inflamação/metabolismo , Células Th1 , Células Th17 , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Pyroptosis is crucial for controlling various immune cells. However, the role of allergen-induced CD11c + dendritic cell (DC) pyroptosis in allergic rhinitis (AR) remains unclear. METHODS: Mice were grouped into the control group, AR group and necrosulfonamide-treated AR group (AR + NSA group). The allergic symptom scores, OVA-sIgE titres, serum IL-1ß/IL-18 levels, histopathological characteristics and T-helper cell-related cytokines were evaluated. CD11c/GSDMD-N-positive cells were examined by immunofluorescence analysis. Murine CD11c + bone marrow-derived DCs (BMDCs) were induced in vitro, stimulated with OVA/HDM, treated with necrosulfonamide (NSA), and further cocultured with lymphocytes to assess BMDC function. An adoptive transfer murine model was used to study the role of BMDC pyroptosis in allergic rhinitis. RESULTS: Inhibiting GSDMD-N-mediated pyroptosis markedly protected against Th1/Th2/Th17 imbalance and alleviated inflammatory responses in the AR model. GSDMD-N was mainly coexpressed with CD11c (a DC marker) in AR mice. In vitro, OVA/HDM stimulation increased pyroptotic morphological abnormalities and increased the expression of pyroptosis-related proteins in a dose-dependent manner; moreover, inhibiting pyroptosis significantly decreased pyroptotic morphology and NLRP3, C-Caspase1 and GSDMD-N expression. In addition, OVA-induced BMDC pyroptosis affected CD4 + T-cell differentiation and related cytokine levels, leading to Th1/Th2/Th17 cell imbalance. However, the Th1/Th2/Th17 cell immune imbalance was significantly reversed by NSA. Adoptive transfer of OVA-loaded BMDCs promoted allergic inflammation, while the administration of NSA to OVA-loaded BMDCs significantly reduced AR inflammation. CONCLUSION: Allergen-induced dendritic cell pyroptosis promotes the development of allergic rhinitis through GSDMD-N-mediated pyroptosis, which provides a clue to allergic disease interventions. Video Abstract.
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Alérgenos , Rinite Alérgica , Animais , Camundongos , Piroptose , Citocinas , Inflamação , Células Dendríticas , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVE: To investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. METHODS: A mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. RESULTS: The tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). CONCLUSIONS: Specific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Receptor Notch2 , Animais , Camundongos , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Antígeno Ki-67/metabolismo , Camundongos Knockout , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linfócitos T Reguladores , Microambiente Tumoral , Receptor Notch2/genética , Receptor Notch2/metabolismoRESUMO
[This corrects the article DOI: 10.7150/thno.33520.].
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STUDY OBJECTIVES: Neuronal intranuclear inclusion disease (NIID) is a rare progressive neurodegenerative disorder, with complex and diverse of clinical manifestations characterized by eosinophilic hyaline inclusions in neurons and somatic cells. Due to the improvement in diagnostic methods, NIID is being increasingly diagnosed. METHODS: Herein, we reported three NIID cases, which were diagnosed by skin biopsy and FMR1 gene, after DWI showed the characteristic corticomedullary junction hyperintensity. Then we reviewed all the published cases of NIID in PubMed, which were diagnosed by the same method. RESULTS: We discussed 15 NIID cases, including three cases diagnosed by us. The average age was 63.4 ± 14.0 years. The average time from onset of symptom to diagnosis was 5.4 ± 7.9 years. Nine cases had dementia or cognitive impairment. Three cases presented with encephalitis. Three cases showed bladder dysfunction and two cases only presented with dizziness and headache. Two cases showed acute neurological deficit mimicking stroke. All cases were diagnosed by skin biopsy, after DWI showed abnormal corticomedullary junction hyperintensity. Ten cases showed inclusions in sweat gland cells, and seven cases in adipocytes, sweat gland cells, and fibroblasts. EMG was performed in five cases, four of whom had abnormal results, showing simultaneous involvement of motor and sensory nerves. CONCLUSIONS: The results indicated that inclusions were more easily detected in sweat gland cells in skin biopsy. The early stage of NIID could only characterized by autonomic nerve function involvement. Combined autonomic nerve dysfunction might be another relatively common manifestation in NIID.
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Encefalite , Doenças Neurodegenerativas , Idoso , Biópsia , Encefalite/patologia , Proteína do X Frágil da Deficiência Intelectual , Humanos , Corpos de Inclusão Intranuclear/patologia , Pessoa de Meia-Idade , Doenças Neurodegenerativas/genéticaRESUMO
Tubules injury and immune cell activation are the common pathogenic mechanisms in acute kidney injury (AKI). However, the exact modes of immune cell activation following tubule damage are not fully understood. Here we uncovered that the release of cytoplasmic spliceosome associated protein 130 (SAP130) from the damaged tubular cells mediated necroinflammation by triggering macrophage activation via miRNA-219c(miR-219c)/Mincle-dependent mechanism in unilateral ureteral obstruction (UUO) and cisplatin-induced AKI mouse models, and in patients with acute tubule necrosis (ATN). In the AKI kidneys, we found that Mincle expression was tightly correlated to the necrotic tubular epithelial cells (TECs) with higher expression of SAP130, a damaged associated molecule pattern (DAMP), suggesting that SAP130 released from damaged tubular cells may trigger macrophage activation and necroinflammation. This was confirmed in vivo in which administration of SAP130-rich supernatant from dead TECs or recombinant SAP130 promoted Mincle expression and macrophage accumulation which became worsen with profound tubulointerstitial inflammation in LPS-primed Mincle WT mice but not in Mincle deficient mice. Further studies identified that Mincle was negatively regulated via miR-219c-3p in macrophages as miR-219c-3p bound Mincle 3'-UTR to inhibit Mincle translation. Besides, lentivirus-mediated renal miR-219c-3p overexpression blunted Mincle and proinflammatory cytokine expression as well as macrophage infiltration in the inflamed kidney of UUO mice. In conclusion, SAP130 is released by damaged tubules which elicit Mincle activation on macrophages and renal necroinflammation via the miR-219c-3p-dependent mechanism. Results from this study suggest that targeting miR-219c-3p/Mincle signaling may represent a novel therapy for AKI.
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Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Inflamação/patologia , Túbulos Renais/patologia , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Fatores de Processamento de RNA/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas/genética , Adulto , Animais , Sequência de Bases , Estudos de Casos e Controles , Morte Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lectinas Tipo C/genética , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Necrose , Células RAW 264.7RESUMO
BACKGROUND: Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. METHODS: Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. RESULTS: The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. CONCLUSIONS: Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.
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Quimiocina CCL21 , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Vesículas Extracelulares , Animais , Aquaporina 2 , Biomarcadores , Quimiocina CCL21/genética , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/genética , Humanos , RNA Mensageiro/genética , RatosRESUMO
Immunoglobulin A (IgA) nephropathy, in the presence of myeloid bodies, has been reported in Fabry disease (FD). In this case, we excluded the diagnosis of FD by demonstrating the absence of mutation in the α-galactosidase Aï¼GLAï¼gene. Our patient also denied any history of use of cationic amphiphilic drugs. Interestingly, we identified a novel missense mutation for Coenzyme Q2ï¼COQ2ï¼ , which is known to cause COQ2 mutation-associated nephropathy. We also found heteromorphic mitochondria and good treatment response in our patient following coenzyme Q10 supplementation. In light of our findings, our patient was diagnosed with COQ2 nephropathy and IgA nephropathy. To our knowledge, this is the first case report of COQ2 nephropathy with pathologic manifestations of myeloid bodies in podocytes.
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Exosomes are increasingly recognized as vehicles of intercellular communication. However, the role of exosome in maintaining cellular homeostasis under stress conditions remained unclear. Here we show that Rab27a expression was upregulated exclusively in tubular epithelial cells (TECs) during proteinuria nephropathy established by adriamycin (ADR) injection and 5/6 nephrectomy as well as in chronic kidney disease patients, leading to the increased secretion of exosomes carrying albumin. The active exosome production promoted tubule injury and inflammation in neighboring and the producing cells. Interferon regulatory factor 1 (IRF-1) was found as the transcription factor contributed to the upregulation of Rab27a. Albumin could be detected in exosome fraction and co-localized with exosome marker CD63 indicating the secretion of albumin into extracellular space by exosomes. Interestingly, inhibition of exosome release accelerated albumin degradation which reversed tubule injury with albumin overload, while lysosome suppression augmented exosome secretion and tubule inflammation. Our findings revealed that IRF-1/Rab27a mediated exosome secretion constituted a coordinated approach to lysosome degradation for albumin handling, which lead to the augment of albumin toxicity as a maladaptive response to maintain cell homeostasis. The findings may suggest a novel therapeutic strategy for proteinuric kidney disease by targeting exosome secretion.
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Albuminas/metabolismo , Exossomos/metabolismo , Nefropatias/metabolismo , Lisossomos/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Adulto , Animais , Comunicação Autócrina , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Inflamação/patologia , Fator Regulador 1 de Interferon/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Nefrectomia , Comunicação Parácrina , Proteinúria/complicações , Ratos Sprague-DawleyRESUMO
Tubulointerstitial inflammation is a common characteristic of acute and chronic kidney injury. However, the mechanism by which the initial injury of tubular epithelial cells (TECs) drives interstitial inflammation remains unclear. This paper aims to explore the role of exosomal miRNAs derived from TECs in the development of tubulointerstitial inflammation. Global microRNA(miRNA) expression profiling of renal exosomes was examined in a LPS induced acute kidney injury (AKI) mouse model and miR-19b-3p was identified as the miRNA that was most notably increased in TEC-derived exosomes compared to controls. Similar results were also found in an adriamycin (ADR) induced chronic proteinuric kidney disease model in which exosomal miR-19b-3p was markedly released. Interestingly, once released, TEC-derived exosomal miR-19b-3p was internalized by macrophages, leading to M1 phenotype polarization through targeting NF-κB/SOCS-1. A dual-luciferase reporter assay confirmed that SOCS-1 was the direct target of miR-19b-3p. Importantly, the pathogenic role of exosomal miR-19b-3p in initiating renal inflammation was revealed by the ability of adoptively transferred of purified TEC-derived exosomes to cause tubulointerstitial inflammation in mice, which was reversed by inhibition of miR-19b-3p. Clinically, high levels of miR-19b-3p were found in urinary exosomes and were correlated with the severity of tubulointerstitial inflammation in patients with diabetic nephropathy. Thus, our studies demonstrated that exosomal miR-19b-3p mediated the communication between injured TECs and macrophages, leading to M1 macrophage activation. The exosome/miR-19b-3p/SOCS1 axis played a critical pathologic role in tubulointerstitial inflammation, representing a new therapeutic target for kidney disease.
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Injúria Renal Aguda/genética , Exossomos/genética , Túbulos Renais/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , MicroRNAs/metabolismo , Injúria Renal Aguda/metabolismo , Adulto , Idoso , Animais , Células Cultivadas , Nefropatias Diabéticas/urina , Células Epiteliais/metabolismo , Exossomos/metabolismo , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/urina , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Nefrite/genética , Nefrite/patologia , Proteinúria/induzido quimicamente , Proteinúria/genética , Proteinúria/metabolismo , Células RAW 264.7 , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismoRESUMO
With continuing damage, both the indigenous cells of the cortex and medulla, and inflammatory cells are involved in the formation and development of renal fibrosis. Furthermore, interactions among the glomerular, tubular, and interstitial cells contribute to the process by excessive synthesis and decreased degradation of extracellular matrix. The morphology of kidney is different from pathological stages of diseases and changes with various causes. At the end stage of the disease, the kidneys are symmetrically contracted with diffuse granules. Most glomeruli show diffuse fibrosis and hyaline degeneration, and intervening tubules become atrophied. Renal interstitium shows obvious hyperplasia of fibrous tissues with marked infiltration of lymphocytes, mononuclear cells, and plasma cells. The renal arterioles are wall thickening frequently because of hyaline degeneration. Morphologic analysis based on Masson staining of the kidney tissues has been regarded as the golden standard to evaluate the visual fibrosis. However, the present studies have found that the evaluation system has poor repeatability. Several computer-aided image analysis techniques have been used to assess interstitial fibrosis. It is possible that the evaluation of renal fibrosis is carried out by the artificial intelligence renal biopsy pathological diagnosis system in the near future.
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Nefropatias/patologia , Rim/patologia , Biópsia , Fibrose , Humanos , Glomérulos Renais/patologiaRESUMO
Although glucocorticoids are the mainstays in the treatment of renal diseases for decades, the dose dependent side effects have largely restricted their clinical use. Microvesicles (MVs) are small lipid-based membrane-bound particles generated by virtually all cells. Here we show that RAW 264.7 macrophage cell-derived MVs can be used as vectors to deliver dexamethasone (named as MV-DEX) targeting the inflamed kidney efficiently. Methods: RAW macrophages were incubated with dexamethasone and then MV-DEX was isolated from the supernatants by centrifugation method. Nanoparticle tracking analysis, transmission electron microscopy, western blot and high-performance liquid chromatography were used to analyze the properties of MV-DEX. The LC-MS/MS was applied to investigate the protein compositions of MV-DEX. Based on the murine models of LPS- or Adriamycin (ADR)-induced nephropathy or in-vitro culture of glomerular endothelial cells, the inflammation-targeting characteristics and the therapeutic efficacy of MV-DEX was examined. Finally, we assessed the side effects of chronic glucocorticoid therapy in MV-DEX-treated mice. Results: Proteomic analysis revealed distinct integrin expression patterns on the MV-DEX surface, in which the integrin αLß2 (LFA-1) and α4ß1 (VAL-4) enabled them to adhere to the inflamed kidney. Compared to free DEX treatment, equimolar doses of MV-DEX significantly attenuated renal injury with an enhanced therapeutic efficacy against renal inflammation and fibrosis in murine models of LPS- or ADR-induced nephropathy. In vitro, MV-DEX with about one-fifth of the doses of free DEX achieved significant anti-inflammatory efficacy by inhibiting NF-κB activity. Mechanistically, MV-DEX could package and deliver glucocorticoid receptors to renal cells, thereby, increasing cellular levels of the receptor and improving cell sensitivity to glucocorticoids. Notably, delivering DEX in MVs significantly reduced the side effects of chronic glucocorticoid therapy (e.g., hyperglycemia, suppression of HPA axis). Conclusion: In summary, macrophage-derived MVs efficiently deliver DEX into the inflamed kidney and exhibit a superior capacity to suppress renal inflammation and fibrosis without apparent glucocorticoid adverse effects. Our findings demonstrate the effectiveness and security of a novel drug delivery strategy with promising clinical applications.
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Vesículas Citoplasmáticas/química , Dexametasona/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nefropatias/tratamento farmacológico , Animais , Sistemas de Liberação de Medicamentos/instrumentação , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibrose/tratamento farmacológico , Fibrose/genética , Fibrose/imunologia , Integrinas/genética , Integrinas/imunologia , Rim/efeitos dos fármacos , Rim/imunologia , Nefropatias/imunologia , Macrófagos/química , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7RESUMO
316: F1006-F1015, 2019. First published March 6, 2019; doi: 10.1152/ajprenal.00413.2018 .-Experimental studies have shown that pharmacological activation of calcium-sensing receptor (CaSR) attenuates renal fibrosis in some animal models beyond modification of bone and mineral homeostasis; however, its underlying mechanisms remain largely unknown. Since excessive collagen deposition is the key feature of fibrosis, the present study aimed to examine whether CaSR was involved in the regulation of collagen expression in rats with adenine diet-induced renal fibrosis and in profibrotic transforming growth factor (TGF)-ß1-treated renal proximal tubular epithelial cells (PTECs). The results showed that the CaSR agonist cinacalcet significantly attenuated renal collagen accumulation and tubular injury in adenine diet-fed rats. Additionally, the in vitro experiment showed that profibrotic TGF-ß1 significantly increased the expression of collagen and decreased CaSR expression at the mRNA and protein levels in a concentration- and time-dependent manner. Furthermore, the CaSR CRISPR activation plasmid and cinacalcet partially abrogated the upregulation of collagen induced by TGF-ß1 treatment. Blockade of CaSR by the CRISPR/Cas9 KO plasmid or the pharmacological antagonist Calhex231 further enhanced TGF-ß1-induced collagen expression. Mechanistic experiments found that Smad2 phosphorylation and Snail expression were markedly increased in PTECs treated with TGF-ß1, whereas the CaSR CRISPR activation plasmid and cinacalcet substantially suppressed this induction. In summary, this study provides evidence for a direct renal tubular epithelial protective effect of CaSR activation in renal fibrosis, possibly through suppression of collagen expression in PTECs.
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Calcimiméticos/farmacologia , Cinacalcete/farmacologia , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Nefropatias/prevenção & controle , Túbulos Renais Proximais/efeitos dos fármacos , Receptores de Detecção de Cálcio/agonistas , Adenina , Animais , Benzamidas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Cicloexilaminas/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Humanos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Fosforilação , Ratos Wistar , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Proteína Smad2/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
IgA nephropathy (IgAN) features variable renal pathology and a heterogeneous clinical course. Our aim was to search noninvasive biomarkers from urinary exosomes for IgAN patients; membrane nephropathy and minimal change disease were included as other glomerulopathy controls. Transmission electron microscopy and nanoparticle tracking analysis confirmed the size and morphology characteristic of urinary exosomes. Exosome markers (Alix and CD63) as well as renal cell markers [aquaporin 2 (AQP2) and nephrin] were detected, which indicate the renal origin of urinary exosomes. Exosome excretion was increased markedly in IgAN patients compared with controls and correlated with levels of proteinuria and tubular injury. More important, urinary exosome excretion correlated with greater histologic activity (mesangial hypercellularity, crescents, and endocapillary hypercellularity). Profiling of the inflammation-related mRNA revealed that exosomal chemokine (C-C motif) ligand 2 (CCL2) was up-regulated in IgAN patients. In a validation study, CCL2 was exclusively highly expressed in IgAN patients compared with healthy controls as well as minimal change disease and membrane nephropathy patients. Also, a correlation between exosomal CCL2 and estimated glomerular filtration rate levels was found in IgAN. Exosomal CCL2 was correlated with tubulointerstitial inflammation and C3 deposition. High CCL2 levels at the time of renal biopsy were associated with subsequent deterioration in renal function. Thus, urinary exosomes and exosomal CCL2 mRNA are promising biomarkers reflecting active renal histologic injury and renal function deterioration in IgAN.
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Biomarcadores/urina , Quimiocina CCL2/urina , Exossomos/metabolismo , Glomerulonefrite por IGA/complicações , Inflamação/diagnóstico , Nefrite Intersticial/diagnóstico , RNA Mensageiro/metabolismo , Adulto , Estudos de Casos e Controles , Quimiocina CCL2/genética , Exossomos/genética , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite por IGA/patologia , Humanos , Inflamação/etiologia , Inflamação/urina , Masculino , Nefrite Intersticial/etiologia , Nefrite Intersticial/urina , RNA Mensageiro/genéticaRESUMO
The study of parathyroid hyperplasia with bone disease as a critical manifestation of chronic kidney disease-mineral and bone disorders (CKD-MBDs) is challenging due to the lack of a suitable research model. Here, we established a rat model with secondary hyperparathyroidism (SHPT) and bone disease induced by adenine and a high phosphorous diet and analyzed the skeletal characteristics. We performed blood analysis, emission computed tomography (ECT), dual energy X-ray absorptiometry (DEXA), micro-computed tomography (micro-CT), bone histomorphometry, and bone mechanical tests. The CKD rats with SHPT induced by adenine and a high phosphorus diet showed severe abnormalities in calcium and phosphorus metabolism and exhibited parathyroid hyperplasia. The bone mineral density (BMD) of femurs and lumbar vertebrae was significantly lower in the CKD rats than in the control (CTL) rats. The cortical and trabecular bone parameters of femurs showed significant bone loss. In addition, we found decreases in ultimate force, work to failure, stiffness, and elastic modulus in the CKD rats. In conclusion, our findings demonstrated that the CKD rats with SHPT induced by adenine and a high phosphorus diet may serve as a useful model for skeletal analysis in CKD with SHPT.
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Doenças Ósseas Metabólicas/patologia , Doenças Ósseas/patologia , Osso e Ossos/patologia , Dieta/efeitos adversos , Hiperparatireoidismo Secundário/patologia , Falência Renal Crônica/patologia , Adenina/efeitos adversos , Animais , Densidade Óssea , Doenças Ósseas/complicações , Doenças Ósseas/etiologia , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/etiologia , Modelos Animais de Doenças , Hiperparatireoidismo Secundário/complicações , Hiperparatireoidismo Secundário/etiologia , Rim/patologia , Falência Renal Crônica/complicações , Falência Renal Crônica/etiologia , Masculino , Fósforo/efeitos adversos , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Albuminuria is a key instigator of tubulointerstitial inflammation associated with CKD, but the mechanism through which filtered albumin propagates renal injury remains unclear. In this study, we explored the role in this process of exosome mRNA released from tubular epithelial cells (TECs). Compared with control mice, acute and chronic kidney injury models had more exosomes containing inflammatory cytokine mRNA, particularly the chemokine CCL2, in kidneys and urine. In vitro stimulation of TECs with BSA recapitulated this finding. Notably, the internalization of purified TEC exosomes by cultured macrophages increased if TECs were exposed to BSA. Macrophage internalization of exosomes from BSA-treated TECs led to an enhanced inflammatory response and macrophage migration, but CCL2 silencing in TECs prevented these effects. Using a GFP-CCL2 fusion mRNA construct, we observed direct transfer of CCL2 mRNA from TEC exosomes to macrophages. Mice subjected to tail vein injection of purified BSA-treated TEC exosomes developed tubular injury with renal inflammatory cell infiltration. However, injection of exosomes from BSA-treated CCL2-deficient TECs induced less severe kidney inflammation. Finally, in patients with IgA nephropathy, the increase of proteinuria correlated with augmented urinary excretion of exosomes with exaggerated expression of CCL2 mRNA. Moreover, the level of CCL2 mRNA in urinary exosomes correlated closely with levels of renal interstitial macrophage infiltration in these patients. Our studies demonstrate that the increasing release of exosomes that transfer CCL2 mRNA from TECs to macrophages constitutes a critical mechanism of albumin-induced tubulointerstitial inflammation.
Assuntos
Injúria Renal Aguda/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Células Epiteliais/metabolismo , Exossomos/metabolismo , Glomerulonefrite por IGA/urina , Túbulos Renais/metabolismo , Macrófagos/metabolismo , RNA Mensageiro/metabolismo , Insuficiência Renal Crônica/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/urina , Adulto , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Exossomos/genética , Feminino , Inativação Gênica , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/patologia , Humanos , Túbulos Renais/citologia , Túbulos Renais/patologia , Macrófagos/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Nefrite/metabolismo , Nefrite/patologia , Proteinúria/etiologia , Proteinúria/patologia , Proteinúria/urina , Ratos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/urina , Soroalbumina Bovina/farmacologia , Adulto JovemRESUMO
Renal fibrosis is a common pathological pathway of progressive chronic kidney disease (CKD). However, kidney function parameters are suboptimal for detecting early fibrosis, and therefore, novel biomarkers are urgently needed. We designed a 2-stage study and constructed a targeted microarray to detect urinary mRNAs of CKD patients with renal biopsy and healthy participants. We analysed the microarray data by an iterative random forest method to select candidate biomarkers and produce a more accurate classifier of renal fibrosis. Seventy-six and 49 participants were enrolled into stage I and stage II studies, respectively. By the iterative random forest method, we identified a four-mRNA signature in urinary sediment, including TGFß1, MMP9, TIMP2, and vimentin, as important features of tubulointerstitial fibrosis (TIF). All four mRNAs significantly correlated with TIF scores and discriminated TIF with high sensitivity, which was further validated in the stage-II study. The combined classifiers showed excellent sensitivity and outperformed serum creatinine and estimated glomerular filtration rate measurements in diagnosing TIF. Another four mRNAs significantly correlated with glomerulosclerosis. These findings showed that urinary mRNAs can serve as sensitive biomarkers of renal fibrosis, and the random forest classifier containing urinary mRNAs showed favourable performance in diagnosing early renal fibrosis.
Assuntos
Nefropatias/urina , RNA Mensageiro/urina , Adulto , Biomarcadores/urina , Estudos de Casos e Controles , Interpretação Estatística de Dados , Feminino , Fibrose , Humanos , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/classificaçãoRESUMO
BACKGROUND: Adverse outcome of chronic kidney disease, such as end stage renal disease, is a significant burden on personal health and healthcare costs. Urinary tubular injury markers, such as NGAL, KIM-1 and NAG, could provide useful prognostic value for the early identification of high-risk patients. However, discrepancies between recent large prospective studies have resulted in controversy regarding the potential clinical value of these markers. Therefore, we conducted the first meta-analysis to provide a more persuasive argument to this debate. METHODS: In the current meta-analysis, based on ten prospective studies involving 29366 participants, we evaluated the role of urinary tubular injury markers (NGAL, KIM-1 and NAG) in predicting clinical outcomes including CKD stage 3, end stage renal disease and mortality. The prognostic values of these biomarkers were estimated using relative risks and 95% confidence interval in adjusted models. All risk estimates were normalized to those of 1 standard deviation increase in log-scale concentrations to minimize heterogeneity. Fixed-effects models were adopted to combine risk estimates. The quality of the research and between-study heterogeneity were evaluated. The level of research evidence was identified according to the GRADE profiler. RESULTS: uNGAL was identified as an independent risk predictor of ESRD (pooled adjusted relative risk: 1.40[1.21 to 1.61], p<0.001) and of overall mortality (pooled adjusted relative risk: 1.10[1.03 to 1.18], p = 0.001) in patients with chronic kidney disease. A borderline significance of uKIM-1 in predicting CKD stage 3 independently in the community-based population was observed (pooled adjusted relative risk: 1.13[1.00 to 1.27], p = 0.057). Only the prognostic value of uNGAL for ESRD was supported by a grade B level of evidence. CONCLUSION: The concentration of uNGAL can be used in practice as an independent predictor of end stage renal disease among patients with chronic kidney disease, but it may be not useful in predicting disease progression to CKD stage 3 among community-based population.