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1.
Life Sci ; 270: 118997, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33453249

RESUMO

Advanced glycation end products (AGEs) play an important role in oxidative stress and inflammation, processes implicated in the development and progression of kidney dysfunction. In the present study, we investigated the participation of the pro-oxidant protein thioredoxin-interacting protein (TXNIP) and of epigenetic mechanisms on kidney tissue (in vivo, in non-diabetic rats) and on terminally differentiated glomerular podocytes (in vitro) chronically exposed to AGEs. AGEs induced total kidney and glomerular TXNIP expression and decreased H3K27me3 content. Concomitant treatment with the antioxidant N-acetyl-cysteine (NAC) reversed only the increased TXNIP expression. TXNIP expression positively correlated with proteinuria and negatively correlated with H3K27me3 content. In vitro studies in podocytes showed that 72 h exposure to AGEs decreased nephrin expression and increased Txnip, Nox4, Col4a1, and epithelial-to-mesenchymal transition (EMT) markers (Acta2, Snail1, and Tgfb1). Podocytes treatment with NAC reversed Nox4, Col4a1, Acta2, and Tgfb1 increased expression but did not abrogate the reduced expression of nephrin. MiR-29a expression was downregulated by AGEs in vivo, but not in vitro. In conclusion, treatment of non-diabetic rats with AGEs induced TXNIP expression and decreased the contents of the repressive epigenetic mark H3K27me3 and of miR-29a, potentially driving injury to glomerular filtration barrier and podocytes dysfunction.


Assuntos
Proteínas de Ciclo Celular/genética , Nefropatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Animais , Antioxidantes/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/genética , Epigênese Genética/genética , Células Epiteliais/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Histonas , Rim/citologia , Rim/metabolismo , Glomérulos Renais/metabolismo , Masculino , Proteínas de Membrana , Estresse Oxidativo , Podócitos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
2.
Life Sci ; 267: 118920, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33352171

RESUMO

This study investigates the role of ranolazine in contrast-associated acute kidney injury (CA-AKI) and potential mechanisms. For in vivo studies, mouse models of CA-AKI and control mice were treated with ranolazine or vehicle. Blood urea nitrogen (BUN) and serum creatinine were detected by spectrophotometry. Anti-T-cell immunoglobulin and mucin domain 1 (TIM 1) and anti-lipocalin 2 antibody (LCN2) were detected by immunofluorescence. Hemodynamic parameters were detected via invasive blood pressure measurement and renal artery color doppler ultrasound, capillary density was measured by CD31 immunofluorescence, vascular permeability assay was performed by Evans blue dye. The expressions of oxidative stress and apoptotic markers were measured and analyzed by immunofluorescence and western blotting. For in vitro studies, intracellular calcium concentration of HUVECs was measured with Fluo 3-AM under confocal microscopy. Results show that compared with control mice, serum BUN, creatinine, TIM 1 and LCN2 levels were elevated in CA-AKI mice, but this effect was alleviated by ranolazine-pretreatment. Safe doses of ranolazine (less than 64 mg/kg) had no significant effect on overall blood pressure, but substantially improved renal perfusion, reduced contrast-induced microcirculation disturbance, improved renal capillary density and attenuated renal vascular permeability in ranolazine-pretreated CA-AKI mice. Mechanistically, ranolazine markedly down-regulated oxidative stress and apoptosis markers compared to CA-AKI mice. Intracellularly, ranolazine attenuated calcium overload in HUVECs. These results indicate that ranolazine alleviates CA-AKI through modulation of calcium independent oxidative stress and apoptosis.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Meios de Contraste/efeitos adversos , Ranolazina/farmacologia , Lesão Renal Aguda/metabolismo , Animais , Apoptose/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Cálcio/metabolismo , Creatinina/análise , Creatinina/sangue , Modelos Animais de Doenças , Receptor Celular 1 do Vírus da Hepatite A/análise , Rim/citologia , Rim/metabolismo , Lipocalina-2/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ranolazina/metabolismo , Artéria Renal/metabolismo
3.
Methods Mol Biol ; 2258: 171-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340361

RESUMO

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.


Assuntos
Diferenciação Celular , Rim/fisiologia , Células-Tronco Pluripotentes/fisiologia , Engenharia Tecidual , Técnicas de Cultura de Células , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Rim/citologia , Microscopia de Fluorescência , Morfogênese , Organoides , Transdução de Sinais , Fatores de Tempo
4.
PLoS One ; 15(12): e0244796, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382808

RESUMO

Tiny membrane-enclosed cellular fragments that can mediate interactions between cells and organisms have recently become a subject of increasing attention. In this work the mechanism of formation of cell membrane nanovesicles (CNVs) was studied experimentally and theoretically. CNVs were isolated by centrifugation and washing of blood cells and observed by optical microscopy and scanning electron microscopy. The shape of the biological membrane in the budding process, as observed in phospholipid vesicles, in erythrocytes and in CNVs, was described by an unifying model. Taking the mean curvature h and the curvature deviator d of the membrane surface as the relevant parameters, the shape and the distribution of membrane constituents were determined theoretically by minimization of membrane free energy. Considering these results and previous results on vesiculation of red blood cells it was interpreted that the budding processes may lead to formation of different types of CNVs as regards the compartment (exo/endovesicles), shape (spherical/tubular/torocytic) and composition (enriched/depleted in particular kinds of molecules). It was concluded that the specificity of pinched off nanovesicles derives from the shape of the membrane constituents and not primarily from their chemical identity, which explains evidences on great heterogeneity of isolated extracellular vesicles with respect to composition.


Assuntos
Membrana Celular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Animais , Apoptose/fisiologia , Linhagem Celular , Cães , Membrana Eritrocítica/ultraestrutura , Rim/citologia , Rim/ultraestrutura , Microscopia Eletrônica de Varredura , Modelos Biológicos
5.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348732

RESUMO

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.


Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Nucleares/antagonistas & inibidores , Traumatismo por Reperfusão/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Rim/citologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Traumatismo por Reperfusão/imunologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
6.
Medicine (Baltimore) ; 99(50): e23277, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33327252

RESUMO

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates the interaction between malignant cells and the innate immune system. Recently, MIF has received attention for its role in tumorigenesis. We evaluated the prognostic role of MIF in clear cell renal cell carcinoma (CCRCC).A total of 152 patients, who underwent nephrectomy for CCRCC were enrolled in this study. Immunohistochemical staining of tissue microarray blocks containing 298 cores-2 cores per CCRCC patient was performed. The relationship between MIF expression and clinicopathological factors was evaluated. Total RNA and protein were extracted from 7 RCC (renal cell carcinoma) cell lines. MIF was knocked down in Caki-2 cells, and a wound healing assay was performed to evaluate migratory activity.Among the 298 cores, 180 (60.4%) were positive for MIF. Multivariate analysis, showed that, CCRCC patients with negative MIF expression exhibited poor disease-free survival (hazard ratio: 2.087, 95% confidence interval: 0.821-5.307, P value: .023) and poor disease-specific survival (hazard ratio: 2.101, 95% confidence interval: 1.009-4.374, P value: .047). The wound healing assay revealed that cell confluence was lower in MIF-deficient Caki-2 cells than in control cells.Negative MIF expression might be an independent prognostic marker for patients with CCRCC.


Assuntos
Carcinoma de Células Renais/diagnóstico , Neoplasias Renais/diagnóstico , Fatores Inibidores da Migração de Macrófagos/análise , Biomarcadores Tumorais/análise , Western Blotting , Carcinoma de Células Renais/química , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Rim/química , Rim/citologia , Rim/patologia , Neoplasias Renais/química , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sobrevida , Análise Serial de Tecidos
7.
Proc Natl Acad Sci U S A ; 117(52): 33404-33413, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376219

RESUMO

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.


Assuntos
Sondas de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização de Ácido Nucleico , RNA/metabolismo , Análise de Célula Única , Animais , Sistemas CRISPR-Cas/genética , Expressão Gênica , Humanos , Íntrons/genética , Células K562 , Rim/citologia , Camundongos , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , Fatores de Tempo
8.
PLoS One ; 15(9): e0238816, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32898157

RESUMO

Interleukin-10 (IL-10), a cytokine with anti-inflammatory effects, is produced by renal parenchymal cells and bone marrow derived cells. Both endogenous and exogenous IL-10 are protective in cisplatin-induced acute kidney injury. However, the source of endogenous IL-10 in cisplatin-induced nephrotoxicity is not clear. Bone marrow chimera experiments in IL10-KO mice indicated that bone marrow derived cells were the primary source of IL-10 in cisplatin nephrotoxicity. Cell specific deletion of IL-10 in T regulatory cells and dendritic cells was accomplished using Foxp3 and CD11c driven cre recombination in IL10flox/flox mice, respectively. Upon treatment with cisplatin, both the IL10flox/flox and the Foxp3YFP-Cre x IL10flox/flox mice developed similar degrees of kidney injury. However, mice with the dendritic cell deletion of IL-10 showed more severe structural and functional changes in the kidney compared to the IL10flox/flox mice. These results indicate that IL-10 from dendritic cells but not from T regulatory cells offers significant endogenous protection against cisplatin induced nephrotoxicity.


Assuntos
Cisplatino/efeitos adversos , Células Dendríticas/metabolismo , Interleucina-10/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/imunologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
9.
Phys Rev Lett ; 125(3): 038003, 2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32745423

RESUMO

Experiments and theory have shown that cell monolayers and epithelial tissues exhibit solid-liquid and glass-liquid transitions. These transitions are biologically relevant to our understanding of embryonic development, wound healing, and cancer. Current models of confluent epithelia have focused on the role of cell shape, with less attention paid to cell extrusion, which is key for maintaining homeostasis in biological tissue. Here, we use a multiphase field model to study the solid-liquid transition in a confluent monolayer of deformable cells. Cell overlap is allowed and provides a way for modeling the precursor for extrusion. When cells overlap rather than deform, we find that the melting transition changes from continuous to first order like, and that there is an intermittent regime close to the transition, where solid and liquid states alternate over time. By studying the dynamics of five- and sevenfold disclinations in the hexagonal lattice formed by the cell centers, we observe that these correlate with spatial fluctuations in the cellular overlap, and that cell extrusion tends to initiate near fivefold disclinations.


Assuntos
Células Epiteliais/química , Células Epiteliais/citologia , Rim/química , Rim/citologia , Modelos Biológicos , Animais , Fenômenos Biofísicos , Movimento Celular/fisiologia , Forma Celular/fisiologia , Cães , Transição Epitelial-Mesenquimal , Células Madin Darby de Rim Canino , Transição de Fase
10.
PLoS One ; 15(8): e0236839, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780746

RESUMO

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Adaptadora GRB2/antagonistas & inibidores , Quinoxalinas/farmacologia , Animais , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Humanos , Células K562 , Rim/citologia , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligação Proteica , Quinoxalinas/química , Quinoxalinas/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Ressonância de Plasmônio de Superfície , Peixe-Zebra , Domínios de Homologia de src
11.
Int J Nanomedicine ; 15: 5043-5060, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764935

RESUMO

Background: Hydroxyapatite (HAP) is a common component of most idiopathic calcium oxalate (CaOx) stones and is often used as a nidus to induce the formation of CaOx kidney stones. Methods: This work comparatively studies the cytotoxicity of four kinds of HAP crystals with different sizes (40 nm to 2 µm), namely, HAP-40 nm, HAP-70 nm, HAP-1 µm, and HAP-2 µm, on human renal proximal tubular epithelial cells (HK-2). Results: HAP crystals reduce the viability and membrane integrity of HK-2 cells in a concentration-dependent manner and consequently cause cytoskeleton damage, cell swelling, increased intracellular reactive oxygen species level, decreased mitochondrial membrane potential, increased intracellular calcium concentration, blocked cell cycle and stagnation in G0/G1 phase, and increased cell necrosis rate. HAP toxicity to HK-2 cells increases with a decrease in crystal size. Conclusion: Cell damage caused by HAP crystals increases the risk of kidney stone formation.


Assuntos
Citotoxinas/química , Citotoxinas/toxicidade , Durapatita/química , Durapatita/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Oxalato de Cálcio/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
12.
J Med Life ; 13(2): 241-248, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32742521

RESUMO

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Vírus da Febre Aftosa/imunologia , Rim/citologia , Vacinas Virais/imunologia , Aminoácidos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Cricetinae , Vírus da Febre Aftosa/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Proteínas/farmacologia , Vitaminas/farmacologia
13.
Vet Microbiol ; 247: 108785, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32768229

RESUMO

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Interferons/antagonistas & inibidores , Animais , Linhagem Celular , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/imunologia , Interferons/imunologia , Intestinos/citologia , Intestinos/virologia , Rim/citologia , Rim/virologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia
14.
J Vis Exp ; (161)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32716365

RESUMO

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney's filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm's Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Podócitos/metabolismo , Diferenciação Celular , Humanos , Rim/metabolismo
15.
J Vis Exp ; (160)2020 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-32658202

RESUMO

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.


Assuntos
Linfócitos T CD8-Positivos/citologia , Citometria de Fluxo , Rim/citologia , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Memória Imunológica/imunologia , Rim/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL
16.
Proc Natl Acad Sci U S A ; 117(29): 16969-16975, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611816

RESUMO

Understanding to what extent stem cell potential is a cell-intrinsic property or an emergent behavior coming from global tissue dynamics and geometry is a key outstanding question of systems and stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially localized niche, giving rise to a stochastic conveyor-belt model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favorably repositioned. Importantly, even when assuming that all cells in a tissue are molecularly equivalent, we predict a common ("universal") functional dependence of the long-term clonal survival probability on distance from the niche, as well as the emergence of a well-defined number of functional stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets of pubertal mammary gland tips and embryonic kidney tips, as well as homeostatic intestinal crypts. Importantly, we find good agreement for the predicted functional dependency of the competition as a function of position, and thus functional stem cell number in each organ. This argues for a key role of positional fluctuations in dictating stem cell number and dynamics, and we discuss the applicability of this theory to other settings.


Assuntos
Linhagem da Célula , Autorrenovação Celular , Nicho de Células-Tronco , Animais , Sobrevivência Celular , Feminino , Homeostase , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Rim/citologia , Rim/crescimento & desenvolvimento , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Modelos Teóricos , Razão Sinal-Ruído , Células-Tronco/citologia , Células-Tronco/fisiologia
17.
Am J Physiol Renal Physiol ; 319(3): F359-F365, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32686523

RESUMO

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that induces nitric oxide (NO) production. IL-10 supplementation has been previously shown to lower blood pressure (BP) in male hypertensive mice, but the effect of exogenous IL-10 in hypertensive female rodents has not been studied. For the present study, we hypothesized that chronic infusion of IL-10 in hypertensive rats would lower BP concomitant with an increase in renal NO synthase (NOS) activity. Male and female spontaneously hypertensive rats (SHRs; 12 wk old) were randomized to receive IL-10 infusion by subcutaneous minipump (3.5 µg·kg-1·day-1) or serve as sham controls (n = 4-6 rats per treatment per sex). BP was measured by tail cuff before and after 2 wk of treatment. Renal T cells and IL-10 were measured by flow cytometry, and NOS activity was determined by conversion of radiolabeled arginine to radiolabeled citrulline. Female SHRs had greater IL-10+ renal cells than male SHRs and greater expression of the IL-10 receptor at baseline. BP did not change in female SHRs treated with IL-10, but BP significantly decreased following IL-10 infusion in male SHRs. Contrary to our hypothesis, NOS enzymatic activity decreased with IL-10 treatment in the renal inner medulla and cortex of both sexes. Renal regulatory T cells also decreased in both sexes after IL-10 treatment. In conclusion, despite male SHRs having less IL-10 and IL-10 receptor expression in the kidney compared with female SHRs, exogenous IL-10 selectively decreased BP only in male SHRs. Furthermore, our data suggest that exogenous IL-10-induced decreases in BP in male SHRs are not dependent on upregulating renal NOS activity.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Interleucina-10/farmacologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Bombas de Infusão , Infusões Subcutâneas , Interleucina-10/administração & dosagem , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/metabolismo , Fatores Sexuais , Linfócitos T/citologia
18.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510495

RESUMO

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV's easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3' arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.


Assuntos
Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/metabolismo , Rim/metabolismo , Infecções por Poxviridae/metabolismo , Poxviridae/genética , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Especificidade de Hospedeiro , Humanos , Rim/citologia , Rim/virologia , Infecções por Poxviridae/virologia , Coelhos , Vírus Vaccinia/genética
19.
Proc Natl Acad Sci U S A ; 117(27): 15827-15836, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571911

RESUMO

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.


Assuntos
Membrana Basal/metabolismo , Bromo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Peroxidase/metabolismo , Animais , Biópsia , Bromatos/metabolismo , Brometos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Iminas/metabolismo , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica
20.
Proc Natl Acad Sci U S A ; 117(26): 15160-15171, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541026

RESUMO

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1ß, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1ß and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.


Assuntos
Reprogramação Celular/fisiologia , Nefrite Lúpica/metabolismo , Animais , Células Cultivadas , Dinoprostona/genética , Dinoprostona/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Glicólise/fisiologia , Humanos , Imunoglobulina G/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Rim/citologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio , Receptores de IgG/genética , Receptores de IgG/metabolismo
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