RESUMO
Kidney disease is poorly understood because of the organ's cellular diversity. We used single-cell RNA sequencing not only in resolving differences in injured kidney tissue cellular composition but also in cell-type-specific gene expression in mouse models of kidney disease. This analysis highlighted major changes in cellular diversity in kidney disease, which markedly impacted whole-kidney transcriptomics outputs. Cell-type-specific differential expression analysis identified proximal tubule (PT) cells as the key vulnerable cell type. Through unbiased cell trajectory analyses, we show that PT cell differentiation is altered in kidney disease. Metabolism (fatty acid oxidation and oxidative phosphorylation) in PT cells showed the strongest and most reproducible association with PT cell differentiation and disease. Coupling of cell differentiation and the metabolism was established by nuclear receptors (estrogen-related receptor alpha [ESRRA] and peroxisomal proliferation-activated receptor alpha [PPARA]) that directly control metabolic and PT-cell-specific gene expression in mice and patient samples while protecting from kidney disease in the mouse model.
Assuntos
Nefropatias/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Estrogênio/deficiência , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Enzima de Conversão de Angiotensina 2/farmacologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Células Cultivadas , Chlorocebus aethiops , Sinergismo Farmacológico , Humanos , Modelos Moleculares , Proteínas Recombinantes/farmacologia , SARS-CoV-2/fisiologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.
Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/farmacologia , Pneumonia Viral/tratamento farmacológico , Proteínas Recombinantes/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/ultraestrutura , Vasos Sanguíneos/virologia , COVID-19 , Chlorocebus aethiops , Humanos , Rim/citologia , Rim/virologia , Camundongos , Organoides/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
Exquisite regulation of energy homeostasis protects from nutrient deprivation but causes metabolic dysfunction upon nutrient excess. In human and murine adipose tissue, the accumulation of ligands of the receptor for advanced glycation end products (RAGE) accompanies obesity, implicating this receptor in energy metabolism. Here, we demonstrate that mice bearing global- or adipocyte-specific deletion of Ager, the gene encoding RAGE, display superior metabolic recovery after fasting, a cold challenge, or high-fat feeding. The RAGE-dependent mechanisms were traced to suppression of protein kinase A (PKA)-mediated phosphorylation of its key targets, hormone-sensitive lipase and p38 mitogen-activated protein kinase, upon ß-adrenergic receptor stimulation-processes that dampen the expression and activity of uncoupling protein 1 (UCP1) and thermogenic programs. This work identifies the innate role of RAGE as a key node in the immunometabolic networks that control responses to nutrient supply and cold challenges, and it unveils opportunities to harness energy expenditure in environmental and metabolic stress.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Termogênese , Proteína Desacopladora 1/metabolismo , Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Metabolismo Energético , Jejum/metabolismo , Jejum/fisiologia , Humanos , Lipólise/genética , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Fosforilação , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Termogênese/genética , Transplante Homólogo , Proteína Desacopladora 1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.
Assuntos
Espaço Extracelular/metabolismo , Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Tecidos/métodos , Diferenciação Celular , Microambiente Celular , Feminino , Humanos , Cinética , Células-Tronco Pluripotentes/metabolismo , Gravidez , Terceiro Trimestre da Gravidez , TranscriptomaRESUMO
Understanding epigenetic mechanisms is crucial to our comprehension of gene regulation in development and disease. In the past decades, different studies have shown the role of epigenetic modifications and modifiers in renal disease, especially during its progression towards chronic and end-stage renal disease. Thus, the identification of genetic variation associated with chronic kidney disease has resulted in better clinical management of patients. Despite the importance of these findings, the translation of genotype-phenotype data into gene-based medicine in chronic kidney disease populations still lacks faithful cellular or animal models that recapitulate the key aspects of the human kidney. The latest advances in the field of stem cells have shown that it is possible to emulate kidney development and function with organoids derived from human pluripotent stem cells. These have successfully recapitulated not only kidney differentiation, but also the specific phenotypical traits related to kidney function. The combination of this methodology with CRISPR/Cas9 genome editing has already helped researchers to model different genetic kidney disorders. Nowadays, CRISPR/Cas9-based approaches also allow epigenetic modifications, and thus represent an unprecedented tool for the screening of genetic variants, epigenetic modifications or even changes in chromatin structure that are altered in renal disease. In this Review, we discuss these technical advances in kidney modeling, and offer an overview of the role of epigenetic regulation in kidney development and disease.
Assuntos
Epigênese Genética , Edição de Genes , Nefropatias/genética , Rim/embriologia , Rim/patologia , Modelos Genéticos , Organoides/metabolismo , Animais , Humanos , Nefropatias/patologiaRESUMO
Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator-activated receptor-γ (PPARG)-responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68(+) macrophages from atherosclerotic plaques of Ldlr(-/-) mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr(-/-) mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Colesterol/metabolismo , Angiopatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Macrófagos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/agonistas , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Transporte Biológico , Linhagem Celular , Células Cultivadas , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/imunologia , Angiopatias Diabéticas/patologia , Produtos Finais de Glicação Avançada/sangue , Humanos , Ligantes , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/genética , Lipoproteínas/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Placa Aterosclerótica/sangue , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Regiões Promotoras Genéticas , Receptor para Produtos Finais de Glicação Avançada/sangue , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
In mammals, changes in the metabolic state, including obesity, fasting, cold challenge, and high-fat diets (HFDs), activate complex immune responses. In many strains of rodents, HFDs induce a rapid systemic inflammatory response and lead to obesity. Little is known about the molecular signals required for HFD-induced phenotypes. We studied the function of the receptor for advanced glycation end products (RAGE) in the development of phenotypes associated with high-fat feeding in mice. RAGE is highly expressed on immune cells, including macrophages. We found that high-fat feeding induced expression of RAGE ligand HMGB1 and carboxymethyllysine-advanced glycation end product epitopes in liver and adipose tissue. Genetic deficiency of RAGE prevented the effects of HFD on energy expenditure, weight gain, adipose tissue inflammation, and insulin resistance. RAGE deficiency had no effect on genetic forms of obesity caused by impaired melanocortin signaling. Hematopoietic deficiency of RAGE or treatment with soluble RAGE partially protected against peripheral HFD-induced inflammation and weight gain. These findings demonstrate that high-fat feeding induces peripheral inflammation and weight gain in a RAGE-dependent manner, providing a foothold in the pathways that regulate diet-induced obesity and offering the potential for therapeutic intervention.
Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Inflamação/metabolismo , Resistência à Insulina , Fígado/metabolismo , Obesidade/metabolismo , Receptores Imunológicos/metabolismo , Animais , Técnica Clamp de Glucose , Inflamação/genética , Resistência à Insulina/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada , Aumento de Peso/genéticaRESUMO
BACKGROUND: The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR). RESULTS: Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR.Here we found that VEGF-A was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with MMP9 in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, VEGF-B, VEGF-C and VEGF-D, together with MMP15 was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, MMP9 correlated positively and VEGF-C, VEGF-D and MMP15 correlated negatively with HOMA-IR, in both SC and OM. CONCLUSION: We hereby propose that the alteration in MMP15, VEGF-B, VEGF-C and VEGF-D gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of VEGF-A in adipose tissue could have a relationship with the prevention of this pathology.
Assuntos
Tecido Adiposo/irrigação sanguínea , Resistência à Insulina/fisiologia , Metaloproteases/metabolismo , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Indutores da Angiogênese , Biomarcadores/metabolismo , Expressão Gênica , Humanos , Metaloproteases/genética , Neovascularização Fisiológica , Obesidade/fisiopatologia , Obesidade Mórbida/metabolismo , OmentoRESUMO
ChREBP is an essential transcription factor for lipogenesis. Its physiological role in adipose tissue has been studied only to a small extent and the control of its expression remains unknown in human adipocytes. We have studied ChREBP mRNA and protein expression levels in the liver and the omental (OM) and subcutaneous (SC) adipose tissues from obese and lean subjects, as well as in human differentiated preadipocytes. Liver and OM and SC adipose tissue biopsies were obtained from lean and obese patients. Human preadipocytes were isolated from the adipose tissues from obese patients and differentiated under adipogenic conditions. ChREBP expression levels were quantified by RT-PCR and Western blot analysis. We found opposing results in terms of ChREBP regulation in the liver and adipose samples. ChREBP increased in the liver from obese compared to lean subjects, whereas the expression decreased in both adipose tissues. The mRNAs of other adipogenic markers were checked in these tissues. The pattern of FASN was similar to the one for ChREBP, ADCY3 decreased in both adipose tissues from obese patients, AP2 decreased only in OM adipose tissue of obese patients and ATGL did not change. The levels of ChREBP mRNA and protein showed dramatic increases during the differentiation of human OM and SC preadipocytes. In conclusion, ChREBP expression has an opposite regulation in the liver and adipose tissue from obese subjects which is compatible with the increased hepatic lipogenesis and decreased adipocytic lipogenesis found in these patients. The dramatic increase of ChREBP mRNA and protein levels during preadipocyte differentiation suggests a role in adipogenesis.
Assuntos
Adipócitos/metabolismo , Adipogenia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fígado/metabolismo , Obesidade/genética , Obesidade/metabolismo , Omento/metabolismo , Gordura Subcutânea/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Adipócitos/citologia , Adulto , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Diferenciação Celular , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Expressão Gênica , Humanos , Lipase/genética , Lipase/metabolismo , Lipogênese , Masculino , Pessoa de Meia-Idade , Obesidade/patologia , Omento/citologia , Cultura Primária de Células , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gordura Subcutânea/citologiaRESUMO
Housekeeping genes frequently used in gene expression studies are highly regulated in human adipose tissue. To ensure a correct interpretation of results, it is critical to select appropriate reference genes. Subcutaneous (SC) and omental (OM) adipose tissue expression was analyzed from lean and obese subjects using whole genome complementary DNA (cDNA) microarrays to identify stably expressed genes and commercial TaqMan low density arrays (LDAs), with 16 common control genes. The best candidate gene from microarrays analysis was F-box and leucine-rich repeat protein-10 (FBXL10) (fold-change 10(-3) P < 0.01), an ubiquitous nucleolar protein evolutionarily conserved. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) and importin 8 (IPO8), were the best reference genes among the 16 genes in the LDAs with coefficient of variation (CV) of 4.51 and 4.55%, respectively. However, when the LDAs data were further analyzed by the geNorm and NormFinder softwares, IPO8, a nuclear protein mediating import of proteins, was the first and the third better reference gene, respectively. IPO8 and FBXL10 were further validated by real-time PCR in additional OM and SC fat samples and primary cultured preadipocytes. According to their CV, IPO8 resulted more suitable than FBXL10 in both adipose tissue depots and SC preadipocytes, whereas FBXL10 performed better than IPO8 in OM cultured preadipocytes. Both genes expression levels did not change throughout adipogenesis. Thus, we provide clear evidence that IPO8 and FBXL10 are good candidates to use as reference genes in gene expression studies in human OM and SC adipose tissues as well as differentiated primary preadipocytes.