RESUMEN
The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies.
Asunto(s)
Vesículas Extracelulares , Proteómica , Células Epiteliales , Vesículas Extracelulares/metabolismo , Riñón/metabolismo , Túbulos Renales Proximales/metabolismoRESUMEN
To find out whether the AGO-miRNA complex is more sensitive to the accessibility of a particular region inside the seed match, we analyze in detail the accessibility of a wide set of miRNA binding sites validated by PAR-CLIP and HITS-CLIP experiments. Our analysis reveals that nucleotides at the 3'-end of bound seed matches are significantly more accessible than nucleotides at the 5'-end as well as nucleotides at any positions in the unbound seed matches. We show that the accessibility of a single nucleotide at the 3'-end is more effective than the accessibility of several nucleotides at the 5'-end in discriminating between functional and nonfunctional binding sites. Analysis of mRNA and protein fold changes induced by miRNA overexpression demonstrates that genes with accessible nucleation regions at the 3'-end are down-regulated more strongly than genes whose accessible nucleation regions are located elsewhere within the seed match. We also observed an increase in the precision of the miRNA target prediction algorithm PACMIT when accessibility toward the 3'-end of the seed match was required. The pronounced sensitivity of the AGO-miRNA complex to the accessibility of the 3'-end of the seed match suggests that, in most cases, nucleation occurs in this region. We show that this conclusion is consistent with previous experimental studies.
Asunto(s)
Emparejamiento Base/fisiología , Inmunoprecipitación de Cromatina/métodos , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión/genética , Biología Computacional/métodos , Reactivos de Enlaces Cruzados/farmacología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , MicroARNs/química , MicroARNs/genética , ARN Mensajero/química , ARN Mensajero/genética , Análisis de Secuencia de ADN/métodos , Termodinámica , Estudios de Validación como AsuntoAsunto(s)
Neoplasias , Biología de Sistemas , Humanos , Polifarmacología , Proteómica , Pirimidinas , SulfonasRESUMEN
Signaling networks are nonlinear and complex, involving a large ensemble of dynamic interaction states that fluctuate in space and time. However, therapeutic strategies, such as combination chemotherapy, rarely consider the timing of drug perturbations. If we are to advance drug discovery for complex diseases, it will be essential to develop methods capable of identifying dynamic cellular responses to clinically relevant perturbations. Here, we present a Bayesian dose-response framework and the screening of an oncological drug matrix, comprising 10,000 drug combinations in melanoma and pancreatic cancer cell lines, from which we predict sequentially effective drug combinations. Approximately 23% of the tested combinations showed high-confidence sequential effects (either synergistic or antagonistic), demonstrating that cellular perturbations of many drug combinations have temporal aspects, which are currently both underutilized and poorly understood.