Development of an in vitro rat proximal tubule cell model as a platform for drug transporter and drug-drug interaction studies

PhD ThesisThe kidney plays a vital role in the elimination of many endogenous metabolites and xenobiotics. Drug transporters expressed in the proximal tubule cells are key factors in the ability of the organ to successfully carry out its function. Previously, primary human proximal tubule cells have been shown to retain a remarkable degree of differentiation in culture and provide a realistic model of the proximal tubule. To address the challenge of extrapolation of drug transporter data from animal and human, this project was set out to develop a parallel rat proximal tubule cell model. This would allow direct comparison of the handling of candidate drugs in both species, and provide better understanding of the mechanisms of drug transport. A technique to isolate primary rat proximal tubule cells (PTCs) was successfully developed using a collagenase digest/Percoll gradient approach. Rat PTCs cultured for 6 days were shown to exhibit cobberstone morphology, typical of many epithelial cells. A range of transport proteins including Mdr1a/b, Bcrp, Mrp2, Oat1, Oct2, Oatp4c1, Slc2a9, Urat1, Mate1, and Mct1 were detected at the mRNA level in these cells. Functional expression of Mdr1a/b, Bcrp, Mrp2, Oct2 and Mct1 was also detected using fluorescence substrate retention assays. In addition, Mdr1a/b, Bcrp and Mrp2 transporters were found localised on the apical membrane of polarised rat PTC monolayer, and Oct2 was found on the basolateral membrane. The handling of urate by rat PTC monolayers was investigated. The monolayers showed absorptive and secretory pathways for urate, although the absorptive pathway was 3.2-fold higher in magnitude. Similarly, 3.4-times more urate was predominant across the apical than across the basolateral membrane. Oat1 and Bcrp were deduced as the transporters responsible for the secretory pathway, and Urat1 and Slc2a9 in the absorptive pathway. This was in accordance with the human PTC monolayers, and both models were representative of urate transport in vivo. Digoxin transport exhibited a net absorptive flux in rat PTC monolayers; absorptive flux was 1.7-fold higher in magnitude than the secretory flux. In contrast, in human PTC monolayers, digoxin secretory flux was 4.2-fold higher than the absorptive flux. In human PTC monolayers, digoxin secretion consisted of OATP4C1-mediated digoxin uptake by the basolateral membrane and MDR1-mediated efflux across the apical membrane. In rat PTC monolayers in addition to these pathways, a significant Oatp-mediated absorptive flux of digoxin located on the apical membrane of rat PTC monolayer was identified as the difference between rat and human digoxin handling, resulting in a dominant absorptive flux of digoxin in rat compared to net secretion in human PTC monolayers. These data alone highlight the importance of developing realistic in vitro human and rat PTC models to understand species difference in renal drug handling

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs

Incorporating microglia‐like cells in human induced pluripotent stem cell‐derived retinal organoids

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina

SEAS: A System for SEED-Based Pathway Enrichment Analysis

Pathway enrichment analysis represents a key technique for analyzing high-throughput omic data, and it can help to link individual genes or proteins found to be differentially expressed under specific conditions to well-understood biological pathways. We present here a computational tool, SEAS, for pathway enrichment analysis over a given set of genes in a specified organism against the pathways (or subsystems) in the SEED database, a popular pathway database for bacteria. SEAS maps a given set of genes of a bacterium to pathway genes covered by SEED through gene ID and/or orthology mapping, and then calculates the statistical significance of the enrichment of each relevant SEED pathway by the mapped genes. Our evaluation of SEAS indicates that the program provides highly reliable pathway mapping results and identifies more organism-specific pathways than similar existing programs. SEAS is publicly released under the GPL license agreement and freely available at http://csbl.bmb.uga.edu/~xizeng/research/seas/