Animal models play a central role in the study of chronic kidney disease (CKD). Researchers have used mice and rats to model kidney damage seen in human patients with diabetes, focal segmental glomerulosclerosis, lupus and fibrosis injury. Although many useful gene and protein targets have emerged from these studies, rodent models are limited for a number of reasons. One is the ethical issues around the numbers of mice in particular that are needed to generate gene knockouts, particularly tissue-specific knockouts (usually in the 100s-1000s). A second limitation is the fact that mice and rat models do not faithfully recapitulate the disease phenotype seen in the human kidney. For example, the most commonly used genetic strain of mice, C57Bl/6, are resistant to diabetes-induced kidney damage. Many gene and proteins that have been identified in rodent models have failed to materialise as bona fide targets when tested in humans. These limitations have led to a growing frustration in academia and industry, and led to the drive to improve our models of CKD.
We propose to establish a new model of kidney injury based on 3D organoids derived from human iPS cells. Our protocol of differentiation triggers the formation of both glomerular and tubular epithelial cells that form themselves into glomerular-like and tubule-like structures (Fig. 2). These organoids will form the basis of the three interrelated project aims, which will use cell biology, microscopy and single cell sequencing to interrogate the genetic profile of the kidney organoid cells (Aim 1). We will then establish a range of new models of CKD exposing organoids to high glucose and other important drivers of kidney disease to induce damage. Cutting-edge single cell sequencing using the 10 x Genomics platform (https://www.10xgenomics.com) will be applied in-house to interrogate the genetic profiles of each of the different kidney-like cells in 3D organoids in normal versus disease conditions. The sequencing data obtained from the various organoid damage models will then be compared to existing datasets from previously used rodent models of disease available on the GEO bioinformatics platform (e.g. https://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS3990). The critical role of Gremlin1 in organoid formation and response to fibrotic insult will be tested using CRISPR/Cas9 gene editing.
Successful completion of this project will achieve a number of scientific objectives. The first is the development of a next generation cell culture organoid model of kidney injury and CKD. The development of 3D organoid models is a common direction of travel in many fields, as the limitations of studying individual cells in isolation are becoming increasingly evident. A major outcome from this proposal will be reduction potential under the NC3Rs framework. Given the numbers of mice used for CKD experiments each year (estimated at approximately 68,000 in 2016, Fig. 1), and the moderate/severe grading of many of these experiments, we believe there is exciting potential to reduce these numbers in the future. Another significant deliverable will be the use of human iPS cells to generate the kidney organoids, which will represent a translationally relevant model of human kidney disease. The potential to modify the ES cells at the gene level using CRISPR/Cas9 gene editing will allow researchers to interrogate the role of their gene of interest not only in kidney development, but also in kidney cell responses to disease insults in a 3D organoid. We will use our networks such as the European Renal Cell Study Group (ERSCG) in the UK, Europe and the US to promote the potential of the organoid model as an alternative to rodent models of CKD. Dissemination of our results at national meetings such as the UK Kidney Week and the American Society of Nephrology will maximise the potential of widespread uptake and create exciting added value for the project.