An NC3Rs project grant, awarded in 2009, has enabled Professor Jamie Davies to explore the use of cell lines to replace the use of mouse embryos in studies of kidney development.
Principal Investigator: Jamie Davies, Professor of Experimental Anatomy
Organisation: University of Edinburgh
Award: £364,044, in 2008, over 36 months
Title: Replacing the use of animals with kidney cell lines
Read more about Professor Davies' research
Kidney disease affects around 40,000 people each year in the UK
Kidney disease, the progressive loss of kidney function, affects around 40,000 people in the UK each year. In adults the most common causes are diabetes, hypertension and glomerulonephritis, that together account for 75% of cases. The disease is classified into five stages of increasing severity. For patients reaching stage five, permanent renal replacement therapy is the only option.
In the UK, 1,667 kidney transplants were performed in the last year. There is a growing gap between the requirement for transplants (currently about 7,000 people) and the availability of donor organs; as a result there is increasing interest within the scientific, clinical and patient communities in the potential of stem cells and tissue engineering to regenerate damaged kidneys or even to provide new organs.
Embryonic mouse kidneys can be grown in vitro
For kidney regeneration to be a viable clinical option, it is necessary to understand how the kidneys develop. This is usually studied in genetically modified mice, either in whole animals or by dissecting kidney rudiments from very early stage mouse embryos and maintaining them in organ culture.
When the kidney rudiment is isolated from a mouse embryo, just 10.5 days into gestation, it consists of three cell types only; the mesenchyme, ureteric bud tip and the ureteric bud stalk. In organ culture, kidney rudiments continue to develop and form an organ that is in most respects anatomically normal, with the ureteric bud tips and stalks branching to form a urinary collecting duct system and the mesenchyme making many excretory nephrons that connect to it. These nephrons include the structures that normally filter blood (glomerular podocytes) and the long tubules that recover valuable molecules from the urine.
Experiments in the laboratory of Professor Jamie Davies, University of Edinburgh, have previously shown that a viable kidney can be produced simply by mixing metanephrogenic mesenchyme and ureteric bud stem cells from an embryo, with no need for them to be arranged in the form of a kidney rudiment. The ability to culture rudimentary kidneys and to reconstitute by mixing different cells types allows cells from different genotypes to be used, cell movement to be tracked and the effect of different added factors (for example, drugs) to be investigated. These studies still, however, depend on isolating embryonic kidney stem cells from freshly killed pregnant mice. It is estimated that around 15,000 female mice are used directly for this purpose in the UK each year, although the number is likely to be higher than this overall given the extensive breeding programmes required to maintain many genetically modified lines.
Conditionally-immortal kidney cell lines as an alternative to using mouse embryos
With NC3Rs funding, Professor Davies has developed a range of conditionally-immortal kidney cell lines from mouse kidney rudiments. These have been characterised for their potential as a high throughput screen of molecules and drugs that affect kidney development. The new cell lines have the appropriate cell morphology and express the correct lineage specific markers.
The ureteric bud-type cell lines have the ability to form branching tubules in three-dimensional culture, and the mesenchyme-type cell lines respond correctly to signals that alter gene expression in the early development of the natural kidney mesenchyme. Direct mixing of the different cells does not, however, result in their forming a kidney. Nevertheless, on its own each cell line behaves similarly enough to the corresponding stem cell-type in normal kidney rudiments that it has the potential to be used for high throughput screening for pathways and molecules that control specific aspects of development.
The effectiveness of this approach as a screen for pathways that control ureteric bud branching has been demonstrated in a proof of concept study. Using the ureteric bud-type cell lines in a simple two-dimensional scrape-healing assay, a set of drugs that affected collective cell movement, the two-dimensional analogue of branching, have been identified. A subset of the drugs was further tested in a three dimensional culture model, showing for the first time that the src-FAK pathway is important in the regulation of ureteric bud branching.
Identifying pathways and molecules that affect kidney development without using animals
This proof-of-concept study has demonstrated that the cell lines can be used as a high throughput screen, replacing the use of animals. An equivalent study using genetically modified mice would use around 600 animals; 150 for the experiments and the remaining for breeding purposes. Such studies may also be associated with suffering because of the impact of some kidney-specific knock-outs on animal welfare. Screening using cultured kidneys dissected from embryos would use approximately 140 mice.
Influencing the wider kidney research community
There have been seven publications, published or in press, arising from this grant to date. The research has also been presented at various international and national conferences including the 2010 World Biobanking and Cellular Therapy summit, the 15th UK Nephrogenesis meeting and the 2011 Harden Conference. The new cell lines are already being used by research groups in the UK and in Nice, and the techniques are being used by groups in Vienna and Brisbane, maximising the potential to further replace the use of animals.
This case study was published in a review of our research portfolio in September 2011.