Davies, Jamie

Hadoke, Patrick

Tarnick, Julia

other

2022-02-11T10:20:22Z

2022-02-11T10:20:22Z

2021-12-07

The best way of treating end-stage renal disease is the transplantation of a donor organ. However, the number of patients requiring a transplant exceeds the number of available kidneys. Growing kidneys from induced pluripotent stem cells could help to close the gap between organ demand and supply and could omit the need to take immunosuppressants if the engineered kidneys were to be generated from the patient’s own cells. Kidney organoids from murine kidney cells are similar to embryonic kidneys but, with traditional culture methods, the organoids, as well as the kidneys themselves, grow rather flat and spread out, which does not represent the overall shape of a kidney grown in vivo. Embedding kidney explants in type 1 collagen resulted in a more spherical shape, but also in central necrosis, most likely caused by hypoxia. Staining of the explants for endothelial markers revealed the presence of capillaries, which remained immature. In some tissues, arterial differentiation is induced by neurons. To analyse whether the renal arterial maturation is controlled by neurons, developing kidneys were isolated and co-stained for vascular and neuronal markers. The co-staining revealed that innervation occurs after formation of the smooth muscle cell lining and during upregulation of calponin 1, which is involved in smooth muscle cell contraction. Several studies link vascular differentiation to mechanical stimuli caused by the onset of blood flow. To detect whether connecting the kidneys to the vasculature of a host would enhance vascular maturity, the explants were grafted onto the chick chorioallantoic membrane. Injection of the chick vessels following isolation and staining of the explant confirmed blood flow through the graft vessels. However, vascular maturity was not improved as indicated by the lack of vascular smooth muscle cells. The use of fertilized eggs makes it difficult to influence the growth environment of the explants. Isolating blood vessels and co-culturing them under perfusion with embryonic kidneys in vitro would allow more control over available growth factors and mechanical cues by adjusting the stiffness of the surrounding matrix as well the flow rate through the blood vessels. For this purpose, I have designed a culture device that allows the long-term perfusion of arteries and veins. Using this device, I have demonstrated that isolated blood vessels respond to proangiogenic treatment by forming sprouts. In co-cultures of the blood vessels with embryonic kidneys, endothelial sprouts were seen between both tissues and appeared to form a connection between the vessel and the explant, which sets the basis for the in vitro vascularisation of kidney explants. Vascularizing kidney explants can aid identifying methods to vascularize kidney organoidsin vitro. This is a critical step in renal tissue engineering as it would improve organoid growth and enable testing their functionality in terms of blood filtration. A functional vasculature will also be essential in generation transplantable renal tissue, which could on day help to treat kidney disease.

https://hdl.handle.net/1842/38555

http://dx.doi.org/10.7488/era/1819

en

The University of Edinburgh

Munro, D. A. D., Wineberg, Y., Tarnick, J., Vink, C. S., Li, Z., Pridans, C., Dzierzak, E., Kalisky, T., Hohenstein, P. and Davies, J. A. (2019). Macrophages restrict the nephrogenic field and promote endothelial connections during kidney development. Elife 8, https://doi.org/10.7554/eLife.43271

kidney disease

type-1 collagen

chorioallantoic membrane

artificially created kidneys

renal arterial maturation

Design and testing of ex vivo vascularization methods for kidney explants in order to overcome the diffusion limit of oxygen and nutrients in 3D-culture-systems

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy