Human Kidney Organoids as a System to Model Genetic Kidney Disease

Abstract

Alport syndrome (AS) is an inherited disorder caused by variants in COL4A3, COL4A4 and COL4A5. These genes encode the a3 4 5 network of type IV collagen, a major component of basement membranes in the kidney, inner ear and eyes. 85% of individuals with AS have variants in COL4A5, which follows an X-linked mode of inheritance and causes kidney failure from early adulthood requiring transplantation. There is currently no cure for AS or human in vitro model suitable for studying the kidney phenotype in the development of disease. Kidney organoids are three-dimensional millimetre-sized kidney structures derived from stem cells over a 25-day protocol. They self-organize following induction with CHIR99021 and FGF9 to recapitulate kidney development and form multiple kidney structures including glomeruli, tubules. They contain more than 10 cell types representing homology to first/second trimester human foetal kidney and they present a candidate system for investigating the kidney phenotype in AS. Using immunofluorescence, I confirmed that human kidney organoids secrete and assemble collagen IV networks, including the a3 4 5 network. I found that patient-derived Alport stem cells differentiate into kidney organoids forming mature basement membranes and expressing key matrix proteins identified with mass spectrometry-based proteomics. Using human embryonic stem cells, I established a COL4A5 knockout line with CRISPR-Cas9 and I demonstrated separation between the knockout cells and isogenic control cells using proteomics. To compare organoids with kidney development in vivo, I characterised foetal kidney tissue from a Col4a5 knockout mouse at E15.5 and E17.5 using proteomics and found minimal differences in cellular and matrix composition at these time points. Finally, I correlated proteomic data from human kidney organoids and mouse foetal kidney and demonstrated medium to strong correlation at E15.5. In conclusion, I demonstrated the potential for human kidney organoids to model early basement membrane assembly in health and disease and this system could be applied to investigate gene therapy in Alport syndrome.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Brian Derby (Supervisor), Susan Kimber (Supervisor) & Rachel Lennon (Supervisor)