Investigating Hepatocyte Nuclear Factor 1B Associated Renal Disease in a Human Kidney Organoid Model

Abstract

Hepatocyte nuclear factor 1B (HNF1B) is a transcription factor expressed in the developing kidney nephron tubule epithelium and implements a variety of critical functions throughout development. Heterozygous mutations in the HNF1B gene are the most prevalent monogenic cause of congenital kidney disease, with the associated kidney disease phenotype including dysplastic nephron development, renal cysts and tubulopathies resulting in electrolyte wasting. Several genetic mutations have been identified and display a phenotypic heterogeneity that does not appear to follow a genotype to phenotype correlation. Mouse models of Hnf1b disease often do not capture the intricacies of the associated human disease and current understanding of the human disease mechanisms is limited. In recent years, human pluripotent stem cell (hPSC) technologies have emerged as valuable tools for improving our understanding of human development and disease. To this end, the work in this thesis describes the development of a human embryonic stem cell (hESC)-derived kidney organoid model of heterozygous HNF1B kidney disease to deepen knowledge of the associated human disease pathobiology and identify potential disease mechanisms. Validation of a CRISPRCas9 gene edited hESC line was performed, confirming a heterozygous frameshift mutation within exon 1 of the HNF1B gene. Following an optimised protocol based on a published method by Takasato and colleagues in 2015, HNF1B mutant and isogenic hESCs were differentiated to kidney organoids. Organoids exhibited the presence of metanephric mesenchyme (MM) derivatives, including glomeruli, proximal and distal tubules, nephron progenitor, stromal and endothelial cells. HNF1B-mutant kidney organoids contained a subset of abnormal multi-layered epithelial structures. HNF1B-mutant tubules of a more typical morphology contained dilated lumens and thicker tubule epithelium with increased proliferation compared to non-mutant organoid tubules. Increased apoptosis and a loss of apicobasal polarity were also found in HNF1B-mutant organoids compared to non-mutant organoids. Bulk RNA sequencing revealed a significant reduction of several transcripts associated with Mendelian kidney tubulopathies in mutant organoids. In addition, mutant organoids demonstrated a reduced capacity to develop dilatations following cAMP stimulation, suggesting that HNF1B mutations result in a developmental delay. Single cell (sc) RNA sequencing was employed to see whether there were differences in cellular composition and detected a 25-fold decrease of mature proximal tubule cells in HNF1B-mutant compared to non-mutant organoids. A subset of cells identified exclusively in HNF1B mutant organoids overexpressed Glutamate receptor ionotropic kainate 3 (GRIK3); the role of which in kidney development and disease is currently unknown. GRIK3 transcripts were abundant in mutant multi-layered tubules and GRIK3 protein levels were significantly increased in mutant organoids. Preliminary antagonist experiments were performed, however future GRIK3 knockdown studies in organoid cultures are required to determine its potential role in HNF1B kidney disease. Together the results presented in this thesis demonstrate that hPSC-kidney organoids can model aspects of human HNF1B related disease and can be used as tools to enable the identification of potential therapeutic targets.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Susan Kimber (Supervisor), Philip Withers (Supervisor) & Adrian Woolf (Supervisor)