Harries' lab: Team RNA

Most recently Jodie Evans, a Graduate Research Assistant, was uncovering new approaches to treat diabetic kidney disease. This research involved humanising three different kidney cell lines (HK-2, HEK293 and HRPTEpC) so they can be cultured without the use of any animal-derived biomaterial. These cell lines were then be treated with the diabetomimetic stressors. Changes in HNRNPD gene and isoform expression (by qPCR analysis) and cellular viability, morphology, damage, and phenotypic markers were closely observed. Later, manipulation of HNRNPD isoform expression was performed, and cells were monitored for signs of resolution of such diabetomimetic stress.

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The idea for exploring diabetic kidney disease followed previous research projects led by Dr Nicky Jeffery and later followed up by Dr Sam Gould. Over a decade ago, Dr Nicky Jeffery assessed the causes of cell identity changes in pancreatic beta cells. To begin, Nicky humanised the EndoC-βH1 beta cell culture method. This allowed us to create a model for diabetes which more closely reflected human physiology. Through this research, we demonstrated that contrary to prior belief, loss of beta cell mass in diabetes derives not only from cell death, but also from changes in the identity of beta cells to other islet cell types induced by diabetes-related stresses, such as hyperglycaemia and altered lipid composition.

 

Following this discovery, Nicky led her second project where we were able to isolate the specific gene responsible for beta cell identity changes, the RNA binding protein hnRNPD (also known as AUF1). We identified this gene by isolating the altered ‘delta’ cell population by fluorescence-assisted cell sorting (FACS), using somatostatin as a marker. Following successful enrichment of the delta cell population, we carried out a whole transcriptome search for genes that demonstrated altered expression specifically in transdifferentiated ‘delta’ cells.  Few coding genes demonstrated altered expression in somatostatin-positive cells, but of those that did, the most dysregulated gene was HNRNPD, which demonstrated a 2.7-fold reduction in expression compared to the somatostatin-negative cells in the culture.

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To begin our exploration into diabetic complications, Dr Sam Gould developed an animal free tissue-engineered retinal barrier model. This involved humanising both the Retinal Endothelial Cells (RECs) and Retinal Pigmented Epitheial Cells (RPEs). This model was developed to look at HNRNPD isoform expression in the retina, following diabetomimetic stimulation, with the view to gain a deeper understanding of the onset of diabetic retinopathy and ascertain whether HNRNPD isoform manipulation could act as a therapeutic option for this disease.