Disputation: Molecular Imaging of Diabetic Kidney Tissue and Binding Studies of Proinsulin C-peptide

Lina Lindfors försvarar sin avhandling.
Opponent: Prof. Fredrik Almqvist, Umeå universitet.

Abstract

Diabetic kidney disease is a serious complication of diabetes with a complex and incompletely understood pathology. In this work, the molecular changes in diabetic rat kidneys at a very early disease stage were studied using nanospray desorption electrospray ionisation mass spectrometry imaging. Our results demonstrate how disease-relevant metabolites and lipids can be conveniently analysed on intact kidney tissue sections. A number of significantly increased metabolites were identified in the diabetic kidney, revealing disturbances in energy metabolism detectable before histological changes.

Proinsulin C-peptide is produced in the pancreas along with insulin and has shown beneficial effects in diabetes, but its mode of action is not yet known. 125I radiolabelled C-peptide was used to study its tissue distribution in healthy and diabetic rats after intravenous injection. The majority of C-peptide accumulated in renal tissues, with lower levels in the diabetic animals, showing that there are significant changes in kidney – C-peptide interactions in early stage diabetes.

The interactions of C-peptide with the orphan receptor GPR146, which has been proposed as its receptor, were also investigated using Chinese hamster ovary cells overexpressing human GPR146. Neither dynamic mass redistribution nor β-arrestin recruitment assays showed any significant response to human or murine C-peptides in the GPR146 overexpressing cells compared to controls. Fluorescence confocal microscopy revealed no surface binding or cellular uptake of C-peptides by GPR146 overexpressing cells compared to controls. These combined results refute the suggestion that GPR146 is the C-peptide receptor.

To further probe the function of C-peptide, 15N-labelled residues were incorporated into the peptide in preparation for nanoscale secondary ion mass spectrometry imaging of cells and intact kidney tissue sections. A number of crosslinking C-peptides were also designed and synthesised for experiments aimed at identifying its binding target. These studies have not yet been completed. Finally, to investigate the structure-activity relationship of C-peptide, a library of modified pentapeptide analogues was created for medium-throughput testing in a cell assay.