A Novel Isolated Glomerular Albumin Permeability Assay Based on Quantitative Fluorescence Microscopy

Abstract

Vascular permeability is disrupted in several pathological conditions. We've taken advantage of the microscope densitometric technique to develop a new method to measure the solute permeability coefficient (Ps) of fluorescently labelled molecules ex vivo in glomeruli. Calibration experiments have been carried out to test the validity of this assay. Glomeruli were pre-perfused with Octadecyl-Rhodamine-B Chloride and Alexa Fluor 488 BSA (AF488-BSA) via whole-organ kidney perfusion in vivo. Following exsanguination and nephrectomy, glomeruli were sieved from the cortex of each kidney. Glomeruli were then secured in an imaging chamber and exposed to an albumin gradient, which drives efflux of fluorescent albumin from glomerular capillaries that can be quantified by time-lapse confocal imaging. Analysis and acquisition parameters were chosen using specific gain values. The fluorescence intensity values were calculated by selecting specific regions of interest inside different loops and in the perfusion bath. Using a similar protocol we also assessed the contribution of the z-stack position, the photobleaching effect, the free AF488 present in our solution, and differences between acellular and intact glomeruli on Ps values. There was no significant difference in photobleaching between AF488-BSA exposed to the laser for 90 min or 0min (n=4, Paired T Test = 0.20). Neither free dye present in our solution (Ps=0 n=4[1]), or z-stack position effected. Ps values were significantly higher in acellular glomeruli compared to non treated glomeruli. This technique is powerful to sensitively measure changes in albumin across a single glomerular capillary wall and has important implications, especially for isolated human glomeruli.