A novel assay provides sensitive measurement of physiologically relevant changes in albumin permeability in isolated human and rodent glomeruli

Sara Desideri,Karen L Onions,Yan Qiu,Raina D Ramnath,Matthew J Butler,Christopher R Neal,Matthew L.R King,Andrew E Salmon,Moin A Saleem,Gavin I Welsh,C Charles Michel,Simon C Satchell,Andrew H.J Salmon,Rebecca R Foster

doi:10.1016/j.kint.2017.12.003

Sara Desideri, Karen L Onions + Show 12 more

Open Access

https://doi.org/10.1016/j.kint.2017.12.003

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Abstract

Increased urinary albumin excretion is a key feature of glomerular disease but has limitations as a measure of glomerular permeability. Here we describe a novel assay to measure the apparent albumin permeability of single capillaries in glomeruli, isolated from perfused kidneys cleared of red blood cells. The rate of decline of the albumin concentration within the capillary lumen was quantified using confocal microscopy and used to calculate apparent permeability. The assay was extensively validated and provided robust, reproducible estimates of glomerular albumin permeability. These values were comparable with previous in vivo data, showing this assay could be applied to human as well as rodent glomeruli. To confirm this, we showed that targeted endothelial glycocalyx disruption resulted in increased glomerular albumin permeability in mice. Furthermore, incubation with plasma from patients with post-transplant recurrence of nephrotic syndrome increased albumin permeability in rat glomeruli compared to remission plasma. Finally, in glomeruli isolated from rats with early diabetes there was a significant increase in albumin permeability and loss of endothelial glycocalyx, both of which were ameliorated by angiopoietin-1. Thus, a glomerular permeability assay, producing physiologically relevant values with sufficient sensitivity to measure changes in glomerular permeability and independent of tubular function, was developed and validated. This assay significantly advances the ability to study biology and disease in rodent and human glomeruli.

Highlights

The ability to measure glomerular permeability is essential in the detection and assessment of glomerular disease
Kidneys were perfused first with mammalian 4% Ringer bovine serum albumin (BSA) to flush out red blood cells
They were perfused with octadecyl rhodamine B chloride (R18, a lipophilic membrane stain) in 4% Ringer BSA, to label cell membranes red in order to distinguish the glomerular capillary walls

Summary

Introduction

The ability to measure glomerular permeability is essential in the detection and assessment of glomerular disease. The development of a reliable, physiologically relevant assay to directly measure glomerular permeability using isolated glomeruli would be very useful and independent of confounding variables. In 1992, Savin et al modified an isolated glomerular oncometric assay, originally developed to assess hydraulic conductivity,[3] to measure glomerular reflection coefficients[4] and further modified the assay in 2015 for higher thoughput.[5] this assay is limited to measuring relative changes in permeability. Daniels et al developed an ex vivo method to quantify the apparent diffusion of fluorophores across individual glomerular capillaries in cross-section,[6] yet permeability values were significantly higher than values previously measured in vivo.[7] Using similar principles we developed an assay to measure apparent, diffusive glomerular albumin permeability in isolated glomeruli deriving physiologically relevant values. The pathophysiological variables tested were: (i) disruption of endothelial glycocalyx,[8,9,10] (ii) exposure to plasma from patients with recurrence of nephotic syndrome posttransplant,[11,12] (iii) early diabetes,[13,14] and (iv) rescue of glomerular permeability in diabetes using angiopoietin-1.15,16

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