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

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.

SUBMITTER: Desideri S

PROVIDER: S-EPMC5912930 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Desideri Sara S Onions Karen L KL Qiu Yan Y Ramnath Raina D RD Butler Matthew J MJ Neal Christopher R CR King Matthew L R MLR Salmon Andrew E AE Saleem Moin A MA Welsh Gavin I GI Michel C Charles CC Satchell Simon C SC Salmon Andrew H J AHJ Foster Rebecca R RR

Kidney international 20180209 5

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 valid ...[more]

PMID: 29433915

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Project description:BackgroundThe ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFN?-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpots cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFN? signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.MethodsWhole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.ResultsAntigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFN?-R signaling allows responses to be detected in as little as 25 uL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.ConclusionsA monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation are required before processing.

Dataset Information

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

Publications

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

Similar Datasets

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