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.

Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease

“…The assay has been extensively validated and provided robust, reproducible estimates of glomerular Ps'alb. 24 We observed an increase in glomerular Ps'alb, confirming reduced glomerular capillary wall integrity in early DKD. The increase in albuminuria and in glomerular permeability was associated with a reduction in endothelial and podocyte glycocalyx depth without affecting other glomerular ultrastructures such as GBM thickness, podocyte slit diaphragm, and foot process width.…”

“…These data are consistent with our previously published work that treatment with vascular endothelial growth factor A 165b and angiopoietin-1 reversed damage to the glomerular endothelial glycocalyx and normalized glomerular permeability in early DKD. 24,27 Moreover, vascular endothelial growth factor C also protected against raised glomerular permeability and ameliorated glycocalyx disruption in diabetic glomeruli. 41 Blockade of MMP2 and -9 attenuated diabetes-induced changes in glomerular endothelial glycocalyx Sdc4 and glomerular SDC4 at the mRNA and protein levels, suggesting a reduction in glomerular endothelial SDC4 shedding.…”

“…43 Our data confirm previous observations that loss of glomerular glycocalyx, in the absence of changes in other glomerular capillary wall components, is associated with albuminuria and glomerular permeability in early DKD. 24,27 The role of the podocyte glycocalyx has not previously been examined in detail so further work is necessary to determine the significance of our observation of podocyte glycocalyx loss in diabetes. Some studies support the = Figure 3 | (continued) week 9 post-STZ showed a significant reduction in glycocalyx SDC4 in diabetes (control, 182.5 AE 12.04, n ¼ 5 mice; diabetes, 64.31 AE 6.513, n ¼ 5 mice; ***P< 0.0001).…”

“…Glomerular Ps'alb assay The glomerular Ps'alb assay was carried out as previously described. 24 Briefly, Ringer-perfused kidney was sieved in 4% bovine serum albumin (BSA) in Ringer solution. Isolated glomeruli were incubated in 36.5 mg/ml Octadecyl rhodamine B chloride (R18, O246; Thermo Fisher Scientific) for 15 minutes, then washed in 4% Ringer BSA to remove unbound R18 followed by 15 minutes' incubation in 30 mg/ml Alexa Fluor 488-BSA (A13100; Thermo Fisher Scientific).…”

“…[6][7][8]18 Our group and others have shown that the glomerular endothelial glycocalyx contributes to the barrier to albumin permeability in cultured cells [18][19][20] and in vivo. [21][22][23][24] Loss of glycosaminoglycan within the glomerular capillary wall is seen in albuminuric diabetic rats 25 and diabetic mice. 26,27 In humans, systemic glycocalyx dimensions are reduced in diabetes.…”