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

The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease.

Endothelial glycocalyx restoration by growth factors in diabetic nephropathy.

The endothelial glycocalyx (eGlx) constitutes the first barrier to protein in all blood vessels. This is particularly noteworthy in the renal glomerulus, an ultrafiltration barrier. Leakage of protein, such as albumin, across glomerular capillaries results in albumin in the urine (albuminuria). This is a hall mark of kidney disease and can reflect loss of blood vessel integrity in microvascular beds elsewhere. We discuss evidence demonstrating that targeted damage to the glomerular eGlx results in increased glomerular albumin permeability. EGlx is lost in diabetes and experimental models demonstrate loss from glomerular endothelial cells. Vascular endothelial growth factor (VEGF)A is upregulated in early diabetes, which is associated with albuminuria. Treatment with paracrine growth factors such as VEGFC, VEGF165b and angiopoietin-1 can modify VEGFA signalling, rescue albumin permeability and restore glomerular eGlx in models of diabetes. Manipulation of VEGF receptor 2 signalling, or a common eGlx biosynthesis pathway by these growth factors, may protect and restore the eGlx layer. This would help to direct future therapeutics in diabetic nephropathy.

VEGFC Reduces Glomerular Albumin Permeability and Protects Against Alterations in VEGF Receptor Expression in Diabetic Nephropathy.

Elevated levels of vascular endothelial growth factor (VEGF) A are thought to cause glomerular endothelial cell (GEnC) dysfunction and albuminuria in diabetic nephropathy. We hypothesized that VEGFC could counteract these effects of VEGFA to protect the glomerular filtration barrier and reduce albuminuria. Isolated glomeruli were stimulated ex vivo with VEGFC, which reduced VEGFA- and type 2 diabetes-induced glomerular albumin solute permeability (Ps'alb). VEGFC had no detrimental effect on glomerular function in vivo when overexpression was induced locally in podocytes (podVEGFC) in otherwise healthy mice. Further, these mice had reduced glomerular VEGFA mRNA expression, yet increased glomerular VEGF receptor heterodimerization, indicating differential signaling by VEGFC. In a model of type 1 diabetes, the induction of podVEGFC overexpression reduced the development of hypertrophy, albuminuria, loss of GEnC fenestrations and protected against altered VEGF receptor expression. In addition, VEGFC protected against raised Ps'alb by endothelial glycocalyx disruption in glomeruli. In summary, VEGFC reduced the development of diabetic nephropathy, prevented VEGF receptor alterations in the diabetic glomerulus, and promoted both glomerular protection and endothelial barrier function. These important findings highlight a novel pathway for future investigation in the treatment of diabetic nephropathy.

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

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.