Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

Abstract

All capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.

Figure 1

Glomerular morphology and endothelial surface layer thickness 10 min after perfusion. Representative pictures of glomerular capillaries from a rat perfused with physiological salt (NS) (A), high osmolarity (HO) (B) and high salt (HS) solution (C). The thickness of the ESL was estimated by measuring the distance between infused intralipid droplets and the endothelial cells. ESL thickness was reduced in the rats perfused with HS (D). Scale bar represents 2 µm. ***P < 0.001, error bars represent SEM.

Figure 2

Glomerular filtration rate (GFR) and fractional clearance of albumin. Differences in GFR over time for three different groups of rats that were perfused for 3 min with physiological salt solution (NS), high osmolarity control (HO) and high salt (HS), respectively (A). 10 min after the perfusion the GFR had decreased to around half of the GFR at the start of the experiment in the rats perfused with HO, and even further in the rats perfused with HS (B). Fractional clearance for the three different groups over time. 40 min after perfusion with HS the rats became anuric (C). 10 min after the perfusion the fractional clearance of albumin was increase in the rats perfused with HS, but not in the rats perfused with HO or NS (D). **P < 0.01, ***P < 0.001, error bars represent SEM.

Figure 3

Concentration of hyaluronan in eluates. The concentration of hyaluronan in the eluates from the rats was measured after perfusion with physiological salt (NS), high osmolarity (HO) and high salt (HS) solution. Perfusing with HS or HO eluted similar amounts of hyaluronan, while NS eluted lower amounts. . ***P < 0.001, error bars represent SEM.

Figure 4

Expression of proteoglycans in the human glomerular endothelium. Immunofluorescence of proteoglycans and Ulex europaeus agglutinin I, a marker for endothelial cells (red), confirmed expression of (in green) lumican, decorin, glypican-4, agrin, collagen 15 alpha 1 chain, collagen 18 alpha 1 chain and CD44 in the glomerulus of human kidney sections. Co-localization is seen as yellow. Scale bars for pictures of full glomeruli represent 20 µm, zoomed in images 10 µm.

Figure 5

Gene expression of proteoglycans in human glomerular endothelial cells in vitro. Relative gene expression of the proteoglycans; LUM, AGRN, DCN, GPC4, COL15A1, COL18A1 and CD44 after treatment of human glomerular endothelial cells with high glucose (HG), palmitate (100 µM) bound to HSA (PA) or a combination of both (HG+PA) for 24 h. *P < 0.05, **P < 0.01, error bars represent SEM.

Figure 6

Protein expression of lumican in human glomerular endothelial cells in vitro. Western blot of lumican expression in cells after treatment of human glomerular endothelial cells with high glucose (HG), palmitate (100 µM) bound to HSA (PA) or a combination of both (HG+PA)for 24 h. The band corresponding to lumican was analyzed for protein abundance. The blot presented is a cropped version without ladder and the full blots are presented in Supplementary Figure 1. *P < 0.05, error bars represent SEM.