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Glomerular charge selectivity has been attributed to anionic heparan sulfate proteoglycans (HSPGs) in the glomerular basement membrane (GBM). Agrin is the predominant GBM-HSPG, but evidence that it contributes to the charge barrier is lacking, because newborn agrin-deficient mice die from neuromuscular defects. To study agrin in adult kidney, a new conditional allele was used to generate podocyte-specific knockouts. Mutants were viable and displayed no renal histopathology up to 9 months of age. Perlecan, a HSPG normally confined to the mesangium in mature glomeruli, did not appear in the mutant GBM, which lacked heparan sulfate. Moreover, GBM agrin was found to be derived primarily from podocytes. Polyethyleneimine labeling of fetal kidneys revealed anionic sites along both laminae rarae of the GBM that became most prominent along the subepithelial aspect at maturity; labeling was greatly reduced along the subepithelial aspect in agrin-deficient and conditional knockout mice. Despite this severe charge disruption, the glomerular filtration barrier was not compromised, even when challenged with bovine serum albumin overload. We conclude that agrin is not required for establishment or maintenance of GBM architecture. Although agrin contributes significantly to the anionic charge to the GBM, both it and its charge are not needed for glomerular permselectivity. This calls into question whether charge selectivity is a

Background and objectives ESRD is accompanied by endothelial dysfunction. Because the endothelial glycocalyx (endothelial surface layer) governs interactions between flowing blood and the vessel wall, perturbation could influence disease progression. This study used a novel noninvasive sidestream-darkfield imaging method, which measures the accessibility of red blood cells to the endothelial surface layer in the microcirculation (perfused boundary region), to investigate whether renal function is associated with endothelial surface layer dimensions.Design, setting, participants, & measurements Perfused boundary region was measured in control participants (n=10), patients with ESRD (n=23), participants with normal kidney function after successful living donor kidney transplantation (n=12), and patients who developed interstitial fibrosis/tubular atrophy after kidney transplantation (n=10). In addition, the endothelial activation marker angiopoietin-2 and shed endothelial surface layer components syndecan-1 and soluble thrombomodulin were measured using ELISA.Results Compared with healthy controls (1.8260.16 mm), ESRD patients had a larger perfused boundary region (+0.23; 95% confidence interval, 0.46 to ,0.01; P,0.05), which signifies loss of endothelial surface layer dimensions. This large perfused boundary region was accompanied by higher circulating levels of syndecan-1 (+57.71; 95% confidence interval, 17.38 to 98.04; P,0.01) and soluble thrombomodulin (+12.88; 95% confidence interval, 0.29 to 25.46; P,0.001). After successful transplantation, the perfused boundary region was indistinguishable from healthy controls (without elevated levels of soluble thrombomodulin or syndecan-1). In contrast, however, patients who developed interstitial fibrosis and tubular atrophy showed a large perfused boundary region (+0.36; 95% confidence interval, 0.09 to 0.63; P,0.01) and higher levels of endothelial activation markers. In addition, a significant correlation between perfused boundary region, angiopoietin-2, and eGFR was observed (perfused boundary region versus GFR: Spearman's r=0.31; P,0.05; perfused boundary region versus angiopoietin-2: Spearman's r=20.33; P,0.05).Conclusion Reduced renal function is strongly associated with low endothelial surface layer dimensions. After successful kidney transplantation, the endothelial surface layer is indistinguishable from control.

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Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate side chains are covalently attached. These heparan sulfate side chains can be modified at different positions by several enzymes, which include N-deacetylases, N- and O-sulfotransferases, and an epimerase. These heparan sulfate modifications give rise to an enormous structural diversity, which corresponds to the variety of biologic functions mediated by heparan sulfate, including its role in inflammation. The HSPGs in the glomerular basement membrane (GBM), perlecan, agrin, and collagen XVIII, play an important role in the charge-selective permeability of the glomerular filter. In addition to these HSPGs, various cell types express HSPGs at their cell surface, which include syndecans, glypicans, CD44, and betaglycan. During inflammation, HSPGs, especially heparan sulfate, in the extracellular matrix (ECM) and at the surface of endothelial cells bind chemokines, which establishes a local concentration gradient recruiting leukocytes. Endothelial and leukocyte cell surface HSPGs also play a role in their direct adhesive interactions via other cell surface adhesion molecules, such as selectins and beta2 integrin. Activated leukocytes and endothelial cells exert heparanase activity, resulting in degradation of heparan sulfate moieties in the ECM, which facilitates leukocyte passage into tissues and the release of heparan sulfate-bound factors. In various renal inflammatory diseases the expression of agrin and GBM-associated heparan sulfate is decreased, while the expression of CD44 is increased. Heparan sulfate or heparin preparations affect inflammatory cell behavior and have promising therapeutic, anti-inflammatory properties by preventing leukocyte adhesion/influx and tissue damage.