High glucose causes dysfunction of the human glomerular endothelial glycocalyx

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.     

An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.     

Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

The structural stability of the endothelial glycocalyx after enzymatic removal of glycosaminoglycans

Rationale

It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.

Objective

To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.

Methods and Results

Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.

Conclusion

All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.     

Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx

Vascular endothelial cells are shielded from direct exposure to flowing blood by the endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans, and associated glycosaminoglycans (GAGs). Recent data indicate that the incorporation of the unsulfated GAG hyaluronan into the endothelial glycocalyx is essential to maintain its permeability barrier properties, and we hypothesized that fluid shear stress is an important stimulus for endothelial hyaluronan synthesis. To evaluate the effect of shear stress on glycocalyx synthesis and the shedding of its GAGs into the supernatant, cultured human umbilical vein endothelial cells (i.e., the stable cell line EC-RF24) were exposed to 10 dyn/cm2 nonpulsatile shear stress for 24 h, and the incorporation of [3H]glucosamine and Na2[35S]O4 into GAGs was determined. Furthermore, the amount of hyaluronan in the glycocalyx and in the supernatant was determined by ELISA. Shear stress did not affect the incorporation of 35S but significantly increased the amount of glucosamine-containing GAGs incorporated in the endothelial glycocalyx [168 (SD 17)% of static levels, P < 0.01] and shedded into the supernatant [231 (SD 41)% of static levels, P < 0.01]. Correspondingly with this finding, shear stress increased the amount of hyaluronan in the glycocalyx [from 26 (SD 24) x 10(-4) to 46 (SD 29) x 10(-4) ng/cell, static vs. shear stress, P < 0.05] and in the supernatant [from 28 (SD 11) x 10(-4) to 55 (SD 16) x 10(-4) ng x cell(-1) x h(-1), static vs. shear stress, P < 0.05]. The increase in the amount of hyaluronan incorporated in the glycocalyx was confirmed by a threefold higher level of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the glycocalyx, which may contribute to its vasculoprotective effects against proinflammatory and pro-atherosclerotic stimuli.     

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease

AimsIdentifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD.MethodsDiabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx.ResultsNeuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.     

The glycocalyx is present as soon as blood flow is initiated and is required for normal vascular development

The glycocalyx, and the thicker endothelial surface layer (ESL), are necessary both for endothelial barrier function and for sensing mechanical forces in the adult. The goal of this study is to use a combination of imaging techniques to establish when the glycocalyx and endothelial surface layer form during embryonic development and to determine the biological significance of the glycocalyx layer during vascular development in quail embryos. Using transmission electron microscopy, we show that the glycocalyx layer is present as soon as blood flow starts (14 somites). The early endothelial glycocalyx (14 somites) lacks the distinct hair-like morphology that is present later in development (17 and 25 somites). The average thickness does not change significantly (14 somites, 182nm±33nm; 17 somites, 218±30nm; 25 somites, 212±32nm). The trapping of circulating fluorescent albumin was used to evaluate the development of the ESL. Trapped fluorescent albumin was first observed at 25 somites. In order to assess a functional role for the glycocalyx during development, we selectively degraded luminal glycosaminoglycans. Degradation of hyaluronan compromised endothelial barrier function and prevented vascular remodeling. Degradation of heparan sulfate down regulated the expression of shear-sensitive genes but does not inhibit vascular remodeling. Our findings show that the glycocalyx layer is present as soon as blood flow starts (14 somites). Selective degradations of major glycocalyx components were shown to inhibit normal vascular development, examined through morphology, vascular barrier function, and gene expression.     

Heterogeneity of glomerular barrier function in early adriamycin nephrosis of MWF rats

Adriamycin (ADR), selectively toxic to glomerular epithelial cells, was administered (5 mg/kg BW, i.v.) to MWF/Ztm rats to study its early effects on glomerular barrier function with respect to albumin and high molecular weight (HMW) proteins. After 7 days of ADR incubation (glomerular filtration rate remains unchanged), protein excretion was significantly increased in treated rats. The proteinuria was due to a nonselective glomerular lesions resulting in an increase in both, but not a changed ratio of HMW proteins to albumin. However, this ADR-induced proteinuria seen in the final urine was not confirmed by free-flow micropuncture studies of superficial glomeruli. The albumin and HMW protein concentrations in samples taken from Bowman's capsular space of ADR-treated rats did not significantly differ from control samples. These data suggest that cortical nephrons are less sensitive to ADR than juxtamedullary nephrons.     

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.     

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography ³, that topography limits adhesive bond formation with other surfaces ⁹, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface ^{3, 7}. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface ⁶. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface ⁴. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo ². The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa ⁵. The thickness and structure of the glycocalyx have been studied using electron microscopy ⁸, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.     

Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits

The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300 nM, 5 min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.     

Structural alteration of glycosaminoglycan side chains and spatial disorganization of collagen networks in the skin of patients with mcEDS-CHST14

Musculocontractural Ehlers-Danlos syndrome (mcEDS) due to CHST14/D4ST1 deficiency (mcEDS-CHST14) is a recently delineated type of EDS caused by biallelic loss-of-function mutations in CHST14, which results in the depletion of dermatan sulfate (DS). Clinical characteristics of mcEDS-CHST14 consist of multiple malformations and progressive fragility-related manifestations, including skin hyperextensibility and fragility. Skin fragility is suspected to result from the impaired assembly of collagen fibrils caused by alteration of the glycosaminoglycan (GAG) chain of decorin-proteoglycan (PG) from DS to chondroitin sulfate (CS). This systematic investigation of the skin pathology of patients with mcEDS-CHST14 comprised both immunostaining of decorin and transmission electron microscopy-based cupromeronic blue staining to visualize GAG chains. Collagen fibrils were dispersed in the affected papillary to reticular dermis; in contrast, they were regularly and tightly assembled in controls. Moreover, the fibrils exhibited a perpendicular arrangement to the affected epidermis, whereas fibrils were parallel to control epidermis. Affected GAG chains were linear, stretching from the outer surface of collagen fibrils to adjacent fibrils; in contrast, those of controls were curved, maintaining close contact with attached collagen fibrils. This is the first observation of compositional alteration, from DS to CS, of GAG side chains, which caused structural alteration of GAG side chains and resulted in spatial disorganization of collagen networks; this presumably disrupted the ring-mesh structure of GAG side chains surrounding collagen fibrils. McEDS-CHST14 provides a critical example of the importance of DS in GAG side chains of decorin-PG during assembly of collagen fibrils in maintenance of connective tissues.     

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.     

Angiopoietin-like protein 3 modulates barrier properties of human glomerular endothelial cells through a possible signaling pathway involving phosphatidylinositol-3 kinase/protein kinase B and integrin alphaVbeta3

Podocytes can influence glomerular endothelial cell (GEnC) barrier properties and take part in the development of proteinuria by some molecules. Angiopoietin-like protein 3 (Angptl3), secreted by podocytes, is a member of the angiopoietin-like protein family that has important biological functions in endothelial cells. In our previous studies, we showed that mRNA expression of Angptl3 increased significantly in kidneys of children with minimal change nephrotic syndrome. And the mRNA level of Angptl3 was increased in the glomerulus of adriamycin rats with the development of proteinuria. It was also found that Angptl3 was expressed in the cytoplasm of cultured podocytes. Thus, Angptl3 might influence the biological functions of GEnCs in a paracrine manner. In this study, we found that Angptl3 could increase the permeability of GEnCs and increase the level of protein kinase B phosphorylation in cultured GEnCs in vitro. LY294002, a phosphatidylinositol-3 kinase inhibitor, could prevent the increase of permeability of GEnCs induced by Angptl3. Our results also indicated that the integrin αVβ3 antibody (LM609) could block the Angptl3-induced protein kinase B phosphorylation.     

Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function

A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3 beta,5 alpha,6 beta-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3 beta,5 alpha,6 beta-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.     

Glomerular lysozyme binding in protein-overload proteinuria

Binding of the cationic molecule lysozyme to the glomerular basement membrane and to the glomerular epithelial cell coat was investigated in the glomerulus of normal female Wistar rats and in rats in which heavy proteinuria was induced by the daily administration of 1 g of bovine serum albumin. In normal rats the binding of lysozyme to the anionic groups in the glomerular basement membrane and the cell coat had no effect on the ultrastructure of the glomerular epithelial cell, in particular the foot processes were unchanged. In the proteinuric rats the lysozyme-binding to the glomerular basement membrane and the epithelial cell coat was completely lost in the damaged glomeruli. In the apparently normal glomeruli present in these proteinuric animals binding was similar to that seen in normal rats. These results suggest that in protein-overload proteinuria there is a loss of glomerular anion and hence a reduction in the glomerular charge barrier. This may account, at least in part, for the increased glomerular leak of negatively charged serum albumin in this experimental model of proteinuria.     

Elevated Urine Heparanase Levels Are Associated with Proteinuria and Decreased Renal Allograft Function

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains, leading to structural modifications that loosen the extracellular matrix barrier and associated with tumor metastasis, inflammation and angiogenesis. In addition, the highly sulfated heparan sulfate proteoglycans are important constituents of the glomerular basement membrane and its permselective properties. Recent studies suggest a role for heparanase in several experimental and human glomerular diseases associated with proteinuria such as diabetes, minimal change disease, and membranous nephropathy. Here, we quantified blood and urine heparanase levels in renal transplant recipients and patients with chronic kidney disease (CKD), and assessed whether alterations in heparanase levels correlate with proteinuria and renal function. We report that in transplanted patients, urinary heparanase was markedly elevated, inversely associated with estimated glomerular filtration rate (eGFR), suggesting a relationship between heparanase and graft function. In CKD patients, urinary heparanase was markedly elevated and associated with proteinuria, but not with eGFR. In addition, urinary heparanase correlated significantly with plasma heparanase in transplanted patients. Such a systemic spread of heparanase may lead to damage of cells and tissues alongside the kidney.The newly described association between heparanase, proteinuria and decreased renal function is expected to pave the way for new therapeutic options aimed at attenuating chronic renal allograft nephropathy, leading to improved graft survival and patient outcome.     

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.     

The focal segmental glomerulosclerosis permeability factor: biochemical characteristics and biological effects

Focal segmental glomerulosclerosis (FSGS) is characterized by steroid resistant nephrotic syndrome and progression to end-stage renal disease. Proteinuria in certain patients with FSGS may be caused by a circulating factor (FSGS permeability factor [FSPF]). The current report documents the biochemical characteristics and the biological and molecular effects of 70% ammonium sulfate supernatant of plasma from patients with recurrence of FSGS after transplantation (FSGS 70% supernatant). FS permeability activity, defined as the capacity of plasma from patients with FSGS to increase albumin permeability (P(alb)) of isolated glomeruli, was assessed in vitro. Permeability activity was not affected by lyophilization. FSPF bound strongly to matrices containing Mono-Q anion exchanger or protein A. It eluted from matrix-bound Cibacron blue F3GA over a wide range of salt concentrations, indicating a potential binding with other proteins, such as albumin. FSPF caused a maximal increase in P(alb) within 2 mins of incubation in vitro. Cellular proteins isolated from glomeruli with increased P(alb) showed decreased tyrosine phosphorylation of focal adhesion kinase, paxillin, and other proteins. Tyrosine phosphatase ]inhibition prevented the increase in P(alb). Intravenous administration of as little as 3 mg protein in FSGS 70% supernatant increased P(alb), while 9 mg or more were required to produce proteinuria. We conclude that FSPF is a low-molecular-weight protein, carries an anionic charge, and binds to protein A. Effects of FSPF on the glomerular permeability barrier are rapid and dose dependent and involve signaling through altered phosphorylation of cellular proteins. Identification of these biochemical and biological characteristics may be used to design strategies for removing FSPF from circulation and for purification and identification of this factor.     

HMGB1 contributes to glomerular endothelial cell injury in ANCA‐associated vasculitis through enhancing endothelium–neutrophil interactions

Our previous studies demonstrated that high mobility group box‐1 (HMGB1), a typical damage‐associated molecular pattern (DAMP) protein, is associated with the disease activity of antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). Moreover, HMGB1 participates in ANCA‐induced neutrophil activation. The current study aimed to investigate whether HMGB1 regulated the interaction between neutrophils and glomerular endothelial cells (GEnC) in the presence of ANCA. Correlation analysis on HMGB1 levels in AAV patients and soluble intercellular cell adhesion molecule‐1 (sICAM‐1) levels or vascular endothelial growth factor (VEGF) levels, which are markers of endothelial cell activation, was performed. The effect of HMGB1 on neutrophil migration towards GEnC, respiratory burst and degranulation of neutrophils in coculture conditions with GEnC was measured. The activation of neutrophils, the activation and injury of GEnC, and the consequent pathogenic role of injured GEnC were evaluated. Plasma levels of HMGB1 correlated with sICAM‐1 and VEGF (r = 0.73, P < 0.01; r = 0.41, P = 0.04) in AAV patients. HMGB1 increased neutrophil migration towards GEnC, as well as respiratory burst and degranulation of neutrophils in the presence of ANCA in the coculture system. In the presence of robust neutrophil activation, GEnC were further activated and injured in the coculture system of GEnC and neutrophils. In addition, injured GEnC could produce TF‐positive leuco‐endothelial microparticles and endothelin‐1 (ET‐1), while NF‐κB was phosphorylated (S529) in the injured GEnC. Plasma levels of HMGB1 correlated with endothelial cell activation in AAV patients. HMGB1 amplified neutrophil activation and the activation and injury of GEnC in the presence of ANCA.