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BASIC RESEARCH |
* Academic Renal Unit, Southmead Hospital; Microvascular Research Laboratories, University of Bristol; Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester; and Department of Vascular Medicine and Diabetes Research, Peninsula Medical School, University of Exeter, Exeter, United Kingdom
Correspondence: Dr. Anurag Singh, Academic Renal Unit, Paul O'Gorman Lifeline Centre, Clinical Sciences at North Bristol, University of Bristol, Southmead Hospital, Bristol, BS10 5NB, UK. Phone/Fax: +44-117-959-5438; E-mail: Anurag.Singh{at}bristol.ac.uk
Received for publication January 28, 2007. Accepted for publication June 18, 2007.
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Introduction |
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Evidence of GEnC damage in conditions such as hemolytic uremic syndrome and preeclampsia, plus strong association of generalized endothelial dysfunction in diabetes and hypertension with the onset of microalbuminuria, suggest that GEnC play an important role in glomerular disease.3, 4 Further emphasizing the role of GEnC in glomerular physiology, we and others have shown that GEnC–podocyte communication via soluble mediators (including vascular endothelial growth factor [VEGF] and angiopoietin-1) is vital to maintaining the functioning of the GFB.5–7
The characteristic fenestrated phenotype of GEnC, along with increasing evidence of heterogeneity in endothelial behavior derived from different species and organs, indicates that it is vital to conduct these experiments in human GEnC.8, 9 Historically, GEnC have been difficult to study in culture because of their poor replicative potential leading to early senescence, in addition to nonavailability of representative GEnC lines. We have addressed this problem successfully by generating a conditionally immortalized (Ci) human GEnC line the behavior of which is comparable with primary culture GEnC, including the expression of fenestrations.10
The luminal aspect of all endothelia is covered by a mesh-like hydrated structure known as glycocalyx, which comprises glycoproteins and, in particular, a special class of heavily glycosylated glycoproteins, proteoglycans (PG). Although glycocalyx is not visible on standard electron microscopy (EM), modification of the EM technique, for example by staining with cationic probes before fixation, overcomes this problem.11, 12 Reports of the thickness of the glycocalyx have varied between 50 and 300 nm, although these may be underestimations as a result of the dehydrating effect of EM fixation and processing. New innovative tools such as quantitative intravital microscopy have provided indirect evidence for a much thicker layer in the systemic microvasculature measuring up to 500 nm and known as endothelial surface layer.13 In addition to the glycocalyx that is physically attached to the endothelial surface, endothelial surface layer includes a dynamic component made of adsorbed plasma proteins. In nonrenal microvascular beds, it is evident that thick glycocalyx has a significant impact on hemodynamics, oxygen transport, coagulation, inflammation,14–18 and above all vascular permeability possessing both size and charge exclusion properties.19
With very few reports describing the relevance of glomerular endothelial glycocalyx in historical literature,20 its role in glomerular microcirculation is yet to be elucidated. Until recently, the fenestrae of the GEnC, a requirement to support the high hydraulic conductivity across the GFB, were considered to be empty; however, scanning EM studies using sophisticated techniques to preserve glycocalyx structure have revealed that the GEnC glycocalyx measures up to 300 nm and covers both the fenestral and the interfenestral domains.21 Such landmark studies confirm, first, that GEnC, like other microvascular endothelia, are covered with a thick coat of glycocalyx and, second, that the GEnC fenestrations are not empty but contain glycocalyx, which may contribute to the barrier to protein passage.
PG are an important constituent of the glycocalyx and have two components: A core protein and covalently bound glycosaminoglycan (GAG) side chains. Heparan sulfate (HS) PG, named after the attached HS GAG side chains, are the most abundant PG in the glycocalyx and are known to be produced by GEnC.22 The contribution of HS GAG to the GFB has been studied in animal models, in which its enzymatic removal leads to increased permeability to macromolecules23; however, because of the presence of HS GAG in both the GBM and glycocalyx,24 it is difficult to conclude from these in vivo studies whether the highly charge-selective restrictive properties of HS GAG are due to its presence in glycocalyx or GBM, most of which is produced by podocytes. Previous studies that have attributed this increase in macromolecular permeability to the loss of HS GAG from the GBM have not considered the possibility of a role for the GEnC glycocalyx.25
Human heparanase (HPSE-1) is an endo-
-d-glucuronidase that degrades HS GAG and is associated with extracellular matrix turnover and angiogenesis.26, 27 Overexpression of HPSE-1 in transgenic mice leads to early proteinuria and renal failure.28 More recently, HPSE-1 expression was shown to be upregulated in a number of animal models of renal disease (passive Heymann nephritis,29 puromycin aminonucleoside nephrosis,30 and anti-GBM nephritis31) and in glomerular podocytes cultured in high glucose milieu.32 In addition, increased HPSE-1 activity has been shown in urine from patients with early diabetes33 (and microalbuminuria) and also in nondiabetic nephrotic syndrome.34 This evidence suggests the involvement of HPSE-1 in diabetic and nondiabetic proteinuric renal disease.
We have now characterized the GEnC glycocalyx in detail and studied its contribution to the permeability properties of a GEnC monolayer using sophisticated in vitro culture systems. We have developed and tested the hypothesis that HPSE-1 can contribute to proteinuria through degradation of the GEnC glycocalyx.
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Discussion |
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The presence of glycocalyx on CiGEnC was confirmed by TEM and the lectin-binding experiments. Our findings of a glycocalyx thickness of 200 nm are consistent with estimates in published work on GEnC in vivo35 and other endothelia in vitro.13 The binding preferences of WGA lectin also indicate the predominance of sialic acid–rich glycoproteins and GAG chains in CiGEnC glycocalyx. Our Western blotting results confirm that nonproliferating CiGEnC express PG core proteins at levels comparable to primary culture GEnC and similar to the description in a recent publication.23 In light of our results, it is plausible that the PG core protein perlecan present in the subendothelial (lamina rara interna) GBM24 is produced by the GEnC, potentially also contributing to the anionic charge of GBM. This could also account for the loss of anionic charge (as a contributor to the onset of albuminuria) reported in conditions such as diabetes,36 in which both generalized endothelial dysfunction and loss of perlecan in the GBM (perhaps also in glycocalyx) are early features.36, 37 Expression of HS GAG by CiGEnC suggests their potential of regulating interactions with the neighboring cells via the extracellular matrix. The method used to disrupt the glycocalyx using enzymes was based on two specific aims: First, in the case of neuraminidase, to remove the majority of the glycocalyx without disturbing the integrity of the cell monolayer, and, second, with heparinase III and recombinant HPSE-1, to degrade a specific constituent of glycocalyx (HS GAG, implicated in renal pathology).
The marked reduction of WGA binding after neuraminidase treatment suggests that sialic acid is a major constituent of the sugar moieties present in CiGEnC glycocalyx. This was previously reported in GEnC and is also consistent with the reports from other microvascular beds.13, 38, 39 Compared with neuraminidase, as expected, heparinase III treatment caused only a small reduction in WGA labeling; however, a specific antibody to HS GAG demonstrated a marked reduction in its expression, confirming the activity of the enzyme.
HPSE-1 has a different mechanism of action compared with heparinase III and cleaves HS GAG into larger fragments of 5 to 7 kD through a different cleavage site compared with the latter.40 It is synthesized as a 65-kD latent enzyme that subsequently undergoes proteolytic cleavage, yielding a 50- and an 8-kD active heterodimer.41 Lysosomes are the main storage site of active HPSE-1 because their acidic milieu (pH 5 to 6.5) is best suited to preserve its maximal activity. In addition to its intracellular housekeeping role in HS metabolism, active HPSE-1 is known to be transported by endosomes to the plasma membrane and extracellular space.42 The recombinant HPSE-1 used in our study possessed maximal activity at pH 5 with a significant activity observed at pH 6.43 The greater activity in an acidic milieu suggests that the majority of the HPSE-1 activity is intracellular, affecting the turnover of HS GAG, which could potentially lead to changes in both GEnC glycocalyx and GBM. This would also be consistent with the observations of high HPSE-1 activity coupled with loss of glomerular HS GAG in models of experimental diabetic nephropathy.44
TEER is used as an indicator of the pathways across a cell layer open to water and small molecules. The cAMP medium and thrombin provided good controls, demonstrating the ability of this system to detect changes in ion flux across CiGEnC monolayers. Our results show that removal of the majority of the glycocalyx by neuraminidase has a significant effect on both the passage of water and small solutes (as measured by TEER) and albumin flux; however, removal of HS GAG alone (using heparinase III or recombinant HPSE-1) has a significant effect on the albumin flux without affecting the pathways open to water and small solutes. We further tested the validity of these observations in the presence of exogenous VEGF, at a concentration and for a length of exposure shown previously to induce fenestrations.10 The results were consistent with experiments performed in usual media, confirming the ability of the glycocalyx to contribute to protein restrictive properties of CiGEnC monolayers in a more fenestrated state as found in vivo.
The ability of HS GAG to restrict differentially the passage of albumin is probably due to its previously described charge-selective properties. This has been demonstrated in vivo, where removal of HS GAG has been shown to increase glomerular permeability to albumin.23 Here glomerular permeability was estimated in cooled isolated perfused kidneys, a technique that inhibits tubular reabsorption and proteolytic degradation of albumin; however, in a recent study on intact rats,45 removal of HS GAG by heparinase did not seem to cause proteinuria. This discrepancy could be a result of compensation of the increase in glomerular leakage by an increase in tubular reabsorption of protein (because this was not inhibited in this model). The evidence linking excess HPSE-1 to proteinuria is strongly suggestive of the contribution of HS GAG27–30 in glomerular permselectivity; therefore, the study on intact rats45 (which concluded that HS GAG in the GBM does not contribute to glomerular permselectivity) is suggestive of an important contribution from the GEnC glycocalyx.
Our results support the belief that enzyme treatments used in our experiments removed the surface glycocalyx rather than basement membrane components23 because the basement membrane components were not directly exposed to enzymes. Furthermore, if the basement membrane components had been affected, then the integrity of the monolayers would have been compromised, yet, in our experiments, the monolayers seemed unchanged by phase contrast microscopy, retention of junctional morphology by VE-cadherin; therefore, the observed changes in macromolecular permeability are attributable to glycocalyx disruption rather than affects on basement membrane components
Our work confirms the presence of GEnC glycocalyx in culture, the structure of which is composed of sialic acid–rich glycoproteins and PG. The structure of this glycocalyx can be disrupted in culture by enzymes. Removal of HS GAG has a significant effect on albumin flux but not on passage of water and solutes. This finding is important for two reasons: First, it provides evidence to support the role of GEnC glycocalyx in selective permeability (restricting passage of macromolecules but allowing passage of water and small solutes); second, it suggests a mechanism by which HPSE-1 may increase permeability to macromolecules and hence contribute to the pathogenesis of proteinuria in clinical diseases. Blocking HPSE-1 in experimental disease may reduce proteinuria by preventing GEnC glycocalyx degradation instead of or as well as blocking effects on GBM. Blockade of HPSE-1 activity has therapeutic potential in a variety of glomerular diseases, particularly diabetic nephropathy, in which both proteinuria and upregulation of HPSE-1 have been described. Emerging data showing systemic loss of endothelial glycocalyx, along with increased microvascular permeability, in diabetes46 both are consistent with the role of GEnC glycocalyx in glomerular permselectivity proposed here and suggest endothelial glycocalyx dysfunction as the missing common link between proteinuria and cardiovascular disease.
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CONCISE METHODS |
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CiGEnC were used for experiments after they were maintained at the nonpermissive temperature between 5 and 7 d. In some experiments, primary culture GEnC (Applied Cell Biology Research Institute, Kirkland, WA) were used for comparison. All GEnC were cultured in endothelial growth medium 2 microvascular (EGM2-MV; Cambrex, Wokingham, UK) containing FCS (5%) and growth factors as supplied with the exception of VEGF, unless otherwise stated. Ci podocytes were cultured in RPMI 1640 as described previously.47
Phase Contrast Microscopy
Morphology of CiGEnC monolayers was examined by phase contrast microscopy after enzyme treatments and compared with monolayers exposed to vehicle only. All images were acquired using a digital camera (Nikon Coolpix E 4500; Nikon UK Ltd, Surrey, UK).
TEM
This technique has been described previously.48 Briefly, monolayers of CiGEnC were fixed in freshly prepared solution of 2.5% glutaraldehyde and 0.5% Alcian blue in 0.1 M cacodylate buffer (pH 7.3). After 6 h, cells were washed three times in 0.1 M cacodylate buffer at 4°C and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer. Cells were washed again three times in cacodylate buffer and then distilled water before dehydration with ethanol. This was followed by embedding in Araldite resin. The cell layer was trimmed, and 0.5-µm survey sections were cut and stained with 1% toluidine blue in 1% aqueous borax for light microscopy. Cell-rich areas of the block were trimmed, and 50-nm-thick sections were cut and stained with 3% aqueous uranyl acetate and Reynolds lead citrate solution. Digital micrographs were taken on a Philips 100CS microscope.
Lectin Binding
Cells were grown to confluence on glass coverslips and fixed in 2% paraformaldehyde for 10 min and thereafter incubated with FITC-WGA (Sigma-Aldrich, St. Louis, MO) at 2 µl/ml for 30 min. WGA (from Triticum vulgaris) binds to sugar moieties (N-acetyl glucosamine and N-acetyl neuraminic acid) of glycoproteins present on the cell surface, the majority of which are likely to be PG constituents of glycocalyx. Nuclear staining was demonstrated using 4,6-diamidino-2-phenylindole. Coverslips were mounted in Vectashield aqueous mountant (Vector Laboratories, Peterborough, UK) and examined using a Leitz DMRB fluorescence microscope (Leica, Solms, Germany) and TCS-NT confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).
Immunofluorescence
After fixation as described previously, coverslips were either incubated directly in the blocking solution (5% FCS and 0.05% Tween20 in PBS) or permeabilized in 0.3% Triton X-100 before blocking. Cells were then incubated with antibodies to HS (HepSS-1; US Biologicals, Swampscott, MA), VE-cadherin (Santa Cruz Biochemicals, Santa Cruz, CA), or von Willebrand factor (vWF; DakoCytomation, Ely, Cams, UK) or with a Texas red–conjugated phalloidin to label actin (Molecular Probes, Eugene, OR). Primary antibody binding was detected using FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) except for HS (Alexa Flour 488 conjugated probe; Molecular Probes). Control cells were incubated with secondary antibodies only.
Generation of Recombinant Human Heparanase
The active 50-/8-kD heterodimer form of HPSE-1 protein was produced in an insect cell expression system as described previously.43 Briefly, protein targeted for secretion was isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity as judged by SDS-PAGE analysis. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity, and its preference for acidic pH.
Enzyme Treatments
Neuraminidase from Clostridium perfringens (EC 3.2.1.18; Sigma Aldrich) hydrolyzes the sialic acid residues of oligosaccharides and glycoproteins. Monolayers of CiGEnC were incubated in serum-free medium (SFM) with neuraminidase at concentrations from 0.125 to 1 U/ml for 60 min at 37°C. Controls were incubated in SFM alone under the same conditions.
Heparinase III from Flavobacterium heparinum (EC 4.2.2.8; Sigma Aldrich) cleaves HS GAG. Monolayers were incubated with concentrations ranging from 0.20 to 1 U/ml in SFM at 37°C for 4 h. Controls were incubated in SFM alone under the same conditions.
For some experiments, both control and test monolayers were cultured in 100 ng/ml recombinant human VEGF (VEGF-A165; R&D Systems, Minneapolis, MN) for 24 h before enzyme treatments with neuraminidase and heparinase III. Using the previously mentioned protocol, we previously showed that VEGF increases the number of fenestrations expressed by CiGEnC.10
HPSE-1 cleaves HS GAG into larger fragments and was used at 200 to 1000 ng/ml in SFM pH 6.0 over 4 h. The control cells were incubated with SFM at pH 6. The optimal pH of incubation buffer (SFM) was determined by a set of preliminary experiments (data not shown), aiming to preserve HPSE-1 activity but with least effect on the integrity and permeability characteristics of CiGEnC monolayers. The results of these experiments confirmed higher activity of HPSE-1 at more acidic pH (pH 5 > 6 > 7).
Cell-Based Fluorescence Assay
A CBF assay, essentially as described previously,6 was used to quantify changes in lectin binding and HS expression after treatments with neuraminidase (0.125 to 1 U/ml) or heparinase III (1.0 U/ml) and recombinant HPSE-1 (500 ng/ml).
Western Blotting
CiGEnC were lysed in Laemmli sample buffer, and protein concentrations were determined (bicinchoninic acid assay; Pierce Chemical Co., Rockford, IL). Lysates of primary GEnC were used as control. Samples were separated by SDS-PAGE and were blotted onto nitrocellulose or polyvinylidene fluoride (Immobilon-P; Millipore Corp., Billerica, MA) membranes. The membranes were blocked in 5% fat-free milk before incubation with antibodies to syndecan-1, syndecan-4, perlecan (all Zymed Laboratories, San Francisco, CA), glypican-1 (Santa Cruz), versican (R&D Systems, Morrisville, NC), and actin to confirm loading of comparable amounts of protein in each lane. After incubation with horseradish peroxidase–conjugated secondary antibodies, bands were detected by using Supersignal West Femto maximum sensitivity chemiluminescence substrate (Pierce Chemical Co.) followed by imaging using a MultiDoc-It imaging System (UVP, Upland, CA).
Measurement of TEER
TEER was measured using an automated bioimpedance sensing system (ECIS; Applied Biophysics, Troy, NY). Compared with conventional tissue culture insert-based methods, this method has added advantages of sensitive, real-time TEER measurements in a controlled environment. The technical details of this method have been described previously.49 CiGEnC were seeded at 100,000 cells/cm2 in each well of the eight-well electrode slide (8W10E) supplied by the manufacturer. The base of an individual well has an array of 10 gold film electrodes that connect ECIS electronics to each of the eight wells. The resistance is reported in 
, and the measurement from each well, at a given time point, is an average of the recordings from 10 electrodes.
The effects of a cAMP analogue and thrombin were examined as controls for the system. Increasing cAMP decreases permeability (hence, increases TEER) as opposed to thrombin, which increases permeability (reduction in TEER) of GEnC.26 A cell membrane-permeable cAMP analogue, 8-(4-chlorophenylthio) pCPT-cAMP (Sigma), was used in combination with a cAMP-specific phosphodiesterase inhibitor, RO-20-1724 (CN Biosciences, Nottingham, UK) to elicit a maximal response. The medium in cell culture wells was replaced with SFM medium containing 20 µM RO-20-1724 and 300 µM pCPTcAMP (cAMP medium) or thrombin (1 U/ml) at the same time as enzyme treatments.
Culture of CiGEnC in Tissue-Culture Inserts
Polycarbonate supports (0.4 µm pore size, 0.5 cm2 surface area) in tissue-culture inserts (1 cm diameter; Nalge Nunc Int., Rochester, NY) were seeded with CiGEnC at 100,000 cells/cm2. Inserts were placed in 24-well plates, and media were changed three times a week.
Measurement of Transendothelial Protein Passage
Transendothelial permeability to macromolecules was assessed by measurement of passage of FITC-labeled BSA (Sigma) across the monolayer using tissue culture inserts as described previously.6 Medium in both wells and inserts, containing CiGEnC monolayers, was replaced with SFM. After 1 h, the medium in the insert was replaced with 500 µl of SFM containing 0.5 mg/ml FITC-labeled BSA; that in the well was replaced with 500 µl of SFM containing 0.5 mg/ml unlabeled BSA. At 1, 2, and 3 h, 100-µl aliquots were removed and replaced with 100 µl of SFM containing unlabeled BSA (0.5 mg/ml). The fluorescence of the aliquots was measured as already described, and the amount of FITC-BSA passing through the monolayer was calculated by reference to a set of standard dilutions.
Statistical Analyses
GraphPad Prism-4 statistical software package (GraphPad Software, San Diego, CA) was used for all analyses, including SEM and ANOVA. P < 0.05 was taken to indicate statistical significance
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DISCLOSURES |
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Acknowledgments |
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Some of the data were presented in abstract form at the annual meeting of the American Society of Nephrology; November 14 through 19, 2006; San Diego, CA.
We thank Dr. Mark Jepson and Alan Leard at the MRC Cell Imaging facility, University of Bristol, and Dr. Gavin Welsh, Academic Renal Unit, University of Bristol, for assistance with confocal laser scanning microscopy.
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