Characterization of Urothelial Inclusions in Male Wistar Han Rats Treated Orally With the Novel α2A-Adrenoceptor Agonist Tasipimidine.

Intracellular inclusions were observed in urinary bladder epithelium of male Wistar rats, following oral treatment with high doses of the α2A-adrenoceptor agonist tasipimidine for 28 days. No cell death or inflammation was associated with the brightly eosinophilic inclusions. Electron microscopy (EM) studies showed that the inclusions represented intact or fragmented red blood cells (RBC) resulting from erythrophagocytosis, further supported by the presence of iron in urothelial cells. In addition, scattered iron-positive macrophages were observed in the submucosa and muscle layer, indicating microvascular leakage, as no major hemorrhage was evident. Despite the presence of inclusions, the urothelium showed normal uroplakin III distribution, normal cell turnover, and an absence of α-2u-globulin. It is, therefore, concluded that the inclusions were not associated with urothelial damage or increased renewal of the epithelium. This finding shows also that urothelial cells have the capability to phagocytize and break down RBCs originating from submucosal microvascular leakage. Similar changes were not observed in tasipimidine-treated beagle dogs (28 days), suggesting these findings were rat specific. The leakage of RBCs into the urothelium is suggested to be a consequence of exaggerated pharmacology leading to vasoconstriction of submucosal blood vessels in combination with transient increased bladder distension and pressure.

Unusual Incidence and Location of Renaut Bodies in Beagle Dogs in a Nonclinical Study.

We identified the presence of Renaut bodies in an unusual location in Beagle dogs on a 3-month nonclinical toxicity study. These peculiar structures are commonly reported as a background finding in the sciatic nerve of dogs. In our study, however, they were also observed in autonomic nerves surrounding the adrenal gland, a location in which they have not been reported before. The incidence in both locations were 8 of 32 Beagle dogs in the sciatic nerve and 6 of 40 Beagle dogs around the adrenal gland in the dosing and/or recovery phases of the study.

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-ß), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.

Estrous cycle-dependent morphology in the reproductive organs of the female Göttingen minipig.

The present study describes the normal histology of female reproductive organs during the estrous cycle in the Göttingen minipig. For this purpose, sexually mature females were sacrificed at different phases of the cycle (follicular/proliferation, ovulation, and early-, mid-, and late-luteal/secretory phase). Ovaries, uterus, cervix, vagina, and mammary gland tissues were processed for microscopic evaluation. Sexual maturity was assured by selecting females in which at least 1 progesterone peak was measured. Stage-distinguishing features in ovaries were the Graafian follicles (disrupted vs. nondisrupted) and corpora lutea of recent and preceding cycles (size, cell morphology, and structural composition). In the uterus, stage-specific markers were epithelial morphology, secretory activity (using periodic acid-Schiff/hematoxylin staining), and epithelial mitosis and/or apoptosis. The other reproductive organs were not suitable to discriminate between the different phases of the cycle due to a high morphologic variability (mammary gland, and vagina) or absence of clear morphologic differences between the phases (cervix). The increased use of young minipigs (frequently immature/peripubertal) in preclinical testing requires more knowledge on the histologic cyclic changes. With the present morphologic description of the morphologic characteristics of the reproductive tract in recently ovulating minipigs, a guidance for staging the estrous cycle and determination of sexual immaturity is provided.

Differential performance of Wistar Han and Sprague Dawley rats in behavioral tests: differences in baseline behavior and reactivity to positive control agents.

Developmental neurotoxicity (DNT) testing assesses potentially adverse effects on the developing nervous system. The present DNT study was conducted to generate historical data with the Wistar Han (WH) and Sprague Dawley (SD) rat strains, commonly used in Europe and the US, respectively. Potential differences between these strains in DNT endpoints have not been extensively investigated. Motor activity, startle response, learning and memory testing, and neurological (quantitative and qualitative) examinations were conducted using three groups of control, prenatally exposed (to Methylazoxymethanol [MAM] on gestation Day 15) and acutely treated (with IDPN, MK-801 or Chlorpromazine) animals for each strain. The positive controls produced clear effects in most endpoints investigated, with limited functional differences in baseline behavior and positive control sensitivity. However, SD rats were considerably more susceptible to MAM-induced learning and memory impairments and neurological damage. These data highlight differential sensitivity between the strains, which may require risk assessment consideration for developmental neurotoxicants.

Characterization of anticoagulant heparinoids by immunoprofiling.

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and-when additional saccharides are present-inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.

Removal of heparan sulfate from the glomerular basement membrane blocks protein passage.

Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid--but not HS--from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall.

Anti-proteinuric effects of glycosaminoglycan-based drugs.

Heparan sulfate (HS) is a member of the family of glycosaminoglycans (GAGs) that is generally bound to a core protein to form a proteoglycan (PG). HSPGs may be cell-membrane associated (glypicans and syndecans) or located within the extracellular matrix (agrin, perlecan and type XVIII collagen). The sulfate and carboxylic groups in HS are responsible for the negative charge of the sugar chain. HS is abundantly present in the filter unit of the kidney, especially in the glomerular basement membrane (GBM), and is assumed to repel negatively charged proteins, including albumin, thereby preventing their filtration. Alterations in HS expression in the GBM have been reported in a number of renal pathologies, including diabetic nephropathy, minimal change nephropathy and membranous glomerulopathy.A decreased HS expression in the GBM generally correlates with an increase in the level of proteinuria. Progressive proteinuria may result in end-stage renal failure when untreated. Based on these findings, GAG-based drugs have been used to treat proteinuria and some, notably sulodexide, have shown beneficial effects. The biosynthesis of HS and its possible role in renal filtration are discussed, an overview of GAG-based drugs and their effect on proteinuria is provided, and possible mechanisms by which GAG-based drugs ameliorate proteinuria are discussed.

Expression of glomerular heparan sulphate domains in murine and human lupus nephritis.

BACKGROUND: Recently, we identified specific N- and 6-O-sulphated heparan sulphate (HS) domains on activated glomerular endothelial cells. In this study, we evaluated in lupus nephritis the expression of different HS domains on glomerular endothelium and in the glomerular basement membrane (GBM). METHODS: The expression of specific glomerular HS domains and the presence of immunoglobulins (Ig) were determined by immunofluorescence staining of kidney sections of patients with nephritis due to systemic lupus erythematosus (SLE) and MRL/lpr lupus mice. The expression/presence of glomerular HS domains and Ig was also evaluated after eluting Ig from renal sections of lupus mice using two elution methods, and in renal sections of lupus mice treated with heparinoids. RESULTS: Both MRL/lpr mice and patients with lupus nephritis showed a decreased expression of HS in the GBM. The expression of N- and 6-O-sulphated HS domains on glomerular endothelium was decreased in MRL/lpr mice, but increased in SLE patients. MRL/lpr mice had more extensive glomerular Ig deposits than SLE patients. After elution of Ig, the glomerular endothelial expression of N- and 6-O-sulphated HS domains in MRL/lpr mice was recovered and even increased above normal levels, while the expression of HS in the GBM was restored to normal levels. Treatment with heparinoids prevented Ig deposition and preserved the expression of glomerular HS domains at normal levels in lupus mice. CONCLUSION: The expression of specific HS domains on glomerular endothelium and in the GBM is changed during lupus nephritis due to masking by Ig deposits and induction of inflammatory N- and 6-O-sulphated HS domains.

Adult and paediatric patients with minimal change nephrotic syndrome show no major alterations in glomerular expression of sulphated heparan sulphate domains.

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is the most frequent form of nephrotic syndrome in childhood. In the glomerular basement membrane (GBM) of adult patients with MCNS, a reduced expression of a specific heparan sulphate (HS) domain has been reported. In children with MCNS, urinary activity of the HS-degrading enzyme heparanase was increased. It is, therefore, possible that a decreased GBM HS expression is associated with the pathogenesis of proteinuria in patients with MCNS. METHODS: In this study, HS in glomeruli of five adult and six paediatric patients with MCNS were analysed by immunofluorescence staining using four different antibodies, each defining a specific sulphated HS domain. The pediatric patients were subdivided into three groups depending on the presence or absence of podocyte foot process effacement, the level of proteinuria and prednisone administration at the time of the biopsy. In addition, kidneys of rats with adriamycin nephropathy (ADRN), a model for MCNS, were included in the study. RESULTS: Expression of sulphated HS domains was not aberrant in adult or paediatric patients compared with control subjects. Children with and without proteinuria had the same HS content. In contrast, rats with ADRN showed a decreased glomerular expression of sulphated HS domains. CONCLUSIONS: These results suggest that in patients with MCNS proteinuria is not associated with major changes in glomerular expression of sulphated HS domains.

Heparan sulfate on activated glomerular endothelial cells and exogenous heparinoids influence the rolling and adhesion of leucocytes.

BACKGROUND: Proliferative glomerulonephritides are characterized by the influx of leucocytes. Heparan sulfate (HS) plays an important role in the recruitment, rolling and firm adhesion of leucocytes to activated endothelium. Recently, we have shown the importance of HS on activated mouse glomerular endothelial cells (mGEnC-1) for the firm adhesion of leucocytes in a static adhesion assay. In the present study, we evaluated the role of HS on glomerular endothelial cells and the effect of adding heparinoids on the leucocyte-glomerular endothelium interaction under dynamic flow conditions. METHODS: The number of rolling and firmly adhering leucocytes, and the rolling velocity of leucocytes was determined on a monolayer of unactivated or TNF-alpha-activated mGEnC-1 under dynamic flow conditions using physiological relevant shear stress rates in a flow chamber system. Furthermore, the effects of removal of HS on TNF-alpha-activated mGEnC-1 by heparinase III treatment, and of different concentrations of heparin, tinzaparin and HS, on the rolling and adhesion of leucocytes were evaluated. RESULTS: At the calculated physiological shear stress rate of 0.8 dynes/cm2 the number of rolling and firmly adhering leucocytes to mGEnC-1 increased 2-fold after activation with TNF-alpha, whereas the rolling velocity of the leucocytes decreased 2-fold. Addition of heparin, tinzaparin or HS, and the removal of HS on mGEnC-1 reduced the number of leucocytes rolling and adhering to activated mGEnC-1 about 2-3-fold, while the rolling velocity increased more than 2-fold. CONCLUSIONS: HS on activated glomerular endothelial cells is important for the interaction with leucocytes under flow conditions, while exogenous heparinoids interfere with this interaction. These results suggest that supplementary treatment of proliferative glomerulonephritides with heparinoids is an interesting option to pursue.

In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria.

Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hyaluronic acid, and keratan sulfate). Modification of the backbone by sulfation, deacetylation, and epimerization results in unique sequences within GAG molecules, which are instrumental in the binding of a large number of proteins. Investigating the exact roles of GAGs has long been hampered by the lack of appropriate tools, but we have successfully implemented phage display technology to generate a large panel of antibodies against CS, DS, HS, and heparin epitopes. These antibodies provide unique and highly versatile tools to study the topography, structure, and function of specific GAG domains. In this chapter, we describe the selection, characterization, and application of antibodies against specific GAG epitopes.

Induction of glomerular heparanase expression in rats with adriamycin nephropathy is regulated by reactive oxygen species and the renin-angiotensin system.

Heparan sulfate (HS) in the glomerular basement membrane (GBM) is important for regulation of the charge-dependent permeability. Heparanase has been implicated in HS degradation in several proteinuric diseases. This study analyzed the role of heparanase in HS degradation in Adriamycin nephropathy (AN), a model of chronic proteinuria-induced renal damage. Expression of heparanase, HS, and the core protein of agrin (to which HS is attached) was determined on kidney sections from rats with AN in different experiments. First, expression was examined in a model of unilateral AN in a time-course study at 6-wk intervals until week 30. Second, rats were treated with the hydroxyl radical scavenger dimethylthiourea (DMTU) during bilateral AN induction. Finally, 6 wk after AN induction, rats were treated with angiotensin II receptor type 1 antagonist (AT1A) or vehicle for 2 wk. Heparanase expression was increased in glomeruli of rats with AN, which correlated with HS reduction at all time points and in all experiments. Treatment with DMTU prevented the increased heparanase expression, the loss of GBM HS, and reduced albuminuria. Finally, treatment of established proteinuria with AT1A significantly reduced heparanase expression and restored glomerular HS. In conclusion, an association between heparanase expression and reduction of glomerular HS in AN was observed. The effects of DMTU suggest a role for reactive oxygen species in upregulation of heparanase. Antiproteinuric treatment by AT1A decreased heparanase expression and restored HS expression. These results suggest involvement of radicals and angiotensin II in the modulation of GBM permeability through HS and heparanase expression.

Aberrant heparan sulfate profile in the human diabetic kidney offers new clues for therapeutic glycomimetics.

BACKGROUND: Diabetic nephropathy poses an increasing health problem in the Western world, and research to new leads for diagnosis and therapy therefore is warranted. In this respect, heparan sulfates (HSs) offer new possibilities because crude mixtures of these polysaccharides are capable of ameliorating proteinuria. The aim of this study is to immuno(histo)chemically profile HSs from microalbuminuric kidneys from patients with type 1 diabetes and identify specific structural HS alterations associated with early diabetic nephropathy. METHODS: Renal cryosections of control subjects and patients with type 1 diabetes were analyzed immunohistochemically by using a set of 10 unique phage display-derived anti-HS antibodies. HS structures defined by relevant antibodies were characterized chemically by means of enzyme-linked immunosorbent assay and probed for growth factor binding and presence in HS/heparin-containing drugs. RESULTS: In all patients, HS structure defined by the antibody LKIV69 consistently increased in basement membranes of proximal tubules. This structure contained N- and 2-O-sulfates and was involved in fibroblast growth factor 2 binding. It was present in HS/heparin-containing drugs shown to decrease albuminuria in patients with diabetes. The HS structure defined by the antibody HS4C3 increased in the renal mesangium of some patients, especially those who developed macroalbuminuria within 8 to 10 years. This structure contained N- and 6-O-sulfates. For 8 other antibodies, no major differences were observed. CONCLUSION: Specific structural alterations in HSs are associated with early diabetic nephropathy and may offer new leads for early diagnosis and the rational design of therapeutic glycomimetics.

Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies.

Glycosaminoglycans (GAG) play an important role in renal homeostasis. They are strongly negatively charged polysaccharides that bind and modulate a myriad of proteins, including growth factors, cytokines, and enzymes. With the aid of specific phage display-derived antibodies, the distribution of heparan sulfate (HS) and chondroitin sulfate (CS) domains in the normal human kidney was studied. HS domains were specifically located in basement membranes and/or surfaces of renal cells and displayed a characteristic distribution over the nephron. A characteristic location in specific parts of the tubular system was also observed. CS showed mainly an interstitial location. Immunoelectron microscopy indicated specific ultrastructural location of domains. Only partial overlap with any of seven different proteoglycan core proteins was observed. Two HS domains, one highly sulfated (defined by antibody HS4C3) and one low sulfated (defined by antibody RB4Ea12), were studied for their cell biologic relevance with respect to the proliferative effect of FGF-2 on human mesangial cells in vitro. Fibroblast growth factor 2 (FGF-2) binding was HS dependent. Addition of purified HS4C3 antibody but not of the RB4Ea12 antibody counteracted the binding and the proliferative effect of FGF-2, indicating that the HS4C3 domain is involved in FGF-2 handling by mesangial cells. In conclusion, specific GAG domains are differentially distributed in the normal human kidney and are likely involved in binding of effector molecules such as FGF-2. The availability of tools to identify and study relevant GAG structures allows the development of glycomimetica to halt, for instance, mesangial proliferation and matrix production as seen in diabetic nephropathy.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

BACKGROUND: The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. METHODS: Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. RESULTS: As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. CONCLUSION: The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

The parietal epithelial cell: a key player in the pathogenesis of focal segmental glomerulosclerosis in Thy-1.1 transgenic mice.

Focal segmental glomerulosclerosis (FSGS) is a hallmark of progressive renal disease. Podocyte injury and loss have been proposed as the critical events that lead to FSGS. In the present study, the authors have examined the development of FSGS in Thy-1.1 transgenic (tg) mice, with emphasis on the podocyte and parietal epithelial cell (PEC). Thy-1.1 tg mice express the Thy-1.1 antigen on podocytes. Injection of anti-Thy-1.1 mAb induces an acute albuminuria and development of FSGS lesions that resemble human collapsing FSGS. The authors studied FSGS lesions at days 1, 3, 6, 7, 10, 14, and 21, in relation to changes in the expression of specific markers for normal podocytes (WT-1, synaptopodin, ASD33, and the Thy-1.1 antigen), for mouse PEC (CD10), for activated podocytes (desmin), for macrophages (CD68), and for proliferation (Ki-67). The composition of the extracellular matrix (ECM) that forms tuft adhesions or scars was studied using mAb against collagen IV alpha2 and alpha4 chains and antibodies directed against different heparan sulfate species. The first change observed was severe PEC injury at day 1, which increased in time, and resulted in denuded segments of Bowman's capsule at days 6 and 7. Podocytes showed foot process effacement and microvillous transformation. There was no evidence of podocyte loss or denudation of the GBM. Podocytes became hypertrophic at day 3, with decreased expression of ASD33 and synaptopodin and normal expression of WT-1 and Thy-1.1. Podocyte bridges were formed by attachment of hypertrophic podocytes to PEC and podocyte apposition against denuded segments of Bowman's capsule. At day 6, there was a marked proliferation of epithelial cells in Bowman's space. These proliferating cells were negative for desmin and all podocyte markers, but stained for CD10, and thus appeared to be PEC. The staining properties of the early adhesions were identical to that of Bowman's capsule, suggesting that the ECM in the adhesions was produced by PEC. In conclusion, the authors propose the following sequence of events leading to FSGS lesions in the Thy1.1 tg mice: (1) PEC damage and denudation of Bowman's capsule segments; (2) podocyte hypertrophy and bridging; and (3) PEC proliferation with ECM production.

Heparan sulfate proteoglycans in glomerular inflammation.

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.