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.

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.

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.

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.

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.

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.

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.