Cystathionine γ-lyase is expressed in human atherosclerotic plaque microvessels and is involved in micro-angiogenesis.

Atherosclerotic plaques are classically divided into stable and vulnerable plaques. Vulnerable plaques are prone to rupture with a risk for infarction. High intraplaque microvessel density predisposes to plaque vulnerability. Hydrogen sulfide (H2S) is a proangiogenic gasotransmitter which is endogenously produced by cystathionine γ-lyase (CSE), and is believed to have vasculoprotective effects. However, due to its proangiogenic effects, H2S may result in pathological angiogenesis in atherosclerotic plaques, thereby increasing plaque vulnerability. The aim of this study was to determine CSE expression pattern in atherosclerotic plaques, and investigate whether CSE is involved in micro-angiogenesis in vitro. Endarterectomy plaques were studied for CSE expression, and the role of CSE in micro-angiogenesis was studied in vitro. CSE is expressed in plaques with similar levels in both stable and vulnerable plaques. CSE co-localized with von Willebrand Factor-positive microvessel endothelial cells and alpha-smooth-muscle actin-positive SMCs. In vitro, inhibition of CSE in HMEC-1 reduced tube formation, cell viability/proliferation, and migration which was restored after culture in the presence of H2S donor GYY4137. CSE is expressed in intraplaque microvessels, and H2S is a stimulator of micro-angiogenesis in vitro. Due to this pro-angiogenic effect, high levels of CSE in atherosclerotic plaques may be a potential risk for plaque vulnerability.

CD16⁺ monocytes with smooth muscle cell characteristics are reduced in human renal chronic transplant dysfunction.

In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal α-SMA(+) smooth muscle-like cells are present in human renal allografts with CTD. Human PBMC contain myeloid cells capable of differentiating into α-SMA(+) cells in vitro; the phenotype of the ancestral subset is as yet unknown. This study aimed to investigate whether monocyte subsets contain cells with smooth muscle-like cell differentiation capacity and whether CTD in renal transplant recipients is associated with a shift in these monocyte subsets. To accomplish this goal, monocyte subsets from healthy controls were sorted based on CD14 and CD16 expression to investigate gene expression levels of mesenchymal markers α-SMA and SM22α. CD14(+)/CD16(++) monocytes displayed increased α-SMA and SM22α mRNA expression compared with CD14(++)/CD16(-) monocytes, suggesting increased differentiation potential toward smooth muscle-like cells. Flow cytometry revealed that in non-CTD transplant recipients the percentage of CD14(+)/CD16(++) monocytes was reduced, with an even further reduction in patients with CTD. To determine a potential correlation between CD14(+)/CD16(++) monocytes and α-SMA(+) cell outgrowth potential in vitro, PBMC of healthy controls and transplant recipients with and without CTD were cultured under fibrotic culture conditions, and indeed a significant correlation (p=0.0002, r=0.62) was observed. Finally, double staining for α-SMA and CD16 revealed presence of α-SMA(+)CD16(+) cells in kidney explants from CTD patients, albeit at very low numbers. Our data represent evidence that, compared to CD14(++)CD16(-) monocytes, CD14(+)CD16(++) monocytes have an increased expression of smooth muscle cell-associated genes. This monocyte subpopulation is reduced in renal transplant patients with CTD, possibly due to selective migration into the allograft.

Hepatic syndecan-1 changes associate with dyslipidemia after renal transplantation.

Syndecan-1 is a transmembrane heparan sulfate (HS) proteoglycan present on hepatocytes and involved in uptake of triglyceride-rich lipoproteins via its HS polysaccharide side chains. We hypothesized that altered hepatic syndecan-1 metabolism could be involved in dyslipidemia related to renal transplantation. In a rat renal transplantation model elevated plasma triglycerides were associated with fivefold increased expression of hepatic syndecan-1 mRNA (p < 0.01), but not protein. Expression of syndecan-1 sheddases (ADAM17, MMP9) and heparanase was significantly up-regulated after renal transplantation (all p < 0.05). Profiling of HS side chains revealed loss of hepatic HS upon renal transplantation accompanied by significant decreased functional capacity for VLDL binding (p = 0.02). In a human renal transplantation cohort (n = 510), plasma levels of shed syndecan-1 were measured. Multivariate analysis showed plasma syndecan-1 to be independently associated with triglycerides (p < 0.0001) and inversely with HDL cholesterol (p < 0.0001). Last, we show a physical association of syndecan-1 to HDL from renal transplant recipients (RTRs), but not to HDL from healthy controls. Our data suggest that after renal transplantation loss of hepatic HS together with increased syndecan-1 shedding hampers lipoprotein binding and uptake by the liver contributing to dyslipidemia. Our data open perspectives toward improvement of lipid profiles by targeted inhibition of syndecan-1 catabolism in renal transplantation.

Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf ß1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf ß1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Therapeutic potential of vasopressin V2 receptor antagonist in a mouse model for autosomal dominant polycystic kidney disease: optimal timing and dosing of the drug.

BACKGROUND: The renoprotective effect of vasopressin V2 receptor antagonist (V2RA) is currently being tested in a clinical trial in early autosomal dominant polycystic kidney disease (ADPKD). If efficacious, this warrants life-long treatment with V2RA, however, with associated side effects as polydipsia and polyuria. We questioned whether we could reduce the side effects without influencing the renoprotective effect by starting the treatment later in the disease or by lowering drug dosage. METHODS: To investigate this, we administered V2RA OPC-31260 at a high (0.1%) and low (0.05%) dose to a tamoxifen-inducible kidney epithelium-specific Pkd1-deletion mouse model starting treatment at Day 21 (early) or 42 (advanced). After 3 and 6 weeks of treatment, we monitored physiologic and potential renoprotective effects. RESULTS: Initiation of V2RA treatment at advanced stage of the disease lacked renoprotective effects and had less pronounced physiologic effects than early initiation. After 3 weeks on a high dose, cyst ratio and kidney weight were reduced versus untreated controls (18 versus 25%, P = 0.05, and 0.33 versus 0.45 g, P = 0.03, respectively). After 6 weeks of treatment, however, this did not reach significance anymore, even at a high dose (cyst ratio 24 versus 27%, P = 0.12, and kidney weight 0.55 versus 0.66 g, P = 0.38). CONCLUSIONS: Our results suggest that intervention with V2RA should be instituted early in ADPKD and that it might be necessary to further increase the dosage of this drug later in the disease to decrease cyst growth.

Tubulointerstitial heparan sulfate proteoglycan changes in human renal diseases correlate with leukocyte influx and proteinuria.

Heparan sulfate proteoglycans (HSPGs) are well known for their proposed role in glomerular filtration. In addition, HSPGs can bind the leukocyte adhesion molecule l-selectin and chemokines, suggesting a role in inflammation. We examined a panel of biopsies representing different human primary kidney diseases for l-selectin and monocyte chemoattractant protein-1 (MCP-1) binding. In various renal diseases, l-selectin and MCP-1 binding to interstitial perivascular matrix HSPGs is increased, which is significantly associated with leukocyte influx. In proteinuric diseases, including membranous glomerulopathy, minimal change disease, but also IgA nephropathy and lupus nephritis, increased binding of l-selectin and MCP-1 to tubular epithelial cell (TEC) HSPGs is observed, which colocalizes with increased basolateral syndecan-1 and anti-heparan sulfate 10E4 staining. Short-hairpin RNA-mediated silencing demonstrates that syndecan-1 on TECs indeed mediates l-Selectin binding. Increased TEC expression of IL-8 in biopsies of proteinuric patients suggests that the increase in luminal protein may activate TECs to increase expression of l-selectin and MCP-1 binding syndecan-1. Strikingly, urinary syndecan-1 from proteinuric patients is less capable of binding l-selectin compared with urinary syndecan-1 from healthy controls, although syndecan-1 concentrations are similar in both groups. Together, our data show pronounced tubulointerstitial HSPG alterations in primary kidney disease, which may affect the inflammatory response.

Feasibility of mesothelial transplantation during experimental peritoneal dialysis and peritonitis.

The mesothelial cell layer lining the peritoneum orchestrates peritoneal homeostasis. Continuous exposure to peritoneal dialysis fluids and episodes of peritonitis may damage the monolayer irreversibly, eventually leading to adhesion formation and fibrosis/sclerosis of the peritoneum. Autologous mesothelial cell transplantation is thought to be one of the options to reduce dysfunction of the peritoneal membrane. In this article we will review the mesothelial cell transplantation experiments performed in the field of peritoneal dialysis and peritonitis. In addition we will focus on the trouble shooting using cultured autologous mesothelial cells for transplantation.

Effect of fixation protocols on in situ detection of L-selectin ligands.

In situ binding of (chimeric) proteins to tissue sections is a widely used method to identify ligands and their localization. Many different protocols for the fixation of frozen tissue sections are used for in situ binding studies. We report the effects of different fixation protocols on the binding pattern observed using in situ binding of an L-selectin-IgM chimeric protein to both rat lymph node and kidney tissue sections. L-selectin is a C-type lectin, expressed on leukocytes and is involved in both lymphocyte homing and migration upon inflammation. We show that different in situ binding patterns in rat kidney are observed using different fixation protocols, including glutaraldehyde, methanol, formaldehyde and acetone fixation. The observed staining is specific, as it can be blocked in the presence of EGTA, an L-selectin blocking antibody or by ligand competition. Enzymatic pre-treatment of the tissue sections using sialidase, heparitinase I or chondroitinase ABC has differential effects on in situ binding depending on tissue type and fixation protocol. These data indicate that special attention should be paid in choosing a fixation protocol for in situ binding studies, especially when using lectins. This could prevent biologically relevant ligands remaining undetected or wrong conclusions being drawn based on the localization of observed binding.

Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells.

Recognition events between hematopoietic progenitor cells (HPC) and bone marrow endothelial cells (BMEC) initiate homing of HPC to the bone marrow. The chemokine SDF-1 is present on BMEC and plays a crucial role in bone marrow engraftment. We studied the role of proteoglycans (PGs) on BMEC in binding and presentation of SDF-1. SDF-1 mRNA was present in three human BMEC cell lines. Competition experiments showed that 125I-SDF-1 alpha binding to the BMEC cell line 4LHBMEC was inhibited by heparins, heparan sulfate (HS) intestinal mucosa, chondroitin and dermatan sulfate (CS/DS), but not by HS bovine kidney. Pretreatment of 4LHBMEC with glycosaminoglycan (GAG)-degrading enzymes or sodium chlorate demonstrated that SDF-1 bound to both HSPGs and CS/DSPGs in a sulfation-dependent manner, as determined with an SDF-1 antibody recognizing the CXCR4-binding site. 4LHBMEC bound four-fold more SDF-1 than HUVEC. Isolated endothelial PGs did not bind SDF-1 in a filter or microplate-binding assay, suggesting the necessity of membrane association. In flow adhesion experiments, endothelial arrest of CXCR4+ KG-1 and not of CXCR4- KG-1a cells increased significantly when SDF-1 was presented on 4LHBMEC. In conclusion, SDF-1 is produced by BMEC and binds to the BMEC cell surface via HS and CS/DS-GAGs, thereby presenting its CXCR4 binding site to HPC contributing to their arrest.

The application of animal models to study the biocompatibility of bicarbonate-buffered peritoneal dialysis solutions.

The application of animal models to study the biocompatibility of bicarbonate-buffered peritoneal dialysis solutions. Patients treated with peritoneal dialysis (PD) are at risk for development of ultrafiltration failure and peritonitis. These two significant complications can result in the termination of PD treatment. The relative unphysiologic composition of the currently used standard peritoneal dialysis fluids (PDF) is considered to be a major cause for the development of morphologic changes of the peritoneal membrane, ultimately resulting in ultrafiltration failure and probably contributing to changes in local defense mechanisms with the associated increased risk of peritonitis. In recent years, a major research focus has become the development of new and improved PD solutions. This has resulted in the development of an amino-acid-based PDF, a glucose polymer-based PDF, and several bicarbonate-buffered PDF. Typically, the first phase of biocompatibility testing of new PD solutions involves in vitro testing, employing isolated cells such as peritoneal macrophages or cell culture systems using human peritoneal mesothelial cells. The results of such evaluations are useful in providing insights into the biocompatibility performance of any given formulation, but suffer from several disadvantages, which can be better addressed using animal models. In vivo studies using animals permit the analysis of biocompatibility under conditions that allow for cell-to-cell interactions and dynamic changes in solution composition that more closely mimic the clinical situation. In this paper, we will review the use of animal models for the study of PDF biocompatibility and their application to the assessment of bicarbonate-buffered PDF.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

Vascular localization of heparan sulfate proteoglycans in retinas of patients with diabetes mellitus and in VEGF-induced retinopathy using domain-specific antibodies.

PURPOSE: The Steno hypothesis (Deckert et al. ) states that in diabetes mellitus (DM), changes in vascular heparan sulfate proteoglycan (HSPG) expression are involved in systemic endothelial dysfunction and increased capillary permeability. In diabetes-induced glomerular capillary leakage, loss of HSPG and its side chains has been documented. This study aimed to investigate whether microvascular leakage in diabetic retinopathy (DR) is also associated with altered expression of HSPG in retinal microvessels. METHODS: Serial cryosections of post-mortem eyes of 22 subjects with DM and 7 controls were stained with antibodies against the core proteins of the basement membrane HSPGs agrin (Abs Bl31 and JM72) and perlecan (Ab 1948), and four antibodies against heparan sulfate side chains (HS) (Abs JM403, HepSS1, JM13, 3G10). Moreover, we investigated Cynomolgus monkey eyes injected with vascular endothelial growth factor (VEGF)-A, as a model of retinal microvas-cular leakage. The endothelial antigen PAL-E was used to detect microvascular leakage. RESULTS: In the retina of all controls and DM cases, agrin and perlecan core proteins and HS as recognized by JM403 and 3G10 were expressed in the walls of microvessels. Staining for JM13 was variable between cases, but unrelated to microvascular leakage as determined by PAL-E. Staining for HepSS1 was absent in all human retinal microvessels. In monkey retinas, HSPG staining was identical to that in human retinal tissues, except for the staining for HepSS1, which was found absent in control monkey eyes but which was positive in VEGF-injected eyes. CONCLUSIONS: Increased microvascular permeability in human DR is not associated with changes in expression of the HSPGs studied, whereas high amounts of VEGF may induce increased expression of the HS side chain epitope recognized by HepSS1. These results suggest that the mechanism underlying retinal leakage is different from diabetic glomerular capillary leakage.

Rat models in peritoneal dialysis.

BACKGROUND: It is widely accepted that the currently used dialysis solutions are not biocompatible with the peritoneal membrane. Therefore, animal studies have been performed to study different aspects of peritoneal dialysis. However, representative models mimicking the human situation are not yet available. METHODS: The effect of a single injection of peritoneal dialysis (PD) fluid on the cellular composition was studied. Thereafter, the effect of a single injection of PD fluid on bacterial clearing was tested over time. Finally, an in vivo rat model was established to study the effects of long-term exposure to PD fluid on the peritoneal membrane and the local host defence (peritoneal cells). RESULTS: In the rat model, long-term daily exposure is possible. The 'drop-out' after 9-10 weeks on the most commonly used PD fluid Dianeal 3.86%, however, is approximately 50% due to omental wrapping. In the remaining study group, large differences were observed (as compared with controls), especially with respect to morphological parameters. CONCLUSIONS: The rat peritoneal continuous exposure model seems to have potential for intervention studies, since it uses no additions, no antibiotics and no omentomectomy, and gives continuous long-term exposure to PD fluid. However, problems still remain: 'drop-out' is quite often seen and this non-uraemic exposure model does not totally mimic the situation present in continuous ambulatory PD patients.

Effect of PD fluid instillation on the peritonitis-induced influx and bacterial clearing capacity of peritoneal cells.

BACKGROUND: The commonly used peritoneal dialysis fluids contain glucose as the osmotic agent. Heat sterilization leads to the formation of glucose degradation products which contribute, together with glucose, to the formation of advanced glycation end-products (AGEs). AGEs have been shown to be present in the peritoneal cavity. Methods have been developed to minimize the amount of glucose degradation products in peritoneal dialysis fluids. In a rat peritoneal dialysis model, we compare the effect of a commonly used peritoneal dialysis fluid, Gambrosol, with a newly developed peritoneal dialysis fluid, PD-Bio, on the influx and functional capacity of the peritoneal cells after 2 weeks of peritoneal dialysis fluid instillation. METHODS: Three groups of animals were used: rats received daily infusion with 15 ml of either 4% Gambrosol (group 1) or 4% PD-Bio (group 2), and a control group of animals did not receive fluid (group 3). After 2 weeks of PD fluid instillation, all the animals were injected with a 0.5 ml suspension containing 3x10(8) colony-forming units of Staphylococcus aureus. The in vivo bacterial clearing capacity was determined after 15 h. RESULTS: A statistically significant higher leukocyte influx was found in the control group compared with both PD fluid-injected groups. No statistical differences in bacterial clearing were observed among the three groups, although the number of bacteria recovered from the PD-Bio group tended to be lower than that from the Gambrosol group. Moreover, in both PD fluid instillation groups, the bacteria tended to be cleared more slowly compared with the control group. The number of mesothelial cells in the PD fluid groups was significantly greater than in the control group. CONCLUSION: No differences were observed in bacterial clearing capacity, leukocyte influx and mesothelial cell number after a 2 week exposure of the peritoneal cavity to Gambrosol vs PD-Bio.

Familial nephropathy differing from minimal change nephropathy and focal glomerulosclerosis.

BACKGROUND: Nephrotic syndrome in childhood is mainly due to minimal change nephropathy. In general, it is characterized by selective proteinuria, by steroid responsiveness, and histologically by podocytic foot process effacement. Familial presentation is rare and mainly restricted to one generation. METHODS: We describe the occurrence of a familial nephropathy in a mother and two daughters. An initial diagnosis of minimal change nephropathy was made, but subsequently unique features became apparent. During follow-up, detailed studies of renal function and urinary protein excretion were performed. Available frozen renal biopsy material was revised and processed for immunofluorescence to detect abnormalities in the expression of heparan sulfate proteoglycans. The latter results were compared with renal biopsies of a control group composed of five adult patients with minimal change nephropathy. RESULTS: The mother and two daughters were proteinuric since their early childhood. The mother revealed a persistent nephrotic syndrome for more than 20 years despite treatment with various immunosuppressive drugs. Likewise, treatment with prednisone was ineffective in the daughters. All three patients retained normal renal function during follow-up. Detailed measurements revealed that the proteinuria was incredibly selective (selectivity index approximately 0.01), and there was no evidence of tubulointerstitial damage, as reflected by a normal excretion of the low-molecular weight proteins beta(2)-microglobulin and alpha1-microglobulin. Renal biopsy performed in the mother and one daughter was thought to be compatible with minimal change nephropathy. However, histologically, two remarkable findings were made. By electron microscopy, there was no evidence of foot process retraction; specifically, the foot process width and slit pore diameter were normal. Furthermore, in contrast to the control patients, the expression of heparan sulfate polysaccharide side chains, as reflected by the staining with monoclonal antibody JM403, was normal. CONCLUSIONS: We propose that this family represents a new familial nephropathy. The molecular basis of the permeability defect remains to be identified.

ACE inhibition preserves heparan sulfate proteoglycans in the glomerular basement membrane of rats with established adriamycin nephropathy.

The gradual onset of the antiproteinuric effects of ACE inhibition suggests that structural effects on the glomerular basement membrane (GBM) may be involved in their renoprotective action. To test this hypothesis, we studied the effects of lisinopril (5 mg/kg/24 h) on proteinuria, focal glomerulosclerosis (FGS) and glomerular heparan sulfate (HS) proteoglycan (HSPG) GBM staining in rats with established Adriamycin nephrosis. Treatment was started 6 weeks after disease induction. As expected, lisinopril reduced blood pressure, proteinuria and the FGS score. In control rats, Adriamycin nephrosis was associated with significantly impaired GBM staining for both HSPG core protein (assessed from BL-31 staining) and HS staining (assessed from JM-403 staining) 12 weeks after disease induction. In rats treated with lisinopril (5 mg/kg/24 h) GBM staining was significantly better preserved for HS as well as for HSPG core protein. These data suggest that structural effects on the GBM, improving glomerular permselectivity, may be involved in the renoprotective effects of ACE inhibition in proteinuria-induced renal damage.

Accumulation of omental mast cells during peritoneal dialysis.

OBJECTIVE: New vessel formation has been reported in various tissues during peritoneal dialysis (PD). In that line, mast cells can produce factors such as tryptase, chymase, or basic fibroblast growth factor that might contribute to the formation of new vessels. In the present study, the association of mast cells with neovascularization during PD was investigated. METHODS: Rats received daily 10 mL infusions of conventional 3.86% glucose-containing PD fluid over a 10-week period. The infusions were delivered through a subcutaneously implanted mini access port that was connected by catheter to the peritoneal cavity. Untreated rats served as a control group. The number of blood vessels and of mast cells in the omentum were counted. Also, the number of peritoneal mast cells was determined. RESULTS: Chronic exposure to PD fluid resulted in an increased number of mast cells in the omentum. However, no clear correlation was found between the elevated number of omental blood vessels and the number of mast cells in the omentum or in the peritoneal cavity. CONCLUSIONS: Omental mast cells accumulated dramatically upon exposure to PD fluid. The actual role of accumulated omental mast cells in the induction of angiogenesis during PD should, however, be further investigated.

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies.

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Glomerular heparan sulfate alterations: mechanisms and relevance for proteinuria.

Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.

Agrin is a major heparan sulfate proteoglycan accumulating in Alzheimer's disease brain.

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimer's type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.