Rat Glomerular Basement Membrane Research Articles

The Nephritogenic Glycopeptide from Rat Glomerular Basement Membrane. X. Synthesis of an N-Triglycosyl Dipeptide and Characteristics of Its cis-trans Isomers.

Isomers of O-alpha-D-glucopyranosyl-(1----6)-O-beta-D-glucopyranosyl- (1----6)-N-[L-aspart-1-oyl-(L-proline)-4-oyl]-alpha-D-glucopyranos ylamine have been prepared, as models for a derivative possibly present in the glomerular basement membrane of rats, by condensation of the corresponding dipeptide derivative (5) with triglycosylamine (4) in the presence of O,O-diethylcyanophosphonate, followed by deprotection of the trisaccharide-dipeptide derivative. During the deprotection process, cis- and trans-isomers containing proline were separated by silica gel column chromatography and also reversed-phase high performance liquid chromatography.

腎糸球体基底膜分子篩の超微構造

Electron microscopic studies with negative staining showed that bovine and rat glomerular basement membranes and rat tubular basement membrane were meshwork composed of fibrils and had numerous pores equal in diameter. The diameter of the pores in glomerular basement membrane was less than the minor axis of an albumin molecule. Therefore, the meshwork of the glomerular basement membrane works as a molecular sieve for ultrafiltration in the glomerulus. The meshwork structure could also be demonstrated by means of the ultrathin sectioning method after specimens were treated with enzymes. The molecular sieve is thus presumed to be a true structure in vivo. Proteinuria in nephrotic syndrome may be caused by the breakage of the molecular sieve.

Soluble Immune Complexes Stimulate Production of Interleukin-1 by Cultured Rat Glomerular Mesangial Cells

Interleukin-1 (IL-1) is a pleiotropic factor, eliciting a broad set of immunologic and inflammatory events. We have previously shown that IL-1 is present in inflamed glomeruli. To evaluate factors that might regulate of IL-1 production, we tested the effects of substances accessible to mesangial cells (MC): immune complexes (IC) are known to modulate MC function. We have attempted to assess the ability of soluble IC derived from rat glomerular basement membrane to induce the production of IL-1. When isolated MC were incubated with the soluble IC, substantial amount of IL-1 could be detected in the supernatants as measured by the mouse thymocyte assay. To block the effect of prostaglandins on the IL-1 assay, we cultured the MC with the addition of indomethacin and assayed IL-1 activity in the culture supernatants. The use of indomethacin resulted in a further increase in IL-1 production. These biological activities were neutralized by a specific antibody to IL-1. In the present report, we show that IC represent important sources of stimulation of MC for the production of IL-1. We speculate that IC could augment local inflammatory responses in the kidney partly due to their capacity to induce the production of IL-1.

Experimental Autoimmune Glomerulonephritis Induced by Homologous and Isologous Glomerular Basement Membrane in Brown-Norway Rats

In order to study disease mechanisms and potential forms of therapy in glomerulonephritis, a model of experimental autoimmune glomerulonephritis (EAG) has been developed in the rat. We have examined the response of Brown-Norway (BN) rats to a single i.m. injection of collagenase-solubilised homologous (Sprague-Dawley, SD) or isologous (BN) glomerular basement membrane (GBM), with and without complete Freund's adjuvant (CFA). There was a dose-dependent circulating anti-GBM antibody response to all preparations of rat GBM. Animals given either antigen alone at a dose of 2 mg/kg developed circulating anti-GBM antibodies, which reached peak values by 6 weeks (63 +/- 5% following SD GBM; 53 +/- 8% following BN GBM), but did not develop glomerular deposits of IgG or nephritis. Animals given 2 mg/kg SD GBM in CFA developed greater concentrations of anti-GBM antibody by 6 weeks (122 +/- 20%) together with linear deposits of IgG on glomerular and tubular basement membranes (TBM), albuminuria (mean 7 mg/24 h), and variable focal segmental necrotising glomerulonephritis with mild interstitial nephritis. The same dose of BN GBM in CFA produced similar concentrations of circulating antibody (144 +/- 26%), with linear deposits of IgG on GBM but rarely TBM, little albuminuria, and variable mild focal glomerulonephritis. Other strains injected with SD GBM in CFA showed a variable circulating anti-GBM antibody response, which was similar to that of BN rats in PVG and DA rats but lower in LEW and WAG rats. Linear deposits of IgG on the GBM were detected in a proportion of PVG and DA rats, but not in LEW or WAG rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional architecture of rat glomerular basement membrane by ultra-high resolution scanning electron microscopy.

We demonstrated the ultrastructure of rat glomerular basement membrane (GBM) by ultra-high resolution scanning electron microscopy. GBM prepared by sonication methods and conductive-staining could be observed without metal coating at magnifications as high as 400,000 times. The GBM showed an irregular meshwork structure composed of various strands and pores. The width of the strands ranged from 6 to 15 nm, and the diameter of pores ranged from 6 to 50 nm. The present study confirmed our molecular sieve theory of the basement membrane.

Effects of leflunomide on glomerulonephritis induced by antibasement membrane antibody in rats

Leflunomide (HWA 486, a novel isoxazol derivative), shown to have potent immunosuppressant and antiinflammatory effects, was evaluated for its inhibitory and therapeutic effects on the glomerulonephritis induced in rats by rabbit antiserum against rat glomerular basement membrane. Leflunomide was administered orally to rats at 0.5 and 2 mg/kg/day for 20 days from 2 days before injection of the rabbit antiserum and at 2 mg/kg/day for 14 days from 5 days after the antibody injection. The present study consisting of 2 experiments for inhibitory (I) and therapeutic (II) effects of leflunomide revealed the following effects at 2 mg/kg: in experiment I, significant decreases in (a) urinary total protein, (b) plasma total cholesterol and fibrinogen and (c) thymus weight, and decreased incidences of fibrin deposits in Bowman's space, adhesion of the glomerulus to Bowman's capsule and deposition of rat IgG and C3; and in experiment II, decreases in (a), (b) and (c), though smaller than in experiment I, and decreases deposition of rat C3. Thus, leflunomide had potent inhibitory and limited therapeutic effects on glomerulonephritis, suggesting that the compound is effective in inhibiting the onset and development of glomerulonephritis.

Synthesis of the α and β anomer of an N-triglycosyl dipeptide

O-α- d-Glucopyranosyl-(1 → 6)- O-β- d-glucopyranosyl-(1 → 6)-1- N-[ l-aspart-1-oyl-( l-proline)-4-oyl]-α- and -β- d-glucopyranosylamine have been prepared, as models for a derivative possibly present in the glomerular basement membrane of rats, by condensation of the corresponding d-glucosyl-dipeptide derivatives with 2, 3, 4, 2′, 3′, 4′, 6′-hepta- O-acetyl-α- d-isomaltopyranosyl bromide in the presence of mercuric cyanide, followed by deprotection of the trisaccharide-dipeptide derivatives.

Effects of gemtamicin (G) on development of rat glomerular basement membrane (6BM)

Effects of gemtamicin (G) on development of rat glomerular basement membrane (6BM)

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [ 3H]-amino acids and [ 35S]sulfate. Two heparan- 35SO 4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan- 35SO 4 proteoglycan eluted with the column void volume and at a K av of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan- 35SO 4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B K av 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan- 35SO 4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan- 35SO 4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan- 35SO 4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan- 35SO 4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan- 35SO 4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B K av 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963–970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.

Glomerular platelet-activating factor levels and origin in experimental glomerulonephritis

The content of platelet activating factor (PAF) in glomeruli isolated from rats with nephrotoxic serum glomerulonephritis (NSGN) was quantified at various stages of the disease and the role of complement, platelets and neutrophils in mediating changes in glomerular PAF levels was evaluated. PAF content was assessed following extraction, isolation and quantification of this alkyl ether lipid using a bioassay based on [3H]-serotonin release from labelled rabbit platelets. Following induction of NSGN using proteinuric doses of rabbit immune serum raised against rat glomerular basement membrane, enhanced glomerular PAF levels were observed at 3 hours, 24 hours and on day 15 following induction of the disease. In complement depleted rats and at three hours following induction of NSGN, glomerular PAF levels were significantly lower than in complement replete controls studied in parallel. At the same time point of the disease, platelet depleted rats with NSGN demonstrated significantly lower glomerular PAF levels than parallel controls, whereas in neutrophil depleted rats glomerular PAF levels were no different than controls. These observations indicate that in infiltrative and complement dependent forms of glomerular immune injury, glomerular PAF levels are increased via a complement mediated mechanism. Infiltrating platelets, but not neutrophils, partially account for the enhanced glomerular PAF levels. The observations are of potential importance in the pathophysiology of glomerulonephritis.

Effects of antithrombin III on glomerulonephritis induced by antibasement membrane antibody in rats.

Antithrombin III (AT III), a plasma inhibitor of coagulation factors, was evaluated for its inhibitory effects on the development of glomerulonephritis induced by antibasement membrane antibody in rats. AT III was administered intravenously at 250 and 1000 Units/kg to rats once a day for 14 days after intravenous injection of rabbit antiserum against the rat glomerular basement membrane. In the rat model, the high dose of AT III reduced the urinary total protein, plasma total cholesterol and plasma phospholipid, and tended to lower the incidence of fibrin deposits in Bowman's space, cell proliferation in the glomeruli, and PAS-positive granules (absorption droplets) in the glomerular epithelium. The above results suggest that AT III is effective in inhibiting the development of glomerulonephritis.

Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (king cobra)

Nget Hong Tan and M. N. Saifuddin. Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (king cobra). Toxicon 28, 385–392, 1990.—The major hemorrhagin (termed hannahtoxin) of the venom of Ophiophagus hannah (king cobra) was purified to electrophoretic homogeneity by DEAE-Sephacel ion exchange chromatography, Sephadex G-200 gel filtration followed by a second DEAE-Sephacel chromatography. Proteolytic activity was associated with the hemorrhagic activity throughout the purification procedures. Hannahtoxin constituted approximately 2% of the crude venom. It had an isoelectric point of 5.3, a carbohydrate content of 12%, a mol. wt of 66,000 as determined by SDS-polyacrylamide gel electrophoresis and 63,000 as determined by gel filtration. It contains 1 mole of Zn per mole of protein. The minimum hemorrhage doses for hannahtoxin are 0.7 μg and 75 μg, respectively, in rabbits and in mice. Hannahtoxin was not lethal to mice at a dose of 2 mg/kg (i.v.) but killed rabbits at doses above 0.18 mg/kg (i.v.). It liberated protein from rabbit glomerular basement membrane but not rat glomerular basement membrane. Treatment of the hemorrhagin with EDTA and 1,10-phenanthroline eliminated both the proteolytic and hemorrhagic activities completely.

Renal antigens in mercuric chloride induced, anti-GBM autoantibody glomerular disease

Two antibody probes were used to characterize the putative renal antigens of HgCl2-induced antiglomerular basement membrane renal disease in Brown Norway (BN) rat. The first probe was the linear immunofluorescence imparting, in vivo bound, nephritogenic antiglomerular-basement-membrane autoantibody (anti-GBM-Ab). The second probe was a rat monoclonal antibody to the B subunit of laminin that was obtained from fusion of spleen cells of HgCl2 injected BN rat. By enzyme-linked immunosorbent assay (ELISA) the anti-GBM-Ab reacted with laminin, type IV collagen, collagenase-resistant noncollagenous portion of glomerular basement membrane (GBM), saline soluble proteins of kidney cortex homogenate and fibronectin. Western blot analysis of laminin indicated that the reactive epitopes detected by both probes were on the B chain subunit but not the A subunit. In nonreduced collagenase-digested GBM the epitopes were present on 27 kD and 42 to 48 kD polypeptides. A similar pattern was seen on collagenase-digested human GBM. On rat and human GBM the patterns obtained with rat autoantibody and autoantibody from a patient with Goodpasture syndrome were similar, suggesting that some of the in vivo bound anti-GBM autoantibodies in HgCl2-induced disease in rat are directed against epitopes which are similar to the Goodpasture antigen of human. Reactive epitopes were also detected on saline soluble proteins of kidney cortex homogenate with the predominant antigen being a 31 kD polypeptide. In the saline soluble proteins the reactive polypeptides including the major 31 kD polypeptide did not originate from laminin, type IV collagen, or the collagenase-resistant noncollagenous part of GBM. The precise structural origin of soluble proteins was not defined.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclosporine on puromycin aminonucleoside-induced nephrotic rats.

The effects of cyclosporine (CY) were determined to see whether CY has the potency of reducing urinary protein excretions due to puromycin aminonucleoside (PAN) in nephrotic rats which are the model of minimal change nephrotic syndrome (MCNS). CY had a marked reduction of urine protein excretion during treatment with this drug, but it was observed that an increment in urinary protein excretion was introduced within a few days after the withdrawal of CY. Immunological and histological studies were performed to know the mechanisms of reducing urinary protein excretion in PAN rats. Firstly, the lymphocyte subsets of peripheral blood were evaluated using monoclonal antibodies of W3/25 and MRCOX/8. We found that the W3/25/MRCOX-8 ratio increased to 3.2 +/- 0.3 in the PAN rats but significantly decreased to 2.8 +/- 0.3 with CY treatment. From these data it was impossible to decide whether the effect with CY had direct activity or not, because the interleukin-2 (IL-2) activity was not measured in rats. Secondly, polyethyleneimine (PEI) staining was performed to detect the sites of anion charge on glomerular basement membrane (GBM), and anionic dots on GBM in the CY-treated rats were clearly defined as nearly normal. Although there persist questions as to why nephrotic rats relapsed after the withdrawal of CY treatment, our results indicate that CY might reduce urinary protein excretion through immunological and non-immunological mechanisms.

Effect of prednisone on nephrotic peripheral blood mononuclear cell mediated increase in 35sulfate uptake in rat glomerular basement membrane.

We have previously described a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal-lesion nephrotic syndrome (IMLNS) in relapse, but not with PBMC of IMLNS patients in remission. In the present study we examined the effect of prednisone therapy on the PBMC-mediated increase in 35sulfate GBM uptake. The GBM 35sulfate uptake after rat glomeruli were cocultured with PBMC from 11 IMLNS patients in relapse (geometric mean 437 cpm/mg dry glomerular weight) was significantly higher than the incorporation observed in glomeruli cultured alone (geometric mean 229 cpm/mg dry glomerular weight; p less than 0.01). However, no significant differences in 35sulfate uptake were seen between glomeruli cultured alone and glomeruli cocultured with PBMC from IMLNS patients when PBMC were obtained from the 11 patients on treatment with prednisone (2 mg/kg/day) or the same patients in remission and off prednisone therapy. Prednisone therapy abolished the PBMC-mediated increased 35sulfate uptake by rat GBM. GBM sulfated compounds seem to play a role in glomerular permeability. The temporal relationship between inhibition of GBM sulfate incorporation by prednisone and resolution of the proteinuria support the hypothesis that PBMC from IMLNS patients in relapse could secrete a lymphokine which by altering the metabolism of the GBM sulfated compounds may subsequently increase glomerular permeability to plasma proteins.

Isolation, Characterization and Immunological Determination of Basement Membrane-Associated Heparan Sulfate Proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.

Molecular sieve of the rat glomerular basement membrane: a transmission electron microscopic study of enzyme-treated specimens.

Isolated rat glomerular basement membrane was treated with elastase and observed by transmission electron microscopy. The treatment with elastase revealed the fundamental structure of the glomerular basement membrane quite clearly, and enabled the observation of a sieve structure within the glomerular basement membrane. This sieve structure may play a major role in the filtration of blood as well as in the production of urine. Treatment with antibody showed that the sieve was mainly constituted of type IV collagen.

Effect of puromycin aminonucleoside on HSPG core protein content of glomerular epithelial cells.

It has been suggested that the glomerular basement membrane heparan sulfate proteoglycan (HSPG) is an important determinant of the glomerular permselectivity barrier. Derangements in the content of basement membrane heparan sulfate have been implicated in alterations in glomerular permselectivity seen in many glomerular diseases such as aminonucleoside nephrosis. The cellular origin and metabolism of the glomerular basement membrane HSPG have not been studied in detail. We have detected the expression of the proteoglycan by cloned glomerular visceral epithelial cells of the rat by employing a specific antibody against the core protein of HSPG isolated from the rat glomerular basement membrane. These findings suggest that in the rat in vivo glomerular visceral epithelial cells are one source of heparan sulfate present in the glomerular basement membrane. The effect of puromycin aminonucleoside (PAN) on the HSPG core protein content of the cloned glomerular epithelial cells was studied. By a quantitative immunoperoxidase method, the aminonucleoside caused a 28% reduction in the core protein content of the epithelial cells (P less than 0.01) following 72 h of incubation. However, the content of Heymann nephritis-related antigen, Fx1A was unchanged. Studies employing [3H]leucine incorporation showed that PAN was a weak inhibitor of de novo protein synthesis at 24 h of incubation, with complete recovery at 48 and 72 h. These data suggest that PAN effect on heparan sulfate core protein cannot be attributed to generalized inhibition of protein synthesis. The precise mechanism underlying the aminonucleoside effect on heparan sulfate core protein remains to be elucidated.

Nephritogenicity and immunocytochemical localization of the 70-kilodalton glycoprotein subunit (gp70) of heymann antigen

Heymann nephritis (HN) is an experimentally induced glomerulonephropathy of the rat characterized by subepithelial immune deposits and proteinuria. Immunization with a complex multimeric glycoprotein, gp600, comprising four subunits gp330, gp140, gp110, and gp70 has been shown to induce the complete form of the disease including proteinuria. Examination of three different batches of heterologous anti-gp600 antisera by immunoblot technique showed that the reactivity toward gp70 was dominant and common to all three antisera. gp70 was isolated from Triton X-100-solubilized Fx1A by lectin Lens culinaris affinity chromatography, and the purity was confirmed by SDS-PAGE. Ten rats were actively immunized with 200 μg of gp70. All 10 animals developed circulating brush border antibody and typical granular IgG deposits in the glomerulus but only 1 10 animals developed abnormal proteinuria. A potent antiserum against gp70 was prepared in the rabbit. It reacted strongly to the glomerular capillary wall and the proximal tubular brush border by immunofluorescence. By Protein A immunogold technique using anti-gp70, gold particles were found associated with the glomerular basement membrane (GBM)-endothelial region. By immunoblot analysis of rat GBM using the same anti-gp70 antiserum, a 70-kDa cross-reactive antigen was demonstrated in GBM preparations. These results show that the smallest subunit, gp70 of the complete HN antigen, gp600 Fx1A can independently induce the lesion of HN, but without proteinuria. The presence of gp70 on the endothelial side of the GBM is consistent with a role for in situ antigen-antibody reactions at sites other than the subepithelial region in the pathogenesis of HN.

Changes in the molecular sieve of the glomerular basement membrane of rats with chronic serum sickness.

In order to explore the pathogenic mechanism of proteinuria in glomerulonephritis, ultrastructural changes of the glomerular basement membrane were investigated in rats with chronic serum sickness induced by repeated intravenous injections of bovine serum albumin (experimental rats). Rats injected with saline served as controls. The animals were sacrificed and examined 13 weeks after treatment, when the mean urinary protein of experimental animals reached 206 mg/24h/100g body weight. Enhanced transcapillary passage of anionic ferritin was observed in experimental rats. Purified glomerular basement membranes of control and experimental rats were examined by electron microscopy after negative staining. The glomerular basement membrane of experimental rats had enlarged pores. The results suggest that an increase in the radius of glomerular pores may be responsible for proteinuria in glomerulonephritis.