Sites In Glomerular Basement Membrane Research Articles

ストレプトゾトシン糖尿病ラットにおける腎糸球体基底膜pore sizeおよびanionic sitesの経時的変化について

ストレプトゾトシン糖尿病ラットにおける腎糸球体基底膜pore sizeおよびanionic sitesの経時的変化について

Ultrastructural Study of Anionic Sites in Glomerular Basement Membranes at Different Perfusion Pressures by Quick-Freezing and Deep-Etching Method

The ultrastructures of anionic sites in rat glomerular basement membranes (GBM) were studied at different perfusion pressures of 100, 150, 200 or 250 cm H₂O by a quick-freezing and deep-etching (QF-DE) method, in addition to conventional fixation methods, using polyethyleneimine (PEI) as a cationic tracer. By the QF-DE method, three-dimensional ultrastructures at each pressure were observed more clearly than those seen on conventional ultrathin sections. When the perfusion pressures were changed from low levels to higher ones, the total thickness of GBM with anionic sites became gradually thinner. Many PEI particles were observed around filaments, not only in the laminae lucidae, but also in the lamina densa. These findings indicated the existence of anionic sites in both laminae lucidae and lamina densa of GBM. The numbers of PEI particles in the lamina rara externa were counted on conventional ultrathin sections for morphometric analyses. The numbers per unit length of GBM were significantly decreased at higher perfusion pressures (200 and 250 cm H₂O) than those seen at both normal (150 cm H₂O) and lower (100 cm H₂O) pressures. It is concluded that the ultrastructures of anionic sites in the GBM may be changed in vivo, depending on the hemodynamics in the glomerular capillary.

Reevaluation of anionic sites in the rat glomerular basement membrane.

We reevaluated the distribution of anionic sites in the glomerular basement membrane with ruthenium red, alcian blue, cationic ferritin, polyethyleneimine, and cationic colloidal gold. Without the accentuation by the metal staining with uranyl acetate and lead citrate, we observed the existence of anionic sites throughout all the layers of the glomerular basement membrane with a similar reticular pattern and the structural difference between the lamina rara and lamina densa, indicating that the lamina rara is an actual structural element.

Composition of the peritrophic matrix of the tsetse fly, Glossina morsitans morsitans.

The three-layered peritrophic matrix of Glossina morsitans morsitans is shown, by histochemistry, to be formed of a mixture of glycosaminoglycans, glycoproteins and chitin. In all three layers the glycosaminoglycans contain GlcNAc-hexuronic and Gal-GlcNAc moieties, together with chitin. Glycosaminoglycans in layer 3 are sulphated and sulphated sites have a mean interspace distance of 53 nm - similar to the spacing of fixed charge sites in glomerular basement membrane suggesting a rôle for these sites in the filtration properties of the peritrophic matrix. O-linked oligo- saccharides are present in all three layers. Layer 1 contains the widest variety of glycoprotein oligosaccharide constructs GlcNac and alphalinked GalNAc possibly as GalNAcalpha1,3GalNAc, the latter apparently distal to Galbeta1-4GlcNAc. Lectin binding suggests that layer 2 contains GalNAcalpha1,3Galbeta1,3GlcNAc and that layer 3 contains GalNAc and Galbeta1,4GlcNAc. The evidence for N-linked oligosaccharides is more equivocal. Two dimensional electrophoresis showed that the peritrophic matrix contains a range of proteins, most of which require relatively harsh treatment for their solubilization.

Anionic sites in glomerular basement membrane of rats with serum sickness nephritis: quick-freezing and deep-etching study.

Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.

Studies of anionic sites of the kidney by re-injection of polyethyleneimine (PEI) after disappearance of PEI-particles by initial administration

As anionic sites of glomerular basement membrane (GBM) stained by intravenous injection of PEI became obscure 2 hours after administration, we studied reappearance of the anionic sites upon re-injection of PEI.The following two experimental groups were prepared: In group A; 10 rats were injected with 0.3ml of 0.5% PEI (MW=1,800) solution, and left nephrectomy was performed after 3 hours (group A-1, N=5) and 5 hours (group A-2, N=5). Each tissue samples was subjected to contrasting/fixation immersion, washing, post-fixation and staining. Immediately after nephrectomy, re-administration of 0.3ml of the same PEI solution was performed on each animal. Right nephrectomy was done 15 minutes after re-injection of PEI, and renal tissue was treated the same way as that of the left kidney (group A-1', group A-2'). In group B; PEI with a molecular weight of 70,000 was used. Injection of PEI solution, nephrectomy and treatment of renal tissues were carried out in the same way as for group A (group B-l and B-l', N=6; group B-2 and B-2', N=8).

Analysis of anionic charge of the glomerular basement membrane in aminonucleoside nephrosis

Although there is growing evidence that suggests the importance of the electrostatically charged barrier in the transglome-rular permeation of macromolecules, a controversy still exists as to whether or not the anionic charge barrier is deteriorated in nephrotic states [1–5]. The disparity of opinions seems to arise from the paucity of established methods for quantitative evaluation of the anionic charge. In the present study, a method to morphologically demonstrate the anionic sites in glomerular basement membrane (GBM) was developed, and anionic charge was quantified. With this method, the change of the charge barrier was estimated in puromycin aminonucleoside (PAN) nephrosis in rats.

Glomerular sieving of high molecular weight proteins in proteinuric rats

To characterize the permeability of the glomerular capillary wall to high molecular weight proteins in normal and proteinuric rats, we determined the glomerular sieving coefficients (GSC) of radioiodinated marker proteins of known size and charge by means of a paired label, tissue accumulation method previously validated in this laboratory. In one group of rats (Series A) the GSCs of 125I-anionic IgG (aIgG-molecular weight [mol wt] 150,000, pI 4.9) and 131I-neutral IgG (nIgG-pI 7.4 to 7.6) were measured simultaneously. In Series B, the GSC of a second anionic marker, 131I-human ceruloplasmin (Crp-mol wt 137,000, pI 4.9) was compared to that of 125I-nIgG. As in the previous report, the labeled proteins were not degraded or deiodinated during the 20 minute clearance period for GSC determination. Within Series A and B, three subgroups of rats were studied: control saline-infused rats, rats made acutely proteinuric by infusion of the polycation hexadimethrine (HDM), and rats with chronic doxorubicin (Adriamycin-Adria) nephrosis. In the control rats, GSCs for the anionic markers aIgG (Series A) or Crp (Series B) were significantly greater than that of nIgG (both series). These large proteins crossed the filtration barrier by a different pathway from that available to smaller neutral molecules the size of albumin, which in our previous study had a much higher GSC than a native, anionic albumin marker. In a third group of control rats only (Series C), the GSCs of native anionic bovine albumin (BSA) and nIgG were compared directly. The GSC of BSA (0.0029) was only slightly larger than the GSC of nIgG (0.0025), indicating that most of the native albumin crosses the glomerular capillary wall via a nonselective pathway similar to that available to nIgG. The results in the control groups are compatible with recently-described heteroporous models of glomerular size selectivity. In the HDM proteinuric rats of both series, the filtration of all three markers was significantly increased, although the GSCs of the anionic markers continued to exceed that of nIgG. The finding of increased nIgG filtration in HDM rats confirms our previous observation that HDM, although it binds to and neutralizes anionic sites in glomerular basement membrane, induces a structural change which leads to altered pore size distribution. Adria rats also had increased GSCs of all markers, although the increase was not as great as in HDM rats, and the GSCs of the anionic and neutral markers became equal.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of immune complexes with glomerular heparan sulfate–proteoglycans

The binding characteristics of cationic and more neutral immune complexes with heparan sulfate-proteoglycan enriched anionic sites of glomerular basement membrane and mesangial matrix were studied. Rat kidneys were treated either with buffers alone or buffers containing heparitinase or chondroitinase-ABC followed by perfusion with cationic or native immune complexes. Tissues were processed for immunofluorescence and transmission electron microscopy after fixation with glutaraldehyde or tannic acid glutaraldehyde. Kidneys perfused with radioiodinated immune complexes were processed for light and electron microscopic autoradiography. In addition, glomeruli from kidneys perfused with radioiodinated immune complexes were isolated and counted for radioactivity. By immunofluorescence the cationic immune complexes deposited linearly along the glomerular basement membrane. By electron microscopy, the cationic complexes localized mainly in the inner and outer layers of the glomerular basement membrane and to a certain extent in the mesangial matrix in a distribution that corresponded to previously documented anionic sites. Whereas heparitinase treatment abrogated the binding of cationic immune complexes in both glomerular basement membrane and mesangial matrix, chondroitinase-ABC treatment did not cause any decrease in binding. In contrast, more neutral immune complexes appeared to be nonspecifically trapped in the mesangium, and their distribution was unaffected by both enzymatic treatments. Light and electron microscopic autoradiography and counts of isolated glomeruli confirmed these findings. The results overall indicate that cationic immune complexes bind electrostatically to the heparan sulfate-proteoglycan enriched anionic sites of the glomerular basement membrane and mesangial matrix, while more neutral immune complexes are nonspecifically trapped in the mesangium of the renal glomerulus.

Unaltered anionic sites of glomerular basement membrane in aminonucleoside nephrosis

AbstractUnaltered anionic sites of glomerular basement membrane in aminonucleoside nephrosis. Quantitative electron microscopic autoradiography was employed to determine the changes in the binding of125I-cationic ferritin (CFI, pI ∼ 7.2 to 7.4) to the anionic sites of the glomerular basement membrane (GBM) of rats following the induction of nephrosis. Animals were rendered nephrotic by a single intravenous injection of puromycin aminonucleoside (PAN) and sacrificed 0, 7, 14, and 21 days after its administration. CFI (10 mg/mCi/100 g body weight) was given intravenously. The kidneys were subsequently fixed by perfusion and processed for electron microscopic autoradiography. The mean grain densities over the GBMs on 0, 7, 14, and 21 days of PAN nephrosis were 1.50 ± 0.04, 1.49 ± 0.05, 1.50 ± 0.05, and 1.51 ± 0.04, respectively. These results indicate that there are no significant alterations in the anionic sites rich in heparan sulfate proteoglycan during the entire course of PAN nephrosis.Non-altération des sites anioniques de la membrane basale glomérulaire dans la néphrose aux aminonucléosides. L'autoradiographie quantitative en microscopie électronique a été utilisée pour déterminer les modifications de la liaison de la ferritine125I cationique (CFI, pI ∼ 7,2–7,4) aux sites anioniques de la membrane basale glomérulaire (GBM) de rats, après induction d'une néphrose. Les animaux ont été rendus néphrotiques par une injection unique intraveineuse de puromycine aminonucléoside (PAN) et sacrifiés 0, 7, 14, et 21 jours après son administration. CFI (10 mg/mCi/100 g poids corporel) a été donné par voie intraveineuse. Les reins ont ensuite été fixés par perfusion, et préparés pour autoradiographie en microscopie électronique. Les densités granulaires moyennes sur les GBM aux jours 0, 7, 14, et 21 dans la néphrose à PAN étaient de 1,50 ± 0,04, 1,49 ± 0,05, 1,50 ± 0,05, et 1,15 ± 0,04, respectivement. Ces résultats indiquent qu'il n'y a pas d'altérations significatives des sites anioniques riches en proteoglycan héparine sulfate pendant la totalité de l'évolution de la néphrose par PAN.

Platelet factor 4 binding to glomerular microvascular matrix

Platelet factor 4, a unique peptide released during platelet aggregation, can bind to natural sulfated glycosaminoglycans from human renal cortex. The glycosaminoglycan isolate contained components sensitive to hyaluronidase, chondroitinase ABC and nitrous acid. Binding was demonstrated by the change in electrophoretic mobility of platelet factor 4 applied in combination with glycosaminoglycan compared to either applied alone. This effect, which occurred at physiologic pH but not at acidic pH or with high ionic strength, was preserved in samples subjected to prior hyaluronidase and chondroitinase digestion. Further demonstration that platelet factor 4 can interact with heparan sulfate anionic sites in the glomerular microvascular matrix was obtained by incubating radiolabeled platelet factor 4 with isolated rat glomeruli, and with purified human and rat glomerular basement membrane, followed by displacement with heparin. Total binding and heparin-released binding were decreased in glomerular basement membrane prepared from diabetic patients and from rats with streptozotocin-diabetes compared to control samples. These findings implicate platelet factor 4 in the pathogenesis of the altered capillary integrity associated with diabetes, and provide novel evidence that heparan sulfate anionic sites in glomerular basement membrane are diminished in human and experimental diabetes.

Suppression of proteinuria by dipyridamole in rats with aminonucleoside nephropathy.

Nephrosis was induced by single injections of puromycin of aminonucleoside to rats which were divided into two groups; the experimental group receiving dipyridamole in addition to aminonucleoside and the control group receiving aminonucleoside alone. 24-hour urinary albumin increased in rats of both groups after aminonucleoside injections. However, the experimental rats excreted significantly less albumin than controls. On the contrary, there was no significant difference in urinary IgG between the two groups. The stainings of anionic sites of glomerular basement membrane using ruthenium red revealed the reduction of anionic charge in the glomerular basement membrane of both groups, but the decrease of anionic charge was suppressed in lamina rara interna of the experimental rats. Considering the role of charge barrier in the glomerular basement membrane, it is concluded that the maintenance of anionic charge in experimental rats is causally related to the suppressed excretion of albumin.

Heparan sulfate--rich anionic sites in the human glomerular basement membrane. Decreased concentration in congenital nephrotic syndrome.

Recent work suggests that the normal barrier to penetration of the renal glomerular basement membrane by anionic plasma proteins may depend in part on the existence of negatively charged sites within the membrane. We describe an in vitro cytochemical method for the quantitative demonstration of anionic sites in the normal human glomerular basement membrane. In five normal subjects, ranging in age from 10 days to 57 years, the sites were distributed at regular intervals in the lamina rara externa, with a frequency of 23.8 +/- 6.8 sites per 1000-nm length of membrane. A similar distribution was observed in the basement membranes from three normal human fetuses. Ex vivo perfusion of one cadaver kidney revealed a similar distribution of anionic sites. The number of anionic sites in the glomerular basement membranes of five patients with the congenital nephrotic syndrome was reduced to 8.9 +/- 3.7 (P less than 0.001). Prior incubation of sections of normal kidney in purified heparinase resulted in a marked reduction in the number of anionic sites. We conclude that congenital nephrosis results from failure of heparan sulfate--rich anionic sites to develop in the lamina rara externa of the glomerular basement membrane.

Distribution of annionic sites in glomerular basement membranes: their possible role in filtration and attachment.

Lysozyme (pI = II) has been used to identify anionic sites in the glomerular capillary wall. A solution of 1-3% lysozyme was perfused into the left kidney at varying rates. After perfusion, the kidney was fixed in situ and processed for electron microscopy. Lysozyme was seen as an electron-dense deposit which was not present when succinylated lysozyme (pI = 4.5) or myoglobin (pI = 6.9) was perfused instead of native lysozyme. First, the epithelial cell plasma membrane was outlined by a 300-400 A electron-dense layer. Second, there were discrete dense deposits in the subepithelial portions (lamina rara externa) of the basement membrane which, in normal sections, extended from the epithelial cell membrane to the lamina densa and, in grazing section, formed a continuous reticular pattern.