Hindered transport of macromolecules in isolated glomeruli. I. Diffusion across intact and cell-free capillaries.

The filtrate formed by renal glomerular capillaries must pass through a layer of endothelial cells, the glomerular basement membrane (GBM), and a layer of epithelial cells, arranged in series. To elucidate the relative resistances of the GBM and cell layers to movement of uncharged macromolecules, we measured the diffusional permeabilities of intact and cell-free capillaries to narrow fractions of Ficoll with Stokes-Einstein radii ranging from 3.0 to 6.2 nm. Glomeruli were isolated from rat kidneys, and diffusion of fluorescein-labeled Ficoll across the walls of single capillary loops was monitored with a confocal microscopy technique. In half of the experiments the glomeruli were treated first to remove the cells, leaving skeletons that retained the general shape of the glomerulus and consisted almost entirely of GBM. The diffusional permeability of cell-free capillaries to Ficoll was approximately 10 to 20 times that of intact capillaries, depending on molecular size. Taking into account the blockage of much of the GBM surface by cells, the contribution of the GBM to the diffusional resistance of the intact barrier was calculated to be 13% to 26% of the total, increasing with molecular size. Thus, the GBM contribution, although smaller than that of the cells, was not negligible. The structure that is most likely to be responsible for the cellular part of the diffusional resistance is the slit diaphragm, which spans the filtration slit between epithelial foot processes. A novel hydrodynamic model was developed to relate the diffusional resistance of the slit diaphragm to its structure, which was idealized as a single layer of cylindrical fibers in a ladder-like arrangement.     

Clogging of the glomerular basement membrane

The negative charges of the sulfated glycosaminoglycans (GAGs) of the glomerular basement membrane (GBM) were differentially neutralized by perfusin with high molarity buffers in order to determine whether or not these charges protect the GBM from being clogged by circulating plasma macromolecules. Progressive elimination of the negative charges resulted in clogging of the GBM by perfused native ferritin (NF) and bovine serum albumin as evidenced ultrastructurally by the increase in accumulation of NF in the GBM. In addition, the permeability of the GBM to 125I-insulin, a macromolecule which is normally freely permeable, and the glomerular filtration rate (as determined by [3H]inulin clearance) were markedly reduced after the GBM had been clogged with NF in the presence of high molarity buffer, thereby indicating that clogging severely reduces the ability of the GMB to act as a selective filter. These findings are consistent with the idea that the sulfated GAGs of the GBM serve as anticlogging agents.     

Virus morphology as an aid for rapid diagnosis.

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).     

Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

DDX6 localizes to nuage structures and the annulus of mammalian spermatogenic cells

The localization of DEAD (Asp-Glu-Ala-Asp) box helicase 6 (DDX6) in spermatogenic cells from the mouse, rat, and guinea pig was studied by immunofluorescence (IF) and immunoelectron microscopy (IEM). Spermatogenic cells from these species yielded similar DDX6 localization pattern. IF microscopy results showed that DDX6 localizes to both the nucleus and cytoplasm. In the cytoplasm of spermatogenic cells, diffuse cytosolic and discrete granular staining was observed, with the staining pattern changing during cell differentiation. IEM revealed that DDX6 localized to the five different types of nuage structures and non-nuage structures, including small granule aggregate and late spermatid annuli. Nuclear labeling was strongest in leptotene and zygotene spermatocytes and moderately strong in the nuclear pocket of late spermatids. DDX6 also localized to the surface of outer dense fibers, which comprise of flagella. The results show that DDX6 is present in nuage and non-nuage structures as well as nuclei, suggesting that DDX6 has diverse functions in spermatogenic cells.     

Organogenesis of the kidney glomerulus

The glomerular basement membrane (GBM) is a crucial component of the kidney’s filtration barrier that separates the vasculature from the urinary space. During glomerulogenesis, the GBM is formed from fusion of two distinct basement membranes, one synthesized by the glomerular epithelial cell (podocyte) and the other by the glomerular endothelial cell. The main components of the GBM are laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogen and the heparan sulfate proteoglycan, agrin. By studying mice lacking specific GBM components, we have shown that during glomerulogenesis, laminin is the only one that is required for GBM integrity and in turn, the GBM is required for completion of glomerulogenesis and glomerular vascularization. In addition, our results from laminin β2-null mice suggest that laminin-521, and thus the GBM, contribute to the establishment and maintenance of the glomerular filtration barrier to plasma albumin. In contrast, mutations that affect GBM collagen IV or agrin do not impair glomerular development or cause immediate leakage of plasma proteins. However, collagen IV mutation, which causes Alport syndrome and ESRD in humans, leads to gradual damage to the GBM that eventually leads to albuminuria and renal failure. These results highlight the importance of the GBM for establishing and maintaining a perfectly functioning, highly selective glomerular filter.     

Organogenesis of the kidney glomerulus: focus on the glomerular basement membrane

The glomerular basement membrane (GBM) is a crucial component of the kidney's filtration barrier that separates the vasculature from the urinary space. During glomerulogenesis, the GBM is formed from fusion of two distinct basement membranes, one synthesized by the glomerular epithelial cell (podocyte) and the other by the glomerular endothelial cell. The main components of the GBM are laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogen and the heparan sulfate proteoglycan, agrin. By studying mice lacking specific GBM components, we have shown that during glomerulogenesis, laminin is the only one that is required for GBM integrity and in turn, the GBM is required for completion of glomerulogenesis and glomerular vascularization. In addition, our results from laminin β2-null mice suggest that laminin-521, and thus the GBM, contribute to the establishment and maintenance of the glomerular filtration barrier to plasma albumin. In contrast, mutations that affect GBM collagen IV or agrin do not impair glomerular development or cause immediate leakage of plasma proteins. However, collagen IV mutation, which causes Alport syndrome and ESRD in humans, leads to gradual damage to the GBM that eventually leads to albuminuria and renal failure. These results highlight the importance of the GBM for establishing and maintaining a perfectly functioning, highly selective glomerular filter.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Glomerular extracellular matrices in rat diabetic glomerulopathy by scanning electron microscopy

Characteristic pathological changes in the glomeruli in diabetic nephropathy include expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM). Using an acellular digestion technique combined with scanning electron microscopy, the three-dimensional ultrastructural changes in glomerular extracellular matrices were studied in rats with diabetic glomerulopathy. Diabetes was induced by the intravenous injection of streptozotocin and morphological analyses were performed 3, 6 and 11 months after the injection. Expansion of mesangial area and GBM thickening became evident with time. After treatment with the series of detergents, all cellular components were completely removed leaving the extracellular matrices intact. In normal controls, the mesangial matrix appeared as fenestrated septa with oval or round stomata between the glomerular capillaries. In diabetic glomerulopathy, expansion of mesangial matrix and narrowing of the mesangial fenestrae were observed. These changes in the mesangial matrices seem to play a vital role in the progression of glomerulosclerosis in rat diabetes. A subendothelial thin layer of the GBM was continuous with the mesangial matrix. One cause of GBM thickening in streptozotocin diabetes may be expansion of the mesangial matrix into the peripheral GBM.     

Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes

Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.     

Experimental Dirofilaria immitis-associated glomerulonephritis induced in part by in situ formation of immune complexes in the glomerular capillary wall

Eight dogs were immunized with an aqueous-soluble extract of adult Dirofilaria immitis. Subsequent to at least 7-fold increases in antibody titer, the left renal artery of each dog was infused with 6 mg of D. immitis antigen. Fourteen days after infusion, the left kidney was compared to the right kidney and preinfusion biopsies. All dogs developed glomerular lesions in the left kidney characterized by 1 or more of the following: mesangial cell proliferation, neutrophil infiltration, increased periodic acid-Schiff-positive staining of the mesangium and glomerular basement membrane (GBM), fibrin deposition, and thickening of the GBM. Left kidney glomerular immunofluorescence was positive in 7 of the 8 dogs using polyclonal antisera for canine IgG and C3 in a linear or fine granular pattern. Ultrastructural lesions were present in the left kidney of all dogs and consisted of irregular GBM thickening, intramembranous and mesangial electron-dense deposits, and mesangial and endothelial cell proliferation. Antibodies directed against D. immitis antigen were demonstrated in all kidney eluates from the left kidney. The right kidneys of 3 of the dogs developed lesions; however, in comparison to the left kidney, the lesions in the right kidneys were inconsistent, mild, and focal. The histologic findings in the left kidney were similar to those observed in dogs with naturally occurring D. immitis infections. In sham-immunized control dogs, renal arterial infusion of D. immitis antigen did not cause consistent immune complex glomerulonephritis; however, antigen adherence to glomerular capillary walls was observed by immunofluorescent microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phases

Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.     

甲肝病毒（龙甲—25株）在Frhk4细胞上传代特征

应用Frhk4细胞增殖HAV,经较系统研究,建立了稳定的细胞传代方法,病毒增殖条件,获得了较大量的甲肝病毒抗原.HAV龙甲-25株在2BS细胞上传至第六代移到Frh4细胞上连续传至九代,用荧光法(IF)、EMISA夹心法,和免疫电镜方法(IEM)进行检测,结果表明龙甲-25不同代次间的病毒增殖情况不同.我们将龙甲-25HAV在Frhk4细胞上从第七代传至第十六代,分别用IF,ELISA,IEM方法检测,结果是细胞在感染病毒后不同天数的IF结果阳性,病毒传代的代次不同在ELISA和IEM检测中出现不同的结果.在第九代至第十一代的HAV′培养过程中我们改变了病毒培养及收获方法,再重复试验时,出现三种实验方法一致的结果.     

Permeation of endogenous IgG with an anionic subpopulation into glomerular basement membrane in rat

Conflicting results of previous electron microscopy studies and concerns about the validity of immunoperoxidase technique employed in those studies to accurately localize endogenous IgG in rat glomerular basement membrane (GBM) prompted us to use other techniques to answer the following question: Does endogenous IgG permeate the matrix of GBM? Immunofluorescence, radioimmunoassay (RIA), isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunodetection on Western blots were used to detect endogenous IgG in GBM. Direct immunofluorescence of normal frozen rat kidney sections prepared from in vivo perfused kidney showed endogenous IgG in a linear pattern of staining in the GBM. RIA for rat IgG found the IgG content of collagenase-solubilized GBM to be 0.48% of the dry weight. Immunodetection for rat IgG on Western blots of SDS-PAGE-separated GBM demonstrated endogenous IgG in purified collagenase-solubilized GBM. IgG was detected as an intact molecule with covalently linked light and heavy chains and not as small immunoreactive catabolic fragments. Isoelectric focusing followed by immunodetection on Western blot showed that part of the endogenous IgG in GBM was anionic. The results clearly show that under normal conditions, endogenous IgG can permeate into the collagen matrix of GBM in rat and that some of this IgG is more anionic than the IgG in serum. These findings may assist in understanding the transit of autoantibodies to subepithelial glomerular antigens located beneath the matrix of GBM in membranous glomerulonephropathy.     

Congenital nephrosis of the Finnish type (CNF): matrix components of the glomerular basement membranes and of cultured mesangial cells

Summary Congenital nephrosis of the Finnish type (CNF) is a hereditary renal disease of unknown aetiology manifested by massive proteinuria of the newborn and unresponsive to any treatment. In this study kidney samples and cultured glomerular mesangial cells from five patients with CNF were studied by indirect immunofluorescence microscopy for the presence and location of major basement membrane matrix (GBM) components. Histological changes of glomeruli ranging from mild thickening of basement membranes to total obliteration and sclerosis were seen. Notably, thickening of the subepithelial layer of Bowman's capsules was regularly seen along with hypercellularity at the juxtaglomerular areas. The matrix components studied (laminin, plasma- and cellular fibronectin, type IV collagen, including the NC-1, alpha-1 and alpha-3 chains, heparan sulphate proteoglycan (HSPG) core protein, thrombospondin) were characteristically seen within the glomeruli. Local thickenings alternating with total loss of epitopes along the GBM were seen, especially with anti-type IV collagen and anti-HSPG antibodies. Sera from CNF patients after transplantation failed to show antibodies against GBM structures in immunofluorescence microscopy, suggesting that no missing epitopes of GBM are introduced with the transplant kidney. Cultured mesangial cells of CNF glomeruli also showed continued in vitro production of the matrix components and their incorporation into the matrix underneath the cell layer.     

Cationic gold staining of glomerular anionic sites in archived tissue, reprocessed from paraffin wax into LR gold resin

Summary Glomerular capillary wall anionic sites have been demonstrated by cationic gold staining of archived renal biopsy tissue (up to 10 years old), obtained from six patients, originally embedded in paraffin wax, and subsequently reprocessed into LR gold resin. The staining patterns at pH 2.5 and pH 7.0, demonstrating different glomerular basement membrane (GBM) anionic constituents, were compared in three patients from whom tissue directly processed into LR gold and reprocessed tissue was available. Ultrastructural preservation was poorer and shrinkage artefact greater in paraformaldehyde-lysine periodate (PLP) as opposed to formol saline-fixed reprocessed tissue. However, GBM anionic site expression was well preserved, or even enhanced (lamina rara externa, pH 7.0) in reprocessed tissue, using either fixative. Although it may not be possible to compare subtle changes in anionic site distribution in variously fixed and processed tissues, due to these artefacts, the technique enables retrospective study of charge status in archived material from disease groups in which there are distinct anionic site aberrations.     

Basal lamina secreted by MDCK cells has size- and charge-selective properties

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ～0.0013 for each 1-? increment in solute radius, compared with a decrease of 0.0023 per ? for the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

TNF-alpha induces actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase.

Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin-associated proteins. Since several glomerulopathies characterized by heavy proteinuria are associated with increased glomerular tumor necrosis factor alpha (TNF-alpha) expression, we studied the interaction of TNF-alpha with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-alpha for variable time periods ranging from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total-actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-alpha rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-alpha. We conclude that TNF-alpha induces substantial reorganization of actin cytoskeleton and focal adhesions. These effects occur simultaneously, with a prompt TNF-alpha-induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, known to regulate actin polymerization and formation of focal adhesions, may be directly involved in the mechanism controlling the observed actin redistribution. These findings suggest that the observed TNF-alpha-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-alpha is associated with disturbances in the attachment of podocytes to the GBM.