The anionic barrier system in the mesonephric renal glomerulus of the arctic lamprey,Entosphenus japonicus (Martens) (Cyclostomata)

Summary To examine the selective permeability of the nephrons of lower vertebrates, the permeability of the glomerulus in the kidney of an arctic lamprey, Entosphenus japonicus (Martens), to native anionic ferritin or cationized ferritin was studied by observing the distribution of ionized anionic groups in renal tissues. The cationized ferritin molecules injected into the dorsal aorta penetrated rapidly into the glomerular basement membrane layer through fenestrae present in the capillary endothelium and were subsequently excreted into the urinary spaces via the interstices between foot processes of the visceral epithelial cells. Native anionic ferritin, on the other hand, passed only minimally through the capillary wall. Cytochemical staining of fixed tissue or perfusion of the kidney in situ with cationic cacodylate-iron colloid revealed that the ionized anionic groups of acid mucopolysaccharides were distributed on both the luminal and abluminal surfaces of endothelial cells, and in the thick fibrous lamina rara interna of the glomerular basement membrane; they were especially dense on the surfaces of visceral epithelial cells and their foot processes. These results suggest that the mesonephric glomerulus of the arctic lamprey possesses a functionally well developed anionic barrier system comparable to that of the mammalian metanephric glomerulus.     

The ultrastructure of the renal corpuscle of a lizard

The few and small renal corpuscles of the lizard Podarcis (= Lacerta) taurica are composed of a tuft of three to four capillaries (glomerulus), Bowman's capsule and mesangium. The thin interdigitated capillary endothelial cells are, in most regions, in contact with the mesangium. In some regions, however, they rest on a bilaminate basement membrane with an electron-dense lamina densa and a less dense lamina rara. Bowman's capsule is composed of visceral and parietal layers. The epithelial cells (podocytes) of the visceral layer bear trabeculae connected to pedicels with microvilli. The pedicels rest on a bilaminate basement membrane which in some regions has a double-layered densa with connecting bands. Generally, this basement membrane is thicker than that of the capillary endothelial cells. The mesangium is composed mostly of irregular satellite cells with large nuclei and cytoplasmic processes, but also has smaller cells with kidney-shaped nuclei and cytoplasmic processes containing microfilaments. The mesangium cells are embedded in a collagenous matrix which extends to invade the area between the epithelial basement membrane and the capillary endothelium. These observations are discussed in relation to the structure and function of vertebrate renal corpuscles with special reference to the mesangium.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.     

Incomplete fusion of the epithelial and endothelial basement membranes in interspecies hybrid glomeruli

Avascular, undifferentiated mouse kidneys transplanted onto quail chorioallantoic membrane differentiate and become vascularized by quail vessels. The glomeruli which form under these conditions consist of mouse podocytes and quail endothelial cells. Immunohistochemistry has shown that the glomerular basement membrane (GBM) has a dual origin, as integral basement membrane components are produced by both podocytes and endothelial cells. In electron microscopy this GBM is composed of two partially separated layers, an epithelial and an endothelial basal lamina which both have a lamina densa and a lamina rara. These two basal laminas are partially fused, but there are large areas where this fusion does not occur. In some places of incomplete fusion, fibrillar extracellular material is seen between and beneath the GBM. It is concluded that basement membrane components derived from the different species can interact partially, but the fusion is incomplete. The abnormal assembly of the epithelial and the endothelial basal laminas might be due to molecular differences between the components produced by the two cell lineages. In spite of the incomplete fusion, the system used serves as a good model-system to study basement membrane formation, since the cells organize in a histiotypic fashion and form true vascularized glomeruli.     

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis

A daily injection protocol with puromycin aminonucleoside (PAN) causes loss of sialic acid from the glomerular filter. These changes have been studied previously by colloidal iron staining, but we have recently shown that phosphotungstic acid (PTA) at low pH allows the demonstration of sialic acid groups in the glomerular basement membrane in ultrathin sections of glycolmethacrylate(GMA)-embedded rat kidney. With this technique the slit diaphragm is seen as a continuation of the luminal cell coat and the method also gives an idea of the sialic acid distribution at the podocyte plasma membrane. The availability of this method made it possible to reevaluate the results obtained earlier in aminonucleoside (PAN) nephrosis indicating a decrease in the sialic acid content of the glomerulus. Although there are changes in the epithelial architecture, the ultrastructural appearances of the basement membrane are only slightly altered in PAN nephrosis. Detachment of epithelial cells was variable in different animals. Seven days after the first injection of PAN, staining with PTA revealed local defects in the lamina rara externa which later became more extensive. In PAN-treated animals the luminal cell coat showed reduced staining and large areas of the plasma membrane were completely devoid of a cell coat. These changes coincided with the onset of heavy proteinuria. The results indicate that both the basement membrane and the epithelial plasma membrane are affected in PAN nephrosis, as revealed by decreased staining for sialic acid-containing molecules in the basement membrane and by changes in the epithelial cell coat. The defects in the cell coat material point to functional alterations at the level of the slit pores and it is suggested that the decrease in sialic acid content of the lamina rara externa may be partly responsible for defects in the size-selective filtration barrier in PAN nephrosis.     

Glomerular ultrastructure of the trout,Salmo gairdneri: Effects of angiotensin II and adaptation to seawater

Summary The effect of angiotensin infusion on the glomerular ultrastructure of freshwater- and seawater-adapted rainbow trout, Salmo gairdneri, has been examined by scanning and transmission electron microscopy. Adaptation of trout to seawater resulted in epithelial podocyte flattening, primary process broadening and apparent loss of foot processes in almost all glomeruli, features which were uncommon in freshwater-adapted trout. Similar changes were induced by infusion of freshwater-adapted animals with angiotensin, suggesting that the renin-angiotensin system plays a role in the modification of glomerular epithelial ultrastructure. Adaptation of trout to seawater also reduced glomerular diameter, but infusion of freshwater-adapted animals with angiotensin did not mirror this effect. Infusion of angiotensin into seawater-adapted animals increased the overall thickness of glomerular basement membrane by increasing the lamina rara interna and lamina densa. This did not occur when freshwater-adapted fish were either infused with angiotensin or adapted to seawater. These findings suggest that other humoral systems are involved in the control of glomerular diameter and basement membrane thickness as part of an integrated response to increased environmental salinity.     

Differences in basement membrane-associated microdomains of type I and type II pneumocytes in the rat and rabbit lung

The basement membrane-associated microdomains of type I pneumocytes in rat and rabbit pulmonary alveoli were found to be uniquely different from those of type II pneumocytes in the specific distribution of cytochemically detectable sulfate esters as demonstrated with the high iron diamine (HID) technique at the electron microscopic level. Aldehyde-fixed frozen or Vibratome sections of neonatal and adult lungs were treated with a mixture of the meta and para isomers of N,N-dimethyl-phenylenediamine-HCl in the presence of ferric chloride, which at low pH (1.0) has been previously shown to be highly specific for sulfate esters of glycosaminoglycans and glycoproteins. Reaction product was subsequently enhanced with a thiocarbohydrazide-silver proteinate, postembedding sequence for electron microscopy. Samples of lung parenchyma treated in this fashion were observed to have discrete, electron-dense silver grains associated with the various microanatomical components of pulmonary basement membranes. In the region of the alveolar basement membrane, the lamina rara externa associated with type I cells was observed to contain an abundance of regularly disposed, cytochemically detectable sulfate esters, while the lamina densa and lamina rara interna were diffusely and sparsely reactive by comparison. Quantitatively, 62% of all reactive sites found in the basement membrane region of type I cells were localized in the lamina rara externa. By contrast, the lamina rara externa of type II cells had less than half as many reactive foci indicative of sulfate esters as the same region of type I cell basement membranes. HID-reactive sulfate esters were found evenly distributed within the laminae associated with the basement membrane of type II cells. This cytochemically detectable difference in the sulfate ester composition of basement membrane-associated sulfate ester composition of basement membrane-associated microdomains of type I compared with that of type II pneumocytes may be highly significant when considering known patterns of epithelial renewal in pulmonary alveoli. Since type II cells are known to divide and either remain type II cells or differentiate into type I cells, regional differences in the molecular composition of the alveolar basement membranes and their associated structures may be key determinants of cell-specific processes of cytodifferentiation in the pulmonary alveolus.     

Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.     

Glomerular anionic charge in congenital nephrotic syndrome of the Finnish type

Summary Decrease of the anionic charge of the glomerular basement membrane and especially the reduced amount of heparan sulphate proteoglycan in the lamina rara externa has been suggested to be the basic pathogenetic defect in congenital nephrotic syndrome. In the present study the anionic charge of glomeruli was examined in the congenital nephrotic syndrome of the Finnish type and in controls using cationic stains (polyethyleneimine, Ruthenium Red) in electron microscopy. Chondroitinase and heparinase treatments were used to characterize further the anionic elements detected. Scanning electron microscopy (SEM) was used in addition to transmission electron microscopy (TEM) to examine the tridimensional structure and secondary changes of podocytes in this syndrome. The number (mean ± SD) of polyethyleneimine granules per 1 μm length of lamina rara externa of the glomerular basement membrane was 24.9 ± 4.5 in control and 2.32 ± 4.3 in congenital nephrotic syndrome subjects. The Ruthenium Red staining pattern was closely similar in syndrome and control kidneys. The granules evident after staining with either cationic stain were seen after chondroitinase but not after heparinase treatment in control as well as in syndrome patient kidney samples. No denuded areas of basement membrane in 42 glomeruli from four syndrome patients were found in SEM. In conclusion, the amount of anionic sites in the lamina rara externa as detected by either cationic stain was comparable to controls. These results do not support the hypothesis of decreased anionic sites in the lamina rara externa of the glomerular basement membrane in congenital nephrotic syndrome of the Finnish type.     

Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis

The sequence of antigen localization and the interaction of immune deposits with the anionic sites of the glomerular basement membrane (GBM) were investigated in an active model of in situ immune complex glomerulonephritis using a cationized ferritin. Three weeks after immunization with native horse spleen ferritin, the left kidneys of rats were perfused with 500 micrograms of cationized ferritin through the left renal artery. One h after renal perfusion, most of ferritin particles localized subendothelially, corresponding to the anionic sites of the lamina rara interna. In the glomerular capillary loops, infiltrating polymorphonuclear leukocytes and monocytes were seen. Some of these monocytes were in direct contact with immune complexes containing ferritin aggregates associated with anionic sites of the lamina rara interna. At 24 h, numerous ferritin aggregates were present subepithelially, preferentially beneath the slit membrane. The subepithelial location of ferritin did not always correspond to the anionic sites of the lamina rara externa. From days 3 to 7, there was remarkable endocapillary cell proliferation in some loops and pronounced effacement of epithelial foot processes. Focal detachment of epithelium from the GBM was observed occasionally. From days 14 to 28, most of ferritin aggregates were located intramembranously and subepithelially. Membranous transformation has already begun around the subepithelial deposits. This morphological study provides insight into the fate of immune deposits and injury to the GBM in the glomerulonephritis.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.     

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.     

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.     

An improved colloidal iron stain for glomerular sialoproteins

Summary The thick sialoglycoprotein coat of the glomerular epithelial cells is a part of the glomerular filtration barrier. It can be demonstrated in paraffin histology by means of the colloidal iron procedure. We have studied the special requirements of tissue processing to obtain reproducible results by this staining method. The requirements include proper fixation, avoiding heat during sectioning and ample rehydration. We have further tested a colloidal iron-PAS procedure for simultaneous demonstration of the glomerular epithelial coat and the basement membrane.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

An improved colloidal iron stain for glomerular sialoproteins

The thick sialoglycoprotein coat of the glomerular epithelial cells is a part of the glomerular filtration barrier. It can be demonstrated in paraffin histoslogy by means of the colloidal iron procedure. We have studied the special requirements of tissue processing to obtain reproducible results by this staining method. The requirements include proper fixation, avoiding heat during sectioning and ample rehydration. We have further tested a colloidal iron-PAS procedure for simultaneous demonstration of the glomerular epithelial coat and the basement membrane.