A quantitative study of glomerular basement membrane changes in IgA nephropathy

A quantitative study of glomerular basement membrane (GBM) changes in 16 cases of IgA nephropathy was carried out on two whole glomeruli per case using a wide-field electron microscope. We found that GBM changes are observed more frequently than previously reported, although some changes are small and uneven in distribution. Changes include (a) attenuation, (b) lytic attenuation, (c) garland-shaped widening of the GBM, (d) dome-shaped widening of the GBM, and (e) disruption of the GBM. There was some correlation between the severity of light microscopic findings and the percentage of GBM affected by complicated changes ((b)-(e)). The percentage of complicated GBM changes correlated with the amount of proteinuria at biopsy. The frequent occurrence of GBM changes in IgA nephropathy suggests that they might be playing a role in the pathogenesis of IgA nephropathy.     

High-Resolution Ultrastructural Study of the Rat Glomerular Basement Membrane in Long-Term Experimental Diabetes

The ultrastructure of the glomerular basement membrane of the long-term diabetic and age-matched control rats was studied with the application of advanced high-resolution microscopy. By using the freeze substitution method for the preparation of the renal tissue, it was possible to observe that the glomerular basement membrane in control and diabetic animals is composed of only a single lamina densa without the presence of a lamina lucida interna or externa. High-resolution electron microscopy of the diabetic glomerular basement membrane showed significant alterations in its morphology and ultrastructure. First, the basement membrane in diabetic condition appeared to be split into two halves, endothelial and epithelial. In the epithelial half of the membrane, the network of distinct strands referred to as cords, which were clearly present in the glomerular basement membrane of age-matched control animals, became less distinct and showed a diffused appearance being evenly replaced by thin filaments. The openings of the network were filled with a granular material. In the endothelial half of the membrane, on the other hand, the cord network was variably lost in diabetic condition and, within the resulting vacant spaces, bundles of fibrils 12 nm in width, identified as basotubules, were deposited. Immunolabeling for type IV collagen was found to be enriched in the endothelial half of the basement membrane being associated with the bundles of basotubules. The ultrastructural changes reported by high-resolution microscopy could be related to the molecular alterations of the basement membrane components and to the loss in permselectivity occurring during diabetes.     

Thickening of Glomerular Capillary Walls: A Guide to Differential Diagnosis by Electron Microscopy

Thickening of capillary walls is a feature of many glomerular diseases. Widening of the wall may be produced by deposits and other changes affecting either subepithelial and subendothelial regions or the glomerular basement membrane itself. Careful light microscopic examination using special stains can distinguish some patterns of capillary wall thickening, but electron microscopy is needed to demonstrate most lesions clearly. In this brief review, a guide to the major causes for capillary wall thickening is discussed, using a simple classification, and some of the patterns are illustrated. Precise delineation of the capillary wall changes in glomerular diseases is important to insure accurate classification and a clear understanding of pathogenetic mechanisms.     

Thickening of Glomerular Capillary Walls: A Guide to Differential Diagnosis by Electron Microscopy

Thickening of capillary walls is a feature of many glomerular diseases. Widening of the wall may be produced by deposits and other changes affecting either subepithelial and subendothelial regions or the glomerular basement membrane itself. Careful light microscopic examination using special stains can distinguish some patterns of capillary wall thickening, but electron microscopy is needed to demonstrate most lesions clearly. In this brief review, a guide to the major causes for capillary wall thickening is discussed, using a simple classification, and some of the patterns are illustrated. Precise delineation of the capillary wall changes in glomerular diseases is important to insure accurate classification and a clear understanding of pathogenetic mechanisms.     

Renoprotection From Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age‐matched OVE diabetic mice to produce bi‐transgenic OVE‐JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE‐JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day‐old OVE‐JTMT mice, blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, endothelial‐specific MT overexpression in OVE‐JTMT mice mitigated several DN complications including significantly increased non‐fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560–576, 2017. © 2016 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Lysis of the glomerular basement membrane in children with IgA nephropathy and Henoch-Shönlein nephritis

An electron-microscopic study of the glomerular basement membrane was made on 242 renal biopsies from 222 children with a variety of renal diseases. Lysis of the basement membrane was observed in 16 of the 25 children with Henoch-Sch?nlein nephritis, 36 of the 72 with IgA nephropathy and one with acute poststreptococcal glomerulonephritis. Lysis was frequently associated with subepithelial dense deposits and polymorphonuclear leukocytes, and these are thought to play a role in the lytic process. There was a significant correlation between the presence of lysis of the basement membrane, the degree of proteinuria and the severity of glomerular changes by light microscopy. These findings suggest that the degree of lysis is an indication of the severity of the disease and lysis is associated with progressive disease and a worse prognosis in Henoch-Sch?nlein nephritis and IgA nephropathy.     

High-Resolution Ultrastructural Study of the Rat Glomerular Basement Membrane in Aminonucleoside Nephrosis

In the initial stages of aminonucleoside nephrosis, functional alterations in the glomerular basement membrane occur, as evidenced by the development of proteinuria. However, it has not been possible to observe important ultrastructural modifications at the level of the basement membrane, probably because the changes are taking place at the molecular level. In this study, by the use of high-resolution electron microscopy, an attempt was made to evaluate such changes in rat glomerular basement membrane during acute aminonucleoside nephrosis. As previously reported, in control animals the glomerular basement membrane is composed of a network of 4-nm-wide irregular anastomosing strands, referred to as “cords,” which are known to contain a core filament of type IV collagen surrounded by a “sheath” of other components, such as laminin and heparan sulfate proteoglycan (HSPG). The most conspicuous ultrastructural alteration of the nephrotic glomerular basement membrane, recognizable only at high magnification, is that the cords were denuded leaving only the core filament through the loss of the sheath material. Thus, the cord network was transformed, with the progress of pathological conditions, into a network of fine filaments. On the other hand, abundance and distribution of HSPG molecules known to be present in the form of 4.5- to 5-nm-wide ribbon-like “double tracks,” were found to be similar in control and nephrotic tissues. Since HSPG is one of the charge proteins of the basement membrane, the little changes observed for HSPG are difficult to interpret in view of reported decreases in basement membrane anionic sites in nephrosis. In conclusion, the glomerular basement membrane in aminonucleoside nephrosis loses its cord network components and replaces them with a more perforated network, which could be a cause for the increased permeability of this basement membrane.     

Ultrastructural Investigations of Finger Pulp Biopsies: A Study of 31 Patients with Raynaud's Syndrome

Finger pulp biopsies of 31 patients suffering from Raynaud's syndrome (RS) were investigated by light and electron microscopy. The patients suffered from various underlying diseases. Interest was focused on the vascular changes and nerve and connective tissue alterations, especially the elastic fiber changes. In the dermal capillaries, endothelial swelling, narrowing of the lumen, increased numbers of intracytoplasmic filaments and Weibel-Palade bodies, and various types of basement membrane alterations were observed. The basement membrane of the unmyelinated nerves showed similar alterations to the capillary basement membrane. Severe degenerative changes were found in the myelin sheath of the myelinated axons. In the dermal connective tissue, severe elastotic degeneration and alterations of the collagen fibers were observed. The data probably represent the ultrastructural picture of the end stage of this syndrome.     

Bedside to bench Alport syndrome research: are human urine‐derived podocytes the answer?†

In a recent issue of The Journal of Pathology, Iampietro et al isolated and characterized several clones of urine‐derived podocytes from three patients with Alport syndrome (AS) and proteinuria and one age‐matched non‐proteinuric control. They reported differential expression of genes involved in cell motility, adhesion, survival, and angiogenesis. The authors found AS podocytes to be less motile and to have significantly higher permeability to albumin compared to control podocytes, highlighting that AS podocytes may retain their phenotype even when losing contact with the glomerular basement membrane. The establishment of urine‐derived podocyte cell lines from patients with different genetic forms of AS may represent a valuable and minimally invasive tool to investigate the cellular mechanisms contributing to kidney disease progression in AS and may allow for the establishment of patient‐specific drug screening opportunities. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

New Techniques Scanning Electron Microscopy of Acellular Glomeruli in Human Glomerulonephritis: Application of a Technique

Scanning electron microscopy (SEM) has been employed in studies of human and experimental glomerulonephritis (GN), a major contribution being the elucidation of podocyte morphology and the process of podocyte foot process retraction in proteinuric conditions. Application of SEM in GN has been limited however by an emphasis on cell surface alterations while the site of major disease processes, the glomeru-lar basement membrane, has remained hidden from view. A previously reported technique for the preparation of acellular glomeruli from fresh tissue has been adapted for use on frozen human renal biopsies. One case of minimal change disease (MCD) and one case of membranous glomerulonephritis (MGN) have been studied. The acellular glomerular basement membrane of MCD has a lightly textured or granular surface whereas in MGN a striking reticular network of basement membrane has formed perpendicular to the native basement membrane. The immune complexes have been removed. This technique provides a graphic visualization of GBM alterations occurring in glomerulonephritis and is applicable to the study of human as well as experimental model of glomerulonephritis.     

New Techniques Scanning Electron Microscopy of Acellular Glomeruli in Human Glomerulonephritis: Application of a Technique

Scanning electron microscopy (SEM) has been employed in studies of human and experimental glomerulonephritis (GN), a major contribution being the elucidation of podocyte morphology and the process of podocyte foot process retraction in proteinuric conditions. Application of SEM in GN has been limited however by an emphasis on cell surface alterations while the site of major disease processes, the glomeru-lar basement membrane, has remained hidden from view. A previously reported technique for the preparation of acellular glomeruli from fresh tissue has been adapted for use on frozen human renal biopsies. One case of minimal change disease (MCD) and one case of membranous glomerulonephritis (MGN) have been studied. The acellular glomerular basement membrane of MCD has a lightly textured or granular surface whereas in MGN a striking reticular network of basement membrane has formed perpendicular to the native basement membrane. The immune complexes have been removed. This technique provides a graphic visualization of GBM alterations occurring in glomerulonephritis and is applicable to the study of human as well as experimental model of glomerulonephritis.     

IgA Nephropathy: An Ultrastructural Study

From a series of 333 renal biopsies examined by immunofluorescence microscopy, 231 specimens corresponded to primary glomerulonephritis unassociated with systemic diseases. Of those 231 biopsies, 39 had diffuse mesangial deposits of IgA (16.8%). Thirty cases of IgA nephropathy had one to several glomeruli in the Epon-embedded tissue. All cases showed mild to moderate increase of mesangial cells and matrix. Mesangial deposits were present in all cases; subendothelial (5/30), intramembranous (3/30), and subepithelial (10/30) deposits were also found. Occasional dense granular deposits involved the basement membrane of the capsule of Bowman (1/30) and the subendothelial region of some extraglomerular arterioles (2/30). Thinning (6/30) and splitting (4/30) of the glomerular lamina densa appeared focally in some cases.     

Incidence of Thin Basement Membrane Nephropathy in 990 Consecutive Renal Biopsies Examined with Electron Microscopy

Thin basement membrane nephropathy is one of the main causes of hematuria in both children and adults. It is often associated with a family history and its true incidence is unknown. Accurate diagnosis of thin basement membrane nephropathy relies on the presence of attenuated glomerular basement membranes, a finding that can be appreciated only by examination in the electron microscope. In Cyprus the department of electron microscopy has received 990 consecutive renal biopsies for diagnosis. The aim of this study is to define the incidence of thin basement membrane nephropathy in this population sample based on the results of electron microscopy.     

Rapid Contrasting of Extracellular Elements in Thin Sections

Standard methods for contrasting ultrathin sections generally have their greatest effect on cells and cellular components, whereas extracellular elements remain relatively electron-lucent. Occasionally, some extracellular elements even fail completely to react with the staining solutions. We describe a method for rendering a uniformly high contrast to extracellular tissue components. This consists of a brief prestaining of grids with diluted tannic acid in distilled water. Simple, rapid, and versatile, this procedure can be routinely applied to all tissue samples examined by electron microscopy. As an additional advantage, the method greatly enhances the electron density of intracellular glycogen. Higher concentrations of tannic acid give increased electron density, especially to elastin, and can therefore be used as an elastin stain.     

Rapid Contrasting of Extracellular Elements in Thin Sections

Standard methods for contrasting ultrathin sections generally have their greatest effect on cells and cellular components, whereas extracellular elements remain relatively electron-lucent. Occasionally, some extracellular elements even fail completely to react with the staining solutions. We describe a method for rendering a uniformly high contrast to extracellular tissue components. This consists of a brief prestaining of grids with diluted tannic acid in distilled water. Simple, rapid, and versatile, this procedure can be routinely applied to all tissue samples examined by electron microscopy. As an additional advantage, the method greatly enhances the electron density of intracellular glycogen. Higher concentrations of tannic acid give increased electron density, especially to elastin, and can therefore be used as an elastin stain.     

Nail-Patella syndrome in a spontaneously aborted 18-week fetus: Ultrastructural and immunofluorescent study of the kidneys

Nail-patella syndrome (NPS), hereditary onycho-osteodysplasia, is an autosomal dominant disorder of nail dystrophy, patellar absence or hypoplasia, incomplete elbow extension, conical posterior iliac horns, and nephropathy. We studied the kidneys of an 18-week spontaneously aborted fetus of a mother with the NPS. Ultrastructural examination of the kidney showed thickening of the capillary walls of the glomeruli and mesangium. There was irregular thickening of basement membranes with subendothelial fibrillar electron-dense deposits. Immunofluorescence showed fibrinogen deposition in glomerular basement membranes. Fibrinogen deposition in utero may ultimately lead to glomerular fibrosis and intrabasement membrane collagen deposition as seen in the adult renal lesion of this syndrome. This is the first report of the NPS in which the renal abnormalities have been studied in a fetus. These findings provide support for possible prenatal diagnosis of NPS by intrauterine kidney biopsy. © 1992 Wiley-Liss, Inc.     

Quantitive changes in the glomerular basement membrane components in human membranous nephropathy

In membranous nephropathy (MN), the glomerular basement membrane (GBM) is thickened due to accumulation of GBM material between and around the subepithelial immune deposits. Alterations in the GBM components in relation to subepithelial deposits and GBM thickening are not clearly defined. The GBM distribution of classical and novel [α4(IV)] chains of type IV collagen, laminin, and fibronectin have been studied in seven patients with MN and in three normal controls by a quantitative immunogold technique. In normal kidneys, the labelling of type IV collagen or fibronectin was distributed predominantly along the endothelial side of the GBM; α4(IV) was found in the lamina densa; and laminin was concentrated in the epithelial zone of the GBM (P<0·01). In MN, there were increased immunogold densities for classical and novel type IV collagen chains, laminin, and fibronectin in the spikes of MN patients compared with controls (P<0·05). Furthermore, gold particle labelling for the α4(IV) collagen chain was increased in the middle zone (P<0·01) and that for fibronectin was increased in the endothelial and middle zones of the GBM (P<0·05) compared with normal controls. These findings suggest that subepithelial immune deposits stimulate glomerular epithelial cells (GEC), resulting in enhanced secretion of classical and novel type IV collagen chains, laminin, and fibronectin, forming spikes in MN; of these newly formed components, only novel type IV collagen appears to migrate towards the middle zone of the GBM, contributing to thickening of this zone. The results also suggest that fibronectin, possibly derived from the circulation, is related to thickening of the endothelial zone of the GBM, which in turn might be related to progressive glomerulosclerosis. © 1997 by John Wiley & Sons, Ltd.     

Fell Muir Lecture: Collagen fibril formation in vitro and in vivo

It is a great honour to be awarded the Fell Muir Prize for 2016 by the British Society of Matrix Biology. As recipient of the prize, I am taking the opportunity to write a minireview on collagen fibrillogenesis, which has been the focus of my research for 33 years. This is the process by which triple helical collagen molecules assemble into centimetre‐long fibrils in the extracellular matrix of animals. The fibrils appeared a billion years ago at the dawn of multicellular animal life as the primary scaffold for tissue morphogenesis. The fibrils occur in exquisite three‐dimensional architectures that match the physical demands of tissues, for example orthogonal lattices in cornea, basket weaves in skin and blood vessels, and parallel bundles in tendon, ligament and nerves. The question of how collagen fibrils are formed was posed at the end of the nineteenth century. Since then, we have learned about the structure of DNA and the peptide bond, understood how plants capture the sun's energy, cloned animals, discovered antibiotics and found ways of editing our genome in the pursuit of new cures for diseases. However, how cells generate tissues from collagen fibrils remains one of the big unsolved mysteries in biology. In this review, I will give a personal account of the topic and highlight some of the approaches that my research group are taking to find new insights.     

Extraglomerular distribution of immunoreactive Goodpasture antigen

The distribution of Goodpasture antigen (GA) was studied in a range of human tissues using indirect immunofluorescence and immunoperoxidase techniques. Frozen sections were stained using (1) a mouse monoclonal antibody (P1) raised against the autoantigenic component of human glomerular basement membrane, (2) autoantibodies eluted from the kidneys of patients with Goodpasture's syndrome, (3) antibodies eluted from the kidneys of a sheep with Steblay nephritis, and (4) mouse monoclonal and guinea pig polyclonal antibodies to human type IV collagen. The same pattern of staining was demonstrated using the eluted antibodies and monoclonal antibody P1. The presence of GA was confirmed in the lung and choroid plexus. GA was also detected in basement membranes at a number of previously unreported sites in the eye, thyroid, pituitary, adrenal, breast, and liver. GA was absent from other sites at which type IV collagen could be demonstrated. Direct immunofluorescence studies of tissue from a patient with Goodpasture's syndrome revealed deposition of IgG in the choroid plexus and eye, as well as in the kidney and lung.     

Ultrastructural Changes of Tubular Basement Membranes in Immunologic Renal Tubular Lesions in Humans

Ultrastmctural changes in the tubular basement membrane (TBM) of 65 renal biopsy specimens from patients with nephropathies or transplant rejection were examined for their potential relationship to deposits observed along the TBM by immu nofluorescence (IF). The TBM showed various alterations: irregularities with thickening, lamella tion, duplication, and clear spaces. Granular vesicles or inclusions were present inside the thickened TBM. The morphologic changes of the TBM occurred regardless of the severity of rejection of transplants and the type of deposits observed on IF. Electron-dense deposits inside or along the TBM were not found in cases of linear deposits, however. Although these changes were not strictly specific to tubular immune diseases, they could reflect a reaction of the TBM to immune complexes or deposits of antibodies to TBM with degeneration of tubular epithelial cells and sometimes regeneration of a new TBM.