Glomerular Basement Membrane Alterations Research Articles

Evaluation of ultrastructural alterations of glomerular basement membrane and podocytes in glomeruli by low-vacuum scanning electron microscopy

BackgroundLow-vacuum scanning electron microscopy (LV-SEM) is applied to diagnostic renal pathology.MethodsTo demonstrate the usefulness of LV-SEM and to clarify the optimal conditions of pathology samples, we investigated the alterations of glomerular basement membrane (GBM) and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM.ResultsOn week 15 following induction of AHN, spike formation on GBM with diffuse deposition of IgG and C3 developed. Using LV-SEM, diffuse crater-like protrusions were clearly noted three-dimensionally (3D) on surface of GBM in the same specimens of light microscopy (LM) and immunofluorescence (IF) studies only after removal coverslips or further adding periodic acid-silver methenamine (PAM) staining. These 3D ultrastructural findings of GBM surface could be detected in PAM-stained specimens by LV-SEM, although true GBM surface findings could not be obtained in acellular glomeruli, because some subepithelial deposits remained on surface of GBM. Adequate thickness was 1.5–5 μm for 10% formalin-fixed paraffin-embedded (FFPE) and 5–10 μm for the unfixed frozen sections. The foot processes and their effacement of podocytes could be observed by LV-SEM using 10%FFPE specimens with platinum blue (Pt-blue) staining or double staining of PAM and Pt-blue. These findings were obtained more large areas in 2.5% glutaraldehyde-fixed paraffin-embedded (2.5%GFPE) specimens.ConclusionOur findings suggest that LV-SEM is a useful assessment tool for evaluating the alterations of GBM and podocytes in renal pathology using routine LM and IF specimens, as well as 2.5%GFPE specimens.

Pathologic glomerular characteristics and glomerular basement membrane alterations in biopsy-proven thin basement membrane nephropathy.

Thin basement membrane nephropathy (TBMN) is diagnosed by diffuse thinning of the glomerular basement membrane (GBM) without any clinical and pathologic findings of Alport syndrome and the other renal diseases. TBMN is characterized clinically by benign familial hematuria but rarely develops into end-stage renal disease. In 27 cases of biopsy-proven TBMN, we evaluated the pathologic characteristics of TBMN, and examined the correlation between these pathologic characterizations and renal dysfunction. All patients had hematuria, and 21 patients (77.8%) had proteinuria. In six patients (28.6%) who were more than 50years of age, the estimated glomerular filtration rate (eGFR) decreased from G3a to G4 in the chronic kidney disease stage. Pathologically, an irregular decrease in intensity of type IV collagenα5(IV) chainwas seen in GBM, and irregular thinning with diffuse rough etched images was observed on the GBM surface with several sizes of holes by low-vacuum scanning electron microscopy. The glomerular morphology of TBMN was characterized by an increased number of small glomerular capillaries with an increased extracellular matrix (ECM). These characteristic morphologic alterations were evident from a young age in patients with TBMN, but were not correlated directly with the decrease of eGFR, the degree of hematuria, and proteinuria. The decrease of eGFR in patients with TBMN who were more than 50years of age might be primarily mediated by arteriolosclerosis-associated glomerulosclerosis and interstitial fibrosis. Characteristic pathological glomerular findings and GBM alterations occurred from a young age but were not associated directly with renal impairment in biopsy-proven TBMN.

Characterization of the early molecular changes in the glomeruli of Cd151\u2212/\u2212 mice highlights induction of mindin and MMP-10

In humans and FVB/N mice, loss of functional tetraspanin CD151 is associated with glomerular disease characterised by early onset proteinuria and ultrastructural thickening and splitting of the glomerular basement membrane (GBM). To gain insight into the molecular mechanisms associated with disease development, we characterised the glomerular gene expression profile at an early stage of disease progression in FVB/N Cd151−/− mice compared to Cd151+/+ controls. This study identified 72 up-regulated and 183 down-regulated genes in FVB/N Cd151−/− compared to Cd151+/+ glomeruli (p < 0.05). Further analysis highlighted induction of the matrix metalloprotease MMP-10 and the extracellular matrix protein mindin (encoded by Spon2) in the diseased FVB/N Cd151−/− GBM that did not occur in the C57BL/6 diseased-resistant strain. Interestingly, mindin was also detected in urinary samples of FVB/N Cd151−/− mice, underlining its potential value as a biomarker for glomerular diseases associated with GBM alterations. Gene set enrichment and pathway analysis of the microarray dataset showed enrichment in axon guidance and actin cytoskeleton signalling pathways as well as activation of inflammatory pathways. Given the known function of mindin, its early expression in the diseased GBM could represent a trigger of both further podocyte cytoskeletal changes and inflammation, thereby playing a key role in the mechanisms of disease progression.

Negative Staining for COL4A5 Correlates With Worse Prognosis and More Severe Ultrastructural Alterations in Males With Alport Syndrome

IntroductionAlport syndrome (AS) is a genetic disorder characterized by progressive hematuric nephropathy with or without sensorineural hearing loss and ocular lesions. Previous studies on AS included mostly children.MethodsTo determine the prognostic value of loss of staining for collagen type IV alpha 5 (COL4A5) and its relationship with the ultrastructural glomerular basement membrane alterations, we performed direct immunofluorescence using a mixture of fluorescein isothiocyanate-conjugated and Texas-red conjugated antibodies against COL4A5 and COL4A2, respectively, on renal biopsies of 25 males with AS (including 16 who were diagnosed in adulthood).ResultsAll patients showed normal positive staining of glomerular basement membranes and tubular basement membranes for COL4A2. Of the 25 patients, 10 (40%) patients showed loss of staining for COL4A5 (including 89% of children and 13% of adults) and the remaining 15 (60%) had intact staining for COL4A5. Compared with patients with intact staining for COL4A5, those with loss of staining had more prominent ultrastructural glomerular basement membrane alterations and were younger at the time of biopsy. By Kaplan-Meier survival analysis and Cox regression analysis, loss of staining for COL4A5 predicted earlier progression to overt proteinuria and stage 2 chronic kidney disease or worse. By multivariate Cox regression analysis, loss of staining for COL4A5 was an independent predictor of the development of overt proteinuria and stage 2 chronic kidney disease or worse.DiscussionThus, the COL4A5 expression pattern has an important prognostic value and it correlates with the severity of ultrastructural glomerular basement membrane alterations in males with AS. Loss of COL4A5 staining is uncommon in patients with AS diagnosed in their adulthood.

CD154 Induces Matrix Metalloproteinase-9 Secretion in Human Podocytes.

Matrix remodeling is a key feature of glomerulosclerosis secondary to diabetes or hypertension. Podocytes contribute to glomerular basement membrane (GBM) turnover by producing matrix components and matrix remodelling enzymes, including matrix metalloproteinases (MMPs). The CD40/CD154 signaling pathway modulates matrix remodeling through the synthesis of MMPs and tissue inhibitors of MMPs. Platelets are a primary blood reservoir of CD154. Here we studied, the impact of the CD154/CD40 pathway on MMP-9 expression by cultured human podocytes. The role of CD40/CD154 was evaluated upon exposure of podocytes to recombinant human CD154 (rhCD154) or activated platelet supernatants from healthy human subjects. We first showed by protein and mRNA expression that CD40 was synthesized by podocytes and detectable on kidney tissue sections. CD40 expression was acquired during podocyte differentiation and enhanced upon exposure to rhCD154. In podocytes, rhCD154 induced an increase of MMP-9 production as shown by RT-PCR, Western blot and and gelatin zymography. Activated platelet supernatants induced MMP-9 mRNA synthesis in podocytes, an effect reduced by anti-CD40 antibody. Our results underscore a potential role for platelets through the CD40/CD154 signaling pathway in the control of GBM synthesis and degradation, via its regulatory role on MMP-9 production. CD154 secretion by activated platelets may contribute to GBM alterations in proteinuric nephropathies. J. Cell. Biochem. 117: 2737-2747, 2016. © 2016 Wiley Periodicals, Inc.

Glomerular disease with idiopathic linear immunoglobulin deposition: a rose by any other name would be atypical

Nasr etal. (2016) report 20 cases of "atypical anti-GBM disease," characterized by bright, linear glomerular basement membrane (GBM) immunoglobulin deposition in patients who lacked anti-GBM antibodies by conventional testing and who had a relatively benign course. Half had light chain restriction. The term "idiopathic linear immunoglobulin deposition" may be preferable until anti-GBM activity is demonstrable, since there are alternative mechanisms of linear deposition in the GBM, including physicochemical affinity for GBM components and alterations of the GBM itself.

Glomerular basement membrane injuries in IgA nephropathy evaluated by double immunostaining for α5(IV) and α2(IV) chains of type IV collagen and low-vacuum scanning electron microscopy.

The glomerulus contains well-developed capillaries, which are at risk of injury due to high hydrostatic pressure, hyperfiltration, hypertension and inflammation. However, the pathological alterations of the injured glomerular basement membrane (GBM), the main component of the glomerular filtration barrier, are still uncertain in cases of glomerulonephritis. We examined the alterations of the GBM in 50 renal biopsy cases with IgA nephropathy (31.8 ± 17.6 years old) using double immunostaining for the α2(IV) and α5(IV) chains of type IV collagen, and examining the ultrastructural alterations by transmission electron microscopy (TEM) and low-vacuum scanning electron microscopy (LV-SEM). The GBM of IgA nephropathy cases showed various morphological and qualitative alterations. In the TEM findings, thinning, gaps, rupture, thickening with a lamellar and reticular structure and double contours were detected in the GBM. Double immunostaining for α5(IV) and α2(IV) showed thickening of the GBM with reduced α5(IV) and increased α2(IV), or mosaic images of α5(IV) and α2(IV), and holes, fractures, spiny projections and rupture of α5(IV) in the GBM. In addition, LV-SEM showed an etched image and multiple holes in a widening and wavy GBM. These findings might be associated with the development of a brittle GBM in IgA nephropathy. Glomerular basement membrane alterations were frequently noted in IgA nephropathy, and were easily evaluated by double immunostaining for α2(IV) and α5(IV) of type IV collagen and LV-SEM. The application of these analyses to human renal biopsy specimens may enhance our understanding of the alterations of the GBM that occur in human glomerular diseases.

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

A Model of Strain-Dependent Glomerular Basement Membrane Maintenance and Its Potential Ramifications in Health and Disease

A model is developed and analyzed for type IV collagen turnover in the kidney glomerular basement membrane (GBM), which is the primary structural element in the glomerular capillary wall. The model incorporates strain dependence in both deposition and removal of the GBM, leading to an equilibrium tissue strain at which deposition and removal are balanced. The GBM thickening decreases tissue strain per unit of transcapillary pressure drop according to the law of Laplace, but increases the transcapillary pressure drop required to maintain glomerular filtration. The model results are in agreement with the observed GBM alterations in Alport syndrome and thin basement membrane disease, and the model-predicted linear relation between the inverse capillary radius and inverse capillary thickness at equilibrium is consistent with published data on different mammals. In addition, the model predicts a minimum achievable strain in the GBM based on the geometry, properties, and mechanical environment; that is, an infinitely thick GBM would still experience a finite strain. Although the model assumptions would be invalid for an extremely thick GBM, the minimum achievable strain could be significant in diseases, such as Alport syndrome, characterized by focal GBM thickening. Finally, an examination of reasonable values for the model parameters suggests that the oncotic pressure drop-the osmotic pressure difference between the plasma and the filtrate due to large molecules-plays an important role in setting the GBM strain and, thus, leakage of protein into the urine may be protective against some GBM damage.

Hypertension and kidney alterations in rat offspring from low protein pregnancies

Low birth weight contributes to the early onset of end-stage renal disease. Therefore, this study was undertaken to investigate early and late glomerular structural alterations in both sexes of Wistar rat offspring from dams submitted to severe low protein intake during gestation. Offspring from dams fed normal protein (19% of protein) or low protein (5% of protein) were studied at days 0, 10, 90 and 180 of age. Inner cortical structure showed immature (comma-shaped and S-shaped forms) and mature corpuscles in different proportions in low protein offspring (less maturity) and normal protein offspring (more maturity). At day 10 (end of the nephrogenesis period), immature corpuscles were observed only in low protein offspring. In adulthood, low protein offspring had higher blood pressure, and showed thicker glomerular basement membrane (GBM) with effacement of the pedicles, and slit diaphragm absent with some podocytes directly adhering to the basal membrane with pedicles absent. The number of renal corpuscles was lower in low protein offspring than in normal protein offspring of the same sex, all age groups (P < 0.001). No interaction was observed between sex and maternal nutrition for the same sex and all age groups. Gestational low protein leads to glomerulogenesis retardation and consequently a lower nephron number with thick GBM and structural alterations in the pedicles of podocytes.

Nonneoplastic Kidney Diseases in Adult Tumor Nephrectomy and Nephroureterectomy Specimens: Common, Harmful, Yet Underappreciated

Nonneoplastic kidney diseases, such as arterionephrosclerosis and/or diabetic nephropathy, are commonly encountered in tumor nephrectomy and nephroureterectomy specimens. Although any nonneoplastic kidney disease may be encountered in these resection specimens by chance, additional diseases that may be related to the underlying neoplasm or its treatment regimen include thrombotic microangiopathy, Amyloid A amyloidosis, membranous nephropathy, immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, pauci-immune crescentic glomerulonephritis, focal segmental glomerulosclerosis, minimal-change disease, acute interstitial nephritis, and xanthogranulomatous pyelonephritis. Given the morbidity of chronic kidney disease and the relatively favorable 5-year survival rates for urothelial and renal cell carcinomas, accurate evaluation of the nonneoplastic kidney parenchyma is important. We will discuss our approach for evaluating the nonneoplastic kidney parenchyma in tumor nephrectomy and nephroureterectomy specimens. The pathologic features of the aforementioned kidney diseases as well as pertinent references will be reviewed. The identification of glomerular abnormalities, including mesangial sclerosis or hypercellularity, segmental sclerosis, crescent formation, glomerulitis, or glomerular basement membrane alterations, should lead to additional immunofluorescence and electron microscopic studies. Safeguards to ensure that the nonneoplastic parenchyma is not overlooked include adding this important parameter to synoptic reports and obtaining periodic acid-Schiff and/or Jones methenamine silver stains prior to microscopic evaluation of the neoplasm. Relevant literature and University of Chicago Medical Center pathology archives. The practicing surgical pathologist should be aware of the importance of both correctly classifying the resected renal or urothelial neoplasm and the concomitant nonneoplastic kidney disease that may be present in these specimens.

Invagination and infolding of podocytes in glomerular basement membrane in the cases of primary membranous nephropathy

The ultrastructural findings of membranous nephropathy (MN) are well described. Recently, podocyte infolding in the glomerular basement membrane (GBM) has been observed to be a unique ultrastructural finding formed from diffuse spherical microparticles and microtubules in the GBM. However, these alterations of glomerular epithelial cells have not been well characterized in MN. We selected 126 renal biopsies of primary MN that were diagnosed by light microscopy and immunofluorescence. In these biopsies, we investigated the ultrastructural alterations of GBM and podocytes, especially the presence of podocyte invagination, podocyte infolding, and spherical microparticles in the GBM. In 98 cases (77.8%) we ultrastructurally detected occasional invagination of podocytes in the GBM within or around electron-dense or lucent deposits in mainly stage II-III of MN. In 40 cases (31.7%), we found spherical microparticles in addition to the podocyte invaginations in the GBM. In our cases, spherical microparticles were divided into three types; podocyte infolding, cell debris and virus-like particle types. Only one case displayed numerous spherical microparticles (microspheres) that were probably caused by infolding of podocytes. These microspheres, about 80 nm in diameter, were covered by unit membrane, and were accompanied by similar-sized microtubules and protrusions of podocytes. The spherical microparticles in the other cases were associated with cell debris (n = 23) or virus-like particles (n = 16) and were not connected with podocytes. Podocyte invagination associated with subepithelial deposits was a common pathological finding of primary MN, especially stage II-III of MN. The spherical microparticles in GBM in the case of MN may be associated with not only podocyte infolding but also cell debris and virus-like particles. The spherical microparticles in GBM due to diffuse podocyte infolding was considered as a new pathology finding of the GBM and may appear to be a new glomerular disease entity termed podocytic infolding glomerulopathy.

Glomerular basement membrane alterations induced by gentamicin administration in rats

The widespread therapeutic use of the aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic side effect, which can lead to acute renal failure. This study aimed at examining effects of high, supratherapeutic doses of gentamicin on morphological, structural and functional alterations of the glomerular basement membrane in adult rats. Experiments were done on 30 male Wistar rats, divided into two experimental groups. GM-group (20 rats) received gentamicin at a dose of 100 mg/kg intraperitoneally during eight consecutive days. Control or C-group (10 rats) received 1 ml/day saline intraperitoneally. For histological analysis, 5 μm thick sections were stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), and Jones methenamine silver. Glomerular basement membrane thickness, glomerular area, major and minor axes, perimeter, diameter, roundness and mean optical density were analyzed. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium and potassium. In GM-group rats, glomeruli were larger and glomerular basement membrane was diffusely and irregularly thickened with neutrophil cell infiltration. Glomerular area, major axis, minor axis, diameter and perimeter were significantly higher in GM-group compared to C-group rats. Opposite to this, glomerular optical density and average roundness were larger in C-group than in gentamicin-treated animals. Our results clearly showed morphological and structural alterations of glomeruli and glomerular basement membrane as well as alterations of proximal tubules in adult rats exposed to high doses of gentamicin.

Matrix metalloproteinase-2, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 in the peripheral blood of patients with various glomerular diseases and their implication in pathogenetic lesions: study based on an enzyme-linked assay and immunohistochemical staining

Various glomerular diseases progress to end-stage renal failure due to an accumulation of the mesangial matrix (MM) and a thickening of the glomerular basement membrane (GBM). Both the MM and GBM are consistently metabolized through the synthesis and destruction of the matrix. Such synthesis is influenced by transforming growth factor-beta (TGF-beta) and other factors, whereas the destruction is presumed to be mediated by both matrix metalloproteinases (MMPs) and inhibitors of matrix metalloproteinases (TIMPs). Based on such evidence, we tried to detect MMP-2, MMP-9, and TIMP-1 in the peripheral blood of patients with various glomerular diseases. Serum was used to detect MMP-2 and TIMP-1, while plasma was used to detect MMP-9. These enzymes were detected using an enzyme-linked assay. The findings showed an increased level of MMP-2 in patients with a alteration of GBM, typically membranous nephropathy (MN), regardless of the differences in their etiological processes. In contrast, MMP-9 did not show a strong association with any specific glomerular abnormalities. However, it mainly tended to increase in patients with MM accumulation. In addition, the localization of MMP-2, MMP-9, and TGF-beta1 was studied using immunohistochemical staining. MMP-2 was demonstrated to exist in the glomerular capillary loop (GCL) as well as in the mesangial cells and the mesangial matrix. MMP-9 was found to exist in mesangial cells and the matrix, GCL, infiltrated neutrophils, and some tubular epithelial cells. Positive staining for TGF-beta1 in GCL was found to be associated with an increased level of MMP-2 in patients with MN, whereas in MM such positive staining was not necessarily associated with an increased level of MMP-9. These results therefore suggest that MMP-2 plays an important role in the degradation of GBM, while MMP-9 only moderately affects the degradation of MM.

Podocyte-Specific Deletion of Integrin-Linked Kinase Results in Severe Glomerular Basement Membrane Alterations and Progressive Glomerulosclerosis

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.

Glomerular Expression of α2(IV) and α5(IV) Chains of Type IV Collagen in Patients with IgA Nephropathy

IgA nephropathy (IgAN) frequently accompanies glomerular basement membrane (GBM) alterations. To clarify the compositional changes of the GBM in IgAN, we examined the glomerular expression of α2(IV) and α5(IV) chains of type IV collagen in 36 patients with IgAN. Increased expression of the α2(IV) chain was observed in the expanded mesangium, and there were segmental sclerotic lesions of glomeruli in which the expression of the α5(IV) chain in the GBM was reduced. These lesions were found in 11 out of the 36 patients (30.6%). The patients with these lesions had more proteinuria, lower creatinine clearance and severer histological changes than the patients without them. On the other hand, a partial reduction of α5(IV) chain in normal-appearing GBM was observed in 15 out of the 36 patients (41.7%). The patients with this lesion had a longer disease period before biopsy than the patients without it. No other clinical and histological parameters were related to the partial reduction of α5(IV) in the GBM. In conclusion, an imbalance of glomerular α2(IV) and α5(IV) chain expression occurred in IgAN. This may be associated with the morphological changes of glomerular sclerosis and GBM alterations in IgAN. Further studies are required to understand the pathogenesis of the abnormal expression of type IV collagen chains.

Distribution of alpha-chains of type IV collagen in glomerular basement membranes with ultrastructural alterations suggestive of Alport syndrome.

In Alport syndrome (AS) impaired production and/or assembly of col IV alpha-chain isoforms results in abnormal structure of glomerular basement membrane (GBM), haematuria and, frequently, progressive renal disease. We investigated the relationship between col IV alpha-chains expression and morphology of GBM, as a possible key to the better understanding of the pathogenesis of renal disease in AS. GBM distribution of col IV alpha1-, alpha3-, and alpha5-chain was investigated by immunohistochemistry in 32 patients (21 males and 11 females, mean age at biopsy of 11.5 years) with ultrastructural findings suggestive of AS. Ten patients had a proven COL4A5 mutation. Based on the severity of ultrastructural findings, the biopsies were grouped in three (I-III) electron microscopy (EM) classes. Significant EM changes of GBM (thinning, thickening, splitting, basket weaving of the lamina densa) were singularly evaluated using a semiquantitative scale (0-3). Col IV alpha1-chain was demonstrated in GBM of all patients. Three patterns of staining for col IValpha3- and alpha5-chains were observed: positive, negative, and alpha3(IV)-positive/alpha5(IV)-negative. By chi(2)-test, EM class III lesions and complete loss of alpha3(IV)- and alpha5(IV)-antigen were significantly more frequent (P<0.05 and P<0.01) in male patients, but no significant relation was observed between EM classes and immunohistochemical patterns. GBM alterations did not correlate with staining for alpha5(IV)-chain. Intensity of alpha3(IV)-chain staining, however, had a negative correlation (P<0.05) with the severity of GBM basket weaving. Our results suggest that the alpha3(IV)-chain-containing col IV-network plays a fundamental role in structural and, possibly, functional organization of GBM. Absence of alpha3(IV)-chain in GBM could indicate a more severe renal disease in AS.

Immunoelectron microscopic study on type I, II and III TGF-beta receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats.

Transforming growth factor (TGF)-beta is a regulator of extracellular matrix accumulation. Both TGF-beta receptors, type I (TbetaRI) and type II (TbetaRII), may be required for signal transduction in the TGF-beta pathway. The aim of this study was to investigate the relationship between the TGF-beta pathways and glomerular basement membrane (GBM) accumulation in vivo. We examined TbetaRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats. In normal control rats TbetaRI was not detected on GEP, TbetaRII expression was very occasionally found on GEP and TbetaRIII was seen in the cytoplasm of the GEP. TbetaRI, TbetaRII, and TbetaRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TbetaRI, TbetaRII, and TbetaRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TbetaRI and TbetaRII on GEP increased in association with GBM thickening. Expression of TbetaRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TbetaRI and TbetaRII on GEP was noted, but from day 7 onwards, expression of TbetaR II on GEP dramatically decreased. Expression of TbetaRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis. The results suggest that persistent upregulation of expression of TbetaRI, TbetaRII and possibly TbetaRIII on GEP may contribute to GBM matrix accumulation in vivo.

Electron microscopy study on alterations of glomerular basement membrane in IgA nephropathy

To elucidate the morphological basis of glomerular hematuria in IgA nephropathy, morphometric analysis of GBM alterations was performed by electron microscopy in 73 cases of IgA nephropathy. These cases were divided into 2 groups by the degree of hematuria. The IgA (1) groups (52 cases) showed 1-30 Red Blood Cells (RBC)/hpf in urinary sediment and the IgA (2) group (21 cases) showed 30 or more RBC/hpf. The GBM alterations were observed in 67% of IgA nephropathy patients. Splitting and focal thinning of GBM were more frequent in the IgA (2) group than in the IgA (1) group. The gap was not associated with the degree of hematuria, but when accompanying splitting, it was with the degree of hematuria. It was concluded that most IgA nephropathy patients had GBM alterations, and splitting of the lamina densa, focal thinning of GBM, and a gap with splitting, which was associated with the degree of hematuria.

Uranyl nitrate-induced glomerular basement membrane alterations in rabbits: a quantitative analysis.

Uranyl nitrate-induced glomerular basement membrane alterations in rabbits: a quantitative analysis.