Morphological diagnosis of Alport syndrome and thin basement membrane nephropathy by low vacuum scanning electron microscopy

Abstract

Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetic disorders caused by mutations of the type IV collagen genes COL4A3, COL4A4, and/or COL4A5. We here aimed to investigate the three-dimensional ultrastructure of the glomerular basement membrane (GBM) in order to introduce a novel method of diagnosing AS and TBMN. The subjects were 4 patients with AS and 6 patients with TBMN. Conventional renal biopsy paraffin sections from AS and TBMN patients were stained with periodic acid methenamine silver (PAM) and observed directly under low vacuum scanning electron microscopy (LVSEM). The PAM-positive GBMs were clearly visible under LVSEM through the overlying cellular components. The GBMs showed characteristic coarse meshwork appearances in AS, and thin and sheet-like appearances in TBMN. At the cut side view of the capillary wall, the GBMs in AS appeared as fibrous inclusions between a podocyte and an endothelial cell, while the GBMs in TBMN showed thin linear appearances. These different findings of GBMs between AS and TBMN were easily observed under LVSEM. Thus, we conclude that three-dimensional morphological evaluation by LVSEM using conventional renal biopsy paraffin sections will likely be useful for the diagnosis of AS and TBMN, including for retrospective investigations.Alport syndrome (AS) is a genetic disorder caused by mutations of the type IV collagen genes COL4A3, COL4A4, and COL4A5. It is characterized by renal, cochlear, and ocular involvement, and AS patients are moreover predisposed to developing end-stage renal disease (1, 6, 12). On the other hand, thin basement membrane nephropathy (TBMN) results from mutations of the COL4A3 and COL4A4 genes, and is associated with few extra-renal abnormalities and a good renal prognosis. One of the characteristic features of AS and TBMN is morphological changes of the glomerular basement membranes (GBMs). A combination of type IV collagen genes, which produce different constitutions of the alpha chain of collagen type IV, is very important in the construction of normal GBMs. Renal findings under transmission electron microscopy (TEM) of AS include partially thickening, splitting, fragmenting, and basket-weave appearances of the GBMs; whereas TBMN is characterized by a reduction of the thickness of the GBMs. Although the diagnosis of AS or TBMN is generally established by detailed family history, urinalysis, biopsy of the kidney or skin, and molecular genetic analysis, the differential diagnosis is often difficult in both these diseases, owing largely to insufficiency of the biopsy samples and the complexity of molecular genetic analysis. Furthermore, if there are no glomeruli in the biopsy samples for immunofluorescence and TEM analyses,

Observation of Corneal Wound Healing and Angiogenesis Using Low-Vacuum Scanning Electron Microscopy

“…The greatest advantage of LV-SEM is that conventional paraffin sections for light microscopy can be used so that ultramicroscopic images can be obtained in the same sections for immunostaining. [4][5][6][7]9,18 This advantage enables to search selectively the area to observe in LV-SEM, which can not be done in TEM. Slides that have been used for light microscopy can be used for LV-SEM so that retrospective analysis of two-dimensional structures is possible.…”

Complexities of the glomerular basement membrane

“…For instance, use of SBF-SEM demonstrated invasion of podocyte foot processes into damaged areas of GBM in various mouse models of glomerular disease 9 . Low-vacuum SEM [G] (LV-SEM), has been used to show the presence of spikes on the GBM and subepithelial electron-dense deposits in biopsy samples from patients with membranous nephropathy 59 , irregular GBM thickening and a basket-weave appearance in samples from patients with Alport syndrome 60 , and GBM thinning and perforation in samples from patients with thin basement membrane nephropathy (TBMN) 60,61 . Another emerging SEM technique called helium ion microscopy [G] (HIM) 62 has been used to reveal striking alterations on the surface of glomeruli from Col4a3 -/mice, including the deposition of long microfilaments, cytoplasmic bleb-like projections, and increased numbers of prominent podocyte bridge-like processes 63 .…”

Low-vacuum scanning electron microscopy may allow early diagnosis of human renal transplant antibody-mediated rejection

“…It is impossible to utilizing TEM for every case of renal transplant biopsy. We recently reported the usefulness of low vacuum scanning electron microscopy (LVSEM) for the rapid three-dimensional analysis of renal biopsy specimens (11,15,16). LVSEM is comparatively less expensive, and it can facilitate convenient and quick observations because it does not require preliminary processing.…”