Resolution of the three dimensional structure of components of the glomerular filtration barrier (original) (raw)
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Imaging of the Porous Ultrastructure of the Glomerular Epithelial Filtration Slit
Journal of the American Society of Nephrology, 2010
The ultrastructure of the glomerular filtration slit is still controversial. In the last 30 years, observations from transmission electron microscopy (TEM) and theoretical analysis of solute clearance produced conflicting results. Here, we used scanning EM with a high-sensitivity detector to image the deepest regions of the filtration slits and report a previously undescribed organization of the slits' ultrastructure. In contrast to previous TEM imaging, we observed circular and ellipsoidal pores in the podocyte junctions mainly located in the central region of the slit diaphragm. The normal mean pore radius estimated by digital morphometric analysis had a log-normal distribution, with an average value of 12.1 nm. In proteinuric pathologic conditions, the mean pore radius values were also log-normally distributed with the presence of some very large pores, exceeding the sizes observed in normal conditions. Our morphologic analysis suggests that the filtration slit is a heteroporous structure instead of the previously proposed zipper-like structure. Selective changes in the ultrastructural organization of the pores may be responsible for the increased filtration of plasma proteins in glomerular disease.
Microvascular Research, 1989
Recent advances in specimen preparation techniques and scanning electron microscope (SEM) design have permitted ultrastructural examination of the glomerular capillary wall in three dimensions using high-resolution scanning electron microscopy (HRSEM). Specimens in which the cytosol and cytoskeleton have been extracted. but cell membranes nuclear structures and organelles left in place, were studied using a Hitachi SEM with a resolution of approximately 3 nm. Each HRSEM micrograph displayed a depth of field and information content equivalent to IS-30 consecutive, ultrathin, transmission electron microscope (TEM) sections viewed simultaneously in perfect serial alignment. The results have confirmed previous ultrastructural observations obtained by use of TEM and, in addition, have revealed new ultrastructural features of the normal rat glomerulus. A morphometric analysis of glomerular endothelium carried out using the HRSEM micrographs revealed that the endothelial cell processes, which lie between the fenestrae, are nearly circular in cross section and that they, as well as the fenestrae, have a diameter of approximately 60 nm. The potential functional role of the fenestrae in controlling access to the underlying basement membrane requires further study. o 1989 Academic press, I~C.
Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries
Microvascular Research, 2004
Recent data from various vascular beds suggest that a layer of mucopolysaccharides covering the endothelial cells play an important role in transport processes, among others. In this study, electron microscopy (EM) was used to explore the presence of an endothelial surface layer (ESL) in rat glomerular capillaries. We adopted various fixation and labeling techniques, as follows: (1) negatively charged lipid particles were used as a tracer that was expected to be excluded from the ESL. The density of intravascular lipid particles in flow-arrested capillaries was 89% lower in a 200-nm periendothelial area than in the rest of the luminal space (n = 6 rats, P < 0.001); (2) podocytes of cryofixed fresh tissue had a 20-nm extramembranous coat, interpreted as the true glycocalyx; the coat was less expressed on the endothelium; (3) on unfixed endothelial cells, colloidal lanthanum labeled a 60-nm-thick layer, occasionally forming lumps; (4) perfusion with a fluorocarbon-based oxygen-carrying fixative, followed by tannic acid contrast enhancement, revealed an extensive (> 200 nm) ESL not previously described; however, this finding was restricted to superficial glomerular capillaries; (5) Cupromeronic Blue cytochemistry displayed a loose proteoglycan network in fenestral openings and, occasionally, a semiordered ESL; (6) ferricyanide-reduced osmication resulted in increased numbers of fenestral diaphragms. In conclusion, this study provides novel morphological evidence to support the presence of a significant glomerular ESL.
Glomerular Filtration Barrier Assembly: An insight
A glomerulus is the network of capillaries that resides in the Bowman's capsule that functions as a filtration unit of kidney. The glomerular function ensures that essential plasma proteins are retained in blood and the filtrate is passed on as urine. The glomerular filtration assembly is composed of three main cellular barriers that are critical for the ultrafiltration process, the fenestrated endothelium, glomerular basement membrane and highly specialized podocytes. The podocytes along with their specialized junctions "slit diaphragm" form the basic backbone of this filtration assembly. The presence of high amounts of protein in urine a condition commonly referred as proteinuria indicates a defective glomerular filtration barrier. Various glomerular disorders including Nephrotic syndrome are characterized by significant alteration in the structure of podocytes that is associated with prolonged increase in the glomerular permeability leading to heavy proteinuria. Recent identification of proteins that are specifically localized at the slit diaphragm whose mutations and knockouts are known to result in loss of renal function has significantly advanced our understanding of the molecular makeup of this filtration assembly. The present review is an effort to summarize the recent developments in this field and highlight our understanding of the glomerular filtration barrier assembly.
AJP: Renal Physiology, 2007
The glomerular filtration barrier (GFB) is generally considered to consist of three layers: fenestrated glomerular endothelium, glomerular basement membrane, and filtration slits between adjacent podocyte foot processes. Detailed anatomical examination of the GFB has revealed a novel abluminal structure, the sub-podocyte space (SPS), identified as the labyrinthine space between the underside of podocyte cell body/primary processes and the foot processes. The SPS covers 50-65% of the filtration surface of the GFB, indicating that SPS may influence glomerular permeability. We have examined the contribution of the SPS to the permeability characteristics of the GFB using multiphoton microscopy techniques in isolated perfused glomeruli and in the intact kidney in vivo. SPS were identified using this technique, with comparable dimensions to SPS examined with electron microscopy. The passage of the intermediate weight molecule rhodamine-conjugated 10kDa-dextran, but not the low weight molecule Lucifer yellow (H450 Da), accumulated in SPS-covered regions of the GFB, as compared with GFB regions not covered by SPS ("naked regions"). Net Lucifer Yellow flux (taken to indicate fluid flux) through identifiable SPS regions was calculated to be 66 -75% of that occurring through naked regions. These observations indicate both ultrafiltration and hydraulic resistance imparted by the SPS, demonstrating the potential physiological contribution of the SPS to glomerular permeability.
3D Reconstruction of the Glycocalyx Structure in Mammalian Capillaries using Electron Tomography
Microcirculation, 2012
Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained preparations to reveal details of the architecture of the glycocalyx just above the endothelial cell layer. One preparation uses the novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method).
SEM studies of acellular glomerular basement membrane in human diabetic glomerulopathy
The Anatomical Record, 1986
Previous transmission electron microscopic studies have demonstrated glomerular basement membrane (GBM) thickening and mesangial matrix (MM) expansion in chronic stages of diabetes. It is difficult, however, to achieve an appreciation of GBM surface features and distribution of MM in planar views. In the current study, autopsy human renal cortical tissue from patients with end-stage diabetic nephropathy were minced and rendered acellular with detergents prior to fixation, cryofracture, and preparation for light microscopic CM), transmission electron microscopic (TEM), and scanning electron microscopic (SEM) observation in an effort to visualize extracellular materials in three dimensions.
Scientific Reports, 2018
This study evaluated endothelial cells and podocytes, both being primary components of the glomerular filtration barrier, in the progression of membranoproliferative glomerulonephritis (MPGN) using modified scanning electron microscopy (mSEM) analysis. BXSB/MpJ-Yaa model mice exhibited autoimmune-mediated MPGN characterised by elevated serum autoantibody levels, albuminuria, renal dysfunctional parameters, and decreased glomerular endothelial fenestrations (EF) and podocyte foot process (PFP) effacement with immune cell infiltration. Similar to transmission electron microscopy, mSEM revealed a series of pathological changes in basement membrane and densities of EF and PFP in BXSB/MpJ-Yaa compared with control BXSB/MpJ at different stages. Further, immunopositive area of endothelial marker (CD34), podocyte functional molecules (Nephrin, Podocin, Synaptopodin, and Wilms’ tumour 1 (WT1)), and vascular endothelial growth factor A (VEGF A) significantly decreased in the glomerulus of BXS...