Resolution of the three dimensional structure of components of the glomerular filtration barrier

BMC Nephrology

Chris R Neal⁴  Dave O Bates³  John M Squire⁴  Andy HJ Salmon⁴  Steve J Harper⁴  C Charles Michel¹  Carlo Knupp⁷  Judith M Mantell²  Tobias Starborg⁵  Klaus Qvortrup⁶  Kenton P Arkill² 

[1] Bioengineering, Imperial College, London, UK;Wolfson Bioimaging Facility and School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK;Cancer Biology, Division of Oncology, School of Medicine, University of Nottingham, Queen’s Medical Centre, Nottingham NG2 7UH, UK;School of Physiology and Pharmacology, University of Bristol, Bristol, UK;Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK;CFIM, Pannum Institute, Copenhagen, Denmark;Optometry and Vision Sciences, Cardiff University, Cardiff, UK

关键词: 3D reconstruction;    Glomerulus;    Electron microscopy;    Focused ion beam (FIB);    Serial block face (SBF);    Tomography;    GFB;    Glycocalyx;    Podocyte;    Urinary space;   

Others  :  1082736
DOI  :  10.1186/1471-2369-15-24

 received in 2013-11-07, accepted in 2014-01-21,  发布年份 2014

【 摘 要 】

Background

The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers – the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 μm) cellular and extracellular elements.

Methods

Here we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 10⁵ μm³ volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 μm³ volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 μm³ volume to identify the fine structure of the glycocalyx.

Results

Tom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM.

Conclusions

These images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.

【 授权许可】

   
2014 Arkill et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Deen WM, Lazzara MJ, Myers BD: Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 2001, 281:F579-F596.
• [2]Salmon AHJ, Neal CR, Harper SJ: New aspects of glomerular filtration barrier structure and function: five layers (at least) not three. Curr Opin Nephrol Hypertens 2009, 18:197-205.
• [3]Avasthi PS, Koshy V: Glomerular endothelial glycocalyx. Contrib Nephrol 1988, 68:104-113.
• [4]Avasthi PS, Koshy V: The anionic Matrix at the rat glomerular enodothelial surface. Anat Rec 1988, 220:258-266.
• [5]Rostgaard J, Qvortrup K: Sieve plugs in fenestrae of glomerular capillaries - site of the filtration barrier? Cells Tissues Organs 2002, 170:132-138.
• [6]Haraldsson B, Nystrom J, Deen WM: Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev 2008, 88:451-487.
• [7]Jeansson M, Haraldsson B: Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes. J Am Soc Nephrol 2003, 14:1756-1765.
• [8]Jeansson M, Haraldsson B: Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier. Am J Physiol Renal Physiol 2006, 290:F111-F116.
• [9]Salmon AHJ, Ferguson JK, Burford JL, Gevorgyan H, Nakano D, Harper SJ, Bates DO, Peti-Peterdi J: Loss of the Endothelial Glycocalyx Links Albuminuria and Vascular Dysfunction. J Am Soc Nephrol 2012, 23(8):1339-1350.
• [10]Neal CR, Crook H, Bell E, Harper SJ, Bates DO: Three-dimensional reconstruction of glomeruli by electron microscopy reveals a distinct restrictive urinary subpodocyte space. J Am Soc Nephrol 2005, 16:1223-1235.
• [11]Salmon AHJ, Toma I, Sipos A, Muston PR, Harper SJ, Bates DO, Neal CR, Peti-Peterdi J: Evidence for restriction of fluid and solute movement across the glomerular capillary wall by the subpodocyte space. Am J Physiol Renal Physiol 2007, 293:F1777-F1786.
• [12]Neal CR, Muston PR, Njegovan D, Verrill R, Harper SJ, Deen WM, Bates DO: Glomerular filtration into the subpodocyte space is highly restricted under physiological perfusion conditions. Am J Physiol Renal Physiol 2007, 293:F1787-F1798.
• [13]Gautier A, Bernhard W, Oberling C: *sur lexistence dun appareil lacunaire peri-capillaire du glomerule de malpighi, revelee par le microscope electronique. Comptes Rendus Des Seances De La Societe De Biologie Et De Ses Filiales 1950, 144:1605-1607.
• [14]Elias H, Allara E, Elias PM, Murthy AS: The podocytes, re-examined. Zeitschrift fur mikroskopisch-anatomische Forschung 1965, 72:344-365.
• [15]Neal CR, Harper SJ, Bates D: Abstract: 3-D reconstruction of the glomerular barrier and overlying podocyte reveals a sub-podocyte space: a new urinary space within the glomerulus? J Physiol 2003, 552P:C26.
• [16]Oltean S, Neal CR, Sison K, Qui Y, Patel P, Ahad T, Alsop C, Lee T, Harper SJ, Bates DO, et al.: VEGF165b overexpression restores normal glomerular filtration barrier function in VEGF164-overexpressing adult mice. Am J Physiol in review 2012, 303(7):F1026-F1036.
• [17]Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM: 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 2012, 19:343-351.
• [18]West JB, Fu ZX, Deerinck TJ, Mackey MR, Obayashi JT, Ellisman MH: Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy. Respir Physiol Neurobiol 2010, 170:202-209.
• [19]Denk W, Horstmann H: Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2004, 2:1900-1909.
• [20]Mantell JM, Verkade P, Arkill KP, Iop: Quantitative Biological Measurement In Transmission Electron Tomography. Electron Microscopy And Analysis Group Conference 2012., 371Journal of Physics Conference Series
• [21]Luther PK, Lawrence MC, Crowther RA: A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 1988, 24:7-18.
• [22]Kremer JR, Mastronarde DN, McIntosh JR: Computer visualization of three-dimensional image data using IMOD. J Struct Biol 1996, 116:71-76.
• [23]Mastronarde DN: Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 1997, 120:343-352.
• [24]Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nature Methods 2012, 9:671-675.
• [25]Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods 2012, 9:676-682.
• [26]Arkill KP, Knupp C, Michel CC, Neal CR, Qvortrup K, Rostgaard J, Squire JM: Similar endothelial glycocalyx structures in microvessels from a range of mammalian tissues: evidence for a common filtering mechanism? Biophys J 2011, 101:1046-1056.
• [27]Squire JM, Chew M, Nneji G, Neal C, Barry J, Michel C: Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering? J Struct Biol 2001, 136:239-255.
• [28]Hawes P, Netherton CL, Mueller M, Wileman T, Monaghan P: Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc-Oxford 2007, 226:182-189.
• [29]Bushby A, Mariggi G, Armer H, Collinson L: Correlative light and volume electron microscopy: using focused ion beam scanning electron microscopy to image transient events in model organisms. Meth Cell Biol 2012, 111:357-482.