title: Quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in Munich Wistar rats.
abstract: Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)(1,2,3). Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis(4). These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability(5). Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis(6). The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported(2). These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.
authors: Medicine/Nephrology, Indiana University School of Medicine.
authors: Ruben M
authors: RM
authors: Sandoval
authors: Bruce A
authors: BA
authors: Molitoris
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics*; D007678:Kidney Glomerulus / Q000737:chemistry* / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics*; D014966:Xanthenes / Q000032:analysis / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D005456:Fluorescent Dyes; D012709:Serum Albumin; D014966:Xanthenes; C034657:Texas red
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