Multiple Factors Influence Glomerular Albumin Permeability in Rats

Abstract

Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC A ) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC A . Multiple factors influenced GSC A , including the kidney depth of image acquisition (10-20 mm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC A in Simonsen Munich Wistar rats from 0.03560.005 to 0.01660.004 (P,0.01). Frömter Munich Wistar rats had a much lower GSC A in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability. Over the past several years, the roles the glomerular filtration barrier and the proximal tubule play in the development of albuminuria have been debated. 1,2 The present textbook model, in which albumin filtration across a normal glomerulus is thought to be minimal, has recently been challenged. A wide array of genetic, molecular, biochemical, and imaging studies are consistent with proximal tubule cells (PTCs) having a role in reclaiming filtered proteins, including albumin, and thus minimizing proteinuria. These studies include the following: knockout mice lacking Na+-H+ exchanger isoform 3 or chloride channel-5; 3 megalin-cubilin complex defects; 4 Rab 38-mediated tubular proteinuria and albuminuria; 5 statin-mediated inhibition of guanosine triphosphatase prenylation with reduced proximal tubule (PT) endocytosis; 4,6-8 mice lacking FcRn, the IgG and albumin receptor; 9,10 and selective PTC injury using D-serine 11 or the selective expression of diphtheria toxin receptor on PTC. 12 In a preliminary communication, Menzel and colleagues showed that PT reabsorption and transcytosis of podocyte produced and secreted albumin labeled with V5 and hemagglutinin tags. 13 Finally, recently published data 14 using advanced scanning electron microscope imaging techniques indicate that the podocyte slit diaphragm has pores that are much larger than previously determined and are of the size required for albumin filtration.The development of in vivo two-photon microscopy has enabled the direct visualization and quantitation of fluorescent compounds as they filter through the glomerulus and are endocytosed by

Cubilin Maintains Blood Levels of HDL and Albumin

“…Support for such a mechanism comes from live renal imaging studies showing transcytosis of albumin in rat PTCs and other studies demonstrating the in vitro transcytosis of megalin ligands. 12,13,[17][18][19]30 The underlying mechanisms by which cubilin-mediated uptake would lead to ligand transcytosis remains to be established. In a recent study, a significant reduction in plasma albumin levels and decreased half-life of albumin in the blood were observed in mice transplanted with kidneys deficient in neonatal Fc receptor (FcRn).…”

“…13 Megalin also mediates transcytosis of thyroglobulin, 14 Shh, 15 and the megalin-cubilin binding protein, receptor-associated protein. 16 Indirect evidence for cubilin playing a similar role in ligand transcytosis comes from findings showing that albumin is transcytosed back to circulation via the proximal tubule epithelium, [17][18][19] as well as the fact that cubilin-mediated uptake of intrinsic factor-B 12 complex in the gut leads to its release into blood in the form of transcobalamin-B 12 . 20 Whether or not renal cubilin-mediated uptake of apoA-I and albumin from the glomerular filtrate might be part of a salvage process that affects levels of these proteins in the blood is not known.…”

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

“…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.…”

New frontiers in intravital microscopy of the kidney

“…It has also been proposed, partly on the basis of imaging studies, that albumin may be trafficked directly across proximal tubule cells and back into the blood stream [29,30]. These surprising findings have huge implications for clinical practice and have inevitably generated much controversy; the picture is further complicated by the fact that different groups using the same imaging techniques have reported very different values for the glomerular sieving coefficient [31,32].…”