Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis

Abstract

Background The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown.Methods To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach.Results Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at singleorganelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology.Conclusions Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases

“…Interestingly, the endogenously produced tracer 1 / 2 vdbp-mCherry is uptaken by the early PT segment, differing from the exogenous tracer b-lactoglobulin-Cy5, which is mainly reabsorbed by the more distal portions of the proximal tubule. This observation could be due either to the chemical differences of the tracers 44 or to the saturation of reabsorption capacity of the early portion of PT by the endogenous protein in physiological conditions-so that any exogenous tracer would be uptaken by the late portion of PT.…”

Iron and Cadmium Entry Into Renal Mitochondria: Physiological and Toxicological Implications

“…Renal diseases causing Fe overload of the kidney involve proteinuria of glomerular origin, that predominantly causes PT damage, or decreased PT reabsorption (e.g., as in Fanconi syndrome due to PT damage) with Fe overload of downstream nephron segments. Overload occurs because the PT (in particular the S1-segment) ( Schuh et al, 2018 ) is equipped with both a receptor for (metallo-)protein endocytosis, megalin:cubilin:amnionless ( Kozyraki et al, 2001 ; Weyer et al, 2011 ) and endo-lysosomal machinery expressing DMT1 ( Abouhamed et al, 2006 ), promoting Fe accumulation and damage of PT cells. Distal nephron segments take up Fe via DMT1 in their apical membranes (DCT > thick ascending limb of LOH > CD) ( Ferguson et al, 2001 ).…”

Advances in Ex Situ Tissue Optical Clearing

“…Until now, only a few studies have focused on development and application of TOC for the kidney . These are, however, advanced and bring novel ideas relevant to both basic science and clinical practice.…”