Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes

Abstract

Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts. podocyte PODOCYTES REPRESENT THE OUTERMOST cell layer of the glomerular filtration barrier. Based on results obtained with conventional imaging techniques, the current model pictures the podocyte cell body as floating in Bowman's space while being anchored to the glomerular basement membrane via its arborizing cellular extensions. The finest branches, the foot processes, are arranged in an interdigitating pattern on the outer surface of the basement membrane (11). Neighboring foot processes are connected by a slit diaphragm which supposedly contains the integral membrane proteins nephrin, Neph1, FAT-1, and Pcadherin. These proteins are linked to the actin cytoskeleton through the adapter proteins podocin, CD2AP, and zonula occludens (ZO)-1 (17). It has been suggested that the urinary space between the podocyte and the glomerular basement membrane, the subpodocyte space, impedes the flow of the primary filtrate into Bowman's space and that therefore the subpodocyte space contributes to the filtration properties of the renal glomerulus (8, 9, 13). Hereditary and acquired podocytopathies lead to the destruction of the intricate cytoarchitecture of podocytes and consequently to a failure of the renal filter. The hallmark of such podocytopathies is the disappearance of foot processes and ultimately a detachment of affected podocytes into the urine, thus causing albuminuria (7, 15).The current view on the structural basis of the renal filtration barrier was derived from classic transmission and scanning electron microscopy. While these techniques have provided valuable information, they also suffer from intrinsic limitations and therefore leave open fundamental questions: 1) What is the dimension of a single podocyte? 2) How are podocytes arranged on the glomerular basement membrane? and 3) Does a podocyte form slit diaphragms between its own foot processes? To answer these questions, we applied novel electron microscopic techniques, i.e., focused ion beam-scanning electron microsco...

New structural insights into podocyte biology

“…FIB-SEM allows the segmentation and 3D reconstruction of large glomerular volumes. In contrast to previous reports, primary processes do not seem to be connected to the GBM by the full sole of their basal aspect but rather are only attached to the GBM by a ridge-like prominence (Burghardt et al 2015; Ichimura et al 2015). Foot processes from neighboring podocytes connect to this structure by their distal ends.…”

A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes

“…Recently, two other groups reported results using block-face SEM (FIB-SEM) where they could resolve the arrangement of neighboring podocyte foot processes and prove that individual podocytes indeed only interdigitate with adjacent podocytes but not with their own processes (33, 34). However, this technique does not have the resolution to study the SD in detail.…”

“…However, this technique does not have the resolution to study the SD in detail. One of these groups, therefore, also used electron tomography, albeit with glutaraldehyde fixed samples (34). This might be the reason this group only found one continuous layer of cell-cell contacts.…”

New approaches in renal microscopy

“…It provides superb resolution, but is technically challenging and more expensive than other EM techniques. Recently, focused ion beam-scanning electron microscopy (FIB-SEM) has been used to image podocytes and the glycocalyx with excellent resolution [10,11,53]. FIB-SEM is capable of providing global high-resolution three-dimensional EM images of the area of interest.…”