Identification and classification of epithelial cells in nephron segments by actin cytoskeleton patterns

Abstract

The basic functional unit in a kidney is the nephron, which is a long and morphologically segmented tubule. The nephron begins with a cluster of capillaries called glomerulus through which the blood is filtered into the Bowman's space. The filtrate flows through the nephron segments. During this flow, electrolytes and solutes are reabsorbed by channels and transport systems into the capillaries wrapped around the nephron. Many questions related to renal function focus on identifying the sites of expression of these systems. In this study, we mapped whole kidney sections by confocal microscopic imaging of fluorescent phalloidin, which binds to actin filaments. In tile scans (composed of hundreds of images) of these sections, the cortex and the medullary regions (outer and inner stripes of the outer medulla, and inner medulla) could be easily identified by their cytoskeletal patterns. At a higher resolution, we identified distinct features of the actin cytoskeleton in the apical, basal, and lateral borders of the cells. These features could be used to identify segments of a nephron (the proximal tubule, thin and thick segments of Henle's loop, and distal tubule), the collecting duct system, the papillary ducts in the papilla, and the urothelium that covers the pelvis. To verify our findings, we used additional markers, including aquaporin isoforms, cytokeratin 8‐18, and WGA lectin. This study highlights the power of high‐resolution confocal microscopy for identifying specific cell types using the simple probe of F‐actin‐binding phalloidin.

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system

“…Lotus lectin (LTL) that binds to Fucose on microvilli of PTEC and is an indicator for mature PTEC 17 was also abundant (around 70%) on d16 (Figure 3(B) ). Moreover, Phalloidin allowed visualization of actin bundles, suggesting the presence of microvilli 18 on the hPSC‐derived PTEC, confirmed by immunofluorescence analysis on d16 (Figure 3(C) ). In contrast, in static culture with the same medium and the same protocol, around 60% of the cells expressed AQP1 and 55% were positive for LTL on d16 (Figure S3(A) ).…”

LGR6 marks nephron progenitor cells

“…At P3, LSR‐3D imaging was performed on thick vibratome sections, including markers for F‐Actin and DAPI, to reveal localization and identity of traced cells. 21 TdTom+ cells were exclusively found in the cortex within the nephrogenic zone, indicative of nascent nephrogenesis (Figure 5C ). In addition, by staining for JAG1, WT1, and CDH1 we were able to further delineate this exclusive distribution within nephrogenic compartments; all tdTom + cells were restricted to PTA, RV, and CSBs, as well as distal, medial and proximal parts of SSBs, coinciding with newly developing epithelial structures surrounding CDH1+ UB (Figure 5D‐F ).…”

Quantitative Analyses of Foot Processes, Mitochondria, and Basement Membranes by Structured Illumination

“…For example, phalloidin, paclitaxel, and MitoTracker ® can label f-actin, tubulin, and mitochondria, respectively. 22,23 However, paclitaxel and MitoTracker ® are not applicable to fixed cells, including paraffin-embedded kidney sections. Conventional tissue staining dyes have also been J o u r n a l P r e -p r o o f reported to possess fluorescent properties.…”