Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy

Highlights

• The location of the initial connecting tubule is identified using 3D computerized tracing.

• The cellular morphology of the initial connecting tubule is revealed using EM, showing the complexity of the involved cells.

• A terminal UB tip frequently connected to two nephrons at different developmental stages.

• Immunohistochemistry revealed expression of the water-salt and acid-base transporters in the connecting tubules.

Abstract

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited.

The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na⁺-Cl⁻ cotransporter, H⁺-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development.

Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

Introduction

Connecting tubules (CNT) is a transitional segment between collecting ducts (CDs) and nephrons [1]. Unlike other tubular segments, where the epithelium consists of a single cell type, the CNT epithelium is composed of four cell types. In addition to the CNT cells proper, the other cell types reflect the morphology and transport properties of the adjacent segments [2]. For example, DCT cells express Na⁺-Cl⁻ Cotransporter (NCC) in the DCT, principal cells express aquaporin (AQP) 2 in the CD, and intercalated cells express H⁺-ATPase in both CDs and the late DCT [[3], [4], [5]]. Thus, the CNT is not only a simple connection between nephrons and CDs, but plays also important physiological roles in fine-tuning the sodium reabsorption and in regulating the water-salt and acid-base balances [[6], [7], [8]]. Improper morphogenesis of the CNT results in partial or complete abnormal morphology and function of the nephrons and the CD system, such as seen in diffuse renal dysplasia or hydronephrosis [9].

CDs and nephrons are of embryonically different origins. The CD is derived from iterative bifid branching of the ureteric bud (UB) arising from the caudal end of the nephric duct (Wolffian duct), while the nephrons originate from the metanephric mesenchyme. At the nephrogenic zone, which is the outer layer of metanephros directly beneath the renal capsule, the terminal tips of the UB release signals to induce the metanephric mesenchymal cells to aggregate and form a densely packed cellular cap that cover the tip. Thereafter, the cellular cap undergoes a so-called mesenchymal-epithelial transition (MET) to form the renal vesicle (RV), which is the initial stage of the nephron. Subsequently, the RV transforms into comma- and then S-shaped bodies by successive formation of the lower (first) and upper (second) clefts. The earliest migration of endothelial progenitor cells into the lower cleft (vascular cleft) of the S-shaped body initiate formation of glomerular capillary loops, thereby transforming the primitive nephron into a capillary-loop-stage nephron, which eventually develops into a mature nephron [8,10,11].

For nearly a decade it has been debated whether the CNT originates from the nephron or from the CD system. Due to their properties some reports that, the CNTs originate from the nephrogenic blastema, while the CDs originates from the UB [[12], [13], [14]]. Others propose that both CNTs and CDs arise from the UB [15]. Schmitt and co-workers raise a third hypothesis suggesting that the CNT develops as a product of mutual induction from adjacent segments leading to a unique hybrid epithelium [16]. Recently, both genetic and protein labeling studies have demonstrated that a group of cap mesenchyme-derived Six2 positive nephron progenitor cells form the distal RV, the precursor for the nephron. Then the initial connection protrude and further break into the lumen of the terminal UB tip, eventually leading to a lumen inside the cellular connection [17,18].

The aim of the present study was to investigate and extend the understanding of the morphogenesis of the nephron-CD connection by computer assisted tubule tracing, studies of the ultrastructure, and immunolabelling of the transporters along the connection during kidney development in mice.