Three-dimensional Reconstruction Of The Mouse Proximal Tubule And Efferent Arteriole

ObjectiveThe nephron is the basic structural and functional unit of the kidney. It consists of the renal corpuscle and the renal tubule. The proximal convoluted tubule plays a major role in the reabsorption of Na+, HCO3-, Cl-,K+, Ca2+, PO43-, water, and organic solutes such as glucose and amino acids. Approximately half of the ultrafiltrate is reabsorbed in the proximal tubule. The proximal tubules originating from the different levels of the renal cortex have different characteristics in their spatial courses and functions. The information of the courses and relationship of the mouse proximal tubules is limited to the identification of the pars convoluta and pars recta from traditional methods. In the literature, structural and functional parameters of the mouse proximal tubule are often correlated to the segmentation well described in rat.The efferent arteriole, which arises from the glomerular capillaries and terminates at the peritubular capillaries in the cortex or at the vasa recta in the medulla, regulates the glomerular outflow resistance. It plays an important role in maintaining the glomerular filtration rate despite fluctuations in blood pressure. The efferent arteriole has a relatively small diameter and large resistance, which are very essential for the reabsorption. As the basis of functional study, the morphology of the efferent arteriole is very important. Since the mouse has pretty thin efferent arterioles, it is very difficult to reconstruct the exact courses of the efferent arterioles.In the present study, a detailed description of the courses of the mouse proximal tubules and efferent arterioles was characterized. The analysis was performed by computer-assisted reconstructions of the proximal tubules and efferent arterioles based on serial sections of Epon812-embedded tissue at the lightmicroscope level through three mouse kidneys.Methods1. Preparation of consecutive sections embedded in Epon812The kidneys from three male C57/BL/6 mice, weighing about 25g, were fixed by perfusion retrograde through the abdominal aorta with 1% glutaraldehyde in 0.06 mol/L sodium cacodylate buffer, pH 7.4 and 4% hydroxyethyl starch. Tissue blocks were cut perpendicular to the longitudinal axis of the kidney and postfixed for 2 hours in 1% OsO4 dehydrated in ethanol and acetone, then embedded in Epon 812. From each kidney, a total of 1200,2.5-μm-thick consecutive sections were obtained from the surface to the outer stripe of the outer medulla using Reichert ultracut S microtome and a diamond histoknife and stained with toluidine blue.2. Image Recordings and AlignmentUsing an Olympus AX 70 microscope equipped with a digital camera (Olympus DP 50; Olympus) attached to a standard PC, every second section was digitized. The final image size was 2596 x 1889 pixels, where each pixel corresponded to 1.16μm×1.16μm. The image recording and alignment procedure was carried out on a Windows-based PC. The relative transformation values were determined between two consecutive images and then summarized into a set of absolute transformation values. Then, the images were "moved" according to these absolute transformation values in order to achieve the consistency in physiology.3. Digital Tracing and 3D PresentationThe renal proximal tubules and efferent arterioles were traced with series of computer programs written in C on the Linux-based PC. The proximal tubules were traced from the urinary poles of glomeruli and ended at the junctions between the pars recta and the descending thin limb of the Henle's loop, while the efferent arterioles were traced from the vascular poles of glomeruli until they were transformed into peritubular capillaries or vasa recta. The x-, y-, and z-coordinates along the courses of the proximal tubules and the efferent arterioles were recorded in a data file. All subsequent data analyses were based on this data file, such as the analysis of the relationship between proximal tubules and efferent arterioles and the measurements of their length. The length of the tubules and arterioles were calculated as the sum of the Euclidian distance between two subsequent markers in the courses of the nephrons (formula below).Results1. The spatial course of the proximal tubuleIn cortical labyrinth, all of the proximal tubules leave their glomeruli ascending toward the renal surface for a distance of 100～1400μm, then return and make coils around their own glomeruli and occupy separate domains which do not intermingle with the neighboring nephron tubules. The proximal tubules originating from the superficial nephrons and midcortical nephrons form tight clusters and occupy smaller volume than the proximal tubules originating from the juxtamedullary nephrons. In medullary rays, the pars recta are arranged in a specific pattern:proximal tubules originating in the superficial cortex are arranged in the center, whereas proximal tubules originating deeper in the cortex become layered more and more peripherally, and in the deepest juxtamedullary nephrons, it is impossible to define a pars recta since the "straight" part does not run straightly in the outer stripe of the outer medulla. All the proximal tubules terminate at the boundary between the outer stripe of the outer medulla and the inner stripe of the outer medulla and transition to descending thin limb of the Henle's loop.2. The spatial course of the efferent arteriole87.5% of the efferent arterioles originating from the superficial nephrons leave their glomeruli ascending toward the renal surface until they transform into peritubular capillary networks.8.3% run toward the deep cortex then transform into peritubular capillary networks, and the remaining 4.2% immediately divide into peritubular capillary networks when left the glomeruli.48.0% of the efferent arterioles originating from the midcortical nephrons leave their glomeruli descending toward the deep cortex then transform into peritubular capillary networks.32.0% run toward the renal surface then transform into peritubular capillary networks, and 8.0% immediately divide into capillary networks when left the glomeruli. Different from the efferent arterioles originating from superfical nephrons, 12.0% of the efferent arterioles originating from the midcortical nephrons left their glomeruli in a short descent and then turned to ascend until they transform into peritubular capillary networks.66.7% of the efferent arterioles originating from the juxtamedullary nephrons leave their glomeruli descending toward the medulla then transform into peritubular networks or vasa recta.22.2% immediately divide into capillary networks or vasa recta when left the glomeruli. The remaining 11.1% left their glomeruli in a short descent and then turned to ascend until they transform into peritubular networks or vasa recta.3. The relationship between the proximal tubules and efferent arteriolesThe proximal convoluted tubule makes coils around its own glomerulus and occupies a certain domain in the cortex. The efferent arteriole originating from the same nephron is located within the domain. However, there's no exact relationship between the absolute courses of the early proximal tubules and the efferent arterioles originating from the same glomeruli.Conclusion1. The spatial courses of the proximal tubules and efferent arterioles originating from the different levels of the renal cortex reveal different characteristics, which may be relative to their functions.2. There's no exact relationship between the absolute courses of the early proximal tubules and the efferent arterioles originating from the same glomeruli.