Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhai, X. Y.
Right arrow Articles by Christensen, E. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhai, X. Y.
Right arrow Articles by Christensen, E. I.
J Am Soc Nephrol 14:611-619, 2003
© 2003 American Society of Nephrology

Digital Three-Dimensional Reconstruction and Ultrastructure of the Mouse Proximal Tubule

Xiao Yue Zhai*, Henrik Birn*, Knud B. Jensen{dagger}, Jesper S. Thomsen*, Arne Andreasen{dagger} and Erik I. Christensen*

*Department of Cell Biology and {dagger}Department of Neurobiology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark.

Correspondence to: Dr. Erik Ilsø Christensen, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: 45-89-42-30-57; Fax: 45-86-19-86-64;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Mice are prime targets of experimental gene modification and have become object of an increasing number of biologic studies in renal physiology, development, and molecular biology. Phenotypic changes in response to gene modification require detailed information on normal structure. However, detailed analyses of normal mouse kidney structure and organization are lacking. This study describes the 3D organization and ultrastructural, segmental variation of the mouse kidney proximal tubule. A total of 160 proximal tubules in three C57/BL/6J mouse kidneys were analyzed on 800 serial sections from each kidney from the surface to the inner stripe of the outer zone of medulla. All tubules were reconstructed in 3D and visualized by interactive computer graphics. A quantitative ultrastructural analysis of the mouse proximal tubule at every 300 to 400 µm was performed. The 3D representation revealed a distinct organization of the mouse proximal tubule, each occupying a separate domain within the cortex. Superficial proximal tubules have long straight parts converging into clusters within the medullary rays. Tubules originating deeper within the cortex become longer and increasingly tortuous. In the medullary rays, these are arranged in layers outside the clusters of more superficial tubules. In contrast to rat and human kidney, no major segmental variation in the ultrastructure of the proximal tubule was identified, and no parameters enabled definition of distinct segments in this strain of mice. In conclusion, significant new information on the 3D organization of the murine proximal tubule has been obtained. Quantitative, ultrastructural analyses of mouse proximal tubules reveal substantial differences compared with other species. E-mail: eic@ana.au.dk


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the last decade, the mouse kidney has become the target for an increasing number of functional and morphologic studies. Gene-deleted and transgenic mice have become important tools for the study of a variety of physiologic and pathophysiologic parameters. To be able to recognize structural changes in genetically modified kidneys, it is therefore pertinent to obtain information on the normal architecture of mouse nephrons.

In the literature, structural and functional parameters of the mouse proximal tubule are often correlated to the segmentation well described in other species. The rat proximal tubule segmentation has been intensively studied at the ultrastructural level, subdividing it into three segments (1); studies on mouse proximal tubules often refer to this segmentation. Although the ultrastructure of mouse proximal tubules has been described to some extent (see reference 2), detailed information, including the verification of any segmental, structural variation is required.

Only little information on the three-dimensional organization of the mouse renal proximal tubule is available, limited mainly to the identification of a convoluted and a straight part, the latter being located in the medullary rays. A more detailed description of the tubular organization may be of interest not only in relation to renal development and function but also for the understanding of certain focal types of renal disease.

The present study reveals the three-dimensional organization of the mouse proximal tubule through the entire renal cortex. The analyses were performed by computer-assisted reconstructions of 160 individual proximal tubules based on serial sections through three mouse kidneys. A detailed quantitative and qualitative morphologic analysis at the light and electron microscope level was performed along the proximal tubule at precisely defined distances from the glomerulus, enabling a precise characterization of any structural segmental variation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Renal Tissue
The kidneys from three male C57/BL/6J mice (a strain frequently used for gene deletions and transfections), 25 g of body weight, were fixed by perfusion through the abdominal aorta with 1% glutaraldehyde in 0.06 M sodium cacodylate buffer, pH 7.4 (adjusted to 300 mosm with sucrose), and 4% hydroxyethyl starch (HAES; MEDA, Copenhagen, Denmark) (modified from reference 3). Tissue blocks cut perpendicular to the longitudinal axis of the kidney and extending from the surface through the papilla were fixed overnight in the same fixative, postfixed for 1 h in 1% OsO4 in 0.1 M sodium cacodylate buffer, én bloc stained with uranyl acetate, dehydrated in ethanol, and embedded in flat molds in Epon 812 (TAAB, Aldermaston, Berks, UK). Serial sections, 2.5-µm-thick, were generated from the surface of the kidneys to the inner stripe of the outer zone of the medulla, about 800 sections from each animal using a Reichert Ultracut S microtome (Reichert, Wienna, Austria) and a Diatome histoknife (Diatome, Biel, Switzerland). Average section thickness was determined by counting the number of sections that contained a spherical structure of measured diameter (glomeruli). The analysis of five glomeruli from each of the three animals gave a mean section thickness of 2.56 µm ± 0.11, n = 3, i.e., not significantly different from 2.5 µm. From each of the 11 tubules traced manually (see below), sections at known intervals from the glomerulus along the axis of the proximal tubule, 300- to 400–µm intervals, were reembedded in Epon and sectioned for ultrastructural analyses.

Light Microscope Images
Images were obtained from each semithin section at a magnification of x50 by a Leica DMR light microscope mounted with a Sony color CCD video camera. Color images were acquired using a frame grabber at a resolution of 768 x 592 pixels and were stored on the hard disk for later analysis. Each pixel equalled 1.7 µm x 1.7 µm. The images from the three animals were transferred to a standard PC (Zitech Gold Pentium II 400 MHz) and aligned into three consistent matrices by an established alignment algorithm (see below). In each matrix, the spatial arrangement of superficial, mid-cortical, and juxtamedullary proximal tubules was described by manual and computer-assisted digital tracing.

Alignment
All alignment procedures were performed in Microsoft Windows 98. The approximately 800 TIFF color images from each animal were converted into grayscale images in Adobe Photoshop 4.0.

The alignment was carried out by a principle as described previously in details (4). In short, the sum of the differences of corresponding pixel values in two identical images is zero when the two images are correctly orientated to each other. If the two images are not identical but closely related, the sum will be minimal at the optimum "fit" position and when the images are less related, the minimum will be less distinct, however, still well-defined. Thus, the sum function can be used as a parameter in an alignment algorithm. The relative transformation values from the alignment of each pair of images were summarized into a set of absolute transformation values for each image (5).

The position of some local structures changed disproportionately from one image to the next, resulting in trend phenomena in the transformation values. To avoid these trend phenomena, the absolute transformation values underwent a high-pass filtering.

Manual Tracing
From each of the three matrices, three to four proximal tubules, chosen at random from the superficial cortex, mid-cortex, and juxtamedullary region, were traced through about 800 hard copies made from the digitized images. The tubules of the superficial cortical nephrons had contact several times with the renal capsule, whereas the juxtamedullary tubules started either just above or beneath the arcuate vessels.

Tracing started at the urinary pole of the glomerulus and ended at the transformation into the thin descending limb. Coordinates (x and y) corresponding to the center of the traced tubule in the matrix were entered into the computer; the z-coordinates were given by the section number. A Corel Quattro Pro (Corel Corp., Ottawa, Ontario, Canada) program was created to calculate the coordinates representing the course of the tubule axis and to obtain information of the spatial position of the tubule in adjacent sections and in relation to the glomerulus. Altogether, 11 proximal tubules, three from outer-cortex, three from mid-cortex, and five from juxtamedullary nephrons were analyzed as described above.

In addition, ten glomeruli representing each of the three nephron levels were selected from the images of each kidney for determination of the glomerular diameter and the glomerular volume using the Cavalieri method (6). The volume V was calculated as V = (1/F) x t x (a/p) x P, where F is the fraction of analyzed sections for a given glomerulus (every sixth sections were counted), t is the section thickness, a/p corresponds to the area of each point in the counting grid, and P is the number of points.

Computer-Assisted Digital Tracing
To facilitate the digital tracing procedures, the walls of the tubules of all images underwent edge enhancement by using plugins and batch operations in Adobe Photoshop 4.0, using the function "Glowing." The action was performed on all images by a batch operation. The aligned and edge enhanced images were transferred to a Linux system for the digital tracing procedures.

From the three kidneys, 160 proximal tubules from different levels, including the 11 manually traced tubules, were traced, starting at the glomerulus and ending at the thin descending limb of Henle, with the help of a custom-made computer program. The computer program was written in C and ran under the Linux operating system (http://www.linux.org).

The program enabled the operator to trace the tubules in three dimensions by browsing through the stack of aligned images. When the path of a tubule was ascertained, each tubule cross-section was filled interactively with a color specific for that tubule, and the computer program recorded the x-, y-, and z-coordinates of each tubule cross-section. The x- and y-coordinates were defined as the coordinates of the center of gravity of the tubule cross-section, whereas the z-coordinate was given by the section number. This generated for each tubule a series of xyz-coordinates describing the tubular path from the glomerulus through the outer stripe of the outer medulla. If a tubular path exited the boundary of the matrix, it was discarded.

The data describing the path of each tubule was then imported into a three-dimensional visualization program AC3D (http://www.ac3 d.org) to visualize the course of the tubules. In addition, this program allowed us to visualize the interrelationship between tubules. The paths of the nephrons were exported from AC3D in VRML format and can be found at http://www.birn.suite.dk.

Graphic representation of the traced tubules revealed a systematic aberration in the xy-plane of all tubules in all three kidneys. This may be due partially to sliding during photographing and partially to the initial alignment procedure. Assuming that the individual tubules should be randomly oriented along the z-axis, this was corrected by shifting all x- and y-coordinates in a direction determined by the average shift in the xy-plane of all evaluated tubule profiles in that plane. The average shift in x- and y-coordinates was only considered valid if 20 or more tubular profiles were identified in a given section. Following this alignment a graphic representation of tubules suggested these to be randomly oriented in the xy-plane as expected. Finally, tubule representations were smoothed by a moving averaging window. Each coordinate was calculated as the average of the current, the preceding three, and the succeeding three coordinates (Figure 3D).



View larger version (47K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. (A) Three-dimensional reconstruction of a tubule originating from the outer cortex (white) revealing a relatively simple convoluted part and a very straight part and a juxtamedullary proximal tubule with an extensive convoluted part and virtually no straight part (green). (B) The three-dimensional organization of tubules originating from different cortical levels. The convoluted parts of the two white tubules (originating 200 and 400 µm from the kidney surface) are very similar to that of the blue tubule (originating 600 µm from the kidney surface). However, the straight part of the blue tubule is obviously more tortuous than those of the white tubules. This pattern becomes more and more apparent in the yellow tubule (originating 750 µm from the kidney surface), the yellow-brown tubule (originating 950 µm from the kidney surface), and the red tubule (originating 1150 µm from the kidney surface). In the two latter tubules, also the convoluted parts become more and more extended. (C) Top view of the three-dimensional organization of all tubules originating 750 to 1000 µm from the surface of kidney 1. The convoluted parts of these tubules are located in the cortical labyrinth and the central hole (white asterisk) corresponds to the medullary ray shown in Figure 1D. (D) Three-dimensional reconstruction illustrating the effects of smoothing and correction for systematic aberration described in Materials and Methods.

 



View larger version (152K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. (A) Three-dimensional reconstruction of 53 mouse proximal tubules from animal 1. The figure shows nineteen white tubules (glomerulus located 0 to 500 µm from renal surface), seventeen blue tubules (500 to 750 µm), ten yellow tubules (750 to 1000 µm), 6 red tubules (1000 to 1250 µm), and 1 green tubule (1300 µm). The horizontal white lines at the right correspond approximately to the levels for the sections shown in panels B through D. (B through D) Kidney cortical sections number 350, 522, and 581, i.e., obtained 875 µm, 1300 µm, and 1450 µm from the renal surface. On these sections, the traced tubules are marked by the same colors as in panel A, except for the white tubules, which are colored black. By comparing the colored tubules from the sections, it is possible to identify several tubules on the three-dimensional reconstruction in panel A. The cluster of black tubules in the right part of the medullary ray in panels B through D is clearly surrounded first by the blue tubules, then by the yellow tubules, and then by red tubules. It also appears that there is more than one cluster of superficial tubules in the medullary ray. Compare this figure to Figure 3C.

 
The proximal tubule straight part was identified as the distal part of the tubule beginning at the most distal point of the smoothed tubule profile in which another part of the same tubule could not be identified 20 µm or more deeper into the kidney in the z-direction. The total tubule length and the lengths of the convoluted and the straight parts, respectively, were determined as the sum of distances between n succeeding points, each representing the location of the tubule in a given section equation


(1)

Both the transition point between the convoluted and straight part, the tubule length, and the extension of the tubule profiles in the x- and y-plane, respectively, were calculated using a custom made Pascal program.

Electron Microscopy
Ultrastructural parameters for the eleven manually traced tubules (three to four tubules from each kidney) were obtained from sections at intervals of 300 to 400 µm along the tubular axis, including the starting-point, 30 µm from the glomerulus, and the end point of the proximal tubule. At every given point of the tubule, three electron micrographs taken at random at a final magnification of x15,600 were used to determine the volume densities of large endosomes including lysosomes > 0.5 µm and mitochondria, by point counting using a lattice square test system (7) with a distance between lines of 10 mm. Determinations of brush border height, cell height exclusive of brush border, and diameter of the lumen were performed on micrographs at a final magnification of x2100 from the tubules taken at the same points along the tubules as described above. The tubular wall volume per tubule length (average cross sectional area of the tubular wall) was calculated from the inner (di) and outer (do) tubular diameter per µm tubule length as:

{pi}/4(do2-di2). The exact electron microscope magnifications were determined using a carbon replica (2,160 lines/mm).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spatial Arrangement and Course of the Proximal Tubule
The reliability of the computer-assisted tracing was tested by comparing the course and spatial arrangement of the 11 manually traced tubules with the same tubules traced using our computer setup as described. This analysis showed no significant difference between the two methods, illustrating that the computer-assisted tracing very precisely reflects the course of the tubules.

Proximal tubules of the superficial nephrons (SN) and mid-cortical nephrons (MN), originating in the outer 60% of the cortex (total thickness of cortex about 1.3 mm), are very similar with respect to length and course; they are, however, significantly different from juxtamedullary nephrons (JN), originating in the inner 40% of the cortex (Figures 1 and 2). The convoluted part of proximal tubules of SN and MN form smaller clusters and have only six to seven convolutions compared with 10 to 15 in the JN, which form much larger clusters. Thus, the proximal tubules of the JN are longer and occupy a much larger cortical volume (Figure 2). The average length of proximal tubules of SN and MN is 3.76 mm compared with 5.38 mm of the JN. The lengths of the convoluted and the straight part of the proximal tubule in SN and MN are approximately 2 mm and 1.6 mm, respectively, whereas the length of the convoluted part in the JN is from 4 to 8 mm, and the pars recta is less than 0.5 mm.



View larger version (103K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. (A through D) Diagrams representing the lengths and the cortical volumes occupied by the convoluted tubules of all 160 proximal tubules analyzed from the three kidneys. The proximal tubule lengths are almost constant in nephrons originating within the outer 60% of the cortex. Proximal tubules of nephrons originating within the inner 40% of the cortex become gradually longer. As seen in panels B and C, this increase is due to an increasing length of the convoluted part. Panel D illustrates the increase in length of the convoluted part in the inner 40% of the cortex shown by the increase in cortical volume occupied by the convolutions. {diamond} = animal 1, {blacksquare} = animal 2, and {triangleup} = animal 3.

 
Tubules originating from the outer 500 µm of the cortex have long and very straight pars rectas (Figures 1 and 3). In tubules originating from about 600 to 750 µm from the kidney surface, the pars recta becomes increasingly tortuous; in the deepest juxtamedullary nephrons, it is virtually impossible to define a pars recta (Figure 3, A and B). At all levels of the cortex, the proximal tubules of individual nephrons virtually do not intermingle (http://www.birn.suite.dk). Only rarely is a convolution from one proximal tubule located inside convolutions of another, suggesting each tubule to occupy its own separate territory in the cortex. Also the proximal tubule straight parts do not intertwine, although they pass together as bundles in the medullary rays. Within these, the proximal tubules are arranged in a specific pattern. Proximal tubules originating in the most superficial cortex are arranged as two or more central bundles, whereas proximal tubules originating deeper in the cortex become layered more and more peripherally (Figure 1, A through D). The three-dimensional reconstructions (Figures 1 and 3) also illustrate the organization of medullary rays stretching into the superficial nephrons being surrounded by the cortical labyrinth (Figure 3C). Two to three medullary rays were identified in each of the three blocks, and it was obvious that the medullary rays converged into the outer stripe of the outer zone of the medulla.

Glomeruli
Analysis of the glomerular diameter, based on 30 glomeruli from each of the three kidneys, 10 randomly selected from each of the three zones, showed that glomeruli of superficial and mid-cortical nephrons have similar diameters and volumes of about 90 µm and 4 x 105 µm3, respectively, compared with 115 µm and 8.6 x 105 µm3 for the juxtamedullary nephrons (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Diameter and volume of mouse renal glomerulia
 
In most glomeruli, with the exception of a few relatively large glomeruli, the parietal squamous epithelial cells of Bowman’s capsule are replaced with cuboidal proximal tubule cells extending approximately one third into the glomerulus from the urinary pole (Figure 4A).



View larger version (139K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. (A) Electron micrograph of an outer cortical glomerulus close to the urinary pole. Proximal tubule cells constitute almost the total circumference limiting the urinary space. A capillary loop (CAP) is seen in the center. Magnification, x1250. (B) Electron micrograph of a JN proximal tubule (2000 µm from the glomerulus). The apical plasma membrane is folded into long and densely packed microvilli, and a well-developed endocytic apparatus occupies the apical part of the cells, endosomes (E). The long mitochondria (M) are oriented perpendicular to the tubular axis along the basolateral plasma membrane invaginations. Magnification, x10,000. The inset illustrates the abundant dense apical tubules (arrows) in the apical cytoplasm. Magnification, x29,000. (C) Electron micrograph of a MN proximal tubule (700 µm from the glomerulus). The apical cytoplasm contains several lipid-loaded lysosomes (L), characteristic of mouse proximal tubule. Magnification, x10,300. (D) Electron micrograph of a MN proximal tubule (1000 µm from the glomerulus). Typical lysosomes (L) with very uneven electron density are located in SN and MN proximal tubules between 900 and 2400 µm from the glomerulus. Magnification, x11,000.

 
General Ultrastructure of the Proximal Tubule
The general ultrastructural characteristics of the mouse proximal tubule do not differ fundamentally from that of other species. Apically, the cells have long and densely packed microvilli. In the apical cytoplasm, numerous small and large endosomes and dense apical tubules are observed (Figure 4B and inset). Abundant lysosomes are identified in the apical and central part of the cells, apparently more apically located than in the rat, for example. Elongated mitochondria oriented radially along the vertical ridges or folds of the basolateral membrane occupy a major part of the cytoplasm (Figure 4B). In the very initial part of the proximal tubule and toward the end of the straight part, the cells display fewer basolateral plasma membrane ridges and grooves and less oriented mitochondria. The straight part ends abruptly at the transition into the thin descending limb. At the start of the proximal tubule inside the glomerulus, the parietal epithelial cells of Bowman’s capsule always appear stretched in part covering the first cell of the proximal tubule.

A remarkably high number of lipid-droplets and lipid-loaded lysosomes are located mainly in the apical part of the cells of the convoluted part of the proximal tubules in all three types of proximal tubules (Figure 4C). Another specific feature of the mouse proximal tubule is the presence of large lysosomes with lamellar or uneven high electron density in a segment of the SN and MN located between 900 and 2400 µm from the glomerulus (Figure 4D). These were not observed in the JN. A smooth paramembraneous reticulum was found in all cells parallel to the lateral plasma membrane.

Quantitative Morphometry
Unlike other species (e.g., the rat), there is very little segmental variation at the ultrastructural level along the tubular axis and no significant differences between nephrons originating in the three selected cortical levels in this strain of mice. Thus, there were no major differences in brush border height along the tubular axis, the mean being about 2.76 µm (Table 2). The luminal diameter varied slightly and irregularly, apparently independent of the distance from the glomerulus, the mean being about 22 µm (Table 2). However, the height of the epithelial cells decreased over the last 1000 µm of the tubules from about 7 to 8 µm to about 4 to 5 µm.


View this table:
[in this window]
[in a new window]

  		Table 2. Morphometric parameters of mouse proximal tubulea
 
This is also reflected in a decreased tubular wall volume from about 600 to 700 µm3 per µm tubule length to about 400 to 500 µm3 per µm tubule length over the last 1000 µm of tubule.

The volume density of large endosomes, including lysosomes varied slightly in the SN and MN with a peak between 750 and 2000 µm from the glomerulus of about 4% followed by a decrease within the last 1000 µm of the proximal tubules to about 2%. Similar variations were identified in the JN. The mean volume density was about 3.1% for the three types of nephrons (Table 2). The volume density of mitochondria did not vary along the tubular axis, the mean being about 44% (Table 2).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study describes in detail the organization and ultrastructure of the C57/BL/6J mouse proximal tubule revealing several new and significant observations. In particular, we found no segmental variations at the ultrastructural level in contrast to the previously described segmentation of rat kidney (1). In many species (e.g., rabbit and dog), the brush border decreases in height from segment 1 (S1) to segment 3 (S3) (8). In the rat, the microvilli are long in S1, decrease in S2, and become very long in S3 (9). In the C57/BL/6J mouse, a similar variation in brush border height is not seen, the mean being 2.76 µm (Table 2). Likewise, no segmental differences in the mitochondrial volume density were identified, the mean being about 44%. This density is high as compared with figures obtained from other mammals. However, it is striking that the volume density of mitochondria appears to be inversely related to the size of the species, indicating that proximal tubule cells of small mammals have a higher metabolic rate. The mitochondria constitute 20 to 30% in rat proximal tubule (9–13), 17 to 21% in rabbit proximal tubule (14), 16% in human proximal tubule (15), and 12% in pig proximal tubule (16).

For all three types of nephron, a slight variation was observed in the volume density of lysosomes and large endosomes constituting about 3% at the start of the proximal tubule, increasing to about 4% at 40% of the tubular length, and decreasing to about 2% at the end of the tubule. These figures are very similar to those published for rabbits (14), but significantly lower than those published for rat, 9 to 10% (9,10,17). This may be related to species-dependent differences in the glomerular filtration of macromolecules and the reabsorptive and degradative capacity of the endocytic apparatus in the proximal tubule.

The lipid-like inclusions observed in apical vacuoles of the convoluted parts of the three types of nephrons appear to be specific for mice. These vacuoles have been shown to contain lysosomal enzymes and were named vacuolated bodies (18). Also, the lamellated bodies identified mainly between 1200 and 2400 µm from the glomerulus in superficial and mid-cortical nephrons, have previously been shown to contain lysosomal enzymes (18).

Within the last 500 to 1000 µm, the epithelial cell height decreased in all three types of nephron, from about 7 µm to about 5 µm. Similar changes are observed in rat (9).

The three-dimensional reconstruction of the proximal tubules, illustrated in Figures 1 and 3 and at http://www.birn. suite.dk, provides new information on tubular organization. It is obvious that individual proximal tubules each occupy a separate volume and that they only rarely intermingle with each other. This observation is based on reconstruction of 30 to 50% of all nephrons in the three kidney cortex matrices, suggesting this finding to be universal. The proximal tubule convoluted part of the SN occupies a large volume close to the surface of the kidney. The proximal tubule pars recta of the SN and MN are arranged in an ordered manner within the medullary rays. The pars recta from the most superficial nephrons are located centrally in the bundle, whereas nephrons originating deeper in the cortex become layered more and more peripherally, as also observed by Koepsell and Kriz by other methods (19). The convoluted part of the SN and MN occupy volumes that are similar in size in the x-, y-, and z-direction. In contrast, the proximal tubules of the juxtamedullary nephrons extend much more widely in the x- and y-directions than in the z-direction. This is consistent with previous observations in mouse (19) and in rat (9). The medullary rays at the cortical-medullar border and in the outer stripe of the outer zone of the medulla are surrounded by the convolutions of the JN, which do not have a well-defined pars recta, although the decrease in cell height, tubular wall volume, decreased volume density in the last part indicate cellular changes also for these nephrons.

The validity of the computer-assisted analysis of the kidney tubular organization is supported by a striking similarity of our results with previous findings on the length of mouse proximal tubules based on microdissection (20). The lengths determined in the present study were obtained after a process of smoothing. The rationale for this was the fact that graphic representation of the fully aligned tubules revealed a zig-zag appearance never observed in longitudinal sectioned proximal tubules. This suggested the material to be over-sampled. The smoothing procedure was defined so that no tubular convolutions would disappear as a result of smoothing.

The length of the proximal tubules originating in the outer 60% of the cortex remains rather constant; however, it then increases with distance to the kidney surface up to about 43% in tubules originating in the innermost 40% of the cortex. The longest tubule measured was >8 mm. Such a variation has not been observed in rat (9,20). The transition from the pars recta of the proximal tubule to the thin descending limb lies within a relatively narrow zone of about 100 to 200 µm, enabling easy identification of the outer and inner stripe of the outer medulla in mouse.

The present study reveals an increase in the diameter and volume of glomeruli associated with JN compared with SN and MN similar to previous observations in mice and in rats (20,21) (for review, see reference 22). This illustrates the morphologic and probably also the functional heterogeneity of the different zones of the renal cortex. The present study also confirms the observation that in mice, and most pronounced in male mice, proximal tubular epithelium constitutes in part the parietal epithelium of Bowman’s capsule (see reference 23 and 24 for references).

In summary, computer-assisted reconstruction has proven a reliable tool for the study of kidney tubules. Reconstruction of mouse proximal tubules suggests that each tubule occupies its own space within the cortex and that SN and MN proximal tubule straight parts are organized in a specific manner within the medullary rays. Ultrastructural analyses reveal no obvious morphologic segmentation of the proximal tubule and no significant morphologic differences between the convoluted and the straight part in this strain of mice. It should be emphasized that these findings do not exclude a possible segmentation based on variations in enzyme/transporter-protein expression between in different parts of the tubule.


   Acknowledgments
 
This work was supported by grants from the Danish Medical Research Council, the Novo Nordic Foundation, the Danish Biotechnology Program, and the University of Aarhus Research Foundation. The study was presented in part at the 33rd Annual Meeting of the American Society of Nephrology, Toronto, Canada, 2000. The authors would like to thank Inger Blenker Kristoffersen and Hanne Sidelmann for excellent technical assistance and Jacob Ilsø Christensen for computer assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Maunsbach AB: Observations on the segmentation of the proximal tubule in the rat kidney. J Ultrastruct Res 16: 239–258, 1966[CrossRef][Medline]
  2. Maunsbach AB, Christensen EI: Functional ultrastructure of the proximal tubule. In: Renal Physiology, 2nd edition, edited by Windhager EE, New York, Published for the American Physiological Society by Oxford University Press, 1992, pp 41–107
  3. Dawson TP, Gandhi R, le Hir M, Kaissling B: Ecto-5'-nucleotidase: Localization in rat kidney by light microscopic histochemical and immunohistochemical methods. J Histochem Cytochem 37: 39–47, 1989[Abstract]
  4. Andreasen A, Ren H: Extending the resolution of light microscopy digitized images with reference to cellular changes after in vivo low oxygen exposure. J Neurosci Methods 122: 157–170, 2003[CrossRef][Medline]
  5. Andreasen A, Drewes AM, Assentoft JE, Larsen NE: Computer-assisted alignment of standard serial sections without use of artificial landmarks. A practical approach to the utilization of incomplete information in 3-D reconstruction of the hippocampal region. J Neurosci Methods 45: 199–207, 1992[CrossRef][Medline]
  6. Pagtalunan ME, Drachman JA, Meyer TW: Methods for estimating the volume of individual glomeruli. Kidney Int 57: 2644–2649, 2000[CrossRef][Medline]
  7. Weibel ER: Stereological Methods. Vol. 1: Practical Methods for Biological Morphometry, New York, Academic Press, 1979
  8. Kriz W, Kaissling B: Structural organization of the mammalian kidney. In: The Kidney: Physiology and Pathophysiology,edited by Seldin DW, Giebisch G, New York, Raven Press, 1985, pp 265–306
  9. Dorup J, Maunsbach AB: Three-dimensional organization and segmental ultrastructure of rat proximal tubules. Exp Nephrol 5: 305–317, 1997[Medline]
  10. Christensen EI, Madsen KM: Renal age changes: Observations on the rat kidney cortex with special reference to structure and function of the lysosomal system in the proximal tubule. Lab Invest 39: 289–297, 1978[Medline]
  11. Pfaller W: Structure function correlation on rat kidney. Quantitative correlation of structure and function in the normal and injured rat kidney. Adv Anat Embryol Cell Biol 70: 1–106, 1982[Medline]
  12. Larsson L, Maunsbach AB: Differentiation of the vacuolar apparatus in cells of the developing proximal tubule in the rat kidney. J Ultrastruct Res 53: 254–270, 1975[CrossRef][Medline]
  13. Larsson L: The ultrastructure of the developing proximal tubule in the rat kidney. J Ultrastruct Res 51: 119–139, 1975[CrossRef][Medline]
  14. Nielsen JT, Christensen EI: Ultrastructure of isolated perfused proximal tubules from rabbit kidney. A comparison with proximal tubules fixed by perfusion in vivo. Lab Invest 49: 400–411, 1983[Medline]
  15. Moller JC, Skriver E: Quantitative ultrastructure of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis). Virchows Arch A Pathol Anat Histopathol 406: 389–406, 1985[CrossRef][Medline]
  16. Moller JC: Proximal tubules in long-term compensatory renal growth. Quantitative light- and electron-microscopic analyses. APMIS Suppl 4: 82–86, 1988[Medline]
  17. Christensen EI, Maunsbach AB: Proteinuria induced by sodium maleate in rats. Effects on ultrastructure and protein handling in renal proximal tubule. Kidney Int 17: 771–787, 1980[Medline]
  18. Miller F, Palade GE: Lytic activities in renal protein absorption droplets. An electron microscopic cytochemical study. J Cell Biol 23: 519–552, 1964[Abstract/Free Full Text]
  19. Kriz W, Koepsell H: The structural organization of the mouse kidney. Z Anat Entwicklungsgesch 144: 137–163, 1974[CrossRef][Medline]
  20. Sperber I: Studies on the mammalian kidney. Zool Bidrag Upps 22: 249–431, 1944
  21. Olivetti G, Anversa P, Rigamonti W, Vitali-Mazza L, Loud AV: Morphometry of the renal corpuscle during normal postnatal growth and compensatory hypertrophy. A light microscope study. J Cell Biol 75: 573–585, 1977[Abstract/Free Full Text]
  22. Bankir L, Bouby N, Trinh-Trang-Tan MM: Heterogeneity of nephron anatomy. Kidney Int 31: S25–S39, 1987
  23. Dietert SC: The columnar cells occurring in the parietal layer of Bowman’s capsule. Cellular fine structure and protein transport. J Cell Biol 35: 435–444, 1967[Abstract/Free Full Text]
  24. Hanker JS, Preece JW, MacRae EK: Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse. I. Studies of the renal glomerular capsule. Histochemistry 44: 225–244, 1975[CrossRef][Medline]
Received for publication September 5, 2002. Accepted for publication November 27, 2002.




This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
F. Darlot, A. Artuso, N. Lautredou-Audouy, and D. Casellas
Topology of Schwann cells and sympathetic innervation along preglomerular vessels: a confocal microscopic study in protein S100B/EGFP transgenic mice
Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1142 - F1148.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. R. Hayden, N. A. Chowdhury, S. A. Cooper, A. Whaley-Connell, J. Habibi, L. Witte, C. Wiedmeyer, C. M. Manrique, G. Lastra, C. Ferrario, et al.
Proximal tubule microvilli remodeling and albuminuria in the Ren2 transgenic rat
Am J Physiol Renal Physiol, February 1, 2007; 292(2): F861 - F867.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
X.-Y. Zhai, J. S. Thomsen, H. Birn, I. B. Kristoffersen, A. Andreasen, and E. I. Christensen
Three-Dimensional Reconstruction of the Mouse Nephron
J. Am. Soc. Nephrol., January 1, 2006; 17(1): 77 - 88.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhai, X. Y.
Right arrow Articles by Christensen, E. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhai, X. Y.
Right arrow Articles by Christensen, E. I.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP