Three-Dimensional Reconstruction of the Mouse Nephron

Xiao-Yue Zhai,* Jesper S. Thomsen,† Henrik Birn,* Inger B. Kristoffersen,* Arne Andreasen,‡ and Erik I. Christensen* Departments of *Cell Biology, †Connective Tissue Biology, and ‡Neurobiology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark

Renal function is crucially dependent on renal microstructure which provides the basis for the regulatory mechanisms that control the transport of water and solutes between filtrate and plasma and the urinary concentration. This study provides new, detailed information on mouse renal architecture, including the spatial course of the tubules, lengths of different segments of nephrons, histotopography of tubules and vascular bundles, and epithelial ultrastructure at well-defined positions along Henle’s loop and the distal convolution of nephrons. Three-dimensional reconstruction of 200 nephrons and collecting ducts was performed on aligned digital images, obtained from 2.5-␮m-thick serial sections of mouse kidneys. Important new findings were highlighted: (1) A tortuous course of the descending thin limbs of long-looped nephrons and a winding course of the thick ascending limbs of short-looped nephrons contributed to a 27% average increase in the lengths of the corre- sponding segments, (2) the thick-walled tubules incorporated in the central part of the vascular bundles in the inner stripe of the outer medulla were identified as thick ascending limbs of long-looped nephrons, and (3) three types of short-looped nephron bends were identified to relate to the length and the position of the nephron and its corresponding glomerulus. The ultrastructure of the tubule segments was identified and suggests important implications for renal transport mechanisms that should be considered when evaluating the segmental distribution of water and solute transporters within the normal and diseased kidney. J Am Soc Nephrol 17: 77–88, 2006. doi: 10.1681/ASN.2005080796

he renal architecture is arranged elaborately to fulfill ical findings have been set up to simulate the urine concentra- the physiologic demands for reabsorption of filtered tion mechanism in rat medulla (8). T substances and for urine concentration. The architec- At present, a large body of basic nephrology research focuses ture includes the formation of the renal zones, the population of on mapping the segmental distributions of a variety of recep- short-looped nephrons (SLN) and long-looped nephrons tors (9), water channels (10), tight junction integral proteins (LLN), the distribution of the tubule segments of nephrons, the (11–13), and ion transporters (14) in mouse kidneys, as mice are tubular-vascular histotopography in medulla, and the epithe- considered to be the most convenient animal model for physi- lial configurations of the different tubule segments. ologic studies and gene manipulation (15,16). However, for From the 1960s to the 1980s, Kriz and others studied the renal obtaining detailed and systematic information on renal archi- microstructure, including the distribution of nephrons, the tu- tecture in mice, further investigations are imperative. Our bular-vascular relations in the medullary zones, and the ultra- study provides a detailed description of the 3D structure of the structure of the epithelia along Henle’s loop. Simultaneously, mouse nephrons, including the course and the lengths of indi- the diversities in renal structure were demonstrated between vidual nephrons, histotopography in outer medulla, and ultra- different species, including rat, mouse, hamster, and rabbit structure in well-defined segments of Henle’s loop, and distal (1–3). In the late 1980s, a standard nomenclature for the kidney convolution. structure, which has been widely adopted, was published (4). With the development of digital techniques, three-dimensional Materials and Methods (3D) representations of the tubule segments of nephrons were Preparation of Renal Tissue performed to a limited extent at the proximal tubules (PT) (5), The tissue preparation has previously been described in detail (17). distal tubule (6), and at the thin limbs (TL) of the LLN in the Briefly, the kidneys from three 8-wk-old male C57/BL/6J mice, 25 g of inner medulla (IM) (7) of rats. Two mathematical regional- body weight, were fixed by perfusion through the abdominal aorta based models combining morphologic and immunohistochem- with 1% glutaraldehyde in 0.06 M sodium cacodylate buffer and 4% hydroxyethyl starch. Tissue blocks cut perpendicular to the longitudi-

nal axis of the kidney were postfixed for1hin1%OsO4, and embedded in Epon 812. From each kidney, a total of 2500, 2.5-␮m-thick consecu- Received August 1, 2005. Accepted October 1, 2005. tive sections were obtained from the surface to the papillary tip and Published online ahead of print. Publication date available at www.jasn.org. stained with toluidine blue. The average section thickness was deter- mined by counting the number of sections that contained a spherical Address correspondence to: Dr. Erik Ilsø Christensen, Department of Cell Biol- ogy, Institute of Anatomy, University of Aarhus, DK-8000 Århus C, Denmark. structure of measured diameter (renal corpuscle). The analysis of five Phone: ϩ45-89-42-30-57; Fax: ϩ45-86-19-86-64; E-mail: [email protected] corpuscles from each of the three kidneys gave a mean section thickness

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1701-0077 78 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 77–88, 2006 of 2.5 ␮m (2.56 ␮m Ϯ 0.11; n ϭ 3.). All animal experiments were carried the spatial relationship between the traced nephrons and the kidney out in accordance with provisions for the animal care license provided boundary. by the Danish National Animal Experiments Inspectorate. The CD and the deeper bends of the LLN exited the tissue blocks in the lower IM. Therefore, these LLN were traced both from the urinary pole of the glomerulus and from the macula densa until they exited the Image Recordings and Alignment block. Using an Olympus AX 70 microscope with a ϫ2 objective and equipped with a digital camera (Olympus DP 50; Olympus, Tokyo, Nephron Length Measurements Japan) attached to a standard PC, every second section was digitized. Each nephron was subdivided into four segments: PT, TL, thick Four overlapping digital recordings from each section were combined ascending limbs (TAL), and distal convoluted tubules (DCT). The into one 24-bit color image by use of “analySIS” (Soft Imaging System, lengths of the TL of the completely traced LLN were subdivided into Version 3.2; Soft Imaging System, Munster, Germany). The final image two continuous parts: Descending thin limb (DTL) and ascending thin size was 2596 ϫ 1889 pixels, whereby each pixel corresponded to 1.16 limb (ATL). The length of each segment was calculated as the sum of ␮m ϫ 1.16 ␮m. The image alignment was based on a previously the Euclidian distance between two subsequent markers in the course established alignment algorithm (18,19). Each color image was split into of the nephrons. For reducing measurement noise arising from the three grayscale images (-r,-g, and -b). The relative transformation alignment procedures and the manual placement of the markers, the values were determined between two consecutive images and then path was smoothed using a triangular moving average window in such summarized into a set of absolute transformation values. The absolute a way that the previous point in the path was weighted with one fourth, transformation values underwent a high-pass filtration to avoid poten- the current point with one half, and the next point in the path with one tial distortions of the image stacks as a result of small but accumulating fourth. “trends.” Then, the grayscale images were “moved” according to these absolute transformation values. Finally the moved -r,-g, and -b gray- scale images were merged to form a set of transformed color images. Electron Microscopy The quality of the alignment was checked in two different ways: In the On the basis of the tracing, sections that represented the different seg- first test, the aligned images were arranged as consecutive images ments of the nephrons and the transitions in distal convolutions were similar to a movie film. A copy of this elongated image was made selected for ultrastructural analysis. These sections were re-embedded in beginning with image 2 instead of image 1. Then the copy was super- Epon, sectioned, and observed in a Philips CM 100 electron microscope imposed onto the original so that the consecutive images could be (FEI Company, Hillsboro, OR). compared two by two. In the second test, the consecutive images were turned into an animated film by the program “gifsicle” and inspected Results by use of “gifview.” In cases in which the alignment was not optimal, General Description of the Spatial Course of Nephrons the alignment was repeated with a new set of initial conditions until the In total, 30 CD, 127 SLN, and 41 LLN were traced. Of these, alignment converted into a global optimum. The image recording and 24 LLN were complete, whereas 17 LLN were incomplete as a alignment procedure was carried out on a Windows-based PC (Mi- result of their deep bends exiting the tissue blocks. The crosoft Windows 98; Intel, Santa Clara, CA), whereas the control pro- nephrons were grouped into two types according to the loca- cedures involving “gifsicle” and “gifview” were made on the Linux tion of Henle’s loop bends (4): SLN, which form their loop platform (Mandrake Linux 10.1; AMD Athlon, Sunnyvale, CA). bends within the outer medulla, and LLN, which have their loop bends in the IM. A Henle’s loop consists of a pars recta of Digital Tracing and 3D Presentation the PT, a DTL, an ATL, and a TAL (pars recta of the distal The tracing of the nephron paths was performed on the Linux-based tubule). The major findings in the spatial course of the various PC. A series of custom-made computer programs were written in C on types of SLN and LLN are illustrated in Figure 1. the Linux system for tracing and for calculation of lengths, distances, Renal Zones. In Figure 2A, the boundary between the etc. outer medulla and the IM was defined clearly by the transition The tracing was conducted among collecting duct (CD) “families.” of the LLN from ATL to TAL. In contrast, the boundary be- Such a “family” is defined as a CD and the nephrons that drain into the CD by the connecting tubules (CNT) at the superficial cortical level. tween the outer stripe of the outer medulla (OSOM) and the Nephron tracing started at the urinary pole of a randomly chosen inner stripe of the outer medulla (ISOM) was less sharply glomerulus and ended at the meeting of the CNT. The CD was traced defined by the transition from the pars recta of the PT to the from the cortex toward the IM. DTL. The thicknesses of the OSOM and the ISOM were approx- A marker was placed manually in the tubule lumen along the course imately 0.5 and 1.2 mm, respectively. The cortical-medullary of the tubule. For each marker, the x-, y-, and z-coordinates were boundary was determined by the location of the arcuate ves- recorded in a data file, where the z-coordinate corresponded to the sels. The thickness of the cortex was approximately 1.3 mm, image number. The data file thus documented the paths of all traced which was subdivided arbitrarily into superficial, middle, and nephrons in three dimensions. All subsequent data analyses were based juxtamedullary cortex, corresponding to outer 30%, middle on this data file. In addition, the transitions between each segment of 30%, and inner 40% of the cortex, respectively. The thickness of the nephrons were recorded manually in a separate data file. The the IM was not determined as a result of the curved papilla, tubular profiles and the transitions between different segments were normally identified from the structure in the digital images. In only a which was not included in the tissue blocks. Ͼ few cases were the original sections inspected in a light microscope to Proximal Tubules. Notably, most ( 90%) of the PT that identify the tubules or the transitions. Furthermore, the surface of the initiated at the superficial cortex left their glomeruli ascending renal capsule and the papillary epithelia was traced to obtain the toward the renal surface, whereas Ͼ90% of the PT that initiated distance from the glomerulus center to the renal capsule and to describe at juxtamedullary cortex left their glomeruli in a short descent J Am Soc Nephrol 17: 77–88, 2006 Reconstruction of the Mouse Kidney 79

domains. The individual domains contained only one nephron, and only a few peripherally situated tubules were found to intermingle with the neighboring nephron tubules from the same “family” from time to time. In this study, none of the LLN was found to originate from the superficial cortex, and none of the SLN was found to originate from the juxtamedullary cortex (Figure 3, A and C). Moreover, no nephrons formed loop bends within the cortex, as opposed to findings in humans, pigs, and occasionally in rab- bits (20). Loops of Henle and Tubular-Vascular Histotopography. The most intriguing observation was that, through the outer part of the ISOM, the tortuous course of the “straight” part of the PT continued into the upper part of the DTL in all LLN and the initial part of the DTL of the SLN, which originated from middle to juxtamedullary cortex. The tortuous course of a DTL increased the total length of the DTL of the LLN by approxi- mately 27%. The epithelium that covered the tortuous course of the DTL was characterized at the electron microscope level (see below). The TAL of the SLN and their CD also ran windingly through the ISOM, where the tortuous course of the DTL of the LLN was seen (Figure 3, B and D). The winding course of the TAL increased the total length of the TAL of the SLN by approximately 27% as well. The corresponding descending and ascending limbs of SLN and LLN that originated from the superficial and middle cortex ran closely together through the medullary rays. The loop limbs from one “family” were found to run together with their com- mon CD. However, at different levels through the OSOM close to the ISOM, the corresponding descending and ascending limbs started to separate, running in parallel. The distance between the corresponding limbs varied up to approximately 100 ␮m. The tubules occasionally shifted positions relative to Figure 1. Schematic representation of the typical nephrons and neighboring tubules from the same “family.” The thick de- collecting duct (CD) organization: Tortuous descending thin scending limbs of LLN from the juxtamedullary cortex occu- limbs (DTL) of the long-looped nephrons (LLN; large arrow); pied a large tissue volume because of the tortuous “straight” winding course of thick ascending limbs (TAL) of short-looped part of the PT, and the corresponding TAL ran nearby (Figure nephrons (SLN) and CD (arrowheads); a piece of TAL inserted e.g. in the DTL of LLN (LLNt; small arrow), which forms its bend 2B, , tubule 0). just beneath the “transitional zone” within the inner medulla In the ISOM, the tubule segments were located in a distinct (IM); and three different types of SLN bends (SLN1, SLN2, and histotopographic position. In the outer part of the ISOM, en- SLN3). On the basis of the distribution of the nephron seg- closing large vascular bundles, single thick-walled tubule pro- ments, the renal zones were defined, including cortex, outer files were constantly found in the central part of the vascular stripe of outer medulla (OSOM), inner stripe of outer medulla bundles (Figure 2C) and identified as TAL of LLN with deeper (ISOM), and IM. The postmacula densa segments and the di- loop bends. However, most TAL of LLN were located periph- rections of the proximal tubules (PT) arising from their glomer- erally. The DTL of SLN were constantly integrated in the outer uli are also illustrated. or middle layer of the vascular bundles, and the tortuous DTL of LLN were located in the interbundle regions, together with the winding TAL of SLN and their CD (Figures 1 and 2C). In and then turned to ascend. The PT that originated within the the inner part of the ISOM, SLN successively looped back, and middle or early juxtamedullary cortex left their glomeruli tak- the vascular bundle split into smaller bundles with both DTL ing a horizontal course but turned to ascend after a short and TAL of LLN and remaining SLN running in the vicinity of distance (Figure 1). the bundles (Figure 2D). The proximal convoluted tubules made coils around their In the IM, the limbs of Henle’s loop were only DTL and ATL own glomeruli and occupied either small, tightly packed do- from LLN, and they ran close to each other and next to their CD mains in the superficial and middle cortex or larger, loosely and vasa recta, which were identified at the electron micro- packed domains in the juxtamedullary cortex. The correspond- scope level. The ATL were constantly closer to their CD than ing DCT and cortical TAL were located within or nearby the the DTL. 80 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 77–88, 2006

Figure 2. All SLN, LLN, and CD traced within one kidney (A) and selected cross-sections showing tubular-vascular histotopog- raphy (B through D). (A) The red lines represent the renal capsule and the papilla. The white dots represent glomeruli (not actual size). Tubule segments are represented in different colors: PT, blue; thin limbs (TL), green; TAL, red; DCT, purple; connecting tubules (CNT), orange; and CD, brown. (B) Level of the OSOM (approximately 1570 ␮m from the renal surface). (C) Level of the initial part of the ISOM (approximately 1925 ␮m). (D) Level of the middle to lower ISOM (approximately 2585 ␮m). White circles highlight the same vascular bundles. Each red number denotes a specific nephron or CD. In the vascular bundle, tubules 84, 88, 92, 96, 102, 103, and 105 are the TAL of the LLN. (C) The DTL of SLN 4, 5, 47, 48, 49, 50, 79, and 93 are integrated into the vascular bundle. In the interbundle region, the elongated tubules 0, 8, 31, and 91 reflect a tortuous course of the DTL of LLN, whereas the elongated tubules 4 and 47 reflect a winding course of the TAL of SLN, likewise the tubule 37, a winding course of the CD. In B and D, the same vascular bundle became smaller (white circles). In D, tubules 25, 45, 50, 75, 82, 93, 100, and 104 are longer SLN with a type 3 bend, e.g., tubule 93 and 104 reveal a prebend TAL segment. Scale ϭ 50 ␮m.

Bend of Henle’s Loop. We identified three types of bends Types 1 and 2 bends were located at the middle level of the of the SLN according to the epithelium lining the bends: Type ISOM, and their corresponding glomeruli were located at the 1 bends (21% of the total SLN) with DTL epithelium covering average outer 17 and 24% of the cortex, respectively (200 and the bends and continuing for a short distance into the ascend- 300 ␮m from the renal surface). Type 3 bends were located at ing limbs (Ͻ60 ␮m), type 2 bends (33%) with a transforming the various levels of the inner half of the ISOM, a few reaching epithelium from DTL to TAL within the bend, and type 3 bends the so-called innermost stripe (1), and their corresponding glo- (47%) with TAL epithelium covering the bend and constituting meruli were located at the average outer 40% of the cortex (500 50 to 450 ␮m of the descending limb (prebend TAL segment). ␮m from the renal surface; Figure 3D). The SLN with type 1 and Generally, the deeper the bends, the longer the prebend TAL type 2 bends were comparable in tubule course, lengths of segments of type 3 bends (Figure 3C). segments, and position of the bends. The lengths of the DTL J Am Soc Nephrol 17: 77–88, 2006 Reconstruction of the Mouse Kidney 81

Figure 3. All LLN (A) and SLN (C) traced in one kidney, together with selected LLN (B) and SLN (D) at higher magnification. (A and B) The glomeruli are placed at different levels within the middle to juxtamedullary cortex. The PT have no straight part, and the tortuous course continues into the initial part of the DTL (arrows). All loop bends are located in the IM. The TAL start at the boundary of ISOM and IM and continue toward the cortex. The DCT and their corresponding proximal convoluted tubules intermingle and coil to form the structure domain. The CNT go upward into the superficial cortex. (C) The glomeruli are located within the superficial and middle cortex. The loop bends are located at different levels within the ISOM. The lower bends have TAL epithelia (red) lining the bends and the prebend descending limbs (prebend TAL segment; one LLN as reference). (D) Three bend types of SLN: “1” with DTL epithelium covering and continuing a short distance in ascending limb; “2” with a transitional epithelium, DTL to TAL; and “3” with a prebend TAL segment of 288 ␮m. “3i” has a type 3 bend (446-␮m prebend TAL segment) that is deeper in the innermost stripe and the terminal part of the loop is winding. The bend types correspond to the position of their glomeruli. The TAL of SLN run windingly through the upper part of the ISOM. Those originating from the middle cortex reveal a tortuous course of the pars recta of the PT and initial part of the DTL. 82 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 77–88, 2006 and TAL of the SLN with a type 3 bend were 8 and 9% longer, Distal Convolution and CD. The distal convolution con- respectively, than the lengths of the same segments of the SLN sists of DCT, connecting tubule (CNT), and initial collecting with type 1 and type 2 bends. Most SLN bends had limbs tubule, which is the top and branched part of the cortical CD separated by approximately 20 ␮m. Broad and flat bends with (4). Similar to the rat (21), the DCT commenced downstream limbs that were separated by a distance of 40 to 50 ␮m were from the macula densa at an average distance of 85 ␮m. The occasionally identified among all bend types. The bends of all DCT of the SLN were more coiled compared with the DCT of fully traced LLN were located at successive levels in the IM. the LLN. The DCT that were situated in the outermost of the A transitional zone was identified around the boundary cortex touched the renal capsule one to two times, compared between ISOM and IM, in which almost no loop bends were with five to seven times for the proximal convoluted tubules. found, either from SLN or from LLN. It comprised the inner- The transition from late DCT to CNT was defined by the most stripe and the initial part (approximately 100 ␮m thick) of decrease in epithelium height of the CNT. The CNT in the the IM. Very few bends from SLN reached the innermost stripe, superficial cortex occasionally ran just beneath the capsule for and these SLN were characterized by a winding terminal loop some distance. The CNT that originated from LLN ascended and a type 3 bend (Figure 3D, “3i”). Only a few LLN looped toward the superficial cortex within medullary rays to form an back just beneath this zone. These LLN were structurally sim- “arcade” (22) (Figure 3A). The CNT of the SLN drained into ilar to the other LLN, except for an approximately 100-␮m-long their own CD either by an arcade or directly into the initial and thick-walled segment inserted in the DTL at the level of the collecting tubule (Figure 4). transitional zone (Figure 1, LLNt). The inserted segment was In total, 30 “families” were traced. According to the com- ultrastructurally characterized by TAL epithelium. pletely traced “families,” a CD collected six to seven nephrons,

Figure 4. Ten CD with the CNT deriving from 10 “families” (A through F). A, B, D, and E consist of two families. The nephrons are omitted to show the pattern that a CD (brown) connects with five to seven nephrons by CNT (different colors) at superficial cortex. G is a higher magnification of cortical part of the C: At the top of the CD (brown) is the branched cortical CD, initial collecting tubule (ICT; pale yellow); yellow is the CNT of an LLN. It ascends in the cortex to form the arcade, and the CNT from SLN drain into the CD by either joining directly into ICT or joining the arcade first and then draining into the ICT. J Am Soc Nephrol 17: 77–88, 2006 Reconstruction of the Mouse Kidney 83

Table 1. The lengths of segments of SLN in mouse kidneysa

SLN PT (␮m) TL (␮m) TAL (␮m) DCT (␮m) Total (␮m) PT (%) TL (%) TAL (%) DCT (%) Kidney 1 (n ϭ 44) 4251 Ϯ 447 831 Ϯ 187 2716 Ϯ 257 798 Ϯ 123 8595 Ϯ 775 49 Ϯ 2.2 9.6 Ϯ 1.7 31.6 Ϯ 1.7 9.3 Ϯ 1.5 Kidney 2 (n ϭ 41) 4151 Ϯ 462 755 Ϯ 135 2472 Ϯ 281 709 Ϯ 137 8087 Ϯ 783 51 Ϯ 2.7 9.3 Ϯ 1.2 30.6 Ϯ 2.2 8.7 Ϯ 1.4 Kidney 3 (n ϭ 42) 4452 Ϯ 335 817 Ϯ 127 2774 Ϯ 189 596 Ϯ 82 8638 Ϯ 581 52 Ϯ 1.6 9.4 Ϯ 1.0 32.1 Ϯ 1.3 6.9 Ϯ 0.9 Mean Ϯ SD 4285 Ϯ 153 801 Ϯ 40 2654 Ϯ 160 701 Ϯ 101 8440 Ϯ 306 50.7 Ϯ 1.2 9.4 Ϯ 0.2 31.4 Ϯ 0.8 8.3 Ϯ 1.2

aSLN, short-looped nephrons; TL, thin limbs; TAL, thick ascending limbs; DCT, distal convoluted tubules.

of which either one or two were LLN, reflecting an 82:18 ratio branes and radially oriented mitochondria (type 2). The epithe- (n ϭ 25 “families”) of SLN to LLN. The cortical CD ran within lium of the DTL of LLN located at the boundary between ISOM medullary rays inward to the medulla without receiving more and IM was characterized by many thin luminal microvilli and nephrons in the middle and the juxtamedullary cortex. The first numerous finger-like infoldings of the basal membrane, fewer joining of two CD often occurred around the boundary between mitochondria and other organelles, and fewer and less shallow ISOM and the IM. Subsequent joinings took place at successive tight junctions (type 3). The epithelium of the lower part of the levels in the IM. DTL, the loop bends, and the ATL was characterized by many lateral processes with tight junctions and dense apical mem- Segment Lengths brane with few luminal microprojections (type 4). The part of The lengths of the four well-defined segments of the nephron the DTL covered by type 4 epithelium was approximately 700 were obtained for SLN and LLN. They were in sequence PT, TL, ␮m long, i.e., a prebend ATL segment. The limbs in the IM of TAL, and DCT. The average length for each segment of the SLN those LLN with the bends located just beneath the transitional was determined (Table 1). zone were covered by type 4 epithelium only. The epithelia The lengths of the different segments for the entirely traced along DTL of the LLN transformed gradually from one type to LLN are shown in Figure 5A. The lengths of the PT and the TL another. that originated from deeper than 70% of the cortex increased The transition from the TAL to the DCT was observed at a with increasing depth of their glomeruli in cortex. The lengths distance of an average of 80 ␮m from the macula densa and of the PT and TL that originated from 40 to 69% of the cortex defined by an abrupt increase in cell height. Ultrastructurally, varied slightly. The lengths of the TAL and DCT were relatively the epithelia of the TAL and the DCT are characterized by constant. Therefore, the increase in total length paralleled the extensive invaginations of basolateral plasma membrane and increase in PT and TL lengths. The TL of LLN was subdivided numerous elongated mitochondria, which were placed basolat- into DTL and ATL, which on average constituted 80 and 20% of erally and perpendicular to the basement membrane (Figure 7). the total length of the TL, respectively. The transition from the DCT to the CNT was defined by the The normalized length for all completely traced nephrons is appearance of CNT cells and an abrupt decrease in epithelial shown in Figure 5B. The relative lengths of the four segments height. Along this segment, four distinct cell types were iden- are constant for all SLN, independent of bend type and inde- tified by their specific morphologic characters (24): DCT cells, pendent of the total nephron length. However, for LLN the intercalated cells, CNT cells, and principal cells. However, prin- relative lengths of the TAL decreased corresponding to an cipal cells were rarely seen in the early CNT. In addition, three increase of TL with increasing total nephron length. The rela- subtypes of intercalated cells were identified: Type A, type B, tive lengths of the DCT remained constant and smaller as and non-A–non-B intercalated cells, based on their ultrastruc- compared with the relative lengths of the SLN. tural and immunochemical characters (Figure 8) (25).

Electron Microscopy We have previously quantitatively described the ultrastruc- Discussion ture along the PT (17). In this study, we therefore focused on This study presents the results of computer-assisted, 3D recon- the TL of Henle’s loops, as well as the distal convolutions. On struction of 200 nephrons and CD from mouse kidneys. In con- the basis of multiple micrographs at successive levels through trast to earlier studies, the digital tracing allowed a detailed char- the renal cortex and medulla, our analyses largely agreed with acterization of the spatial structure and interrelation of a large and expanded previous findings. number of nephrons and enabled for the first time an ultrastruc- Four ultrastructurally distinct segment epithelia along the TL tural characterization of the tubule epithelium at well-defined described in different species (3,23) were observed in mice as distances from the glomerulus and relevant tubule segment tran- well (Figure 6). The DTL of most SLN were lined by a uni- sitions. The study reveals several new, important features of formly thin and simple epithelium (type 1). The DTL of LLN mouse kidney microstructure with substantial influence on renal were lined by three distinctly differentiated epithelia: The epi- function. However, it should be emphasized that the findings of thelium of the upper tortuous DTL of LLN and the SLN with this study are based on C57/BL/6J mice only. the deepest bends within the ISOM was characterized by com- A correlation has been established between the position of plex and highly differentiated apical and basolateral mem- the glomeruli within the renal cortex and the point of origin 84 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 77–88, 2006

The ratio of the number of SLN to LLN was 82:18, which may be compared with previous observations—70:30 in rats (32), 34:66 in rabbits (22), 0:100 in dogs (33), and 85:15 in humans (34)—reflecting species differences in urine-concentrating mechanisms. Our findings of the spatial and histotopographic arrange- ment of nephron tubules within the medulla provide crucial structural basis for elucidating the formation of countercurrent multiplier in mouse kidney and for evaluating species differ- ences in the ability of concentrating urine. (1) A tortuous course of the upper part of the DTL of LLN and a few longer SLN, together with a winding course of TAL of SLN and CD were located in the interbundle regions through the ISOM. These windings increased the corresponding segment lengths by ap- proximately 27%. (2) Single thick-walled tubules constantly found in the central part of the larger vascular bundles were identified as TAL of LLN, whereas most of the TAL of LLN were located in the periphery of the vascular bundles through the outer medulla; the DTL of SLN were always integrated into the outer or middle layers of the vascular bundles. (3) A clas- sification was introduced to describe three different SLN bends relating to the lengths and the positions of the tubule segments and the corresponding glomeruli. The epithelium lining the tortuous part of the tubules of the DTL of LLN was ultrastructurally characterized by a highly specialized apical and basolateral membrane, described as type 2 epithelium (35). This highly differentiated membrane has been confirmed to have a high permeability to sodium, chlo- ride, and water by studies in rabbits, rats, and hamsters (36). The lateral cell interdigitations with a high number of single- strand tight junctions are consistent with the distribution of a subtype of a tight-junction integral protein, claudin 2, which is believed to be a component or channel for the solute paracel- lular transport (13). The epithelia lining the DTL and TAL contribute to different solute compositions in the interbundle regions exchanging solutes with the vascular bundles because of different permeability properties. Therefore, it is fair to as- sume that the increase in length as a result of the tortuous or Figure 5. (A) Bar chart showing the lengths of the four segments winding course of the tubules and increase in membrane sur- of the completely traced LLN. They are ordered according to face of DTL owing to the specialized membrane arrangement the distance (in percentage) of their glomeruli from the renal correspond to a similarly increased functional capacity in coun- surface. The lengths of the TAL and DCT are constant. The tercurrent multiplier mechanisms. increase in total length corresponds to an increase in length of The TAL of LLN in relation to the vascular bundles through the PT and the TL. (B) Bar chart showing the relative lengths of all completely traced nephrons. The nephrons are ordered ac- the outer medulla may result from the layer-arranged tubule cording to absolute length within each of the four nephron development (37). However, the finding of the TAL of LLN types: SLN1 (n ϭ 27), SLN2 (n ϭ 41), SLN3 (n ϭ 59), and LLN within the center of the vascular bundles probably reflects local (n ϭ 24). physiologic demands in the active transport by TAL. The tu- bules that integrated into the vascular bundles, e.g., the DTL of SLN, have been proposed to be involved in generating the and the direction of the PT from the glomeruli. The course osmotic gradient in both the tubule lumen and the medullary taken by the PT is similar to observations of efferent arterioles interstitium by exchanging important solutes with vasa recta from superficial nephrons in dog and rat kidneys (26,27). The (38,39). Moreover, the identification of three types of bends DCT was surrounded by proximal convolutions of the same gives us a better understanding of the formation of the osmotic nephron, forming a relatively separate structural domain. This gradients in the ISOM. In rats and rabbits, the bends of the SLN structural domain in the cortex may be related morphologically are located roughly at the same level of the ISOM near the to the regulation of ion reabsorption in the proximal-distal junction to the IM, and the TAL epithelium continues for a tubules by tubuloglomerular feedback (28), as observed in renal short distance in the descending limb (20). This study on mice physiologic studies and computer models (29–31). revealed that the bends of the SLN were situated at successive J Am Soc Nephrol 17: 77–88, 2006 Reconstruction of the Mouse Kidney 85

Figure 6. Four distinct epithelia along the TL of both SLN and LLN. The figure comprises two columns of images representing lower and higher magnifications (right). Type 1 is a cross-section of the DTL of an SLN (440 ␮m from the transition of the PT to the DCT); higher magnification of type 1 shows a simple and thin epithelium with few microprojections, abundant ribosomes, and sparse mitochondria throughout the cytoplasm. Type 2 is a cross-section of the DTL of an LLN (370 ␮m from the same transition as above); higher magnifications of type 2 show a highly specialized epithelium: Abundant short and plump microvilli, basolateral infoldings, and the highly interdigitated cellular processes with the shallow tight junctions (small arrows) and the radially oriented mitochondria. Type 3 is a cross-section of a DTL of LLN around the boundary between the ISOM and the IM (2790 ␮m from the transition of PT to DTL); higher magnifications of type 3 show the highly differentiated membranes: Numerous relatively long microvilli, abundant finger-like basolateral infoldings throughout the cytoplasm (large arrows), and the less shallow tight junctions (small arrow) and few mitochondria. Type 4 is a cross-section of the ATL of an LLN (700 ␮m from the transition of the ATL to the TAL); higher magnifications of type 4 show the bold and dense apical membrane, basolateral infoldings, cellular processes with tight junctions (small arrows), and various organelles with sparse ribosomes throughout the cytoplasm. Scales ϭ 1 ␮m. 86 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 77–88, 2006

Figure 7. The transition from TAL to DCT. Electron micrographs show the transition from TAL (40 ␮m beyond the macula densa) to the DCT. (A) The lower lumen is the TAL and the upper is DCT, corresponding to B and D, respectively. (C) One of the TAL cells showing a centrally placed nucleus and less mitochondria in the basal compartment. A cilium is visible. (E) One of the DCT cells showing an apically placed nucleus and tightly packed mitochondria and membrane infoldings in the basal compartment. Scales for A, B, and D ϭ 5 ␮m; scales for C and E ϭ 1 ␮m.

levels within the middle and inner half of the ISOM. In the volution and CD have been studied intensively morphologi- inner part of the ISOM, where the vascular bundles decreased cally and physiologically in different species (14,24,44). The in size, the length of the prebend TAL segment of type 3 bends DCT started at an average distance of 80 ␮m from the macula increased with increasing depth of the bends (Figure 2D). This densa, compared with a distance of 0 to 500 ␮m for the type 3 bend may be assumed to play an important role in the nephrons of a variety of mammalian species (22). The transition formation of higher osmolality in the surrounding interstitium, from DCT to CNT was determined by the appearance of CNT because TAL epithelium is impermeable to water, but trans- cells during tracing. However, an agreement on the definition ports different electrolytes (40). Our study also identified a of the transition has not been reached between the morphologic transitional zone. The representation of the deepest type 3 and physiologic criteria (45). The multiple cell types in the CNT bends that reach the innermost stripe and the LLNt (Figure 1) suggest the variety of its physiologic functions in fine-tuning revealed an extra contribution to the higher osmolality by the transcellular electrolyte transport, as well as hormone-regu- exclusive TAL epithelium covering the loop limbs around the lated water transport (14). transitional zone. In conclusion, the presented 3D representation of the mouse We believe that these findings in the outer medulla may have kidney has provided a detailed characterization of the spatial functional implications in the formation of the countercurrent arrangement and interrelationship of a large number of multiplier. Therefore, it is important to take the spatial courses and nephrons, of the tubular-vascular histotopography in the renal the epithelial characteristics of the tubules into consideration medulla, and of the ultrastructure of the tubule epithelia in when modeling the urine concentrating mechanism in mice. well-defined segments. New, important features of mouse kid- In the IM, the formation of the gradient osmolality is also ney microstructure with potential functional implications have structurally associated with the epithelia covering the limbs and been revealed in addition to findings that are consistent with bends of the LLN. The type 3 epithelium covering the DTL was our basic knowledge of the human and animal kidneys. This gradually replaced with type 4 epithelium at a distance of approx- information is essential for the development of accurate math- imately 700 ␮m before the bends. With increasing depth of the ematical models simulating urine concentration mechanisms bends in the IM, more tubules are covered with type 4 epithelium and tubular permeability properties. than with type 3 epithelium. The ATL is assumed to contribute to the higher osmolality in the middle to deep IM by passive NaCl absorption, urea permeability, and water impermeability (41,42). Acknowledgments This assumption is corroborated by the findings from a study in This work was supported by grants from the Danish Medical Re- 3D functional reconstruction of the IM in rats (43). search Council, the Novo Nordic Foundation, the Danish Biotechnol- The structural-functional relationships along the distal con- ogy Program, Daloon Foundation, the European Commission (EU J Am Soc Nephrol 17: 77–88, 2006 Reconstruction of the Mouse Kidney 87

Figure 8. The transition from DCT to CNT of an LLN. (A) The tubular transformation from the late DCT into the CNT (upper lumen). (B through E) Different cell types constituting the wall of the CNT: CNT cell (B), type A intercalated cells (C and D), part of DCT cell in C (the short arrow), and non-A–non-B intercalated cell (E). The wall of the late DCT is composed mainly of DCT cells; however, a few intercalated cells are interspersed. Compared with DCT cells, the segment-specific cells, CNT cells, were obviously lower and characterized by a centrally placed nucleus, randomly oriented mitochondria, basolateral invaginations of the plasma membrane, and a relatively smooth lateral margin. Type A and non-A–non-B intercalated cells were commonly characterized by numerous microvilli covering the apical surface, abundant vacuoles, and mitochondria. Compared with type A, the mitochondria in non-A–non-B intercalated cells were mainly located in the bulging apical area above the nucleus, and smaller vesicles were distributed throughout the cytoplasm. Scale for A ϭ 5 ␮m; scales for B, C, D, and E ϭ 1 ␮m.

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