J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis Is Induced by Partial Nephrectomy in the Elasmobranch Leucoraja erinacea

MARLIES ELGER,*† HARTMUT HENTSCHEL,†‡ JENNIFER LITTERAL,*† MAREN WELLNER,†§ TORSTEN KIRSCH,*†§ FRIEDRICH C. LUFT,§ and HERMANN HALLER*† *Department of Nephrology, Hannover Medical School, Hannover, Germany; †Mount Desert Island Biological Laboratory, Salsbury Cove, Maine; ‡Max Planck Institute of Molecular Physiology, Dortmund, Germany; §HELIOS Klinikum-Berlin, Franz Volhard Clinic and Max Delbru¨ck Center for Molecular Medicine, Humboldt University, Berlin, Germany.

Abstract. The mammalian kidney responds to partial nephrec- matic epithelial cells; stage III, segmental nephron segregation; tomy with glomerular and tubular hypertrophy, but without stage IV, functioning nephron. The stages were analyzed after renal regeneration. In contrast, renal regeneration in lower partial nephrectomy. In addition, cell proliferation was as- vertebrates is known to occur. Understanding the underlying sessed by incorporation of bromo-deoxyuridine (BrdU). New mechanisms of renal regeneration is highly important; how- nephrons developed in animals undergoing partial nephrec- ever, a serviceable animal model has not been developed. A tomy. Growth was greatly stimulated in the nephrogenic zone, neonephrogenic zone has been identified in the European lesser both in the remnant tissue and in the contralateral kidney spotted dogfish, Scyliorhinus caniculus (Hentschel H. Am J within 10 wk. Mesenchymal cell aggregates increased signif- Anat 190: 309–333, 1991), as well as in the spiny dogfish icantly per renal cross-section compared with controls (stage I, Squalus acanthias and the little skate, Leucoraja erinacea. The 0.64 Ϯ 0.28 versus 0.27 Ϯ 0.25; P Ͻ 0.005; n ϭ 10 animals zone features the production of new nephrons complete with a per group). The same was the case for S-shaped body-like cysts countercurrent system. To analyze this nephrogenic region of (stage II, 0.24 Ϯ 0.19 versus 0.08 Ϯ 0.09; P Ͻ 0.02). Cellular elasmobranch fish further, a renal reduction model was estab- proliferation in the neonephrogenic zone of the contralateral lished. The neonephrogenic zone in the adult kidney of the kidney was also greatly enhanced (14.42 Ϯ 3.26 versus 2.64 Ϯ little skate resembles the embryonic metanephric kidney and 1.08 BrdU-positive cells per cross-section, P Ͻ 0.001). It is contains stem cell-like mesenchymal cells, tips of the branch- concluded that the skate possesses a nephrogenic zone contain- ing collecting duct system, and outgrowth of the arterial sys- ing stem cell-like mesenchymal cells during its entire life. tem. Four stages of nephron development were analyzed by Partial nephrectomy induces renal growth by accelerating serial sections and defined: stage I, aggregated mesenchymal nephrogenesis. This unique model may facilitate understanding cells; stage II, S-shaped body-like structure with high-pris- renal regeneration.

In mammals, nephron formation is terminated at birth or and tubular growth. Large-scale nephron loss is therefore shortly thereafter (1). Subsequent growth is generally re- not accompanied by new nephron generation (“neo” nephro- stricted to tubular epithelial replacement by proliferation genesis) in mammals. However, lower vertebrates have su- and/or to angiogenic processes involved in vascular recon- perior abilities in that regard. Adult sexually mature bony struction (2,3). The limited regenerative potential results in fish (teleosts) can develop new nephrons as shown in light a greatly reduced capacity to cope with renal disease. In- and electron microscopic studies (6,7). Formation of de jured glomeruli do not regenerate, and glomerular destruc- novo nephrons has been reported in goldfish kidneys after tion is the major cause of final nephron loss (4,5). The the administration of nephrotoxic substances (8). We previ- remaining nephrons respond with compensatory glomerular ously found nephrogenic tissue in the little skate, Leucoraja erinacea, an elasmobranch cartilaginous fish (9), as well as in the spiny dogfish, Squalus acanthias. Furthermore, a Received July 31, 2002. Accepted February 15, 2003. detailed analysis of nephron anlagen and developing H. Hentschel and M. Elger contributed equally to this publication. nephrons in the lesser spotted dogfish, Scyliorhinus canicu- Correspondence to Prof. Dr. Hermann Haller, Department of Nephrology, lus, has been published (10,11). Because elasmobranch kid- Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Phone: 49-511-532-6319; Fax: 49-511-552366; E-mail: haller.hermann@ neys exhibit great structural complexity (12) and resemble mh-hannover.de or [email protected]. by this and other morphologic features mammalian kidneys, 1046-6673/1406-1506 these animals could serve as a model for nephrogenesis. We Journal of the American Society of Nephrology therefore studied nephrogenesis in adult skate kidney and Copyright © 2003 by the American Society of Nephrology tested the hypothesis that this process is stimulated by renal DOI: 10.1097/01.ASN.0000067645.49562.09 size reduction. J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1507

Materials and Methods surgical thread was used in all instances. After the operation, the fish Animals were placed in their holding tanks. They recovered from anesthesia Female skates, Leucoraja erinacea (Mitchell 1825), were netted in after 5 to 10 min. Frenchman Bay, Maine, or purchased from the Marine Station Woods For the second technique, the animals were laid on their ventral Hole, MA, USA. The body size as measured by the disc diameter (fin surface. The gills were dripped with anesthetic via the spiracles. A span) ranged from 20 to 32 cm. The body weight ranged from 310 to longitudinal incision was made through the skin and underlying 790 g. The animals were kept in large circular tanks with running musculature (longissimus dorsalis) in the pelvic region, 2 cm aerated seawater and were acclimatized for a few days before the lateral from the midline. The dorsal surface of the right kidney was experiments. The animals were fed a diet of frozen shrimp. thereby directly exposed. Two thirds of this kidney was removed For reduction of renal mass, we surgically removed renal tissue. without injury to the peritoneal membrane. The incision was closed The animals were anesthetized by adding 0.1 g/L tricaine (Sigma) to by a double suture. Sham operations were performed in one group the seawater. During anesthesia, the seawater was cooled with ice, and of animals. Unoperated fish served as additional controls. Fish deep anesthesia was achieved after 40 min. We developed two oper- were maintained in their tanks with constant feeding. After 10 wk, ations. Fish were mounted upside-down on a styrofoam board. The 20 animals (10 nephrectomized [NX] and 10 control skates) were gills were superfused with a tricaine drip (0.05 g/L seawater) via the sacrificed, and their kidneys were examined. oral cavity. The body was covered with paper towels soaked in ice-cold seawater. The posterior body cavity was opened by a midline Preservation of Kidney Tissue incision through the ventral body wall. The pelvic girdle was The kidneys were fixed by vascular perfusion via the conus transected, and the intestine and gonads were carefully laid aside to arteriosus as described previously (12). In brief, the vascular expose the right kidney. The large efferent intrarenal veins of the renal system was rinsed with modified elasmobranch Ringer solution portal system, which were visible through the peritoneal epithelium, (13) at a pressure of 100 cm H2O for several minutes. For mini- were cauterized. A 2/3 nephrectomy in one kidney was performed mizing osmotic effects during the clearing of the vasculature, 2% with scissors after cauterization of the intrarenal arteries and veins. polyvinyl pyrrolidone was added. After the blood was cleared from Great care was taken not to injure the multiple renal arteries entering the circulatory system, the fixation solution was introduced with- the kidney from the dorsal aorta. The viscera and the pelvic girdle out a pressure drop and perfused for approximately 5 min at the were repositioned. The body wall was closed with two sutures. One same pressure as the rinsing solution. The chilled fixation fluid ran in the subdermal musculature and one in the skin. A nonresorbable contained paraformaldehyde (3 to 4%) and picric acid (0.5%) in

Figure 1. (A) Ventral view of the two kidneys in the little skate Leucoraja erinacea. The peritoneum containing pigment cells is partly removed to uncover the efferent veins (arrows). (B) Relationship between renal weight and body weight. A linear correlation over a large range of body weight was observed (y ϭ 0.025 ϩ 0.293 · x, r ϭ 0.91). 1508 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1506–1518, 2003

Figure 2. (A) Histologic renal cross-section. The nephroneogenic zone (arrow) with newly developing glomeruli is located along the ventrolateral border of the bundle zone. Here, new nephrons are added and effect the growth of renal tissue in a lateral direction. Paraffin section, Masson-Goldner, light micrograph (LM). (B) Schematic cross-section. The two major zones termed “lateral bundles” (i.e., the renal countercurrent system, shown in pink) and “mesial tissue” (blue), and the “nephrogenic tissue” (yellow) are shown. Intrarenal arteries deriving from multiple renal arteries (RA) give rise to bundle arteries (BA) and afferent glomerular arterioles (aff A). The renal portal system enters the kidney by afferent renal and intrarenal veins (ARV, AIRV), supplies the mesial tissue by capillary sinusoids (small arrows), and leaves by the efferent intrarenal and renal veins (EIRV and ERV). eff A, efferent arterioles. The nephrons, including the glomeruli, decrease in size and age from the medial to the lateral side. The nephrogenic tissue is the site of new glomerular and tubular formation. The course and segmentation of a mature nephron is shown at the left: EDT LDT, early and late distal tubule segments; IS, intermediate segment; NS, neck segment; PI and PII, proximal tubule segments I and II. The collecting tubule runs through the bundle and joins the collecting duct (CD) tree. The youngest branches of the CD system are located adjacent to the nephrogenic tissue (adapted from reference 12). (C) Nephrogenic zone at higher resolution. The nephrogenic zone (NZ) is located between the coiled countercurrent bundles (LB) of individual nephrons and the tubules of the mesial tissue (MT). A mature glomerulus (GL) is seen in the central part of the figure. Semithin (0.5 ␮m) plastic section, toluidene blue staining, LM. (D) The nephrogenic zone contains mesenchymal cells in different stages of development. The aggregated mesenchymal cells of the first developmental stage (arrow) and the corresponding end branch of the collecting duct system (CDT) are shown. The developmental stage is embedded in a collagen-rich interstitial tissue (Co). Semithin (0.5 ␮m) plastic section, toluidene blue staining, LM. (E) Mesenchymal cells of developmental stage I adjacent to the collecting duct tip (CDT). The cells (asterisks) are enveloped by laminated cell processes of fibroblasts (FB). Thin (60 nm) plastic section, transmission electron micrograph. J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1509

Figure 2. (Cont’d)

0.1MSo¨rensen phosphate buffer, pH 7.4. The osmolality of the eight to ten blocks and processed for paraffin embedding. Serial buffer of the fixative was adjusted with sodium chloride and cross-sections (3 ␮m) were prepared from each block and stained with sucrose to that of the pre-rinse (950 mOsmol/L). After fixation, the Masson-Goldner trichrome stain. In these sections, developmental body was opened, the kidneys were exposed, and the size and stages I to IV were identified in accordance to developmental stages shape of the kidneys were photographed in situ from the ventral in dogfish, Scyliorhinus caniculus (10). The amount of developmental side. The organs were carefully removed, weighed, and docu- stages was estimated in ten nephrectomized and ten control animals. mented from the dorsal side. The group of nephrectomized animals consisted of five animals op- erated from the dorsal side and five animals operated from the ventral Histology and Quantification of Developmental side. The controls included five sham-operated and five non-operated Nephron Stages animals. The comparison was made between the intact contralateral The excised kidneys were postfixed in fresh fixative for an addi- kidneys of the nephrectomized animals and one kidney of the sham- tional hour. Each kidney was cross-sectioned with razor blades in operated and non-operated controls. Serial cross-sections (8 ␮m) were 1510 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1506–1518, 2003 prepared from five blocks per kidney. Three sections per tissue block (lateral bundles) located on the mesial tissue mass (panel A). at a distance greater than 300 ␮m were used to determine the number These bundles represent the renal countercurrent system. They of developmental stages I and II per renal cross-section. are situated along the dorsolateral surface of the kidney in a continuous zone beginning at a distance of approximately 1 Electron Microscopy mm from the medial edge of the kidney and extending around For transmission electron microscopy, tissue pieces of perfusion- the lateral margin to the ventral surface for up to 1 mm. The fixed kidneys were postfixed in 1.5% glutaraldehyde and 1.5% para- afferent intrarenal veins and the glomeruli separate the bundle formaldehyde solution, followed by fixation in 1% OsO4 solution, and zone and the mesial tissue zone. The nephrons were heteroge- subsequent embedding in Epon resin. Thin sections were obtained neous. The most elaborate nephrons with the largest glomeruli with an ultramicrotome OMU3 (Reichert, Vienna) and viewed with were observed at the medial location. The small nephrons were Zeiss electron microscope EM10 (Zeiss, Oberkochen). located at the lateral edge of the kidneys (see also reference 9). Developing tubules and nephron anlagen were frequently Cell Proliferation present in the vicinity of the outermost small glomeruli. By Proliferative activity of the renal tissue was estimated by indirect careful stereomicroscopic examination of the decapsulated re- immunohistochemical detection of incorporated 5-bromo-2'-deoxyuri- nal surface, a narrow band of whitish tissue was identified dine (BrdU) in a separate set of ten adult female little skate (body size, along the convex lateral surface of the kidney. This tissue 24 to 27 cm; body weight, 500 to 690 g). Ten days after partial demarcated the border between the pale bundle zone and the nephrectomy of the left kidney in five animals (NX) and sham operation in five control skates, the animals received an intraperito- reddish-brown mesial zone. Because the band contained neal injection of 1.5 mg/100 g BW BrdU dissolved in PBS. After 12 h, nephron anlagen, we termed this zone “nephrogenic tissue.” In the kidneys were fixed by perfusion as described above. Four tissue young animals, the nephrogenic tissue formed a ribbon-like blocks from the middle portion toward the caudal end of one kidney structure along the entire kidney. In older animals, the band of control skates and of the contralateral kidney of NX skates were was discontinuous. embedded in paraffin, and six to eight independent cross-sections per Figure 2B shows a schematic drawing of a renal cross- animal were used for numerical determination of BrdU-labeled cells. section. Blood supply and the nephron course are demonstrated The deparaffinized sections were treated with 0.01% trypsin in PBS in the two major kidney zones: the lateral bundle zone and the for 1 min at RT and Universal Blocking Reagent (BioGenex, San mesial tissue. Several renal arteries arise from the dorsal aorta. Remon) for 10 min and were incubated with mouse monoclonal Intrarenal arteries give rise to afferent glomerular arterioles and anti-BrdU antibody (clone BMG 6H8) containing nucleases for DNA to bundle arteries. The mesial tissue is supplied by capillary denaturation (Roche) diluted 1:20 in PBS for 30 min at 37°C, fol- lowed by anti-mouse Ig-alkaline phosphatase (Roche) for 30 min at sinusoids of the renal portal system, which enters the kidney by RT. The reaction was visualized by color substrate solution containing afferent renal and intrarenal veins. The bundle vessels, includ- nitroblue tetrazolium (NBT) salt and 5-bromo-4-chloro-3-indolyl ing the central vessels of the individual bundles and the effer- phosphate (BCIP). Cell nuclei labeled with BrdU were counted per ent arterioles, join the mesial sinusoid system. The efferent kidney cross-section. Three tissue blocks from the ipsilateral kidney intrarenal veins drain the sinusoid system. The zone of neo- of the NX animals were used to study the distribution of proliferative nephrogenesis with newly developing glomeruli is located near cells in the operated kidney. Parallel sections were stained with the most peripheral branches of the arterial system (compare trichrome stain after Masson-Goldner for identification of develop- also reference 9). mental stages. Figures 2C and 2D show a nephrogenic zone at the lateral margin of the kidney at higher resolution. The nephrogenic Statistical Analyses zone was wedged between the ventrolateral margin of the Statistical difference was tested between nephrectomized and con- bundle zone and the mesial tissue. Blind ends of the col- trol animals by t test. lecting duct system invaded the nephrogenic tissue. They frequently showed mitosis of epithelial cells. Occasionally, Results two tips originating from the collecting duct ends were In Figure 1, the ventral aspect of the kidneys is shown in situ observed. Growth of the collecting duct system by forma- after removal of the intestine (panel A). Two separate flat tion of new tips in conjunction with formation of nephron roughly bean-shaped organs are located in the caudal retroperi- anlagen appeared to be exclusively located in this nephro- toneum. We determined the renal mass in fifteen female skates genic zone. We did not observe nephrogenesis in other (300 to 800 g body weight). Total kidney weight ranged from regions of the kidney. 1 to 3 g corresponding to 0.3 to 0.4% of body weight. Kidney The nephrogenic zone contained mesenchymal cells and weight and body weight were closely correlated, and a linear prospective tubular cells in different stages of development correlation over a large range of body weight was observed (r (Figure 2D). Around the collecting duct tips, undifferentiated ϭ 0.91, P Ͻ 0.001) (panel B). mesenchymal cells aggregated to form the first developmental The renal architecture, nephron segmentation, the relation- stage (see below). The developmental stages were adjacent to ship of the nephrons to the renal zones, the renal arterial and a prominent mass of collagen-rich interstitial tissue, which was venous portal systems, and the structure of the nephrogenic part of the band of whitish tissue along the ventrolateral border zone are shown in Figure 2. Cross-sections revealed a flat seen in excised kidneys (as mentioned above). The region elliptical shape and demonstrated a cap of tubular bundles around the early stages was poorly vascularized. The early J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1511

Figure 3. Stages of nephron development in the nephrogenic zone. (A) Stages I and II. A newly formed collecting duct (CD) tip surrounded by aggregated mesenchymal cells (I) can be seen on the left. Stage II was defined as the subsequent development of tubular structures from early cyst formation until the appearance of the glomerular anlage. A late stage II (II) is delineated by square brackets. The prospective podocyte layer (arrow) is evident in the glomerular anlage. Notice the extremely high columnar cells of the prospective proximal tubular portion. (B) Stages III and IV. Differentiation of the tubule into segments and appearance of vascular structures in the developing glomerulus (III) can be observed. Stage IV is present with a young well-perfused glomerulus (GL), a lateral bundle (LB) with two tubular hairpin loops, and two short tubular coils added to the mesial tissue (MT). Paraffin sections, Masson-Goldner staining, LM. 1512 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1506–1518, 2003 aggregating mesenchymal cells were nonpolarized cells with a comparatively large nucleus and a narrow cytoplasmic zone (Figure 2E). They were tightly surrounded by flat, laminate cell processes of adjacent fibroblasts or by neighboring mesenchy- mal cells. For further definition of the developmental stages in the skate kidney, and as a basis for quantification of nephrogenic responses to different stimuli, we arranged the developmental stages according to their size and complexity. We developed a system of four major stages with further subdivisions into early and late phases to grade our findings, as shown in Figures 3 and 4. Stage I was defined as a distinct small structure of less than 100 ␮m in diameter, consisting of a condensed mass of mesenchymal cells in the vicinity of a new bud of the collect- ing duct system (Figures 3A and 4A) . In the first phase (stage I-1), the mesenchymal cells were irregularly arranged. In stage I-2, cells at the very tip of the collecting duct bud began to align and to epithelialize with formation of basement mem- brane and development of apical tight junctions. Stage II was defined as the subsequent development of tubular structures from early cyst formation and the appearance of the glomerular anlage. We defined three subsequent phases of these structures according to their length. In the first phase (stage II-1), the cyst was an ovoid structure with a central lumen. At the presumptive proximal end, a mass of unpolarized mesenchymal cells formed a plug. In the second phase (stage II-2), the elongated cysts usually performed two bends like the letter S and an additional bend at each end (S-shaped body-like cyst). The most advanced cysts (stage II-3) displayed four alternating tubular loops closely adhering to each other (Fig- ures 3A and 4B). The gap in the proximal end of the cyst was closed by the formation of the glomerular epithelial primordia. An accumulation of undifferentiated mesenchymal cells was still attached to the prospective Bowman’s capsule. The ex- Figure 4. Subdivision of the early developmental nephron stages I and ceptionally high epithelial cells (cell height, Ͼ50 ␮m) and the II. (A) Phase I-1: At the blind end of a collecting duct (CD) tip, nonpolar mesenchymal cells accumulate. Phase I-2: Adjacent to the presence of a distinct lumen in the proximal portion were CD tip, the mesenchymal cells begin to epithelialize. (B) Phase II-1: helpful for identification of all phases of stage II on single A vesicle-like structure is formed, which connects to the CD tip. sections. Phase II-2: The cyst begins to elongate and fold up. A gap filled by Stage III was characterized by the progressive differentiation mesenchymal cells is still present. Phase II-3: A S-shaped body-like of nephron segments, including the occurrence of vascular structure with four narrow bends is formed. For better understanding structures in the developing glomerulus (Figure 3B). Pro- of the epithelial structure, the tubular limbs are projected into a nounced elongation of the tubule was accompanied by growth two-dimensional plane. At the proximal end, the glomerular anlage of two loops in direction of the renal capsule to become the with the presumptive podocyte and parietal layers are evident. Mes- hairpin loops of one individual bundle. The growth of the two enchymal cells are present close to the glomerular anlage. Two of the opposite loops was directed to the mesial tissue. Stage IV bends indicate the prospective loops of the mesial tissue, and the two consisted of young nephrons with well-perfused glomeruli and opposite bends represent the prospective loops of the countercurrent system in the lateral bundle. well-differentiated tubular segments (Figure 3B). Stages III and IV proved difficult to quantify. Although the tubular profiles and glomeruli were easily discernible by their lesser diameter and smaller size compared with adult nephrons, the After defining the different stages of nephron development, length of the mesial convolutions and the bundle were already we then tested the hypothesis that the nephrogenesis can be too large to allow quantification by numerical counts. For induced by renal mass reduction. Figure 5 shows a schematic example, the growing countercurrent bundle tended to coil. diagram, the operation itself, and the kidney 10 wk after partial Thus, in any given section, one to five cross-sections appeared nephrectomy. The loss of renal mass was still manifest, and the that were not always identifiable as belonging to one nephron. mean renal weight was approximately half that of control In addition, the developing mesial coils could not be assigned kidneys (Table 1). As a result, a considerable gap partly filled to the same nephron’s loop bundles. with pale connective tissue was visible. The contralateral kid- J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1513 ney did not differ macroscopically from the roughly bean- tissue, a conspicuously large number of developmental stages shaped control kidneys. were evident in partially nephrectomized animals. Two to five Cross-sections of the contralateral kidneys revealed no profiles of nephrogenic stages were seen on a renal cross- changes in areas of mature tissue. However, in the nephrogenic section as shown in Figure 6. All phases of stages I through IV

Figure 5. Nephrectomy in the skate. (A) Schematic view of ventral aspect of the kidneys. The nephrogenic zone runs in a small band along the ventrolateral edge (arrows). Multiple renal arteries (RA) arise from the aorta (A), and several renal veins join the cardinal vein (V). Two thirds of one kidney were surgically removed, leaving the medial margin containing the large renal vessels in place. (B) Operated skate. The right kidney is exposed by an incision through the dorsal body wall. A major portion of the kidney with the lateral nephrogenic zone was surgically removed. (C and D) Kidneys of control animal and NX animal with partial nephrectomy 10 wk after surgery, dorsal aspects. The surface of the kidneys is smooth with scarce lobulation. The intrarenal (endocrine) organs are located at the medial margin (asterisks). Ten weeks after surgery, the operated kidney has begun to rebuild from the caudal portion, where remnants of developmental tissue were intact (arrow). Perfusion-fixed kidneys. 1514 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1506–1518, 2003

Table 1. Kidney weightsa revealed for stage I a mean value of 0.46 Ϯ 0.40 and for stage II a mean value of 0.42 Ϯ 0.39 (mean Ϯ SD, n ϭ 10 animals). Animals Weight (g/kg body weight) Proliferation of the developmental stages was vigorous, as Control animals (Co) many cells in developing tubules and glomeruli displayed left kidney weight 1.727 Ϯ 0.116 BrdU incorporation. Serial sectioning of these ipsilateral, op- right kidney weight 1.764 Ϯ 0.175 erated kidneys did not show signs of proliferation in areas of total kidney weight 3.491 Ϯ 0.261 mature tissue. Specifically, the dilated tubules were essentially Nephrectomized animals (NX) devoid of BrdU labeling. left operated remnant kidney weight 0.872 Ϯ 0.124b right non-operated kidney weight 1.998 Ϯ 0.261c Discussion total kidney weight 2.869 Ϯ 0.279d We have characterized a nephrogenic zone in the adult kidneys from the skate. The nephrogenic zone represents a a Results are mean Ϯ SD. n ϭ 5 animals per group; t test. niche within the kidney where stem cell-like cells reside. The b Ͻ P 0.001 versus means of left and right kidney of Co. Ten tissue responds to partial reduction of renal mass with the weeks after surgery, the weight of operated kidneys of NX animals was still lower than the kidney weight of control animals. formation of new nephrons. The morphogenic process that we cP Ͻ 0.1 (n.s.) veresus means of left and right kidney of Co. term neonephrogenesis appears to be an important mechanism The contralateral non-operated kidney showed a tendency to for renal growth, as well as for repair of injured kidney. Renal increase in weight. hypertrophy, which is a common response to renal mass re- d Ͻ P 0.01 versus total kidney weight of Co. duction in higher vertebrates and man (14–16), contributed only slightly to the reconstitution of renal mass in the skate. Our morphologic analyses demonstrated that a zone of embry- were enhanced. The ramifying blind collecting duct ends were onic renal tissue persists in adult skates. Evidently, the ad- thicker than in control animals. The condensed mesenchymal vanced complexity of elasmobranch kidneys does not preclude stage I masses formed extensive caps with generally more than nephrogenesis in the adult kidney. 200 cells around the collecting duct tips. The nephrogenic zone of the skate contains the essential Growth quantification in the nephrogenic zone was per- structures characteristically found in the embryonic mamma- formed (a) on the frequency of developmental nephron stages lian metanephros. We defined four stages of nephron develop- I and II and (b) on cell proliferation. The renal cross-sections ment. The evaluation of the four stages in the adult skate of controls and intact contralateral kidneys from partially ne- corroborated previous light and electron microscopic results in phrectomized animals revealed values (n ϭ 10 animals per the European dogfish, Scyliorhinus caniculus (10). Because group; mean Ϯ SD) for stage I of 0.27 Ϯ 0.25 and 0.64 Ϯ 0.28, certain stages of nephrogenesis have been observed in adult respectively (P Ͻ 0.005). Mean values for stage II were 0.08 Ϯ specimen of several other fish species, neonephrogenesis is 0.09 for controls and 0.24 Ϯ 0.19 for partially nephrectomized probably a common feature in elasmobranchs and teleosts animals (P Ͻ 0.02) (Figure 7). Small but distinct islands of (6–10). lymphomyeloid cells were observed in the vicinity of the The nephrogenic mesenchyme is present in stages I-1 and nephrogenic tissue in the contralateral kidney of partially ne- I-2. These stages correspond to the two transient mesenchymal phrectomized animals in addition to islands with blood forming condensations that precede the comma-shaped and S-shaped tissue adjacent to large intrarenal arteries. body in the embryonic mammalian metanephros (1,17,18). Cellular proliferation in the contralateral kidney and in the Stage I-1 resembles the cap condensate and stage I-2 is very operated kidney was studied by immunohistochemical demon- similar to the pretubular condensate (17,19,20). The collecting stration of incorporation of BrdU 10 d after nephrectomy. duct tips that are in contact with the stage I structures are the Control skates showed very rarely BrdU-labeled cells in the structural homologues to the tips of the ureteric bud branches. mature portion of the tissue. A low number of reactive cells The collecting duct tip of the skate differs in several morpho- were present in the nephrogenic zone. Many histologic sections logic features from the mammalian ureteric bud branches. The were lacking dividing cells entirely. In the contralateral kidney collecting duct tip is usually a solid structure, compared with of nephrectomized animals, the mean number of labeled nuclei the ampulla-like tip in rodents and humans (20,21). The tip is per kidney cross-section was significantly increased (Figure 8). encompassed by a sheath of fibroblasts that becomes the bun- The majority of the reactive cells were located in the center of dle sheath of the elasmobranch countercurrent system (10). the nephrogenic zone and in adjacent young nephrons. Stage II was defined as S-shaped body-like cysts in agree- We also analyzed the growth and proliferative response in ment with our observations of Scyliorhinus. Our results the ipsilateral, operated kidney. As expected, regions of mas- showed considerable variability in this developmental stage, sive degeneration, areas of dilated tubules, and foci with ap- apparently representing different phases. We suggest that at the parently healthy nephrons were present. However, in the small end of stage Ib, the pretubular condensate grows into a roughly caudal portion where both the mature and the original neph- spherical cyst that is in contact with the collecting duct tip at rogenic zone were basically intact and not disturbed by the the distal end. The proximal end features a plug of unpolarized operative procedure, an increase in the number of developmen- cells. This renal vesicle-like structure grows rapidly to become tal stages was found. Renal cross-sections of this portion an S-shaped body-like cyst with two additional bends. The J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1515

Figure 6. Nephrogenic zone from control animal (panel A) and of contralateral non-operated kidney from NX animal (panel B). (A) In control animals, few developmental nephron stages are present. A stage I is located adjacent to the lateral bundle (LB) of a stage III to IV. Three cross-sections of the same bundle (consisting of five tubular profiles each) are seen. A young glomerulus (GL) of stage IV is located nearby in the mesial tissue (MT). (B) Growth rate and formation of new nephrons is enhanced in the nephrectomized animal. The mass of aggregated mesenchymal cells (MES) is enhanced, and several stages II (II) and at least one stage III (III) are present. The mesial tissue (MT, delineated by a bracket) is irrigated by venous sinusoids (VS), whereas the zone containing the mesenchymal aggregates is virtually devoid of vasculature. Paraffin sections, Masson-Goldner staining, LM. 1516 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 1506–1518, 2003

Figure 7. Quantification of growth in the nephrogenic zone of adult control animals and of the contralateral non-operated kidney of NX animals. The number of sectional profiles per renal cross-section is shown for developmental nephron stages I and II. The formation of Figure 8. Numerical determination of proliferating cells. The number new nephrons is greatly stimulated in the NX animals. In NX animals, of BrdU-positive nuclei per renal cross-section is significantly in- on average, a profile of stage I can be found on more than 50% of the creased in contralateral kidneys of partially nephrectomized animals. sections; a stage II on every third section in. Results are mean Ϯ SD, Results are mean Ϯ SEM, n ϭ 36 sections per group; t test, P Ͻ n ϭ 10 animals per group. t test: P Ͻ 0.005 for stage I, P Ͻ 0.02 for 0.001. stage II. epithelia of the collecting duct tip and the adjacent distal end of derived from a continuous pool of embryonic stem cell-like the cyst merge. Very marked growth soon leads to a structure cells within the nephrogenic zone of the elasmobranch kidney. with four narrow bends. Thus, in this phase of stage II, the cyst We suggest that these cells are precursors of competent stage far exceeds the typical S-shape. The presumptive proximal end I mesenchyme. We further suggest that the mesenchyme pro- still displays a plug of unpolarized cells. In the terminal phase vides self-renewing cells for ongoing nephrogenesis. Such of stage II, a portion of the unpolarized cells epithelializes and cells would fulfill requirements of stem cells (26). Future becomes the glomerular epithelia of stage III. A feature com- studies involving lineage studies will address these hypotheses. mon to all phases of stage II is that the high prismatic epithelial The contralateral kidney of the partially nephrectomized cells appeared uniform. The numerous mitotic figures and the skates increased in size, a common finding in mammalian incorporation of BrdU give evidence for rapid growth in all kidneys (16,27–29). Information from lower vertebrates is regions of the cyst. limited. A nephrectomy study in the common carp, Cyprinus Stage III is characterized by the segregation into tubular carpio, a teleost, was focused on blood cell formation. The segments and the appearance of vessels. The specific architec- contralateral kidney of the carp showed distinct hypertrophic ture of the elasmobranch nephron (22,23) is established at this growth after several months, and the excised portion in the stage. Further growth leads to stage IV, the young nephron. kidney undergoing partial nephrectomy did not regenerate This stage is relatively immature with respect to cell size and (30). Similar results were reported for the newt, Notophthalmus tubular length; however, the segments can now be considered viridescens, a urodelic amphibian. In this animal, nephrogen- as a functioning excretory unit. The addition of newly formed esis was not observed (31). nephrons in the periphery of the elasmobranch kidney, as In this study, we were able to confirm our earlier findings originally uncovered in the young skate (9), was described in that neonephrogenesis indeed occurs in the adult elasmobranch detail in adolescent young dogfish (10). The present study (9,10). Our new observations of BrdU incorporation in skate demonstrates the potential of nephron growth and differentia- provide further evidence for formation of nephrons de novo in tion in the adult skate kidney. the nephrogenic zone. Mitotic activity as visualized by this Mesenchymal cells capable of nephrogenesis must establish technique was high in various stages of nephron development contact with collecting duct tips for growth and branching to and young nephrons of nephrectomized animals. Older por- occur (24,25). It can be anticipated that mesenchymal cells tions of the kidney were virtually devoid of BrdU labeling. competent for nephron development presumably provide the Similar results were obtained in the remnant portion of the progenitor cells also in the skate. These cells are presumably operated kidney. This clearly shows that it is only the lateral J Am Soc Nephrol 14: 1506–1518, 2003 Nephrogenesis 1517 zone that can be stimulated to substantial renal regeneration 4. Kriz W, Elger M, Nagata M, Kretzler M, Uiker S, Koeppen- with production of new nephrons. In this region, nephron Hagemann I, Tenschert S, Lemley KV: The role of podocytes in development and growth of the collecting duct system together the development of glomerular sclerosis. Kidney Int 45(Suppl with growth of the renal vasculature can be stimulated. We 45): S64–S72, 1994 suggest that the undifferentiated cells residing in the mesen- 5. Kriz W, Hosser H, Ha¨hnel H, et al: From segmental glomerulo- chyme of the lateral kidney zone can provide targets for signals sclerosis to total nephron degeneration and interstitial fibrosis: that initiate cell division after stimulation by nephrectomy. A histopathological study in rat models and human glomeru- This will be followed by the formation of initial stages of lopathologies. Nephrol Dial Transplant 13: 2781–2798, 1998 nephron development. The formation of new nephrons was 6. Hentschel H: Vergleichend histologisch-histochemische Unter- also observed in adult teleosts (6,7,32). In contrast, nephrogen- suchungen an den Harnorganen von Stichlingen. Thesis Han- esis in mammals has been described to terminate shortly after nover, Universita¨t, 1973 birth. The reasons for loss of this ability in higher vertebrates 7. Mourier JP: Incorporation of 3H-thymidine in the nephron of are unclear. During prenatal growth of the metanephric mam- Gasterosteus aculeatus L. and its stimulation by methyl testos- malian kidney, nephrogenesis can be greatly augmented ac- terone. Cell Tiss Res 201: 249–262, 1979 cording to recent uninephrectomy experiments with unborn 8. Reimschuessel R, Bennett RO, May EB, Lipsky MM: Renal sheep (33). In these animals, compensatory renal growth of the tubular cell regeneration, cell proliferation and chronic nephro- contralateral kidney is associated with an increase in final toxicity in the goldfish Carassius auratus following exposure to nephron number due to prolonged nephrogenic growth in the a single sublethal dose of hexachlorobutadiene. Diseas Aquat embryonic metanephros. Our experiments suggest that the Organ 8: 211–224, 1990 elasmobranch kidney is equipped with a nephrogenic zone, in 9. Hentschel H: Renal blood vascular system in the Elasmobranch, which similar “embryonic growth” persists life-long. The Raja erinacea Mitchill, in relation to kidney zones. Am J Anat nephrogenic potential can be activated by removal of renal 183: 130–147, 1988 tissue. 10. Hentschel H: Developing nephrons in adolescent dogfish, Scyli- We believe that our findings provide exciting perspectives. orhinus caniculus (L.), with reference to ultrastructure of early We provide a model in which new nephron production can be stages, histogenesis of the renal countercurrent system, and observed. We believe that identifying the stem cells capable of nephron segmentation in marine elasmobranchs. Am J Anat 190: nephrogenesis in this model is possible. Their identification 309–333, 1991 11. Hentschel H, Walther P: Heterogenous distribution of glycocon- will facilitate studying the signaling molecules responsible. jugates in the kidney of dogfish Scyliorhinus caniculus (L.), with Gene expression studies should be helpful in this regard. Be- reference to changes in the glycosylation pattern during ontoge- cause teleost fish also possess nephrogenic potential, our stud- netic development of the nephron. Anat Rec 235: 21–32, 1993 ies may be transferable to zebrafish, the genome of which is 12. Hentschel H, Storb U, Teckhaus L, Elger M: The central vessel currently available. However, elucidation of the elasmobranch of the renal countercurrent bundles of two marine elasmobranchs genome also stands on the horizon. We suggest that models - dogfish (Scyliorhinus caniculus) and skate (Raja erinacea)-as such as these will improve our understanding of basic functions revealed by light and electron microscopy with computer-as- and eventually will contribute to novel therapies. sisted reconstruction. Anat Embryol 198: 73–89, 1998 13. Forster RP, Schweikert SA, Goldstein L: Osmotic diuresis and Acknowledgments elevated clearances in Squalus acanthias: Effect of epinephrine. We thank the skillful technical assistance of M. 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Sariola H: Nephron induction revisited: From caps to conden- Forschungsgemeinschaft (M.E.: EL-92) and by Baxter, USA (H. sates. Curr Opin Nephrol Hypertens 11: 17–21, 2002 Haller). 18. Ekblom P: Renal development. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW, Giebisch G, New York, References Raven, 1992, pp 475–501 1. Saxen L: Organogenesis of the Kidney. Cambridge, 1987 19. Bard JBL, Gordon A, Sharp L, Sellers WI: Early nephron formation 2. Hammermann MR, Safirstein R, Harris RC, Toback FG, Humes in the developing mouse kidney. J Anat 199: 385–392, 2001 HD: Acute renal failure. III. The role of growth factors in the 20. Zamboni L: Embryology of the urinary system. In: Textbook of process of renal regeneration and repair. Am J Physiol Renal 279: Nephrology, 4 ed., edited by Massry SG, Glassock RJ, Philadel- F3–F11, 2000 phia, Lippincott Williams & Wilkins, 2001, pp 3–9 3. Carmeliet P, Collen D: Molecular analysis of blood vessel for- 21. 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See related editorial, “The Skate Weighs in on Kidney Regeneration,” on pages 1704–1705.