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 Delbrück Center for Molecular Medicine, Humboldt University, Berlin, Germany.
Correspondence to Prof. Dr. Hermann Haller, Department of Nephrology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Phone: 49-511-532-6319; Fax: 49-511-552366;
ABSTRACT. The mammalian kidney responds to partial nephrectomywith glomerular and tubular hypertrophy, but without renal regeneration.In contrast, renal regeneration in lower vertebrates is knownto occur. Understanding the underlying mechanisms of renal regenerationis highly important; however, a serviceable animal model hasnot been developed. A neonephrogenic zone has been identifiedin the European lesser spotted dogfish, Scyliorhinus caniculus(Hentschel H. Am J Anat 190: 309–333, 1991), as well asin the spiny dogfish Squalus acanthias and the little skate,Leucoraja erinacea. The zone features the production of newnephrons complete with a countercurrent system. To analyze thisnephrogenic region of elasmobranch fish further, a renal reductionmodel was established. The neonephrogenic zone in the adultkidney of the little skate resembles the embryonic metanephrickidney and contains stem cell-like mesenchymal cells, tips ofthe branching collecting duct system, and outgrowth of the arterialsystem. Four stages of nephron development were analyzed byserial sections and defined: stage I, aggregated mesenchymalcells; stage II, S-shaped body-like structure with high-prismaticepithelial cells; stage III, segmental nephron segregation;stage IV, functioning nephron. The stages were analyzed afterpartial nephrectomy. In addition, cell proliferation was assessedby incorporation of bromo-deoxyuridine (BrdU). New nephronsdeveloped in animals undergoing partial nephrectomy. Growthwas greatly stimulated in the nephrogenic zone, both in theremnant tissue and in the contralateral kidney within 10 wk.Mesenchymal cell aggregates increased significantly per renalcross-section compared with controls (stage I, 0.64 ±0.28 versus 0.27 ± 0.25; P < 0.005; n = 10 animalsper group). The same was the case for S-shaped body-like cysts(stage II, 0.24 ± 0.19 versus 0.08 ± 0.09; P <0.02). Cellular proliferation in the neonephrogenic zone ofthe contralateral kidney was also greatly enhanced (14.42 ±3.26 versus 2.64 ± 1.08 BrdU-positive cells per cross-section,P < 0.001). It is concluded that the skate possesses a nephrogeniczone containing stem cell-like mesenchymal cells during itsentire life. Partial nephrectomy induces renal growth by acceleratingnephrogenesis. This unique model may facilitate understandingrenal regeneration. E-mail: haller.hermann@mh-hannover.de orm.elger@gmx.de.
In mammals, nephron formation is terminated at birth or shortlythereafter (1). Subsequent growth is generally restricted totubular epithelial replacement by proliferation and/or to angiogenicprocesses involved in vascular reconstruction (2,3). The limitedregenerative potential results in a greatly reduced capacityto cope with renal disease. Injured glomeruli do not regenerate,and glomerular destruction is the major cause of final nephronloss (4,5). The remaining nephrons respond with compensatoryglomerular and tubular growth. Large-scale nephron loss is thereforenot accompanied by new nephron generation ("neo" nephrogenesis)in mammals. However, lower vertebrates have superior abilitiesin that regard. Adult sexually mature bony fish (teleosts) candevelop new nephrons as shown in light and electron microscopicstudies (6,7). Formation of de novo nephrons has been reportedin goldfish kidneys after the administration of nephrotoxicsubstances (8). We previously found nephrogenic tissue in thelittle skate, Leucoraja erinacea, an elasmobranch cartilaginousfish (9), as well as in the spiny dogfish, Squalus acanthias.Furthermore, a detailed analysis of nephron anlagen and developingnephrons in the lesser spotted dogfish, Scyliorhinus caniculus,has been published (10,11). Because elasmobranch kidneys exhibitgreat structural complexity (12) and resemble by this and othermorphologic features mammalian kidneys, these animals couldserve as a model for nephrogenesis. We therefore studied nephrogenesisin adult skate kidney and tested the hypothesis that this processis stimulated by renal size reduction.
Animals
Female skates, Leucoraja erinacea (Mitchell 1825), were nettedin Frenchman Bay, Maine, or purchased from the Marine StationWoods Hole, MA, USA. The body size as measured by the disc diameter(fin span) ranged from 20 to 32 cm. The body weight ranged from310 to 790 g. The animals were kept in large circular tankswith running aerated seawater and were acclimatized for a fewdays before the experiments. The animals were fed a diet offrozen shrimp.
For reduction of renal mass, we surgically removed renal tissue.The animals were anesthetized by adding 0.1 g/L tricaine (Sigma)to the seawater. During anesthesia, the seawater was cooledwith ice, and deep anesthesia was achieved after 40 min. Wedeveloped two operations. Fish were mounted upside-down on astyrofoam board. The gills were superfused with a tricaine drip(0.05 g/L seawater) via the oral cavity. The body was coveredwith paper towels soaked in ice-cold seawater. The posteriorbody cavity was opened by a midline incision through the ventralbody wall. The pelvic girdle was transected, and the intestineand gonads were carefully laid aside to expose the right kidney.The large efferent intrarenal veins of the renal portal system,which were visible through the peritoneal epithelium, were cauterized.A 2/3 nephrectomy in one kidney was performed with scissorsafter cauterization of the intrarenal arteries and veins. Greatcare was taken not to injure the multiple renal arteries enteringthe kidney from the dorsal aorta. The viscera and the pelvicgirdle were repositioned. The body wall was closed with twosutures. One ran in the subdermal musculature and one in theskin. A nonresorbable surgical thread was used in all instances.After the operation, the fish were placed in their holding tanks.They recovered from anesthesia after 5 to 10 min.
For the second technique, the animals were laid on their ventralsurface. The gills were dripped with anesthetic via the spiracles.A longitudinal incision was made through the skin and underlyingmusculature (longissimus dorsalis) in the pelvic region, 2 cmlateral from the midline. The dorsal surface of the right kidneywas thereby directly exposed. Two thirds of this kidney wasremoved without injury to the peritoneal membrane. The incisionwas closed by a double suture. Sham operations were performedin one group of animals. Unoperated fish served as additionalcontrols. Fish were maintained in their tanks with constantfeeding. After 10 wk, 20 animals (10 nephrectomized [NX] and10 control skates) were sacrificed, and their kidneys were examined.
Preservation of Kidney Tissue
The kidneys were fixed by vascular perfusion via the conus arteriosusas described previously (12). In brief, the vascular systemwas rinsed with modified elasmobranch Ringer solution (13) ata pressure of 100 cm H2O for several minutes. For minimizingosmotic effects during the clearing of the vasculature, 2% polyvinylpyrrolidone was added. After the blood was cleared from thecirculatory system, the fixation solution was introduced withouta pressure drop and perfused for approximately 5 min at thesame pressure as the rinsing solution. The chilled fixationfluid contained paraformaldehyde (3 to 4%) and picric acid (0.5%)in 0.1 M Sörensen phosphate buffer, pH 7.4. The osmolalityof the buffer of the fixative was adjusted with sodium chlorideand sucrose to that of the pre-rinse (950 mOsmol/L). After fixation,the body was opened, the kidneys were exposed, and the sizeand shape of the kidneys were photographed in situ from theventral side. The organs were carefully removed, weighed, anddocumented from the dorsal side.
Histology and Quantification of Developmental Nephron Stages
The excised kidneys were postfixed in fresh fixative for anadditional hour. Each kidney was cross-sectioned with razorblades in eight to ten blocks and processed for paraffin embedding.Serial cross-sections (3 µm) were prepared from each blockand stained with Masson-Goldner trichrome stain. In these sections,developmental stages I to IV were identified in accordance todevelopmental stages in dogfish, Scyliorhinus caniculus (10).The amount of developmental stages was estimated in ten nephrectomizedand ten control animals. The group of nephrectomized animalsconsisted of five animals operated from the dorsal side andfive animals operated from the ventral side. The controls includedfive sham-operated and five non-operated animals. The comparisonwas made between the intact contralateral kidneys of the nephrectomizedanimals and one kidney of the sham-operated and non-operatedcontrols. Serial cross-sections (8 µm) were prepared fromfive blocks per kidney. Three sections per tissue block at adistance greater than 300 µm were used to determine thenumber of developmental stages I and II per renal cross-section.
Electron Microscopy
For transmission electron microscopy, tissue pieces of perfusion-fixedkidneys were postfixed in 1.5% glutaraldehyde and 1.5% paraformaldehydesolution, followed by fixation in 1% OsO4 solution, and subsequentembedding in Epon resin. Thin sections were obtained with anultramicrotome OMU3 (Reichert, Vienna) and viewed with Zeisselectron microscope EM10 (Zeiss, Oberkochen).
Cell Proliferation
Proliferative activity of the renal tissue was estimated byindirect immunohistochemical detection of incorporated 5-bromo-2'-deoxyuridine(BrdU) in a separate set of ten adult female little skate (bodysize, 24 to 27 cm; body weight, 500 to 690 g). Ten days afterpartial nephrectomy of the left kidney in five animals (NX)and sham operation in five control skates, the animals receivedan intraperitoneal injection of 1.5 mg/100 g BW BrdU dissolvedin PBS. After 12 h, the kidneys were fixed by perfusion as describedabove. Four tissue blocks from the middle portion toward thecaudal end of one kidney of control skates and of the contralateralkidney of NX skates were embedded in paraffin, and six to eightindependent cross-sections per animal were used for numericaldetermination of BrdU-labeled cells. The deparaffinized sectionswere treated with 0.01% trypsin in PBS for 1 min at RT and UniversalBlocking Reagent (BioGenex, San Remon) for 10 min and were incubatedwith mouse monoclonal anti-BrdU antibody (clone BMG 6H8) containingnucleases for DNA denaturation (Roche) diluted 1:20 in PBS for30 min at 37°C, followed by anti-mouse Ig-alkaline phosphatase(Roche) for 30 min at RT. The reaction was visualized by colorsubstrate solution containing nitroblue tetrazolium (NBT) saltand 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Cell nucleilabeled with BrdU were counted per kidney cross-section. Threetissue blocks from the ipsilateral kidney of the NX animalswere used to study the distribution of proliferative cells inthe operated kidney. Parallel sections were stained with trichromestain after Masson-Goldner for identification of developmentalstages.
Statistical Analyses
Statistical difference was tested between nephrectomized andcontrol animals by t test.
In Figure 1, the ventral aspect of the kidneys is shown in situafter removal of the intestine (panel A). Two separate flatroughly bean-shaped organs are located in the caudal retroperitoneum.We determined the renal mass in fifteen female skates (300 to800 g body weight). Total kidney weight ranged from 1 to 3 gcorresponding to 0.3 to 0.4% of body weight. Kidney weight andbody weight were closely correlated, and a linear correlationover a large range of body weight was observed (r = 0.91, P< 0.001) (panel B).
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 = 0.025 + 0.293 · x, r = 0.91).
The renal architecture, nephron segmentation, the relationshipof the nephrons to the renal zones, the renal arterial and venousportal systems, and the structure of the nephrogenic zone areshown in Figure 2. Cross-sections revealed a flat ellipticalshape and demonstrated a cap of tubular bundles (lateral bundles)located on the mesial tissue mass (panel A). These bundles representthe renal countercurrent system. They are situated along thedorsolateral surface of the kidney in a continuous zone beginningat a distance of approximately 1 mm from the medial edge ofthe kidney and extending around the lateral margin to the ventralsurface for up to 1 mm. The afferent intrarenal veins and theglomeruli separate the bundle zone and the mesial tissue zone.The nephrons were heterogeneous. The most elaborate nephronswith the largest glomeruli were observed at the medial location.The small nephrons were located at the lateral edge of the kidneys(see also reference 9). Developing tubules and nephron anlagenwere frequently present in the vicinity of the outermost smallglomeruli. By careful stereomicroscopic examination of the decapsulatedrenal surface, a narrow band of whitish tissue was identifiedalong the convex lateral surface of the kidney. This tissuedemarcated the border between the pale bundle zone and the reddish-brownmesial zone. Because the band contained nephron anlagen, wetermed this zone "nephrogenic tissue." In young animals, thenephrogenic tissue formed a ribbon-like structure along theentire kidney. In older animals, the band was discontinuous.
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 µm) 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 µm) 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.
Figure 2B shows a schematic drawing of a renal cross-section.Blood supply and the nephron course are demonstrated in thetwo major kidney zones: the lateral bundle zone and the mesialtissue. Several renal arteries arise from the dorsal aorta.Intrarenal arteries give rise to afferent glomerular arteriolesand to bundle arteries. The mesial tissue is supplied by capillarysinusoids of the renal portal system, which enters the kidneyby afferent renal and intrarenal veins. The bundle vessels,including the central vessels of the individual bundles andthe efferent arterioles, join the mesial sinusoid system. Theefferent intrarenal veins drain the sinusoid system. The zoneof neonephrogenesis with newly developing glomeruli is locatednear the most peripheral branches of the arterial system (comparealso reference 9).
Figures 2C and 2D show a nephrogenic zone at the lateral marginof the kidney at higher resolution. The nephrogenic zone waswedged between the ventrolateral margin of the bundle zone andthe mesial tissue. Blind ends of the collecting duct systeminvaded the nephrogenic tissue. They frequently showed mitosisof epithelial cells. Occasionally, two tips originating fromthe collecting duct ends were observed. Growth of the collectingduct system by formation of new tips in conjunction with formationof nephron anlagen appeared to be exclusively located in thisnephrogenic zone. We did not observe nephrogenesis in otherregions of the kidney.
The nephrogenic zone contained mesenchymal cells and prospectivetubular cells in different stages of development (Figure 2D).Around the collecting duct tips, undifferentiated mesenchymalcells aggregated to form the first developmental stage (seebelow). The developmental stages were adjacent to a prominentmass of collagen-rich interstitial tissue, which was part ofthe band of whitish tissue along the ventrolateral border seenin excised kidneys (as mentioned above). The region around theearly stages was poorly vascularized. The early aggregatingmesenchymal cells were nonpolarized cells with a comparativelylarge nucleus and a narrow cytoplasmic zone (Figure 2E). Theywere tightly surrounded by flat, laminate cell processes ofadjacent fibroblasts or by neighboring mesenchymal cells.
For further definition of the developmental stages in the skatekidney, and as a basis for quantification of nephrogenic responsesto different stimuli, we arranged the developmental stages accordingto their size and complexity. We developed a system of fourmajor stages with further subdivisions into early and late phasesto grade our findings, as shown in Figures 3 and 4. Stage Iwas defined as a distinct small structure of less than 100 µmin diameter, consisting of a condensed mass of mesenchymal cellsin the vicinity of a new bud of the collecting duct system (Figures 3A and 4A).In the first phase (stage I-1), the mesenchymalcells were irregularly arranged. In stage I-2, cells at thevery tip of the collecting duct bud began to align and to epithelializewith formation of basement membrane and development of apicaltight junctions.
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.
Figure 4. Subdivision of the early developmental nephron stages I and 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 CD tip, the mesenchymal cells begin to epithelialize. (B) Phase II-1: A vesicle-like structure is formed, which connects to the CD tip. Phase II-2: The cyst begins to elongate and fold up. A gap filled by mesenchymal cells is still present. Phase II-3: A S-shaped body-like structure with four narrow bends is formed. For better understanding of the epithelial structure, the tubular limbs are projected into a two-dimensional plane. At the proximal end, the glomerular anlage with the presumptive podocyte and parietal layers are evident. Mesenchymal cells are present close to the glomerular anlage. Two of the bends indicate the prospective loops of the mesial tissue, and the two opposite bends represent the prospective loops of the countercurrent system in the lateral bundle.
Stage II was defined as the subsequent development of tubularstructures from early cyst formation and the appearance of theglomerular anlage. We defined three subsequent phases of thesestructures according to their length. In the first phase (stageII-1), the cyst was an ovoid structure with a central lumen.At the presumptive proximal end, a mass of unpolarized mesenchymalcells formed a plug. In the second phase (stage II-2), the elongatedcysts usually performed two bends like the letter S and an additionalbend at each end (S-shaped body-like cyst). The most advancedcysts (stage II-3) displayed four alternating tubular loopsclosely adhering to each other (Figures 3A and 4B). The gapin the proximal end of the cyst was closed by the formationof the glomerular epithelial primordia. An accumulation of undifferentiatedmesenchymal cells was still attached to the prospective Bowman’scapsule. The exceptionally high epithelial cells (cell height,>50 µm) and the presence of a distinct lumen in theproximal portion were helpful for identification of all phasesof stage II on single sections.
Stage III was characterized by the progressive differentiationof nephron segments, including the occurrence of vascular structuresin the developing glomerulus (Figure 3B). Pronounced elongationof the tubule was accompanied by growth of two loops in directionof the renal capsule to become the hairpin loops of one individualbundle. The growth of the two opposite loops was directed tothe mesial tissue. Stage IV consisted of young nephrons withwell-perfused glomeruli and well-differentiated tubular segments(Figure 3B). Stages III and IV proved difficult to quantify.Although the tubular profiles and glomeruli were easily discernibleby their lesser diameter and smaller size compared with adultnephrons, the length of the mesial convolutions and the bundlewere already too large to allow quantification by numericalcounts. For example, the growing countercurrent bundle tendedto coil. Thus, in any given section, one to five cross-sectionsappeared that were not always identifiable as belonging to onenephron. In addition, the developing mesial coils could notbe assigned to the same nephron’s loop bundles.
After defining the different stages of nephron development,we then tested the hypothesis that the nephrogenesis can beinduced by renal mass reduction. Figure 5 shows a schematicdiagram, the operation itself, and the kidney 10 wk after partialnephrectomy. The loss of renal mass was still manifest, andthe mean renal weight was approximately half that of controlkidneys (Table 1). As a result, a considerable gap partly filledwith pale connective tissue was visible. The contralateral kidneydid not differ macroscopically from the roughly bean-shapedcontrol kidneys.
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.
Cross-sections of the contralateral kidneys revealed no changesin areas of mature tissue. However, in the nephrogenic tissue,a conspicuously large number of developmental stages were evidentin partially nephrectomized animals. Two to five profiles ofnephrogenic stages were seen on a renal cross-section as shownin Figure 6. All phases of stages I through IV were enhanced.The ramifying blind collecting duct ends were thicker than incontrol animals. The condensed mesenchymal stage I masses formedextensive caps with generally more than 200 cells around thecollecting duct tips.
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.
Growth quantification in the nephrogenic zone was performed(a) on the frequency of developmental nephron stages I and IIand (b) on cell proliferation. The renal cross-sections of controlsand intact contralateral kidneys from partially nephrectomizedanimals revealed values (n = 10 animals per group; mean ±SD) for stage I of 0.27 ± 0.25 and 0.64 ± 0.28,respectively (P < 0.005). Mean values for stage II were 0.08± 0.09 for controls and 0.24 ± 0.19 for partiallynephrectomized animals (P < 0.02) (Figure 7). Small but distinctislands of lymphomyeloid cells were observed in the vicinityof the nephrogenic tissue in the contralateral kidney of partiallynephrectomized animals in addition to islands with blood formingtissue adjacent to large intrarenal arteries.
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 new nephrons is greatly stimulated in the NX animals. In NX animals, on average, a profile of stage I can be found on more than 50% of the sections; a stage II on every third section in. Results are mean ± SD, n = 10 animals per group. t test: P < 0.005 for stage I, P < 0.02 for stage II.
Cellular proliferation in the contralateral kidney and in theoperated kidney was studied by immunohistochemical demonstrationof incorporation of BrdU 10 d after nephrectomy. Control skatesshowed very rarely BrdU-labeled cells in the mature portionof the tissue. A low number of reactive cells were present inthe nephrogenic zone. Many histologic sections were lackingdividing cells entirely. In the contralateral kidney of nephrectomizedanimals, the mean number of labeled nuclei per kidney cross-sectionwas significantly increased (Figure 8). The majority of thereactive cells were located in the center of the nephrogeniczone and in adjacent young nephrons.
Figure 8. Numerical determination of proliferating cells. The number of BrdU-positive nuclei per renal cross-section is significantly increased in contralateral kidneys of partially nephrectomized animals. Results are mean ± SEM, n = 36 sections per group; t test, P < 0.001.
We also analyzed the growth and proliferative response in theipsilateral, operated kidney. As expected, regions of massivedegeneration, areas of dilated tubules, and foci with apparentlyhealthy nephrons were present. However, in the small caudalportion where both the mature and the original nephrogenic zonewere basically intact and not disturbed by the operative procedure,an increase in the number of developmental stages was found.Renal cross-sections of this portion revealed for stage I amean value of 0.46 ± 0.40 and for stage II a mean valueof 0.42 ± 0.39 (mean ± SD, n = 10 animals). Proliferationof the developmental stages was vigorous, as many cells in developingtubules and glomeruli displayed BrdU incorporation. Serial sectioningof these ipsilateral, operated kidneys did not show signs ofproliferation in areas of mature tissue. Specifically, the dilatedtubules were essentially devoid of BrdU labeling.
We have characterized a nephrogenic zone in the adult kidneysfrom the skate. The nephrogenic zone represents a niche withinthe kidney where stem cell-like cells reside. The tissue respondsto partial reduction of renal mass with the formation of newnephrons. The morphogenic process that we term neonephrogenesisappears to be an important mechanism for renal growth, as wellas for repair of injured kidney. Renal hypertrophy, which isa common response to renal mass reduction in higher vertebratesand man (14–16), contributed only slightly to the reconstitutionof renal mass in the skate. Our morphologic analyses demonstratedthat a zone of embryonic renal tissue persists in adult skates.Evidently, the advanced complexity of elasmobranch kidneys doesnot preclude nephrogenesis in the adult kidney.
The nephrogenic zone of the skate contains the essential structurescharacteristically found in the embryonic mammalian metanephros.We defined four stages of nephron development. The evaluationof the four stages in the adult skate corroborated previouslight and electron microscopic results in the European dogfish,Scyliorhinus caniculus (10). Because certain stages of nephrogenesishave been observed in adult specimen of several other fish species,neonephrogenesis is probably a common feature in elasmobranchsand teleosts (6–10).
The nephrogenic mesenchyme is present in stages I-1 and I-2.These stages correspond to the two transient mesenchymal condensationsthat precede the comma-shaped and S-shaped body in the embryonicmammalian metanephros (1,17,18). Stage I-1 resembles the capcondensate and stage I-2 is very similar to the pretubular condensate(17,19,20). The collecting duct tips that are in contact withthe stage I structures are the structural homologues to thetips of the ureteric bud branches. The collecting duct tip ofthe skate differs in several morphologic features from the mammalianureteric bud branches. The collecting duct tip is usually asolid structure, compared with the ampulla-like tip in rodentsand humans (20,21). The tip is encompassed by a sheath of fibroblaststhat becomes the bundle sheath of the elasmobranch countercurrentsystem (10).
Stage II was defined as S-shaped body-like cysts in agreementwith our observations of Scyliorhinus. Our results showed considerablevariability in this developmental stage, apparently representingdifferent phases. We suggest that at the end of stage Ib, thepretubular condensate grows into a roughly spherical cyst thatis in contact with the collecting duct tip at the distal end.The proximal end features a plug of unpolarized cells. Thisrenal vesicle-like structure grows rapidly to become an S-shapedbody-like cyst with two additional bends. The epithelia of thecollecting duct tip and the adjacent distal end of the cystmerge. Very marked growth soon leads to a structure with fournarrow bends. Thus, in this phase of stage II, the cyst farexceeds the typical S-shape. The presumptive proximal end stilldisplays a plug of unpolarized cells. In the terminal phaseof stage II, a portion of the unpolarized cells epithelializesand becomes the glomerular epithelia of stage III. A featurecommon to all phases of stage II is that the high prismaticepithelial cells appeared uniform. The numerous mitotic figuresand the incorporation of BrdU give evidence for rapid growthin all regions of the cyst.
Stage III is characterized by the segregation into tubular segmentsand the appearance of vessels. The specific architecture ofthe elasmobranch nephron (22,23) is established at this stage.Further growth leads to stage IV, the young nephron. This stageis relatively immature with respect to cell size and tubularlength; however, the segments can now be considered as a functioningexcretory unit. The addition of newly formed nephrons in theperiphery of the elasmobranch kidney, as originally uncoveredin the young skate (9), was described in detail in adolescentyoung dogfish (10). The present study demonstrates the potentialof nephron growth and differentiation in the adult skate kidney.
Mesenchymal cells capable of nephrogenesis must establish contactwith collecting duct tips for growth and branching to occur(24,25). It can be anticipated that mesenchymal cells competentfor nephron development presumably provide the progenitor cellsalso in the skate. These cells are presumably derived from acontinuous pool of embryonic stem cell-like cells within thenephrogenic zone of the elasmobranch kidney. We suggest thatthese cells are precursors of competent stage I mesenchyme.We further suggest that the mesenchyme provides self-renewingcells for ongoing nephrogenesis. Such cells would fulfill requirementsof stem cells (26). Future studies involving lineage studieswill address these hypotheses.
The contralateral kidney of the partially nephrectomized skatesincreased in size, a common finding in mammalian kidneys (16,27–29).Information from lower vertebrates is limited. A nephrectomystudy in the common carp, Cyprinus carpio, a teleost, was focusedon blood cell formation. The contralateral kidney of the carpshowed distinct hypertrophic growth after several months, andthe excised portion in the kidney undergoing partial nephrectomydid not regenerate (30). Similar results were reported for thenewt, Notophthalmus viridescens, a urodelic amphibian. In thisanimal, nephrogenesis was not observed (31).
In this study, we were able to confirm our earlier findingsthat neonephrogenesis indeed occurs in the adult elasmobranch(9,10). Our new observations of BrdU incorporation in skateprovide further evidence for formation of nephrons de novo inthe nephrogenic zone. Mitotic activity as visualized by thistechnique was high in various stages of nephron developmentand young nephrons of nephrectomized animals. Older portionsof the kidney were virtually devoid of BrdU labeling. Similarresults were obtained in the remnant portion of the operatedkidney. This clearly shows that it is only the lateral zonethat can be stimulated to substantial renal regeneration withproduction of new nephrons. In this region, nephron developmentand growth of the collecting duct system together with growthof the renal vasculature can be stimulated. We suggest thatthe undifferentiated cells residing in the mesenchyme of thelateral kidney zone can provide targets for signals that initiatecell division after stimulation by nephrectomy. This will befollowed by the formation of initial stages of nephron development.The formation of new nephrons was also observed in adult teleosts(6,7,32). In contrast, nephrogenesis in mammals has been describedto terminate shortly after birth. The reasons for loss of thisability in higher vertebrates are unclear. During prenatal growthof the metanephric mammalian kidney, nephrogenesis can be greatlyaugmented according to recent uninephrectomy experiments withunborn sheep (33). In these animals, compensatory renal growthof the contralateral kidney is associated with an increase infinal nephron number due to prolonged nephrogenic growth inthe embryonic metanephros. Our experiments suggest that theelasmobranch kidney is equipped with a nephrogenic zone, inwhich similar "embryonic growth" persists life-long. The nephrogenicpotential can be activated by removal of renal tissue.
We believe that our findings provide exciting perspectives.We provide a model in which new nephron production can be observed.We believe that identifying the stem cells capable of nephrogenesisin this model is possible. Their identification will facilitatestudying the signaling molecules responsible. Gene expressionstudies should be helpful in this regard. Because teleost fishalso possess nephrogenic potential, our studies may be transferableto zebrafish, the genome of which is currently available. However,elucidation of the elasmobranch genome also stands on the horizon.We suggest that models such as these will improve our understandingof basic functions and eventually will contribute to novel therapies.
Acknowledgments
We thank the skillful technical assistance of M. Beese, P. Berkefeld,and M. Schlöter-Kregeler. Artwork and photographic assistancewere provided by H. Ryland, Documentation, MHH, and C. Pieczka,G. Schulte and E. Schlüter, ZE-DOK, MPI Dortmund. Electronmicroscopy was performed in the Dept. of Cell Biology and ElectronMicroscopy, MHH, and in the Institute for Peptide Research Pharmaceuticals,Hannover. The expert technical assistance of W. Posselt andG. Preiss with the electron microscopes is greatly acknowledged.The work was supported by a grant of the Deutsche Forschungsgemeinschaft(M.E.: EL-92) and by Baxter, USA (H. Haller).
Footnotes
H. Hentschel and M. Elger contributed equally to this publication.
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Received for publication July 31, 2002.
Accepted for publication February 15, 2003.
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Abstract
Introduction
