Postembryonic Nephrogenesis and Persistence of Six2-Expressing Nephron Progenitor Cells in the Reptilian Kidney

Abstract

New nephron formation (nephrogenesis) ceases in mammals around birth and is completely absent in adults. In contrast, postembryonic nephrogenesis is well documented in the mesonephric kidneys of fishes and amphibians. The transient mesonephros in reptiles (including birds) and mammals is replaced by the metanephros during embryogenesis. Thus, one may speculate that postembryonic nephrogenesis is restricted to the mesonephric kidney. Previous reports have suggested the metanephros of non-avian reptiles (hereafter reptiles) may continually form nephrons throughout life. We investigated the presence of adult nephrogenesis in reptiles by examining adult kidneys from several species including Trachemys scripta, Chrysemys picta, Boa constrictor, Tupinambis tegu, Anolis carolinensis, and Alligator mississipiensis among others. We found that all major reptilian groups (Testudines, Crocodylia, and Squamates) showed the presence of adult nephrogenesis. The total amount of nephrogenesis varied greatly between species with turtles displaying the highest density of nephrogenesis. In contrast, we were unable to detect adult nephrogenesis in monotremes, and in the iguanid A. carolinensis. Nephron progenitor cells express the transcription factor Six2, which in mammals, becomes downregulated as the progenitor cell population is exhausted and nephrogenesis ends. Using the alligator as a model, we were able to detect Six2-positive cap mesenchyme cells in the adult kidney, which spatially correlated with areas of nephrogenesis. These results suggest that the metanephric kidney of reptiles has maintained the ability to continually grow new nephrons during postembryonic life, a process lost early in mammalian evolution, likely due to the persistence of a Six2-expressing progenitor cell population.

Fig 1. Examples of reptilian post-embryonic nephrogenesis.

(A-H) Kidney tissue sections stained with H&E showing zones of nephrogenesis. (A-F) High magnification of zones of nephrogenesis from juvenile American alligator (A. mississippiensis, A), adult turtles: red-eared slider (T. scripta, B) and painted turtle (C. picta, C), adult tegu (T. teguixin, D), adult Egyptian Mastigure (U. aegyptia, E), and adult boa constrictor (B. constrictor, F). Corresponding lower magnification images are shown in S1 Fig. Scale bar = 50 μm. (G-H) Low magnification images showing tissue organization and variability in arrangement of zones of nephrogenesis between American alligator (G) and red-eared slider (H). Arrows denote zones of nephrogenesis, arrowheads point to mature glomeruli. Scale bar = 100 μm. (I-K) Embryonic alligator kidney (stage 18–19). (I) Wide-field view of embryonic alligator mesonephros (bottom; asterisks label mesonephric tubules) and metanephros (top). Zone of nephrogenesis is indicated by the arrow. Scale bar = 100 μm. (J-K) High magnification of nephrogenesis in embryonic alligator metanephric kidney. Scale bar = 20 μm. Ub = tip of the ureteric bud branch, cm = metanephric cap mesenchyme, c = capsule.

Fig 2. Nephron formation in adult reptiles resembles embryonic nephrogenesis.

(A-G) Adult alligator kidney tissue sections stained with H&E. (A) Wide field view of zones of nephrogenesis on the periphery of the kidney just under the capsule. Scale bar = 100 μm. (B-D) Higher magnification of nephrogenic events as denoted in (A). Early stage nephron formation in (B) which progresses to developed condensed mesenchyme in (C) and a later stage nephrogenesis event in (D) with condensing mesenchyme, ureteric bud-like branch tips (ub) and a newly formed immature nephron (arrow). (E-G) Nephrogenesis in adult alligator is reminiscent of embryonic nephron formation. (E) Metanephric cap mesenchyme undergoing MET to form early nephron structure (arrow). (F) S-shaped developing nephron (arrow) similar to S-shaped bodies detected during embryonic nephron formation. (G). Maturing glomeruli. Newly formed glomerulus (arrow) at the end of a newly formed tubule with progressively more mature glomeruli (arrowheads). Scale bar = 50 μm. Ub = tip of the ureteric bud branch, cm = metanephric cap mesenchyme. The lower magnification images corresponding to (E-G) are shown in S2 Fig.

Fig 3. Species surveyed for post-embryonic nephrogenesis.

Cladogram displaying species tested for presence of nephrogenesis in juvenile (J) or adult (A) kidney. Two species of monotremes, Tachyglossus aculeatus (short-beaked echidna) and Ornithorhynchus anatinus (platypus), were used for comparison as the most basal mammalian group. As with other mammals, no evidence of nephrogenesis was detected in the monotreme species. Post-embryonic nephrogenesis was detected in all major reptilian groups surveyed. Species names in light gray did not display evidence of nephrogenesis. NT = not tested.

Fig 4. Kidney growth occurs by hypertrophy or nephrogenesis in reptiles.

(A-C) Green anole (A. carolinesis) kidney measurements compared to body mass. (A) Scatter plot of kidney mass related to body mass with line of best fit. (B) Scatter plot of estimated glomerular number related to body mass. (C) Scatter plot of the ratio of glomerular number/kidney mass compared to body mass. Number of biological samples = 10. (D-E) Scatter plots of glomerular size compared to body mass in green anole (D) or body length in American alligator (A. mississippiensis) (E). (F) Histology of adult green anole kidney (left) and adult American alligator kidney (right). Arrow denotes zone of nephrogenesis in the American alligator. (g) = grams. (cm) = centimeters.

Fig 5. Extent of nephrogenesis in reptiles.

The number of nephrogenic zones in each species was normalized to the effective surface area of each section analyzed (methods). The ratio of the total number of nephrogenic zones and the effective surface area was then used to compare the extent of post-embryonic nephrogenesis between different reptile species. Only the material with sufficient preservation to accurately estimate the number of nephrogenesis events was used for analysis. Species analyzed were: A. mississippiensis (3 juvenile (J), 1 adult (A)), B. constrictor, C. picta, T. scripta, U. aegyptia. The corresponding density of nephrogenesis: (A. mississippiensis: 2.1 ± 1.1 (0.89m juvenile), 1.8 ± 1.1 (1.04m juvenile), 1.3 ± 0.7(1.63m juvenile), 0.86 ± 0.35(1.98m adult). B. constrictor: 0.9 ± 0.9. C. picta: 6.7 ± 0.9. T. scripta: 8.6 ± 4.3. U. aegyptia: 0.7 ± 0.3).

Fig 6. Nephrogenic zones express kidney progenitor marker Six2.

(A-I) Confocal immunofluorescence imaging of American alligator sections stained with Six2 antibodies. (A-C) Juvenile alligator kidney stained with Six2 (A) and wheat germ agglutinin (WGA) Alexa Fluor 555, used as a non-specific fluorescent counter-stain (B), merged image with DAPI in (C). (D-F) Adult alligator kidney stained with Six2 (D), WGA555 (E), and merged image with DAPI (F). (G-I) Juvenile alligator kidney stained with Alexa Fluor 488 secondary antibody alone (F), WGA555 (H), and merged image with DAPI in (I). (J-L) Kidney from mouse embryonic day 15 (E15) were similarly stained with Six2 antibodies (J) and WGA555 (K). Image merged with DAPI stain is shown in L. (M-O) Adult mouse kidney stained with Six2 antibodies (M) and WGA555 (N) using the same protocol. Image merged with DAPI stain is shown in O. The apparent cell clustering in (A,C, J and L) is due to freeze artifact. Ub = tip of the ureteric bud branch, cm = metanephric cap mesenchyme, c = capsule. Scale bar = 50 μm.

Fig 7. Alligator kidney topology suggests tight correlation between nephrogenesis and branching of the collective system.

(A-B) cross-section of near parallel collecting ducts that transverse the kidney in the sub-capsular location (asterisks), giving off near-perpendicular terminal branches that drain the nephrons (glomeruli can be seen in (A) inside the dotted area. (C-D) Longitudinal section through individual collecting ducts near their tips, where new nephrogenesis can be seen (further detailed in F-G). (E) Section taken parallel to and just under the capsule shows near-parallel row of collecting ducts that occasionally split along their course (white arrows). However, most branching events result in generation of terminal duct branches that are seen in cross-section. (F-G) Higher magnification longitudinal sections through individual collecting ducts near the tips. Black arrows point to zones of nephrogenesis. Arrowheads label glomeruli. White arrows show splitting of terminal collecting branches. (H-I) 3D model of Alligator kidney topology at a single lobe level (only the bottom half of the lobe is shown: it is mirrored on top). Each duct (red) gives off sequential terminal branches (grey), each draining a nephron (represented by yellow spheres). Examples of past branching events are marked with white arrows. At the tips of collecting ducts new nephrons are formed (red caps). Scale bars in (A-G) = 100 μm.