The long-term label retaining population of the renal papilla arises through divergent regional growth of the kidney

Abstract

Long-term pulse chase experiments previously identified a sizable population of BrdU-retaining cells within the renal papilla. The origin of these cells has been unclear, and in this work we test the hypothesis that they become quiescent early during the course of kidney development and organ growth. Indeed, we find that BrdU-retaining cells of the papilla can be labeled only by pulsing with BrdU from embryonic (E) day 11.25 to postnatal (P) day 7, the approximate period of kidney development in the mouse. BrdU signal in the cortex and outer medulla is rapidly diluted by cellular proliferation during embryonic development and juvenile growth, whereas cells within the papilla differentiate and exit the cell cycle during organogenesis. Indeed, by E17.5, little or no active proliferation can be seen in the distal papilla, indicating maturation of this structure in a distal-to-proximal manner during organogenesis. We conclude that BrdU-retaining cells of the papilla represent a population of cells that quiesce during embryonic development and localize within a region of the kidney that matures early. We therefore propose that selective papillary retention of BrdU arises through a combination of regionalized slowing of, and exit from, the cell cycle within the papilla during the period of ongoing kidney development, and extensive proliferative growth of the juvenile kidney resulting in dilution of BrdU below the detection level in extra-papillary regions.

Fig. 1.

Papilla cells quiesce during development of the metanephric kidney. A: scheme for BrdU treatments: animals were pulsed either before [embryonic (E) day 10], during [E14, postnatal (P) day 3], or after (P12) the period of active embryonic development of the permanent kidney. B: immunofluorescent detection of BrdU (red), with DAPI nuclear counterstain (blue) to detect BrdU-retaining cells in papillas of 3-mo-old animals. A BrdU pulse at E10 does not result in retention of labeled cells in the papilla at 3 mo of age, whereas pulse of cells at either E14 or P3 does. A BrdU pulse administered after the period of active kidney development (P12) does not lead to retention of labeled cells at 3 mo. Inset: enlargements of boxed areas in each panel show overlap of BrdU with DAPI, demonstrating nuclear localization of signal. Dashed line in E14.5 panel outlines the kidney papilla.

Fig. 2.

Short-term BrdU retention in the developing kidney. BrdU incorporation is detected by immunohistochemical staining (dark brown nuclei). To visualize tissue morphology, sections have been lightly counterstained with hematoxylin (blue). A: scheme of BrdU pulsing and harvest. B: 12 h after pulse, the majority of cells of the developing kidney (outlined) are BrdU labeled, demonstrating the highly proliferative nature of the tissue. C: 3 days after the BrdU pulse, the majority of cells are BrdU negative, with BrdU-positive cells concentrated to the capsule and the nascent papilla (C'). Six days after pulse, little BrdU labeling is seen in the kidney, with the exception of cells of the kidney capsule, and the nascent papilla (D', arrows). C, capsule; MM, metanephric mesenchyme; NZ, nephrogenic zone; RC, renal calyx; U, ureter; UB, ureteric bud.

Fig. 3.

Localization of BrdU-retaining cells by staining with the collecting duct marker DBA lectin. Papillas were harvested from 3-mo-old animals administered BrdU at E11.5. Adjacent sections were stained for DBA lectin (A) or BrdU (B). Enlargements of boxed areas are shown in A' and B'. A and A': DBA-lectin stained cells (brown) can be seen lining collecting duct tubules of the papilla, as well as the RC. B and B': outline of DBA-stained cells is shown in yellow. Nuclear BrdU staining (dark brown) can be seen in nuclei of collecting duct cells (solid arrows), DBA-negative presumptive loops of Henle (open arrowheads), and presumptive interstitial cells that are not directly associated with tubule lumena (solid arrowheads). P, proximal papilla; D, distal papilla.

Fig. 4.

Papilla represents a cell compartment within the juvenile kidney with very limited turnover. A: BrdU dosing and organ harvest scheme. Three individual animals were dosed and harvested per time point. B: quantification of BrdU-stained nuclei in equivalent areas of tissue from cortex, outer medulla, and papilla reveals that cellular proliferation in the papilla is extremely rare compared with other regions of the kidney. Sections from 3 separate individuals were analyzed, and error bars in the graph represent the standard deviation of measurements. C: representative sections from BrdU-stained cortex, outer medulla, and papilla showing widespread distribution of proliferating cells in cortex and outer medulla, and a paucity of proliferating cells in the papilla.

Fig. 5.

BrdU administered at E17.25 shows limited incorporation in the distal papilla. After administration of a single pulse of BrdU at E17.25, kidney papillas were stained for BrdU retention at E17.5, P3, and P28. At all 3 times of harvest, little BrdU retention can be seen in the distal papilla (square bracket).

Fig. 6.

Localization of BrdU-retaining cells within the papilla following administration at time points during papilla development and growth. BrdU was administered at either E11.5, E14.5, or P2, and papillas were harvested for analysis at 3 mo of age. Staining of serial papilla sections reveals that the majority of BrdU-retaining cells from the E11.5 administration are located within the distal papilla, whereas the majority of retaining cells from the P2 administration can be found in the proximal papilla. Administration at E14.5 results in distribution of retaining cells throughout the papilla.