Patterns of differentiation of renin lineage cells during nephrogenesis

Abstract

Developmentally heterogeneous renin-expressing cells serve as progenitors for mural, glomerular, and tubular cells during nephrogenesis and are collectively termed renin lineage cells (RLCs). In this study, we quantified different renal vascular and tubular cell types based on specific markers and assessed proliferation and de novo differentiation in the RLC population. We used kidney sections of mRenCre-mT/mG mice throughout nephrogenesis. Marker positivity was evaluated in whole digitalized sections. At embryonic day 16, RLCs appeared in the developing kidney, and the expression of all stained markers in RLCs was observed. The proliferation rate of RLCs did not differ from the proliferation rate of non-RLCs. RLCs expanded mainly by de novo differentiation (neogenesis). Fractions of RLCs originating from the stromal progenitors of the metanephric mesenchyme (renin-producing cells, vascular smooth muscle cells, and mesangial cells) decreased during nephrogenesis. In contrast, aquaporin-2-positive RLCs in the collecting duct system, which embryonically emerges almost exclusively from the ureteric bud, expanded postpartum. The cubilin-positive RLC fraction in the proximal tubule, deriving from the cap mesenchyme, remained constant. In summary, RLCs were continuously detectable in the vascular and tubular compartments of the kidney during nephrogenesis. Therein, various patterns of RLC differentiation that depend on the embryonic origin of the cells were identified.NEW & NOTEWORTHY The unifying feature of the renal renin lineage cells (RLCs) is their origin from renin-expressing progenitors. RLCs evolve to an embryologically heterogeneous large population in structures with different ancestry. RLCs are also targets for the widely used renin-angiotensin-system blockers, which modulate their phenotype. Unveiling the different differentiation patterns of RLCs in the developing kidney contributes to understanding changes in their cell fate in response to homeostatic challenges and the use of antihypertensive drugs.

Keywords: differentiation; immunofluorescence; nephrogenesis; renin lineage cells.

Graphical abstract

Figure 1.

The renin lineage cell population. We quantified green fluorescent protein and red fluorescent protein positivity in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. A: quantification of renin lineage cells in a kidney section showed expansion of the renin lineage cell (RLC) population throughout nephrogenesis. B: quantification of de novo differentiated RLCs in a kidney section showed a peak of de novo differentiation per section at postpartum days 6–10. C: the fraction of de novo differentiated RLCs in RLCs declined throughout nephrogenesis. Data are shown as scatter dot plots with means and SD; n = 3–7. e16, embryonic day 16; e17–19, embryonic days 17–19; pp1–5, postpartum days 1–5, pp6–10, postpartum days 6–10.

Figure 2.

Quantification of proliferation and de novo differentiation. We quantified green fluorescent protein and red fluorescent protein positivity in combination with staining for proliferating cell nuclear antigen (PCNA) as a marker of proliferation in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. A: fractions of PCNA-positive cells in renin lineage cells (RLCs) were considered negligible except for embryonic days 17–19 (e17–19) and postpartum days 1–5 (pp1–5). B: fractions of PCNA-positive cells in the section were considered negligible except for e17–19 and pp1–5. The proliferation rate in RLCs and the section was comparable (A and B). C and D: fractions of RLCs in PCNA-positive cells were comparable (C) to fractions of de novo differentiated RLCs in PCNA-positive cells (D) for embryonic day 16 (e16), e17–19, and pp1–5. Data are shown as scatter dot plots with means and SD; n = 3–7. pp6–10, postpartum days 6–10.

Figure 3.

Quantification of renin lineage vascular cells: renin-producing cells, vascular smooth muscle cells, and mesangial cells. We quantified renin, α-smooth muscle actin (aSMA), and α₈-integrin (a8integrin) positivity in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. A: the fraction of renin-positive cells in renin lineage cells (RLCs) decreased throughout nephrogenesis. B: the fraction of aSMA-positive cells in RLCs decreased throughout nephrogenesis. C: the fraction of a8integrin-positive cells in RLCs decreased after postpartum days 1–5 (pp1–5). Data are shown as scatter dot plots with means and SD; n = 3–7. e16, embryonic day 16; e17–19, embryonic days 17–19; pp6–10, postpartum days 6–10.

Figure 4.

Quantification of renin lineage cells (RLCs) in stromal mesenchyme descendants. A: fractions of cells that were assigned to a certain cell type by marker positivity [renin, α-smooth muscle actin (aSMA), and α₈-integrin (a8integrin)] in RLCs decreased throughout nephrogenesis. B: normalized to stromal mesenchymal descendants, their fractions remained stable. Data are shown as stacked bar plots showing mean fractions; n = 3–7. e16, embryonic day 16; e17–19, embryonic days 17–19; pp1–5, postpartum days 1–5; pp6–10, postpartum days 6–10.

Figure 5.

Quantification of renin lineage cells (RLCs) of the proximal tubule. We quantified cubilin positivity in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. The fraction of cubilin-positive cells in RLCs remained stable throughout nephrogenesis. Data are shown as scatter dot plots with means and SD; n = 3–7. e16, embryonic day 16; e17–19, embryonic days 17–19; pp1–5, postpartum days 1–5; pp6–10, postpartum days 6–10.

Figure 6.

Quantification of renin lineage cells (RLCs) of the collecting duct. We quantified aquaporin-2 (AQP2) positivity in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. The fraction of AQP2-positive cells in RLCs increased throughout nephrogenesis. Data are shown as scatter dot plots with means and SD; n = 3–7. e16, embryonic day 16; e17–19, embryonic days 17–19; pp1–5, postpartum days 1–5; pp6–10, postpartum days 6–10.

Figure 7.

Summary of all stained markers in a section. We quantified renin, α-smooth muscle actin (aSMA), α₈-integrin (a8Integrin), aquaporin 2 (AQP2), and cubilin positivity in whole kidney sections obtained from mRenCre-mT/mG mice at different times of nephrogenesis. A: we could assign roughly 40% of renin lineage cells (RLCs) to a certain cell type by marker positivity. Data are shown as stacked bar plots showing mean fractions; n = 3–7. B: we aligned images of five distinct sections at postpartum day 5. Each section was stained for green fluorescent protein (label for RLCs, displayed in green) and one of the markers [α₈-integrin (a8integrin), renin, α-smooth muscle actin (aSMA), aquaporin-2 (AQP2), and cubilin, displayed in magenta]. Red arrows indicate AQP2-negative RLCs in the collecting duct. Scale bar = 1,000 µm. The image detail (width equals 1,000 µm) shows a magnification of AQP2-negative RLCs in the collecting duct system. e16, embryonic day 16; e17–19, embryonic days 17–19; pp1–5, postpartum days 1–5; pp6–10, postpartum days 6–10.