Identification of adult nephron progenitors capable of kidney regeneration in zebrafish

Abstract

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

Figure 1. The adult zebrafish kidney undergoes nephrogenesis throughout life and after injury

a, Zebrafish kidney and nephron model (scale bar, 1 mm). b, Graph showing average number of renal corpuscles (RC) relative to body length (inset shows an RC labelled with dn-fgfr1-EGFP) (error bar, one standard deviation; n = 3 fish per time point). RC, renal corpuscle; N, neck; PI, proximal tubule I; PII, proximal tubule II; DE, distal early; DL, distal late. c, d, Kidney sections showing gentamicin-damaged nephrons (asterisks; scale bar, 10 μm). H&E, haematoxylin and eosin; d.p.i., days post-injection; DT, distal tubule. e–h, Expression of slc20a1a in gentamicin-damaged kidneys (scale bar, 0.5 mm). i, Immature nephron with dividing cells (arrow) (scale bar, 10 μm). j–m, Uptake of 40 kDa fluorescein isothiocyanate (FITC)-conjugated dextran by gentamicin-damaged kidneys (inset in l shows an immature nephron; arrow in l marks a positive nephron; arrow in m indicates an immature nephron; scale bar, 30 μm). n, Serial sections showing that immature basophilic nephrons take up 40 kDa dextran-rhodamine. M&B, methylene blue and basic fuchsin.

Figure 2. The adult zebrafish kidney contains transplantable progenitors that form functional nephrons

a, Overview of the transplantation assay. b, A primary transplanted fish at 18 d.p.t. with cdh17:EGFP⁺ donor-derived nephrons (arrow; inset, higher magnification view; scale bar, 0.5 mm). c, Average number of donor-derived nephrons over time (error bar, one standard deviation; n, total fish per time point). d, Head kidney of a recipient at 34 d.p.t. showing expansion of renal tissue caused by cdh17:mCherry⁺ donor-derived nephrons (arrow; scale bar, 0.5 mm). e, A cdh17:mCherry⁺ donor-derived nephron showing functional uptake of 40 kDa FITC-conjugated dextran (scale bar, 30 μm). f, Connection of donor-derived nephrons (cdh17:mCherry⁺) with the cdh17:EGFP⁺ recipient’s renal system (scale bar, 10 μm). g, A mosaic nephron arising from the co-injection of a mixture of cdh17:EGFP- and cdh17:mCherry-labelled nephron progenitors. h, Overview of the serial transplantation assay. i–k, Donor-derived nephrons (cdh17:EGFP⁺, arrows) in primary-, secondary- and tertiary-engrafted recipients (scale bar, 0.5 mm).

Figure 3. Expression of lhx1a:EGFP and other renal factors in the adult kidney

a, Single mesenchymal cells labelled with lhx1a:EGFP (scale bar, 10 μm). b, Small aggregates labelled with lhx1a:EGFP (scale bar, 10 μm). c, An untreated kidney showing lhx1a:EGFP⁺ aggregates in a portion of the trunk region; scale bar, 0.5 mm). d, Co-expression of wt1b:mCherry and lhx1a:EGFP in large aggregates (asterisk, autofluorescence; scale bar, 10 μm). e, f, Expression of wt1b in untreated and gentamicin-damaged kidneys (scale bar, 0.5 mm). g–i, Expression of wt1b in a large aggregate (g), a comma-shaped body (h) and an immature nephron (i) in gentamicin-damaged kidneys (scale bar, 10 μm). j, Expression of pax2a, wt1a and fgf8a in large aggregates or renal vesicles in gentamicin-damaged kidneys (scale bar, 10 μm).

Figure 4. lhx1a:EGFP⁺ cells form nephrons during adult kidney development and after transplantation

a, Lateral view of a Tg(lhx1a:EGFP;cdh17:mCherry) larva showing the first lhx1a:EGFP⁺ cell to appear on top of the cdh17:mCherry⁺ embryonic kidney tubules (arrow and inset). b, Lateral view of a Tg(lhx1a:EGFP) larva showing the extent of lhx1a:EGFP⁺ cell migration (arrows) and their aggregation (arrowheads). c, Laser-ablation of an lhx1a:EGFP⁺ aggregate (arrow) inhibits nephron formation without affecting nephrogenesis of an adjacent aggregate (arrowhead). d, Time course of a Tg(lhx1a:EGFP;cdh17:mCherry) larva demonstrating that lhx1a:EGFP⁺ cells coalesce into an aggregate and differentiate into a nephron. e, Time course of a Tg(wt1b:EGFP;pax8:DsRed) larva showing development of a wt1b:EGFP⁺ aggregate into a nephron. f, Bright field and fluorescent merge of an aggregate from a Tg(lhx1a:EGFP;cdh17:mCherry) donor. g, Donor-derived cdh17:mCherry⁺ nephrons (one indicated by arrow) and multiple single lhx1a:EGFP⁺ cells (arrowhead) resulting from the transplantation of the aggregate shown in f (scale bar, 30 μm). PT, pronephric tubule; sb, swim bladder; larvae shown with anterior to the left.