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Loss of kidney function underlies many renal diseases1. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones2,3. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury4,5, 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.
Zebrafish nephrons in the adult kidney are similar to those found in the embryonic kidney6, except that they are highly branched and drained by two central collecting ducts (Fig. 1a and Supplementary Fig. 2a–j). We confirmed that zebrafish nephron number increases with age (Fig. 1b), similar to other fish4,5. To identify the source of new nephrons in adult zebrafish, we first characterized the effects of gentamicin injection, an established nephrotoxin7. Intraperitoneal injection of gentamicin induced nephron damage, downregulated the proximal tubule marker slc20a1a and resulted in a failure to take up filtered 40 kDa fluorescent dextran8 by 1 day post-injection (Fig. 1c–f, n = 6/6; Fig. 1j–k, n = 8/8; Supplementary Fig. 2k–p). Around 4 days post-injection, partial restoration in nephron function was observed, suggesting some nephrons recovered from the injury (Fig. 1g, l, arrow). At this stage we also detected small, but appropriately proportioned, nephrons that were dextran-positive, proliferating and basophilic, which are characteristic features of immature nephrons7 (approximately 15 per kidney; Fig. 1i, l inset, n). By 15 days post-injection the damaged nephrons had recovered to near-normal levels, although immature nephrons could still be detected (Fig. 1h, m, arrow).
If the adult kidney contains nephron progenitors responsible for the formation of new nephrons, then these cells might be amenable to transplantation. To test this, we developed a transplantation assay (Fig. 2a and Supplementary Fig. 3a–k) in which recipient fish were immunocompromised by radiation to prevent graft rejection9 and then injected with gentamicin. Unpurified whole-kidney marrow cells (WKM), mostly comprising non-tubular interstitial cells9, were prepared from Tg(cdh17:EGFP)10 or Tg(cdh17:mCherry) donors that express fluorescent reporters in the distal nephron. Injection of approximately 5 × 105 of these cells resulted in donor-derived nephrons in 100% of the recipients (n = 6) by 18 days post-transplantation (d.p.t.), with an average of 24 donor-derived nephrons (Fig. 2b, arrow, inset). Donor nephron number increased with time, reaching an average of 70 nephrons by 59 d.p.t. (Fig. 2c) and greatly expanded the head kidney on the injected side (Fig. 2d, arrow). At these later time points, we also found donor-derived nephrons in locations distant from the site of injection, which suggests that the transplanted cells are migratory (Supplementary Fig. 3l, arrowheads).
To regenerate damaged tissue successfully, newly created structures must incorporate into existing tissue. To determine whether the donor-derived nephrons were capable of blood filtration, we injected 40 kDa fluorescent dextran into transplant recipients that had received WKM from Tg(cdh17:mCherry) donor fish and dissected out individual nephrons. All of the donor-derived nephrons examined (n = 5) were dextran-positive (Fig. 2e), indicating that they had integrated into the recipient’s blood supply. These results show that nephron progenitors are present in the adult kidney and that after transplantation they are capable of forming new functional nephrons within the host’s renal tissue.
Cell transplantation experiments can be confounded by the fusion of donor and recipient cells. To address this, we injected WKM from Tg(cdh17:mCherry) donors into Tg(cdh17:EGFP) recipients. If fusion occurred, we would expect to find nephrons positive for both mCherry and enhanced green fluorescent protein (EGFP). An analysis of engrafted recipients (n = 6) revealed that all of the mCherry-positive nephrons were EGFP-negative, providing evidence that they had not formed by cell–cell fusion. In addition, we identified the connection of the donor-derived nephrons with the host’s renal tubules, providing further evidence that the engrafted nephrons had successfully integrated into the recipient’s renal system (Fig. 2f).
Lineage labelling studies in the developing mouse kidney have revealed that multiple Six2+ cap mesenchyme cells, the source of nephron progenitors, contribute to a single nephron11. To explore this in zebrafish, we transplanted a 1:1 mix of Tg(cdh17:EGFP) and Tg(cdh17:mCherry) WKM cells into conditioned recipients. Mosaic nephrons containing both EGFP-positive and mCherry-positive cells were found in 27% of the engrafted fish (n = 15; Fig. 2g), although the remaining nephrons were either all EGFP-positive or all mCherry-positive. Thus multiple nephron progenitors can contribute to an individual nephron.
Mammalian Six2+ cap mesenchyme cells are also characterized by their stem-cell-like self-renewal properties11. Serial transplantation is used to distinguish haematopoietic stem cells from progenitors12. We investigated whether we could obtain donor-derived nephrons after serial transplantation of WKM from engrafted recipients (Fig. 2h). We transplanted WKM from primary fish containing 2–89 cdh17:EGFP+ donor-derived nephrons and achieved a 48% (n = 21) engraftment rate in secondary fish, with the number of donor-derived nephrons ranging from 1 to 53 by 41 d.p.t. The WKM from one of these secondary fish, containing 53 engrafted nephrons, was transplanted again and successfully engrafted a third time, giving rise to 12 donor-derived nephrons in the tertiary recipient at 35 d.p.t. (a total of 135 days from primary to tertiary fish; Fig. 2i–k). These results demonstrate that nephron progenitors possess significant proliferative potential, consistent with self-renewing capabilities.
We next sought to identify the cells responsible for nephron progenitor activity. We noted that approximately 0.1% of the WKM from Tg(cdh17:EGFP) fish is EGFP-positive (Supplementary Fig. 4a). To test whether cdh17:EGFP+ cells could contribute to new nephrons, we sorted and transplanted this fraction (approximately 5,000 cells per fish) but failed to observe engraftment (n = 0/7). We subsequently explored other markers of nephron progenitors. In mammals, nephrogenesis initiates with the formation of ‘pre-tubular aggregates’ that undergo a mesenchymal-to-epithelial transition into renal vesicles13. These structures express several transcription factors including Lhx1/Lim1 (ref. 14) and Wt1 (ref. 15). We therefore examined the Tg(lhx1a:EGFP)16 and Tg(wt1b:mCherry) transgenic lines to determine whether these reporters mark nephron progenitors. Kidneys from untreated Tg(lhx1a:EGFP) adults were found to contain three distinctive EGFP-positive cell populations: (1) single cells with a mesenchymal morphology (Fig. 3a) that make up approximately 0.02% of the WKM (Supplementary Fig. 4b), (2) homogeneous aggregates of lhx1a:EGFP+ mesenchymal cells ranging from a few to approximately 30 cells (Fig. 3b, c and Supplementary Fig. 4i) (approximately 100 aggregates per kidney) and (3) renal vesicle-like bodies (0–2 per kidney; Fig. 3d). The last two populations were highly reminiscent of pre-tubular aggregates and renal vesicles in mammals.
An examination of Tg(lhx1a:EGFP;wt1b:mCherry) double transgenic kidneys revealed that only the large aggregates and renal vesicles, but not the other lhx1a:EGFP+ populations, were wt1b:mCherry+ (Fig. 3d). We hypothesized that the lhx1a:EGFP+/wt1b:mCherry+ renal vesicle-like bodies, which were rare in untreated kidneys, constitute primitive nephrons. Consistent with this, gentamicin treatment greatly induced the formation of lhx1a:EGFP+/wt1b:mCherry+ double-positive cells (data not shown) and activated the endogenous expression of the early-acting renal genes pax2a, fgf8a, wt1a and wt1b in similar structures (Fig. 3e–j and Supplementary Fig. 4c–h). We failed to detect the expression of mature nephron markers in structures resembling either lhx1a:EGFP+ aggregates or wt1b+ renal vesicles (Supplementary Fig. 5a–c and Supplementary Table 1). Similarly, quantitative PCR analysis of purified lhx1a:EGFP+ and cdh17:EGFP+ cells showed that lhx1a:EGFP+ cells express considerably lower levels of mature nephron markers than cdh17:EGFP+ cells (Supplementary Fig. 5d). These findings suggest that lhx1a:EGFP labels nephron progenitors and lhx1a:EGFP/wt1b:mCherry labels early-stage nephrons.
To clarify the lineage relationships between lhx1a:EGFP+ and lhx1a:EGFP+/wt1b:mCherry+ cells, we took advantage of the optical transparency of larval fish to visualize nephrogenesis in vivo. By observing Tg(cdh17:EGFP) larvae as well as using wholemount in situ hybridization, we found that adult kidney formation initiates at the 5.2-mm stage (approximately 13 days post-fertilization). The first nephron appears consistently on the embryonic (pronephric) renal tubules just posterior to the swim bladder (Supplementary Fig. 6a–e and data not shown). lhx1a:EGFP+ cells appeared before this, at the 4-mm stage (approximately 10 days post-fertilization) (Fig. 4a, arrow, inset), rapidly migrated along the pronephric tubules (Fig. 4b, arrows), and formed into aggregates (Fig. 4b, arrowheads). An in vivo time course of Tg(lhx1a:EGFP;cdh17:mCherry) and Tg(lhx1a:EGFP;wt1b:mCherry) larvae showed that the lhx1a:EGFP+ aggregates arose from the coalescence of three or four lhx1a:EGFP+ cells that expanded to form a renal vesicle and activated expression of wt1b:mCherry (Fig. 4d and Supplementary Fig. 6f). Similar time courses of Tg(lhx1a:EGFP;cdh17:mCherry) and Tg(wt1b:EGFP;pax8:DsRed) larvae demonstrated that the renal vesicle elongated into a cdh17+ nephron, with lhx1a+ cells becoming restricted to the point of fusion with the pronephric tubules, pax8 initiating in the distal tubule and wt1b labelling the glomerulus and proximal tubule (Fig. 4e and Supplementary Fig. 6g). To demonstrate a requirement of lhx1a:EGFP+ cells for nephrogenesis, we ablated single lhx1a:EGFP+ aggregates with a laser (Fig. 4c, arrow), resulting in aborted nephrogenesis in the targeted region without affecting neighbouring nephrons (Fig. 4c, arrowhead) (n = 2/2).
Next we tested whether lhx1a:EGFP+ cells had nephron-forming activity. Transplantation of single lhx1a:EGFP+ cells failed to engraft conditioned recipients. However, transplantation of individual lhx1a:EGFP+ aggregates resulted in successful engraftment in 33% (n = 15) of transplanted fish (Fig. 4f, g). In one case, a single aggregate contributed to 16 nephrons, 27 aggregates and numerous individual cells (Supplementary Fig. 7a–c), consistent with lhx1a:EGFP+ cells having extensive proliferative and self-renewing capabilities. Transplantation of lhx1a:EGFP+/wt1b:mCherry+ renal vesicles failed to engraft (n = 0/10), suggesting that nephron-forming potential is restricted to lhx1a:EGFP+ aggregates. These findings demonstrate that lhx1a:EGFP+ aggregates contain nephron progenitors and support our observation that multiple nephron progenitors are needed to form a nephron.
To determine how similar lhx1a:EGFP+ cells are to Six2+ mouse cap mesenchyme cells, we conducted a microarray analysis, comparing the genes upregulated in lhx1a:EGFP+ cells (relative to cdh17:EGFP+ epithelial cells) with those upregulated in mouse Six2+ cells (relative to mouse proximal tubule epithelial cells). At a global level, the respective gene sets that are upregulated in lhx1a:EGFP+ cells and Six2+ cells are not significantly similar (Supplementary Tables 2–4 and Supplementary Fig. 8a). However, there is conservation of several factors implicated in renal development and/or stem-cell self-renewal. Notably, orthologues of Six2 (six2a) and Wt1 (wt1a), which are essential for cap mesenchyme maintenance, are upregulated both in lhx1a:EGFP+ cells and in Six2+ cells. Quantitative PCR confirmed that six2a and wt1a are expressed over 15-fold higher in lhx1a:EGFP+ cells than cdh17:EGFP+ cells (Supplementary Fig. 8b). Several other potentially important regulators were also identified in the comparison, including Meis2, Ezh2 and Tcf3, which are implicated in Wnt signalling and/or stem-cell function (Supplementary Table 4). These results suggest that, despite having distinct molecular identities, zebrafish lhx1a:EGFP+ cells and Six2+ cells share a core set of regulatory genes that may be important for conferring renal stem/progenitor cell potential.
In conclusion, we have identified an adult population of nephron progenitors that reside in small aggregates throughout the zebrafish kidney. These cells are uniquely defined by their ability to form new functional nephrons during zebrafish growth, injury and after transplantation. Nephron progenitors can be serially transplanted, consistent with stem-cell capabilities, although confirmation of this awaits direct lineage-tracing experiments. Our in vivo imaging of nephrogenesis and chimaeric transplantation results demonstrated that nephrogenic aggregates form by the coalescence of multiple lhx1a:EGFP+ cells (Supplementary Fig. 1). This process is reminiscent of nephrogenesis in mammals and suggests that similar mechanisms govern nephron formation in both species. Consistent with this, lhx1a:EGFP+ cells express six2a and wt1a, two critical regulators of mammalian nephron progenitors. Our observation that only aggregates of lhx1a:EGFP+ cells, but not single cells, are capable of engraftment suggests that nephron progenitor potential may depend upon a ‘community effect’17, a phenomenon whereby continued cell contact is necessary for cells to respond to an inductive signal. The failure of renal vesicles to engraft suggests that nephron-forming potential is lost upon epithelial differentiation.
With our data in hand, it is now possible to pursue whether the mammalian adult kidney contains an equivalent population of nephrogenic aggregates. If present, these cells are most probably dormant or their regenerative abilities blocked, given that nephrogenesis ceases around birth. Using zebrafish to understand the molecular identity of nephron progenitors and the pathways that regulate them may lead to therapeutic ways to activate, or artificially engineer, the mammalian counterpart and augment human renal regeneration. With the rise in chronic kidney disease becoming a serious worldwide healthcare issue, a nephron-progenitor-based regenerative therapy will have a major clinical impact.
For WKM transplants, adult recipient fish were conditioned with intraperitoneal injection of gentamicin (80 μg g−1), then immunocompromised with sub-lethal γ-irradiation (25 Gy) to prevent graft rejection9. Unpurified WKM cells were prepared as previously described9 from Tg(cdh17:EGFP)10 or Tg(cdh17:mCherry) donors that express fluorescent reporter proteins in the distal nephron. For the lhx1a:EGFP+ single cell and aggregate transplants, dissected kidneys from Tg(lhx1a:EGFP;cdh17:mCherry) fish were treated with 10% collagenase/dispase for 15 min and cells/aggregates manually transferred with a mouth pipette to a drop of 1X PBS/2% fetal calf serum on a glass slide. A single cell or aggregate was serially passaged through three droplets of PBS/fetal calf serum until free of non-positive cells just before transplantation.
We thank E. C. Liao for help with suturing, and R. Ethier and L. Gyr for zebrafish care. A.J.D. was supported by the Harvard Stem Cell Institute, the American Society of Nephrology and the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (P50DK074030).
Author Contributions C.Q.D. and A.J.D. designed the experimental strategy, analysed data, prepared the manuscript, and generated and characterized the Tg(cdh17:EGFP), Tg(cdh17:mCherry) and Tg(wt1b:mCherry) lines. C.Q.D. performed the regeneration, transplants, time course and ablation experiments. C.Q.D., D.M. and R.I.H. made the initial observation that nephron progenitors can be transplanted. N.A.H. generated the Tg(lhx1a:EGFP) line (R01DK069403), F.B. and C.E. generated the Tg(wt1b:EGFP) line, and T.I. and F.O. provided the Tg(pax8:DsRed) line. N.A., R.A.W., G.D. and B.L. analysed kidney expression. H.Z. provided sections of regenerating kidneys. R.C.D., T.M.H., R.W.N., and C.A.C. performed quantitative PCR and microarray analyses. All authors commented on the manuscript.
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