The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.
Monica Chang-Panesso, Farid F. Kadyrov, Matthew Lalli, Haojia Wu, Shiyo Ikeda, Eirini Kefaloyianni, Mai M. Abdelmageed, Andreas Herrlich, Akio Kobayashi, Benjamin D. Humphreys
Submitter: Paola Romagnani | paola.romagnani@unifi.it
Authors: Paola Romagnani, Elena Lazzeri, Maria Lucia Angelotti, Anna Peired, Letizia De Chiara, Benedetta Mazzinghi, Laura Lasagni
University of Florence, Florence, Italy
Published December 11, 2019
The kidney was long considered a highly regenerative organ, where stochastic proliferation of remnant tubular epithelial cells (TECs) drives regeneration upon acute kidney injury (AKI). However, this concept was evidenced using cell cycle markers unable to prove true cell divisions (1).In contrast, we and others recently demonstrated that after AKI, similar to heart and liver repair (1), cell cycle marker positivity also relates to mononuclear polyploid cells generated via endoreplication in mice (2,3) and humans (2), while proliferation is limited to a subset of scattered TECs with progenitor features (2,4,5).
Chang-Panesso, et al. developed the Kim1-GFPCreERt2 mouse line to reconfirm the traditional concept of widespread stochastic proliferation of “dedifferentiated” TECs (6). Kim1 is absent in healthy kidneys and upregulated in injured TECs (6). Based on this observation, the authors induced transgene activation by administering tamoxifen after AKI and observed an increase in Kim1+TECs at day 14 that they interpreted as “clonal expansion of dedifferentiated TECs”. We question this interpretation for several reasons: (i). strong Kim1 upregulation persists for days after AKI (2,7,8), and tamoxifen activity, even at low doses, lasts >1 week (9). Therefore, Cre recombination continues and can label any new Kim1-expressing cell beyond day 2, in particular adjacent TECs of injured S3 segments that intensely express Kim1, making the conclusion of a clonal expansion of Kim1+TECs inaccurate (Figure 2B-C); (ii). the RNA sequencing and FACS analysis document the absence of “cycling” Kim1+TECs after day 2 (Figure S3D and Figure 3D), while Figure 1C clearly shows that the number of Kim1+TECs increases after day 3, indicating de novo Kim1 expression in previously Kim1 negative TECs, not clonal expansion of Kim1+TECs.
Furthermore, the authors showed absence of a double (4n) DNA content in Kim1+TECs, excluding both endoreplication and proliferation at day 14 and 30 (Figure 3D). However, Kim1+TECs selectively localize in S3-segments (6), while endoreplication occurs in non-injured TECs of S2-segments (2). As their data are limited to Kim1+TECs, the authors’ conclusions on the absence of endoreplication in proximal tubule is invalid.
In conclusion, the authors cannot corroborate the concept of stochastic TEC proliferation nor invalidate our discoverythat kidney repair after AKI involves proliferation of tubular progenitors in the injured segment and hypertrophy of the remnant differentiated tubular cells in the other segments. Rather, based on a correct interpretation of data, the authors demonstrate that injured and “dedifferentiated” Kim1+TECs do not proliferate nor endoreplicate.
References
Submitter: Benjamin Humphreys | humphreysbd@wustl.edu
Authors: Monica Chang-Panesso and Benjamin D. Humphreys
Washington University School of Medicine
Published December 11, 2019
Clonal analysis allows unambiguous tracing of the proliferative history of a single labeled cell (1). A “coherent clone” is a group of cells, descended from a common progenitor through cell division that subsequently remain adjacent to each other (2).
Residual tamoxifen could cause misclassification of de novo recombination as cell division if the recombination occurred adjacent to a previously labeled cell. Reinert and colleagues demonstrated that 3 mg tamoxifen results in a very low level of de novo recombination at 48 hours - 4.9% (3). We used one third of this dose – 1 mg tamoxifen (4). Furthermore, the Kim-1-CreERt2 line is inefficient, with a 30 mg dose labeling 4.1% of all Kim-1+ cells (Fig. 1F). We can use this frequency to calculate the probability that residual tamoxifen explains our right shifted clone size-frequency analysis (Fig. 2C). With a 30 mg tamoxifen dose, any Kim-1+ cell has a 4.1% chance of undergoing recombination. To generate an artifactual coherent two cell clone from recombination (rather than mitosis), either of the two cells adjacent to a previously labeled cell would need to undergo recombination. There is a 4.1 x 2 = 8.2% chance of that happening. The probability of a three cell clone arising by this mechanism is 8.2%^2, or 0.7%. And the probability of a four cell clone arising is 8.2%^3, or 0.06%. These low probabilities (which are gross overestimates since we actually used 1 mg tamoxifen, not 30 mg) are not consistent with our patch size-frequency results (Fig. 2C).
We are perplexed by the suggestion that our data indicates that there is no proliferation after day 2, since the next timepoint we tested was day 7.
The author suggests that endoreplication is limited to the S1 and S2 segments (although the author has previously published that 7.8 + 2.2% of epithelial cells in the outer stripe of the outer medulla undergo endoreplication (5)). Our prior lineage analysis of the S1 and S2 segments during repair (using the Slc34a1-CreERt2 driver), revealed clonal expansion of S1 and S2 segment epithelia (6). This is not consistent with endoreplication in those segments.
Our results demonstrate that injured and dedifferentiated Kim-1+ cells undergo proliferative expansion during repair, consistent with prior work (6-8). We find no evidence to support a role for endoreplication in this process.
References
1. Humphreys BD, and DiRocco DP. Lineage-tracing methods and the kidney. Kidney Int. 2014;86(3):481-8.
2. Buckingham ME, and Meilhac SM. Tracing cells for tracking cell lineage and clonal behavior. Dev Cell. 2011;21(3):394-409.
3. Reinert RB, Kantz J, Misfeldt AA, Poffenberger G, Gannon M, Brissova M, and Powers AC. Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice. PLoS One. 2012;7(3):e33529.
4. Chang-Panesso M, Kadyrov FF, Lalli M, Wu H, Ikeda S, Kefaloyianni E, Abdelmageed MM, Herrlich A, Kobayashi A, and Humphreys BD. FOXM1 drives proximal tubule proliferation during repair from acute ischemic kidney injury. J Clin Invest. 2019.
5. Lazzeri E, Angelotti ML, Peired A, Conte C, Marschner JA, Maggi L, Mazzinghi B, Lombardi D, Melica ME, Nardi S, et al. Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury. Nature communications. 2018;9(1):1344.
6. Kusaba T, Lalli M, Kramann R, Kobayashi A, and Humphreys BD. Differentiated kidney epithelial cells repair injured proximal tubule. Proc Natl Acad Sci U S A. 2014;111(4):1527-32.
7. Humphreys BD, Czerniak S, Dirocco DP, Hasnain W, Cheema R, and Bonventre JV. Repair of injured proximal tubule does not involve specialized progenitors. Proc Natl Acad Sci U S A. 2011;108(22):9226-31.
8. Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, Duffield JS, McMahon AP, and Bonventre JV. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2008;2(3):284-91.