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Acute kidney injury: what role for Kim1+ tubular epithelial cells?

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

1. Lazzeri E, Angelotti ML, Conte C, Anders HJ, Romagnani P. Surviving Acute Organ Failure: Cell Polyploidization and Progenitor Proliferation.Trends Mol Med. 2019 May;25(5):366-381.

2. Lazzeri E, Angelotti ML, Peired A, Conte C, Marschner JA, Maggi L, Mazzinghi B, Lombardi D, Melica ME, Nardi S, Ronconi E, Sisti A, Antonelli G, Becherucci F, De Chiara L, Guevara RR, Burger A, Schaefer B, Annunziato F, Anders HJ, Lasagni L, Romagnani P. Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury. Nat Commun. 2018 Apr 9;9(1):1344.

3. Manolopoulou M, Matlock BK, Nlandu-Khodo S, Simmons AJ, Lau KS, Phillips-Mignemi M, Ivanova A, Alford CE, Flaherty DK, Gewin LS. Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules. Am J Physiol Renal Physiol. 2019 May 1;316(5):F847-F855.

4. Kang HM, Huang S, Reidy K, Han SH, Chinga F, Susztak K. Sox9-positive progenitor cells play a key role in renal tubule epithelial regeneration in mice. Cell Rep. 2016;14(4):861–871.

5. Rinkevich Y, et al. In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration. Cell Rep. 2014;7(4):1270–1283.

6. Chang-Panesso M, Kadyrov FF, Lalli M, Wu H, Ikeda S, Kefaloyianni E, Abdelmageed MM, Herrlich A, Kobayashi A, Humphreys BD. FOXM1 drives proximal tubule proliferation during repair from acute ischemic kidney injury. J Clin Invest. 2019 Nov 11. pii: 125519.

7. Menke J1, Iwata Y, Rabacal WA, Basu R, Yeung YG, Humphreys BD, Wada T, Schwarting A, Stanley ER, Kelley VR. CSF-1 signals directly to renal tubular epithelial cells to mediate repair in mice. J Clin Invest. 2009 Aug;119(8):2330-42. 

8. Yin C, Wang N. Kidney injury molecule-1 in kidney disease. Ren Fail. 2016 Nov;38(10):1567-1573. 

9. Reinert RB, Kantz J, Misfeldt AA, Poffenberger G, Gannon M, Brissova M, Powers AC. Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice. PLoS One. 2012;7(3):e33529.

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Response to Romagnani et al.

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-CreER^t2 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-CreER^t2 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.