Generation of new cells during

Generation of new cells during kidney repair has long been assumed to result from proliferation of surviving terminally differentiated cells. However, much of the modern evidence has used lineage tracing such as Six2 (Humphreys et al., 2008) or a terminally differentiated marker (such as the proximal tubule phosphate transporter) (Kusaba et al., 2014). Such studies cannot eliminate the possibility that nephron segments might contain progenitor cells that express these markers or are derived from this lineage. Indeed, we found that some Six2-derived cells in the papilla expressed protrudin, as were some pLRCs, and together with Zfyve27-marked cells, protrudin-expressing cells (as well as pLRCs) were found in all three currently known embryonic cell lineages present in papilla: interstitial as well as the two epithelial cells (Six2-derived and collecting ducts). Thus, each different pool of pLRCs (or Zfyve27-marked) cells might generate progeny restricted to their parent compartment; therefore the new tubules containing tdTomato cells found after kidney repair most likely derive from the epithelial protrudin-expressing cells. This finding is compatible with results showing that during kidney repair Six2-marked cells showed no dilution of the marker after kidney regeneration (Humphreys et al., 2008). Moreover, recent studies by Rinkevich et al. (2014) using the Nutlin3a promoter to generate random single-cell clones in the adult kidney showed that some marked cells with high proliferation capacity expanded and generated tracing events restricted to specific nephron segments, indicating that the adult kidney likely contains precursor cells that are segment specific. They also found that cells with high proliferation capacity were Wnt responsive, raising the question of whether the Zfyve27-CreERT2-labeled cells (likewise with high proliferative capacity after KI) might be the same population as the Wnt-responsive cells identified by Rinkevich et al. (2014), a line of inquiry that we plan to pursue in the future. The full power of the Zfyve27-CreERT2-marked cells to regenerate tubules was observed only after severe KI, since milder injury known to induce apoptosis did not induce the cells to proliferate and there was only modest proliferation by the cells after 30 min of kidney ischemia, a potent stimulus for widespread cell cycling in the kidney (Forbes et al., 2000; Oliver et al., 2009). It is thus possible that recovery from modest injury depends on proliferation of another pool of progenitor cells that belongs to a different lineage. These might include the aforementioned segment-specific or other types of progenitors or even terminally differentiated cells, as occurs in the liver (Yanger et al., 2014). Severe injury might deplete these latter pools of progenitors, as was found elsewhere (Tian et al., 2011; Vaughan et al., 2015). The medullary restriction of the Zfyve27-CreERT2-labeled cells emphasizes that the kidney, like other organs (Page et al., 2013; Donati and Watt, 2015), appears to have several precursor cell populations to generate new cells. For instance, stem cells in different segments of an organ, such as the gut, possess cell-autonomous region specificity (Wang et al., 2015) and, while the vertebrate kidney is seen as an organ, it is made up of semi-autonomous units called nephrons (containing different cellular regions), which in more ancient animals are fully individual organs (Hazelton et al., 1988; Scimone et al., 2011). Characterization of the Zfyve27-CreERT2-marked cells in the kidney remains to be completed, but work in other organs suggests several possibilities. They could be bona fide precursor/stem cells that in addition to multilineage potential are able to self-renew and are solely activated during severe organ injury, but to prove multipotency and self-renewal will require analysis of individual cells inside their niche in vivo. Another possibility could be that if the kidney papilla is, as we previously suggested (Oliver et al., 2004), a precursor/stem cell niche, severe injury might have ablated resident stem cells and signals from the niche would then induce Zfyve27-CreERT2-marked (and other) cells to repopulate the niche and, by changing cell identity, become facultative stem cells, as found in regeneration of several organs (Tian et al., 2011; van Es et al., 2012).