NDT Advance Access published August 25, 2014 Nephrol Dial Transplant (2014) 0: 1–8 doi: 10.1093/ndt/gfu281
Full Review Who regenerates the kidney tubule? Rafael Kramann1,2,3, Tetsuro Kusaba1,2 and Benjamin D. Humphreys1,2,4 1
Brigham and Women’s Hospital, Boston, MA, USA, 2Harvard Medical School, Boston, MA, USA, 3Division of Nephrology,
Correspondence and offprint requests to: Benjamin D. Humphreys; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
The kidney possesses profound regenerative potential and in some cases can recover completely ‘restitutio at integrum’ following an acute kidney injury (AKI). Emerging evidence strongly suggests that sometimes repair is incomplete, however, and, in this situation, an episode of AKI leads to future chronic kidney disease (CKD). Understanding the tubular response after AKI will shed light on the relationship between incomplete repair and future risk of CKD. The first repair phase after AKI is characterized by robust proliferation of epithelial cells in the proximal tubule. The exact source of these proliferating cells has been a source of controversy for the last decade. While nearly everyone now agrees that reparative cells arise within the proximal tubule, there is disagreement about whether all surviving cells possess an equivalent repair capacity through dedifferentiation, or alternatively whether a pre-existing intratubular stem cell population [socalled scattered tubular cells (STC)] is responsible for repair. This review will summarize the evidence on both sides of this issue and will discuss very recent genetic fate-tracing data that strongly points against the existence of intratubular stem cells but rather indicates that terminally differentiated proximal tubule epithelial cells undergo dedifferentiation upon injury to replace lost neighboring tubular epithelial cells through proliferative self-duplication. This new evidence includes data clearly indicating that STC are not committed tubular stem cells but instead represent individual dedifferentiated tubular epithelial cells that transiently express putative stem cell markers.
Acute kidney injury (AKI) is a very common clinical syndrome defined as a rapid decrease in kidney function (ranging from hours to days) characterized by reduced glomerular filtration rate as reflected by an increase in creatinine or blood urea nitrogen (BUN). The most common etiology of AKI is acute tubular necrosis as a consequence of ischemic or nephrotoxic damage [1, 2]. The proximal tubular epithelial cells are the main segment of injury following both ischemic and toxic AKI. Proximal tubular cells rely on aerobic respiration, have high metabolic demands due to fluid and electrolyte reabsorptive functions and, therefore, they are uniquely very susceptible to ischemic injury [3, 4]. Ischemia reperfusion injury (IRI) in most animal models induces the most severe injury in the S3 segment of the proximal tubule, whereas, in humans, a controversy exists whether the extent of injury is more severe in proximal or distal tubular epithelial cells [5, 6]. Indeed, growing evidence suggests that, in human AKI, both the proximal and distal tubular cells are injured with urinary increase of biomarkers for proximal tubular epithelial cells as kidney injury molecule-1 (KIM1) and distal tubule markers such as neutrophil gelatinase-associated lipocalin [6]. The tubule has tremendous regenerative potential and can repair completely after mild-to-moderate injury; however, there is growing evidence that patients following AKI are at high risk of developing chronic and end-stage renal disease (CKD/ESRD) with dramatically increased mortality. One of the reasons for this ‘AKI to CKD transition’ might be the incomplete repair of the renal tubules after AKI, eventually triggering interstitial renal fibrosis. For example it has been shown that selective epithelial injury results in secondary interstitial fibrosis and capillary
Keywords: acute kidney injury, dedifferentiation, stem cell, tubular repair
© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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RWTH Aachen University, Aachen, Germany and 4Harvard Stem Cell Institute, Cambridge, MA, USA
DO TUBULAR STEM CELLS EXIST? A stem cell is an undifferentiated cell that can self-renew indefinitely but can also differentiate into more specialized mature cell types. Stem cells can be (i) pluripotent such as embryonic stem cells or induced pluripotent stem cells which can differentiate into any somatic cell type, (ii) multipotent, which can differentiate into several other cell types, but not all, such as hematopoietic stem cells for example, or (iii) unipotent which can only differentiate into one terminally differentiated cell type for example skin stem cells. Since the proximal tubule consists of only one differentiated cell type, if a stem cell existed in this niche then it would have to be a unipotent stem cell, unless it had the capacity to generate cells that could either migrate across the basement membrane into interstitium, or along the nephron to different segments. Such a unipotent stem cell would primarily be
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distinguished from neighboring differentiated proximal tubule cells by certain markers, and by the ability to self-renew indefinitely. Indeed, over the last decade, several groups have tried to identify a kidney stem cell niche based on cell proliferation. The first attempts used injection of the thymidine analog 5bromo-2-deoxyuridine (BrdU) to identify—so-called—labelretaining cells (LRC), cells that retain BrdU because they divide so slowly that nuclear label is not diluted. This approach relies on the observation that many stem cell populations cycle very slowly. Along these lines, Maeshima et al. [13] injected BrdU daily over 1 week in rats followed by a chase for 2 weeks. They identified scattered LRC in the proximal and distal tubule, following IRI these cells became positive for proliferating cell nuclear antigen and expressed the ‘immature marker’ vimentin as well as the epithelial marker E-Cadherin [13]. The authors concluded that these LRCs might be a progenitor population within the tubule and contribute to repair. A problem with this approach, however, is that proximal tubule turnover is extraordinarily low during homeostasis. Thus, a 2-week chase period is insufficient to allow proliferation of cells and dilution of label. Indeed, this approach is likely to simply label cells that happened to be proliferating during the pulse, with no subsequent dilution at all. Subsequently a second group, using a similar strategy with BrdU injection in rats, observed a large number of slow cycling cells in the renal papilla [14]. Because, following IRI these papillary LRCs quickly entered the cell cycle and disappeared from the papilla, the authors concluded that the cells migrated to the site of injury [14]. Again, BrdU labeling can only label the fraction of cells that are in the S phase of the cell cycle during the BrdU pulse. To overcome this hurdle, the same group used transgenic mice with a tetracycline-regulated GFP-histone 2B fusion protein to further study the papillary LRC population [15]. They described two distinct populations of LRCs in either collecting duct epithelial cells or in a subset of papillary interstitial cells [15]. Further experiments with in vivo labeling of papillary cells using a dye suggested that papillary LRCs might have the capability to migrate toward cortex and medulla after injury with some cells even integrated into tubules [15]. Subsequent in vitro studies with isolated cells from the same transgenic mouse suggested that stromal cell-derived factor 1 (SDF-1 or CXCL12) might be involved in the migration of these cells from the papilla to toward the medulla [16]. Because pharmacologic inhibition of the SDF-1 receptor CXCR4 following IRI in rats resulted in a higher number of papillary BrdU+ LRCs and increased creatinine the authors concluded that SDF-1–CXCR4 signaling is important for migration of papillary LRCs to the medulla and subsequent repair mechanisms [16]. These findings do not reconcile with our previous work showing that extratubular cells do not migrate into the tubule during repair [10]. Integration of papillary interstitial progenitors into the proximal tubule is at most an extremely rare event, then. What about intratubular LRCs? To address this issue, we have used a DNA analog-based lineage analysis to track sequential rounds of proliferation following IRI by injecting separate thymidine analogs 5-chloro-2-deoxyrudine
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rarefaction [7]. The authors used bigenic Six2GFPCre;iDTR mice which selectively activate expression of the human simian diphtheria toxin receptor in renal epithelial cells, therefore allowing injury of epithelial cells via diphtheria toxin injection [7]. Furthermore, we have demonstrated that conditional overexpression of the acute injury gene Kim1 in renal epithelial cells is sufficient to induce interstitial inflammation and fibrosis in the absence of an injury stimulus [8]. Given the link between AKI and CKD, understanding the cells responsible for normal repair is critical. Moreover, development of new targeted therapies to reduce injury and promote repair in AKI require a precise understanding of which are the proper cells to target. There has been little consensus on this issue until recently, however. The proliferating cells within the proximal tubule after AKI have been characterized either as resident dedifferentiated tubular epithelial cells, resident kidney stem cells or even homed stem cells from outside the kidney such as mesenchymal stem cells or hematopoietic stem cells [9]. This latter possibility has been excluded. We used the Six2GFPCre driver line crossed to either Rosa26LacZ or Z/Red reporter mice resulting in heritable expression of either β-galactosidase (LacZ) or red fluorescent protein (RFP) in nearly all kidney epithelial cells (94–95%) following Cre-mediated recombination [10]. In contrast, no extratubular cells were labeled. After IRI, we did not observe any dilution of the fate marker (β-Gal or RFP). This indicated that all reparative epithelial cells originated from within the tubule [10]. These findings did not rule out the possibility that a committed intratubular stem cell population might account for repair, however, since the genetic strategies would have labeled these cells too [11, 12]. This review will discuss recent insight into tubular repair mechanisms following AKI and the controversy about key cellular players in tubular proliferation. Understanding the kidney cell types that promote tubule repair after AKI and identifying mechanisms of their activation will be essential to guide the development of novel targeted therapeutics, promoting kidney repair and reducing the risk of future CKD after AKI.
Romagnani’s group was the first to describe a population of progenitor cells among parietal epithelial cells (PECs) of the Bowman’s capsule, defined by the expression of CD133 and CD24 [20]. Once isolated these cells showed a multipotent differentiation capability toward osteoblasts, adipocytes, endothelial cells, various renal cells and even acquired some functional
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S C AT T E R E D T U B U L A R C E L L S I N T U B U L A R R E G E N E R AT I O N
properties of neurons [20, 21]. A subsequent publication from the same group reported that, within this CD133+, CD24+ progenitor pool of PECs different populations exist. CD133+, CD24+, podocalyxin (PDX)− cells that are located at the urinary pole of the Bowman’s capsule were described to differentiate into both tubular epithelial cells and podocytes, whereas CD133+, CD24+ and PDX+ cells that localize between the urinary and vascular pole only showed a unipotent differentiation potential toward podocytes [22]. Lindgren et al. [23] next demonstrated that in the human kidney outside the Bowman’s capsule CD24+, CD133+ epithelial cells exist in the proximal tubule and suggested that they also represent a scattered tubular cell (STC) progenitor population. The authors determined the mRNA expression of cortical cell suspensions from human kidneys and used the aldehyde dehydrogenase (ALDH) enzymatic activity of STCs for enrichment via fluorescence-activated cell sorting [23]. ALDH was used based on various previous reports that ALDH expression might be specific for stem cells and plays a role in stem cell maintenance by converting retinal to retinoic acid [23]. Microarray analysis pointed toward a distinct transcriptional profile of these enriched STCs (ALDHhigh) when compared with fully differentiated proximal tubular cells (ALDHlow), with decreased expression of genes involved in apoptosis and membrane transports among various others [23]. Subsequently, Romagnani’s group further characterized the CD133+CD24+ cell population using vascular endothelial adhesion molecule 1 (VCAM1 or CD106). They isolated CD133+CD24+CD106+ cells localized at the urinary pole of Bowman’s capsule and showed multipotent differentiation capability toward tubular epithelial cells and podocytes [24]. In contrast, the previously described STCs showed an expression of CD133+CD24+CD106−, were located in the proximal tubule but also in the distal convoluted tubule and possessed unipotent potential toward tubular epithelial cells [24]. The authors performed xenograft experiments and injected human CD133+CD24+CD106+ as well as CD133+CD24+CD106− or CD133−CD24− cells into the tail vein of mice with severe combined immunodeficiency and rhabomyolysis-induced AKI [24]. Only CD133+CD24 +CD106+ or CD133+CD24+CD106− cells but not CD133 −CD24− improved the kidney function and reduced renal fibrosis severity [24]. Interestingly, only injection of CD133 +CD24+CD106+ cells resulted in a significant reduction of BUN at Day 3 after AKI when compared with CD133+CD24 +CD106− or CD133−CD24− cell injections [24]. Moreover, the injected cells proliferated in the mouse kidney and appeared to generate tubular structures [24]. These are very intriguing results but they did not employ genetic fate tracing. Clearly, the STC population is a distinct population of intratubular epithelial cells, now described independently by several groups [23–25]. However, whether these STCs are true stem cells is an altogether different question. Important recent work bears directly on this critical distinction. Smeets et al. [26] studied the STC population intensively in human and rat kidneys. Interestingly, they reported that the vast majority of CD24+ cells did not express markers for differentiated functional proximal tubular cells such as a Lotus tetragonolobus agglutinin (labeled) brush border or the protein
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(CldU) and 5-iodo-2-deoxyuridine (IdU) during repair [17]. This allowed us to identify cells that were rapidly cycling, as would be expected for a subpopulation of intratubular stem cells, or epithelial cells that were proliferating randomly, as would be expected in dedifferentiation. We confirmed the existence of LRCs among epithelial cells in renal papilla, primarily in the collecting ducts. However, these LRCs neither migrated during repair from IRI nor did they selectively proliferate in this setting [17]. These findings ruled out a role for papillary LRCs in direct repopulation of proximal tubule after IRI. To directly address whether proximal tubule proliferation is explained by an intratubular stem cell versus self-duplication of fully differentiated epithelia, we next treated mice with a single injection of CldU at 24 h after IRI and a subsequent injection of IdU at 45 h after IRI with sacrifice 3 h later. The results revealed a very small fraction of double-labeled cells. Rather, one group of cells had incorporated CldU and a different subset had incorporated IdU. This result indicates that proximal tubule cell division is stochastic. If a stem cell population existed, we should have observed a large population of double-labeled cells, since this would reflect the rapid proliferation of a predetermined epithelial subset. Kitamura et al. [18] isolated single nephrons from rat kidneys and diluted outgrowing cells until single clones were established. One of these single clones showed a potent proliferative potential, expressed vimentin and c-met on the protein level and ‘progenitor markers’ Sca-1, c-kit and Pax2 on mRNA level. They demonstrated that dye-labeled cells of this clone, when injected under the renal capsule, integrated into tubules of the corticomedullary region following IRI [18]. However, although these are intriguing results, studies of in vivo cell tracking by dye labeling must be interpreted carefully, because the dye might be integrated into neighboring cells following death of injected cells. Genetic lineage tracing remains the goldstandard approach to define cell hierarchies in vivo. Indeed, the presence of an intratubular progenitor cell population defined by the expression of NFATc1 has been proposed by Langworthy et al. [19]. The authors performed fatetracing studies of NFAT1cCre;Rosa26LacZ mice and reported tubular LacZ+ cells at Day 5 following HgCl2-induced kidney injury with a further increase of LacZ+ cells at 10 days after injury [19]. Because the LacZ+ cells did proliferate after injury (gaining BrdU), the authors concluded that they might be a tubular progenitor cell population. However, an alternative interpretation of this result is that NFAT1c is simply an injury marker, expressed by injured, dedifferentiated tubular cells, which then proliferate in order to repair the tubule.
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Rinkevich et al. [12] recently used a ubiquitous Actin Cre driver crossed against the stochastic multicolor Cre-dependent Brainbow 2.1 mouse to randomly label cell clones with one out of four colors. Clonal analysis of these clones during injury and repair following IRI revealed that intratubular fate restricted cells repair the tubule within segment-specific borders [12]. After IRI (2 months), single-colored clones appeared in the cortex, medulla and papilla and were restricted to their tubular/epithelial segment, i.e. proximal, distal and collecting duct [12]. The clones expanded in longitudinal and perpendicular axis of the tubule but did not cross to other nephrons or a different segment within the nephron. These results indicate that each segment of the nephron is responsible for its own repair, confirming our results in the proximal tubule but also extending them to other tubular segments [12].
D I R E C T E V I D E N C E T H AT T E R M I N A L LY D I F F E R E N T I AT E D E P I T H E L I A L C E L L S ARE RESPONSIBLE FOR TUBULAR R E G E N E R AT I O N The classical concept for tubular repair after injury is that surviving epithelial cells dedifferentiate, proliferate and replace lost neighboring cells [9]. Indeed, Vogetseder et al. [31] demonstrated that the majority of tubular epithelial cells in the S3 segments enter the cell cycle after injury, suggesting that not a small population of progenitor cells is responsible for the repair but rather surviving terminally differentiated epithelial cells. Our recent work also addresses the issue of whether an intratubular stem cell might exist in the proximal tubule. Where Berger et al. performed fate tracing of dedifferentiated STCs, we took a complementary approach and performed fate tracing of fully differentiated epithelial cells. The concept underlying this experiment was that if an intratubular stem cell population exists in the proximal tubule, then it would not express a marker for full proximal tubule differentiation, since by definition stem cells are undifferentiated. We generated a knock-in mouse with a tamoxifen-inducible Cre recombinase (CreERt2) under the control of the sodium dependent inorganic phosphate transporter SLC34a1 [32]. It has been previously reported that SLC34a1 is specifically expressed on the apical side of terminally differentiated tubular epithelial cells, and therefore this protein would not be expressed in an undifferentiated stem cell [33]. We crossed the SLC34a1 mice against the Rosa26Tomato reporter line and observed specific and efficient genetic labeling of tubular epithelial cells of the S1 and S2 segment and to a lesser extent in the S3 segment of the proximal tubule [32]. Importantly, there was absolute proximal tubule specificity: we never observed recombination in any extratubular cell, and indeed never observed extrarenal recombination. With the ability to perform genetic fate mapping of fully differentiated proximal tubule cells, we first asked whether these cells were capable of proliferating after injury. We administered submaximal tamoxifen doses to induce single-cell recombination, and then performed clonal analysis during injury and repair (Figures 1 and 2A). Fourteen days after injury, clone size had increased substantially, and the clone
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receptor megalin [26]. Electron microscopic analyses confirmed that these CD24+ tubular cells, when compared with their neighboring CD24− cells, showed less cytoplasm, fewer mitochondria, lack of basolateral invaginations and absence of a mature brush border [26]. Furthermore, the number of STC increased following injury in human acute tubular necrosis biopsies with complete tubules positive for CD24. In fact, most proliferating tubular cells also were positive for CD24 following injury [26]. Importantly, the authors demonstrated that STC co-express the dedifferentiation markers vimentin, integrin alpha V, annexin 3 and the injury marker Kim-1. Since these proteins have never been associated with a stem cell phenotype but rather reflect cellular dedifferentiation and injury, these results suggested an alternative explanation for the existence of STCs, that they reflect individual cells undergoing a spontaneous cycle of injury, dedifferentiation and redifferentiation. This model assumes that, during normal homeostasis, individual epithelial cells might become injured even without an external injury such as ischemia or nephrotoxin [26]. This is a potentially compelling reconciliation of the published results; however, the work is also limited by histologic snapshots rather than genetic fate tracing. Very recently, Berger et al. [27] performed fate-tracing experiments of STCs after IRI. They discovered that the doxycycline-inducible PEC-specific PEC-reverse-tetracycline transactivator (rtTA) transgenic mouse labels not only PECs but also the very rare population of STCs [27]. Interestingly, the authors already noted the labeling of epithelial cells in the thin part of the loop of Henle and the collecting duct in their original publication about the PEC-rtTA mouse [28]. In their recent study, they report that the tubular cells labeled by the PEC-rtTA mouse co-express Kim-1, annexin A3, srcsuppressed C-Kinase substrate and CD44—all previously reported markers of STCs [26, 27]. It should be noted that the commonly used STCs markers CD133 and CD24 cannot be used in the mouse kidney. For CD24, no adequate antibodies exist for mouse tissue whereas the AC133 mAb detects glycosylated human CD133 protein but this antibody does not cross-react with mouse CD133, which carries a different glycosylation pattern [29, 30]. Berger et al. [27] showed that PECrtTA-labeled STCs increase in number following injury. In fact, after repetitive injections of doxycycline and BrdU during the recovery of injury, more than 80% of BrdU-labeled tubular cells showed expression of PEC-rtTA [27]. To directly assess whether STCs represent a fixed intratubular stem cell pool, the authors next genetically labeled the PEC-rtTA-labeled STCs prior to injury then performed IRI and allowed repair to occur. After repair, there was no increase in the number of genetically marked tubular cells—indicating that the STCs are definitively not a fixed intratubular progenitor population [27]. In contrast, when doxycycline was administered during and following the injury, significantly more tubular cells were genetically labeled indicating that terminally differentiated tubular epithelial cells induce expression of PEC-rTA following injury [27]. These results provide strong evidence against the existence of an intratubular stem cell population and support the idea that STCs represent individual epithelial cells spontaneously dedifferentiating.
F I G U R E 2 : Clonal and dilution analysis of SLC34a1GCE/+, Rosa26tomato/+ mice. (A) In uninjured contralateral kidney, cells were labeled solely
by low-dose tamoxifen (left panel). In the IRI kidney, clone size of labeled cells expanded after repair (right panel). (B) Dilution analysis of SLC34a1GCE/+, Rosa26tomato/+ mice. These representative images indicate complete labeling of the proximal tubule in uninjured kidneys and remaining labeling (no dilution) after injury. Staining for BrdU (green) indicates the proliferating cells of the proximal tubule indeed came from the labeled (red) terminally differentiated tubular cells. (C and D) Immunostaining of dedifferentiation markers; vimentin and Pax2. In noninjured kidneys terminally differentiated labeled epithelial cells (red) did never stain for vimentin or Pax2. Whereas, following injury (IRI) these terminally differentiated cells became flattened and expressed both vimentin and Pax2, indicating dedifferentiation.
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single-cell labeling in terminally differentiated proximal tubular epithelial cells by very low-dose tamoxifen injection into SLC34a1GCE/+ mice. Therefore, on a clone size–frequency curve, nearly all clones exist as single-cell clones (blue curve in both panels). If a stem cell population contributes to renal repair, the clone size will remain single and clone frequency will decrease after injury, due to death of differentiated cells after injury (green curve in left panel). If repair is by self-duplication of terminally differentiated cells (= labeled cells), the clone size will expand through proliferation of single-cell clones during repair (red curve in right panel), resulting in a rightward shift of the clone size–frequency curve. (B) To determine whether an intratubular stem cell contributes to repair at all, we performed dilution analysis. Complete cell labeling in the terminally differentiated proximal tubular was accomplished by high-dose tamoxifen administration. A round of IRI and repair follows. Upon analysis of repaired tubules, if only terminally differentiated cells contribute to renal repair, the labeling will remain undiluted by unlabeled cells (Option 1). If an unlabeled progenitor population contributes to repair of the genetic label will be diluted by progeny of these cells (Option 2). Our results support Option 1, self-duplication, as the only mechanism of proximal tubule repair.
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F I G U R E 1 : Rationale of clonal and dilution analysis using SLC34a1−GFPCreER (SLC34a1GCE/+), Rosa26tomato/+ mice. (A) We first induce
size–frequency curve had shifted smoothly to the right. In Sham kidneys, all clones, were either one or two cells but, after injury, there were few single-cell clones and increased numbers of two-, three- and four-cell clones and some clones reaching more than five cells, indicating that, on average, terminally differentiated cells had undergone one to three cellular divisions following injury [32]. This result strongly suggested that not only can terminally differentiated tubular cells contribute to repair after injury, but that this is a property shared by most and probably all differentiated cells—because of the smooth
rightward shift of the clone size–frequency curve (Figures 1 and 2A). We next asked whether a putative intratubular stem cell population made any contribution at all to epithelial repair after injury. We injected high-dose tamoxifen prior to injury to achieve maximal recombination. In this experimental design, if an unlabeled progenitor cell contributes to proximal tubular repair this would lead to dilution of the labeled terminally differentiated tubular cells following injury. However, we did not observe such a dilution and the fraction of labeled
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F I G U R E 3 : Dueling models for epithelial repair after injury. (A) In the stem/progenitor model, scattered progenitors are located adjacent to
differentiated proximal tubule epithelium. The progenitors express CD24, CD133, Kim-1 and Vimentin, among other markers. After injury, these cells preferentially survive and selectively proliferate. Their progeny differentiate into proximal tubular epithelial cells. (B) In the selfduplication model, any fully differentiated cell that survives the injury has an equivalent capacity to dedifferentiate and proliferate. The progeny then re-differentiate into proximal tubular tubule epithelium. In this case, CD24, CD133, Kim-1 and vimentin are not markers of a separate stem cell compartment, but are injury markers transiently expressed by a dedifferentiated epithelial cell. Thus, new epithelia derive from their fully differentiated neighbors in a process of self-duplication.
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CURRENT QUESTIONS
Table 1. Point-by-point summary of the main conclusions • Following AKI terminally differentiated proximal tubular cells undergo dedifferentiation into STC • Therefore, STCs are not a fixed progenitor population but arise from any surviving proximal tubular cell following injury • Dedifferentiation of tubular epithelial cells results in expression of Kim1, vimentin, annexin A3, src-suppressed C-Kinase substrate, CD44, CD133 and CD24; some of these markers have been previously described as being specific for tubular progenitor cells. • Any surviving dedifferentiated tubular epithelial cell (STC) enters the cell cycle and undergoes a limited number of cell divisions replacing lost neighbor cells during the injury repair process. • The molecular pathways involved in the dedifferentiation, proliferation and redifferentiation process of tubular epithelial cells remain incompletely understood and are the challenge for ongoing and future research.
Who regenerates the kidney tubule?
AC K N O W L E D G E M E N T This work was supported by NIH DK088923 (to B.D.H.), by an Established Investigator Award of the American Heart Association (to B.D.H.) and by a fellowship the Deutsche Forschungsgemeinschaft (to R.K., KR 4073/1-1).
C O N F L I C T O F I N T E R E S T S TAT E M E N T The results presented in this paper have not been published previously in whole or part, except in abstract format.
REFERENCES 1. Basile DP, Anderson MD, Sutton TA. Pathophysiology of acute kidney injury. Compr Physiol 2012; 2: 1303–1353 2. Coca SG, Singanamala S, Parikh CR. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. Kidney Int 2012; 81: 442–448 3. Uchida S, Endou H. Substrate specificity to maintain cellular ATP along the mouse nephron. Am J Physiol 1988; 255(5 Pt 2): F977–F983 4. Lieberthal W, Nigam SK. Acute renal failure. I. Relative Importance of Proximal vs. Distal Tubular Injury. Am J Physiol 1998; 275(5 Pt 2): F623–F631 5. Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 2011; 121: 4210–4221 6. Heyman SN, Rosenberger C, Rosen S. Experimental ischemia-reperfusion: biases and myths-the proximal vs. distal hypoxic tubular injury debate revisited. Kidney Int 2010; 77: 9–16 7. Grgic I, Campanholle G, Bijol V et al. Targeted proximal tubule injury triggers interstitial fibrosis and glomerulosclerosis. Kidney Int 2012; 82: 172–183 8. Humphreys BD, Xu F, Sabbisetti V et al. Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis. J Clin Invest 2013; 123: 4023–4035 9. Kusaba T, Humphreys BD. Controversies on the origin of proliferating epithelial cells after kidney injury. Pediatr Nephrol 2014; 29: 673–679 10. Humphreys BD, Valerius MT, Kobayashi A et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2008; 2: 284–291 11. Little MH, Bertram JF. Is there such a thing as a renal stem cell? J Am Soc Nephrol 2009; 20: 2112–2117 12. Rinkevich Y, Montoro DT, Contreras-Trujillo H et al. In vivo clonal analysis reveals lineage-restricted progenitor characteristics in Mammalian kidney development, maintenance, and regeneration. Cell Rep 2014; 7: 1270–1283
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Understanding the mechanisms of tubular injury and repair is the first step for development of novel targeted therapeutics in AKI and its transition into CKD. The therapeutic strategy used to target a pre-existing epithelial stem cell population might be quite different than one aimed at harnessing the proliferative capacity of differentiated tubular cells. The recent evidence strongly points toward terminally differentiated tubular epithelial cells which dedifferentiate upon injury and acquire a STC phenotype with expression of Kim1, vimentin, annexin A3, src-suppressed C-Kinase substrate, CD44 and upregulation of CD133 and CD24 on mRNA level (summarized in Table 1). Therefore, these markers might not be seen as progenitor cell markers but rather markers that indicate dedifferentiation and ‘reprogramming’ of tubular epithelial cells toward a prorepair, antiapoptosis and regenerative phenotype. In addition to our own fate-tracing paper in kidney injury
[32], two other recent papers in stomach and trachea suggest that fully committed mature epithelial cells respond to injury by dedifferentiating, proliferating and redifferentiating into fully functional mature epithelial cells [35, 36]. In fact, these groups present evidence that dedifferentiation represents a state of acquired stemness. This recent work clearly challenges the dogma that stem cell hierarchies are a one-way street from undifferentiated progenitor cells to fully differentiated mature cells and points toward mature epithelial cells that dedifferentiate and acquire a stem cell-like state upon injury. Looking to the future, an important challenge will be to understand this dedifferentiation process and to uncover the molecular pathways that drive the injury and repair of dedifferentiated epithelial cells.
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tubular epithelial cells remained unchanged after injury despite robust proliferation (BrdU incorporation) among genetically labeled cells (Figure 2B) [32]. During the injury phase, labeled proximal tubular cells also gained expression of Pax2 and vimentin protein (Figure 2C and D). By performing fluorescence-activated cell sorting of tdTomato-positive cells, we could also measure upregulated mRNA expression of the putative stem cell markers CD133, CD24 and Kim1 in these cells. This evidence reconciles the prior work on STCs and provides direct evidence that fully differentiated epithelial cells upregulate expression of CD24 and CD133 after injury (Figure 3). Thus, these genes are injury markers—not stem cell markers, as previously concluded [32]. Differences in marker expression between humans and rodents exist and, therefore, it can be argued that the existence of intratubular stem cells has not been disproven in larger mammals [34]. However, the study by Smeets et al. [26] clearly points toward dedifferentiation of terminally differentiated tubular epithelial cells following injury in human kidneys; these dedifferentiated cells coexpress Kim-1, CD133 and CD24 [26], consistent with our results in mouse.
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25. Kim K, Park BH, Ihm H et al. Expression of stem cell marker CD133 in fetal and adult human kidneys and pauci-immune crescentic glomerulonephritis. Histol Histopathol 2011; 26: 223–232 26. Smeets B, Boor P, Dijkman H et al. Proximal tubular cells contain a phenotypically distinct, scattered cell population involved in tubular regeneration. J Pathol 2013; 229: 645–659 27. Berger K, Bangen JM, Hammerich L et al. Origin of regenerating tubular cells after acute kidney injury. Proc Natl Acad Sci USA 2014; 111: 1533–1538 28. Appel D, Kershaw DB, Smeets B et al. Recruitment of podocytes from glomerular parietal epithelial cells. J Am Soc Nephrol 2009; 20: 333–343 29. Kemper K, Sprick MR, de Bree M et al. The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation. Cancer Res 2010; 70: 719–729 30. Angelotti ML, Lazzeri E, Lasagni L et al. Only anti-CD133 antibodies recognizing the CD133/1 or the CD133/2 epitopes can identify human renal progenitors. Kidney Int 2010; 78: 620–621; author reply 621 31. Vogetseder A, Picard N, Gaspert A et al. Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells. Am J Physiol Cell Physiol 2008; 294: C22–C28 32. Kusaba T, Lalli M, Kramann R et al. Differentiated kidney epithelial cells repair injured proximal tubule. Proc Natl Acad Sci USA 2014; 111: 1527–1532 33. Madjdpour C, Bacic D, Kaissling B et al. Segment-specific expression of sodium-phosphate cotransporters NaPi-IIa and -IIc and interacting proteins in mouse renal proximal tubules. Pflugers Arch 2004; 448: 402–410 34. Romagnani P. Of mice and men: the riddle of tubular regeneration. J Pathol 2013; 229: 641–644 35. Tata PR, Mou H, Pardo-Saganta A et al. Dedifferentiation of committed epithelial cells into stem cells in vivo. Nature 2013; 503: 218–223 36. Stange DE, Koo BK, Huch M et al. Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 2013; 155: 357–368
Received for publication: 21.6.2014; Accepted in revised form: 27.7.2014
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13. Maeshima A, Yamashita S, Nojima Y. Identification of renal progenitorlike tubular cells that participate in the regeneration processes of the kidney. J Am Soc Nephrol 2003; 14: 3138–3146 14. Oliver JA, Maarouf O, Cheema FH et al. The renal papilla is a niche for adult kidney stem cells. J Clin Invest 2004; 114: 795–804 15. Oliver JA, Klinakis A, Cheema FH et al. Proliferation and migration of label-retaining cells of the kidney papilla. J Am Soc Nephrol 2009; 20: 2315–2327 16. Oliver JA, Maarouf O, Cheema FH et al. SDF-1 activates papillary labelretaining cells during kidney repair from injury. Am J Physiol Renal Physiol 2012; 302: F1362–F1373 17. Humphreys BD, Czerniak S, DiRocco DP et al. Repair of injured proximal tubule does not involve specialized progenitors. Proc Natl Acad Sci USA 2011; 108: 9226–9231 18. Kitamura S, Yamasaki Y, Kinomura M et al. Establishment and characterization of renal progenitor like cells from S3 segment of nephron in rat adult kidney. FASEB J 2005; 19: 1789–1797 19. Langworthy M, Zhou B, de Caestecker M et al. NFATc1 identifies a population of proximal tubule cell progenitors. J Am Soc Nephrol 2009; 20: 311–321 20. Sagrinati C, Netti GS, Mazzinghi B et al. Isolation and characterization of multipotent progenitor cells from the Bowman’s capsule of adult human kidneys. J Am Soc Nephrol 2006; 17: 2443–2456 21. Lazzeri E, Crescioli C, Ronconi E et al. Regenerative potential of embryonic renal multipotent progenitors in acute renal failure. J Am Soc Nephrol 2007; 18: 3128–3138 22. Ronconi E, Sagrinati C, Angelotti ML et al. Regeneration of glomerular podocytes by human renal progenitors. J Am Soc Nephrol 2009; 20: 322–332 23. Lindgren D, Bostrom AK, Nilsson K et al. Isolation and characterization of progenitor-like cells from human renal proximal tubules. Am J Pathol 2011; 178: 828–837 24. Angelotti ML, Ronconi E, Ballerini L et al. Characterization of renal progenitors committed toward tubular lineage and their regenerative potential in renal tubular injury. Stem Cells 2012; 30: 1714–1725
Full Review Who regenerates the kidney tubule? Rafael Kramann1,2,3, Tetsuro Kusaba1,2 and Benjamin D. Humphreys1,2,4 1
Brigham and Women’s Hospital, Boston, MA, USA, 2Harvard Medical School, Boston, MA, USA, 3Division of Nephrology,
Correspondence and offprint requests to: Benjamin D. Humphreys; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
The kidney possesses profound regenerative potential and in some cases can recover completely ‘restitutio at integrum’ following an acute kidney injury (AKI). Emerging evidence strongly suggests that sometimes repair is incomplete, however, and, in this situation, an episode of AKI leads to future chronic kidney disease (CKD). Understanding the tubular response after AKI will shed light on the relationship between incomplete repair and future risk of CKD. The first repair phase after AKI is characterized by robust proliferation of epithelial cells in the proximal tubule. The exact source of these proliferating cells has been a source of controversy for the last decade. While nearly everyone now agrees that reparative cells arise within the proximal tubule, there is disagreement about whether all surviving cells possess an equivalent repair capacity through dedifferentiation, or alternatively whether a pre-existing intratubular stem cell population [socalled scattered tubular cells (STC)] is responsible for repair. This review will summarize the evidence on both sides of this issue and will discuss very recent genetic fate-tracing data that strongly points against the existence of intratubular stem cells but rather indicates that terminally differentiated proximal tubule epithelial cells undergo dedifferentiation upon injury to replace lost neighboring tubular epithelial cells through proliferative self-duplication. This new evidence includes data clearly indicating that STC are not committed tubular stem cells but instead represent individual dedifferentiated tubular epithelial cells that transiently express putative stem cell markers.
Acute kidney injury (AKI) is a very common clinical syndrome defined as a rapid decrease in kidney function (ranging from hours to days) characterized by reduced glomerular filtration rate as reflected by an increase in creatinine or blood urea nitrogen (BUN). The most common etiology of AKI is acute tubular necrosis as a consequence of ischemic or nephrotoxic damage [1, 2]. The proximal tubular epithelial cells are the main segment of injury following both ischemic and toxic AKI. Proximal tubular cells rely on aerobic respiration, have high metabolic demands due to fluid and electrolyte reabsorptive functions and, therefore, they are uniquely very susceptible to ischemic injury [3, 4]. Ischemia reperfusion injury (IRI) in most animal models induces the most severe injury in the S3 segment of the proximal tubule, whereas, in humans, a controversy exists whether the extent of injury is more severe in proximal or distal tubular epithelial cells [5, 6]. Indeed, growing evidence suggests that, in human AKI, both the proximal and distal tubular cells are injured with urinary increase of biomarkers for proximal tubular epithelial cells as kidney injury molecule-1 (KIM1) and distal tubule markers such as neutrophil gelatinase-associated lipocalin [6]. The tubule has tremendous regenerative potential and can repair completely after mild-to-moderate injury; however, there is growing evidence that patients following AKI are at high risk of developing chronic and end-stage renal disease (CKD/ESRD) with dramatically increased mortality. One of the reasons for this ‘AKI to CKD transition’ might be the incomplete repair of the renal tubules after AKI, eventually triggering interstitial renal fibrosis. For example it has been shown that selective epithelial injury results in secondary interstitial fibrosis and capillary
Keywords: acute kidney injury, dedifferentiation, stem cell, tubular repair
© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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RWTH Aachen University, Aachen, Germany and 4Harvard Stem Cell Institute, Cambridge, MA, USA
DO TUBULAR STEM CELLS EXIST? A stem cell is an undifferentiated cell that can self-renew indefinitely but can also differentiate into more specialized mature cell types. Stem cells can be (i) pluripotent such as embryonic stem cells or induced pluripotent stem cells which can differentiate into any somatic cell type, (ii) multipotent, which can differentiate into several other cell types, but not all, such as hematopoietic stem cells for example, or (iii) unipotent which can only differentiate into one terminally differentiated cell type for example skin stem cells. Since the proximal tubule consists of only one differentiated cell type, if a stem cell existed in this niche then it would have to be a unipotent stem cell, unless it had the capacity to generate cells that could either migrate across the basement membrane into interstitium, or along the nephron to different segments. Such a unipotent stem cell would primarily be
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distinguished from neighboring differentiated proximal tubule cells by certain markers, and by the ability to self-renew indefinitely. Indeed, over the last decade, several groups have tried to identify a kidney stem cell niche based on cell proliferation. The first attempts used injection of the thymidine analog 5bromo-2-deoxyuridine (BrdU) to identify—so-called—labelretaining cells (LRC), cells that retain BrdU because they divide so slowly that nuclear label is not diluted. This approach relies on the observation that many stem cell populations cycle very slowly. Along these lines, Maeshima et al. [13] injected BrdU daily over 1 week in rats followed by a chase for 2 weeks. They identified scattered LRC in the proximal and distal tubule, following IRI these cells became positive for proliferating cell nuclear antigen and expressed the ‘immature marker’ vimentin as well as the epithelial marker E-Cadherin [13]. The authors concluded that these LRCs might be a progenitor population within the tubule and contribute to repair. A problem with this approach, however, is that proximal tubule turnover is extraordinarily low during homeostasis. Thus, a 2-week chase period is insufficient to allow proliferation of cells and dilution of label. Indeed, this approach is likely to simply label cells that happened to be proliferating during the pulse, with no subsequent dilution at all. Subsequently a second group, using a similar strategy with BrdU injection in rats, observed a large number of slow cycling cells in the renal papilla [14]. Because, following IRI these papillary LRCs quickly entered the cell cycle and disappeared from the papilla, the authors concluded that the cells migrated to the site of injury [14]. Again, BrdU labeling can only label the fraction of cells that are in the S phase of the cell cycle during the BrdU pulse. To overcome this hurdle, the same group used transgenic mice with a tetracycline-regulated GFP-histone 2B fusion protein to further study the papillary LRC population [15]. They described two distinct populations of LRCs in either collecting duct epithelial cells or in a subset of papillary interstitial cells [15]. Further experiments with in vivo labeling of papillary cells using a dye suggested that papillary LRCs might have the capability to migrate toward cortex and medulla after injury with some cells even integrated into tubules [15]. Subsequent in vitro studies with isolated cells from the same transgenic mouse suggested that stromal cell-derived factor 1 (SDF-1 or CXCL12) might be involved in the migration of these cells from the papilla to toward the medulla [16]. Because pharmacologic inhibition of the SDF-1 receptor CXCR4 following IRI in rats resulted in a higher number of papillary BrdU+ LRCs and increased creatinine the authors concluded that SDF-1–CXCR4 signaling is important for migration of papillary LRCs to the medulla and subsequent repair mechanisms [16]. These findings do not reconcile with our previous work showing that extratubular cells do not migrate into the tubule during repair [10]. Integration of papillary interstitial progenitors into the proximal tubule is at most an extremely rare event, then. What about intratubular LRCs? To address this issue, we have used a DNA analog-based lineage analysis to track sequential rounds of proliferation following IRI by injecting separate thymidine analogs 5-chloro-2-deoxyrudine
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rarefaction [7]. The authors used bigenic Six2GFPCre;iDTR mice which selectively activate expression of the human simian diphtheria toxin receptor in renal epithelial cells, therefore allowing injury of epithelial cells via diphtheria toxin injection [7]. Furthermore, we have demonstrated that conditional overexpression of the acute injury gene Kim1 in renal epithelial cells is sufficient to induce interstitial inflammation and fibrosis in the absence of an injury stimulus [8]. Given the link between AKI and CKD, understanding the cells responsible for normal repair is critical. Moreover, development of new targeted therapies to reduce injury and promote repair in AKI require a precise understanding of which are the proper cells to target. There has been little consensus on this issue until recently, however. The proliferating cells within the proximal tubule after AKI have been characterized either as resident dedifferentiated tubular epithelial cells, resident kidney stem cells or even homed stem cells from outside the kidney such as mesenchymal stem cells or hematopoietic stem cells [9]. This latter possibility has been excluded. We used the Six2GFPCre driver line crossed to either Rosa26LacZ or Z/Red reporter mice resulting in heritable expression of either β-galactosidase (LacZ) or red fluorescent protein (RFP) in nearly all kidney epithelial cells (94–95%) following Cre-mediated recombination [10]. In contrast, no extratubular cells were labeled. After IRI, we did not observe any dilution of the fate marker (β-Gal or RFP). This indicated that all reparative epithelial cells originated from within the tubule [10]. These findings did not rule out the possibility that a committed intratubular stem cell population might account for repair, however, since the genetic strategies would have labeled these cells too [11, 12]. This review will discuss recent insight into tubular repair mechanisms following AKI and the controversy about key cellular players in tubular proliferation. Understanding the kidney cell types that promote tubule repair after AKI and identifying mechanisms of their activation will be essential to guide the development of novel targeted therapeutics, promoting kidney repair and reducing the risk of future CKD after AKI.
Romagnani’s group was the first to describe a population of progenitor cells among parietal epithelial cells (PECs) of the Bowman’s capsule, defined by the expression of CD133 and CD24 [20]. Once isolated these cells showed a multipotent differentiation capability toward osteoblasts, adipocytes, endothelial cells, various renal cells and even acquired some functional
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S C AT T E R E D T U B U L A R C E L L S I N T U B U L A R R E G E N E R AT I O N
properties of neurons [20, 21]. A subsequent publication from the same group reported that, within this CD133+, CD24+ progenitor pool of PECs different populations exist. CD133+, CD24+, podocalyxin (PDX)− cells that are located at the urinary pole of the Bowman’s capsule were described to differentiate into both tubular epithelial cells and podocytes, whereas CD133+, CD24+ and PDX+ cells that localize between the urinary and vascular pole only showed a unipotent differentiation potential toward podocytes [22]. Lindgren et al. [23] next demonstrated that in the human kidney outside the Bowman’s capsule CD24+, CD133+ epithelial cells exist in the proximal tubule and suggested that they also represent a scattered tubular cell (STC) progenitor population. The authors determined the mRNA expression of cortical cell suspensions from human kidneys and used the aldehyde dehydrogenase (ALDH) enzymatic activity of STCs for enrichment via fluorescence-activated cell sorting [23]. ALDH was used based on various previous reports that ALDH expression might be specific for stem cells and plays a role in stem cell maintenance by converting retinal to retinoic acid [23]. Microarray analysis pointed toward a distinct transcriptional profile of these enriched STCs (ALDHhigh) when compared with fully differentiated proximal tubular cells (ALDHlow), with decreased expression of genes involved in apoptosis and membrane transports among various others [23]. Subsequently, Romagnani’s group further characterized the CD133+CD24+ cell population using vascular endothelial adhesion molecule 1 (VCAM1 or CD106). They isolated CD133+CD24+CD106+ cells localized at the urinary pole of Bowman’s capsule and showed multipotent differentiation capability toward tubular epithelial cells and podocytes [24]. In contrast, the previously described STCs showed an expression of CD133+CD24+CD106−, were located in the proximal tubule but also in the distal convoluted tubule and possessed unipotent potential toward tubular epithelial cells [24]. The authors performed xenograft experiments and injected human CD133+CD24+CD106+ as well as CD133+CD24+CD106− or CD133−CD24− cells into the tail vein of mice with severe combined immunodeficiency and rhabomyolysis-induced AKI [24]. Only CD133+CD24 +CD106+ or CD133+CD24+CD106− cells but not CD133 −CD24− improved the kidney function and reduced renal fibrosis severity [24]. Interestingly, only injection of CD133 +CD24+CD106+ cells resulted in a significant reduction of BUN at Day 3 after AKI when compared with CD133+CD24 +CD106− or CD133−CD24− cell injections [24]. Moreover, the injected cells proliferated in the mouse kidney and appeared to generate tubular structures [24]. These are very intriguing results but they did not employ genetic fate tracing. Clearly, the STC population is a distinct population of intratubular epithelial cells, now described independently by several groups [23–25]. However, whether these STCs are true stem cells is an altogether different question. Important recent work bears directly on this critical distinction. Smeets et al. [26] studied the STC population intensively in human and rat kidneys. Interestingly, they reported that the vast majority of CD24+ cells did not express markers for differentiated functional proximal tubular cells such as a Lotus tetragonolobus agglutinin (labeled) brush border or the protein
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(CldU) and 5-iodo-2-deoxyuridine (IdU) during repair [17]. This allowed us to identify cells that were rapidly cycling, as would be expected for a subpopulation of intratubular stem cells, or epithelial cells that were proliferating randomly, as would be expected in dedifferentiation. We confirmed the existence of LRCs among epithelial cells in renal papilla, primarily in the collecting ducts. However, these LRCs neither migrated during repair from IRI nor did they selectively proliferate in this setting [17]. These findings ruled out a role for papillary LRCs in direct repopulation of proximal tubule after IRI. To directly address whether proximal tubule proliferation is explained by an intratubular stem cell versus self-duplication of fully differentiated epithelia, we next treated mice with a single injection of CldU at 24 h after IRI and a subsequent injection of IdU at 45 h after IRI with sacrifice 3 h later. The results revealed a very small fraction of double-labeled cells. Rather, one group of cells had incorporated CldU and a different subset had incorporated IdU. This result indicates that proximal tubule cell division is stochastic. If a stem cell population existed, we should have observed a large population of double-labeled cells, since this would reflect the rapid proliferation of a predetermined epithelial subset. Kitamura et al. [18] isolated single nephrons from rat kidneys and diluted outgrowing cells until single clones were established. One of these single clones showed a potent proliferative potential, expressed vimentin and c-met on the protein level and ‘progenitor markers’ Sca-1, c-kit and Pax2 on mRNA level. They demonstrated that dye-labeled cells of this clone, when injected under the renal capsule, integrated into tubules of the corticomedullary region following IRI [18]. However, although these are intriguing results, studies of in vivo cell tracking by dye labeling must be interpreted carefully, because the dye might be integrated into neighboring cells following death of injected cells. Genetic lineage tracing remains the goldstandard approach to define cell hierarchies in vivo. Indeed, the presence of an intratubular progenitor cell population defined by the expression of NFATc1 has been proposed by Langworthy et al. [19]. The authors performed fatetracing studies of NFAT1cCre;Rosa26LacZ mice and reported tubular LacZ+ cells at Day 5 following HgCl2-induced kidney injury with a further increase of LacZ+ cells at 10 days after injury [19]. Because the LacZ+ cells did proliferate after injury (gaining BrdU), the authors concluded that they might be a tubular progenitor cell population. However, an alternative interpretation of this result is that NFAT1c is simply an injury marker, expressed by injured, dedifferentiated tubular cells, which then proliferate in order to repair the tubule.
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Rinkevich et al. [12] recently used a ubiquitous Actin Cre driver crossed against the stochastic multicolor Cre-dependent Brainbow 2.1 mouse to randomly label cell clones with one out of four colors. Clonal analysis of these clones during injury and repair following IRI revealed that intratubular fate restricted cells repair the tubule within segment-specific borders [12]. After IRI (2 months), single-colored clones appeared in the cortex, medulla and papilla and were restricted to their tubular/epithelial segment, i.e. proximal, distal and collecting duct [12]. The clones expanded in longitudinal and perpendicular axis of the tubule but did not cross to other nephrons or a different segment within the nephron. These results indicate that each segment of the nephron is responsible for its own repair, confirming our results in the proximal tubule but also extending them to other tubular segments [12].
D I R E C T E V I D E N C E T H AT T E R M I N A L LY D I F F E R E N T I AT E D E P I T H E L I A L C E L L S ARE RESPONSIBLE FOR TUBULAR R E G E N E R AT I O N The classical concept for tubular repair after injury is that surviving epithelial cells dedifferentiate, proliferate and replace lost neighboring cells [9]. Indeed, Vogetseder et al. [31] demonstrated that the majority of tubular epithelial cells in the S3 segments enter the cell cycle after injury, suggesting that not a small population of progenitor cells is responsible for the repair but rather surviving terminally differentiated epithelial cells. Our recent work also addresses the issue of whether an intratubular stem cell might exist in the proximal tubule. Where Berger et al. performed fate tracing of dedifferentiated STCs, we took a complementary approach and performed fate tracing of fully differentiated epithelial cells. The concept underlying this experiment was that if an intratubular stem cell population exists in the proximal tubule, then it would not express a marker for full proximal tubule differentiation, since by definition stem cells are undifferentiated. We generated a knock-in mouse with a tamoxifen-inducible Cre recombinase (CreERt2) under the control of the sodium dependent inorganic phosphate transporter SLC34a1 [32]. It has been previously reported that SLC34a1 is specifically expressed on the apical side of terminally differentiated tubular epithelial cells, and therefore this protein would not be expressed in an undifferentiated stem cell [33]. We crossed the SLC34a1 mice against the Rosa26Tomato reporter line and observed specific and efficient genetic labeling of tubular epithelial cells of the S1 and S2 segment and to a lesser extent in the S3 segment of the proximal tubule [32]. Importantly, there was absolute proximal tubule specificity: we never observed recombination in any extratubular cell, and indeed never observed extrarenal recombination. With the ability to perform genetic fate mapping of fully differentiated proximal tubule cells, we first asked whether these cells were capable of proliferating after injury. We administered submaximal tamoxifen doses to induce single-cell recombination, and then performed clonal analysis during injury and repair (Figures 1 and 2A). Fourteen days after injury, clone size had increased substantially, and the clone
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receptor megalin [26]. Electron microscopic analyses confirmed that these CD24+ tubular cells, when compared with their neighboring CD24− cells, showed less cytoplasm, fewer mitochondria, lack of basolateral invaginations and absence of a mature brush border [26]. Furthermore, the number of STC increased following injury in human acute tubular necrosis biopsies with complete tubules positive for CD24. In fact, most proliferating tubular cells also were positive for CD24 following injury [26]. Importantly, the authors demonstrated that STC co-express the dedifferentiation markers vimentin, integrin alpha V, annexin 3 and the injury marker Kim-1. Since these proteins have never been associated with a stem cell phenotype but rather reflect cellular dedifferentiation and injury, these results suggested an alternative explanation for the existence of STCs, that they reflect individual cells undergoing a spontaneous cycle of injury, dedifferentiation and redifferentiation. This model assumes that, during normal homeostasis, individual epithelial cells might become injured even without an external injury such as ischemia or nephrotoxin [26]. This is a potentially compelling reconciliation of the published results; however, the work is also limited by histologic snapshots rather than genetic fate tracing. Very recently, Berger et al. [27] performed fate-tracing experiments of STCs after IRI. They discovered that the doxycycline-inducible PEC-specific PEC-reverse-tetracycline transactivator (rtTA) transgenic mouse labels not only PECs but also the very rare population of STCs [27]. Interestingly, the authors already noted the labeling of epithelial cells in the thin part of the loop of Henle and the collecting duct in their original publication about the PEC-rtTA mouse [28]. In their recent study, they report that the tubular cells labeled by the PEC-rtTA mouse co-express Kim-1, annexin A3, srcsuppressed C-Kinase substrate and CD44—all previously reported markers of STCs [26, 27]. It should be noted that the commonly used STCs markers CD133 and CD24 cannot be used in the mouse kidney. For CD24, no adequate antibodies exist for mouse tissue whereas the AC133 mAb detects glycosylated human CD133 protein but this antibody does not cross-react with mouse CD133, which carries a different glycosylation pattern [29, 30]. Berger et al. [27] showed that PECrtTA-labeled STCs increase in number following injury. In fact, after repetitive injections of doxycycline and BrdU during the recovery of injury, more than 80% of BrdU-labeled tubular cells showed expression of PEC-rtTA [27]. To directly assess whether STCs represent a fixed intratubular stem cell pool, the authors next genetically labeled the PEC-rtTA-labeled STCs prior to injury then performed IRI and allowed repair to occur. After repair, there was no increase in the number of genetically marked tubular cells—indicating that the STCs are definitively not a fixed intratubular progenitor population [27]. In contrast, when doxycycline was administered during and following the injury, significantly more tubular cells were genetically labeled indicating that terminally differentiated tubular epithelial cells induce expression of PEC-rTA following injury [27]. These results provide strong evidence against the existence of an intratubular stem cell population and support the idea that STCs represent individual epithelial cells spontaneously dedifferentiating.
F I G U R E 2 : Clonal and dilution analysis of SLC34a1GCE/+, Rosa26tomato/+ mice. (A) In uninjured contralateral kidney, cells were labeled solely
by low-dose tamoxifen (left panel). In the IRI kidney, clone size of labeled cells expanded after repair (right panel). (B) Dilution analysis of SLC34a1GCE/+, Rosa26tomato/+ mice. These representative images indicate complete labeling of the proximal tubule in uninjured kidneys and remaining labeling (no dilution) after injury. Staining for BrdU (green) indicates the proliferating cells of the proximal tubule indeed came from the labeled (red) terminally differentiated tubular cells. (C and D) Immunostaining of dedifferentiation markers; vimentin and Pax2. In noninjured kidneys terminally differentiated labeled epithelial cells (red) did never stain for vimentin or Pax2. Whereas, following injury (IRI) these terminally differentiated cells became flattened and expressed both vimentin and Pax2, indicating dedifferentiation.
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single-cell labeling in terminally differentiated proximal tubular epithelial cells by very low-dose tamoxifen injection into SLC34a1GCE/+ mice. Therefore, on a clone size–frequency curve, nearly all clones exist as single-cell clones (blue curve in both panels). If a stem cell population contributes to renal repair, the clone size will remain single and clone frequency will decrease after injury, due to death of differentiated cells after injury (green curve in left panel). If repair is by self-duplication of terminally differentiated cells (= labeled cells), the clone size will expand through proliferation of single-cell clones during repair (red curve in right panel), resulting in a rightward shift of the clone size–frequency curve. (B) To determine whether an intratubular stem cell contributes to repair at all, we performed dilution analysis. Complete cell labeling in the terminally differentiated proximal tubular was accomplished by high-dose tamoxifen administration. A round of IRI and repair follows. Upon analysis of repaired tubules, if only terminally differentiated cells contribute to renal repair, the labeling will remain undiluted by unlabeled cells (Option 1). If an unlabeled progenitor population contributes to repair of the genetic label will be diluted by progeny of these cells (Option 2). Our results support Option 1, self-duplication, as the only mechanism of proximal tubule repair.
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F I G U R E 1 : Rationale of clonal and dilution analysis using SLC34a1−GFPCreER (SLC34a1GCE/+), Rosa26tomato/+ mice. (A) We first induce
size–frequency curve had shifted smoothly to the right. In Sham kidneys, all clones, were either one or two cells but, after injury, there were few single-cell clones and increased numbers of two-, three- and four-cell clones and some clones reaching more than five cells, indicating that, on average, terminally differentiated cells had undergone one to three cellular divisions following injury [32]. This result strongly suggested that not only can terminally differentiated tubular cells contribute to repair after injury, but that this is a property shared by most and probably all differentiated cells—because of the smooth
rightward shift of the clone size–frequency curve (Figures 1 and 2A). We next asked whether a putative intratubular stem cell population made any contribution at all to epithelial repair after injury. We injected high-dose tamoxifen prior to injury to achieve maximal recombination. In this experimental design, if an unlabeled progenitor cell contributes to proximal tubular repair this would lead to dilution of the labeled terminally differentiated tubular cells following injury. However, we did not observe such a dilution and the fraction of labeled
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F I G U R E 3 : Dueling models for epithelial repair after injury. (A) In the stem/progenitor model, scattered progenitors are located adjacent to
differentiated proximal tubule epithelium. The progenitors express CD24, CD133, Kim-1 and Vimentin, among other markers. After injury, these cells preferentially survive and selectively proliferate. Their progeny differentiate into proximal tubular epithelial cells. (B) In the selfduplication model, any fully differentiated cell that survives the injury has an equivalent capacity to dedifferentiate and proliferate. The progeny then re-differentiate into proximal tubular tubule epithelium. In this case, CD24, CD133, Kim-1 and vimentin are not markers of a separate stem cell compartment, but are injury markers transiently expressed by a dedifferentiated epithelial cell. Thus, new epithelia derive from their fully differentiated neighbors in a process of self-duplication.
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CURRENT QUESTIONS
Table 1. Point-by-point summary of the main conclusions • Following AKI terminally differentiated proximal tubular cells undergo dedifferentiation into STC • Therefore, STCs are not a fixed progenitor population but arise from any surviving proximal tubular cell following injury • Dedifferentiation of tubular epithelial cells results in expression of Kim1, vimentin, annexin A3, src-suppressed C-Kinase substrate, CD44, CD133 and CD24; some of these markers have been previously described as being specific for tubular progenitor cells. • Any surviving dedifferentiated tubular epithelial cell (STC) enters the cell cycle and undergoes a limited number of cell divisions replacing lost neighbor cells during the injury repair process. • The molecular pathways involved in the dedifferentiation, proliferation and redifferentiation process of tubular epithelial cells remain incompletely understood and are the challenge for ongoing and future research.
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AC K N O W L E D G E M E N T This work was supported by NIH DK088923 (to B.D.H.), by an Established Investigator Award of the American Heart Association (to B.D.H.) and by a fellowship the Deutsche Forschungsgemeinschaft (to R.K., KR 4073/1-1).
C O N F L I C T O F I N T E R E S T S TAT E M E N T The results presented in this paper have not been published previously in whole or part, except in abstract format.
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Understanding the mechanisms of tubular injury and repair is the first step for development of novel targeted therapeutics in AKI and its transition into CKD. The therapeutic strategy used to target a pre-existing epithelial stem cell population might be quite different than one aimed at harnessing the proliferative capacity of differentiated tubular cells. The recent evidence strongly points toward terminally differentiated tubular epithelial cells which dedifferentiate upon injury and acquire a STC phenotype with expression of Kim1, vimentin, annexin A3, src-suppressed C-Kinase substrate, CD44 and upregulation of CD133 and CD24 on mRNA level (summarized in Table 1). Therefore, these markers might not be seen as progenitor cell markers but rather markers that indicate dedifferentiation and ‘reprogramming’ of tubular epithelial cells toward a prorepair, antiapoptosis and regenerative phenotype. In addition to our own fate-tracing paper in kidney injury
[32], two other recent papers in stomach and trachea suggest that fully committed mature epithelial cells respond to injury by dedifferentiating, proliferating and redifferentiating into fully functional mature epithelial cells [35, 36]. In fact, these groups present evidence that dedifferentiation represents a state of acquired stemness. This recent work clearly challenges the dogma that stem cell hierarchies are a one-way street from undifferentiated progenitor cells to fully differentiated mature cells and points toward mature epithelial cells that dedifferentiate and acquire a stem cell-like state upon injury. Looking to the future, an important challenge will be to understand this dedifferentiation process and to uncover the molecular pathways that drive the injury and repair of dedifferentiated epithelial cells.
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tubular epithelial cells remained unchanged after injury despite robust proliferation (BrdU incorporation) among genetically labeled cells (Figure 2B) [32]. During the injury phase, labeled proximal tubular cells also gained expression of Pax2 and vimentin protein (Figure 2C and D). By performing fluorescence-activated cell sorting of tdTomato-positive cells, we could also measure upregulated mRNA expression of the putative stem cell markers CD133, CD24 and Kim1 in these cells. This evidence reconciles the prior work on STCs and provides direct evidence that fully differentiated epithelial cells upregulate expression of CD24 and CD133 after injury (Figure 3). Thus, these genes are injury markers—not stem cell markers, as previously concluded [32]. Differences in marker expression between humans and rodents exist and, therefore, it can be argued that the existence of intratubular stem cells has not been disproven in larger mammals [34]. However, the study by Smeets et al. [26] clearly points toward dedifferentiation of terminally differentiated tubular epithelial cells following injury in human kidneys; these dedifferentiated cells coexpress Kim-1, CD133 and CD24 [26], consistent with our results in mouse.
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Received for publication: 21.6.2014; Accepted in revised form: 27.7.2014
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