Identification of Renal Progenitor-Like Tubular Cells that Participate in the Regeneration Processes of the Kidney
Akito Maeshima,
Shin Yamashita and
Yoshihisa Nojima
Third Department of Internal Medicine, Gunma University, School of Medicine, Maebashi, Japan
Correspondence to Dr. Akito Maeshima, Third Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa, Maebashi 371-8511, Japan. Phone: +81-27-220-8166; Fax: +81-27-220-8173; E-mail: amaeshima{at}ucsd.edu
ABSTRACT. The present study was conducted to explore renal progenitor-likecells that are actively engaged in tubular regeneration afterinjury. For addressing this issue, the existence of label-retainingcells (LRC; slow-cycling cells) in normal rat kidneys by invivo bromodeoxyuridine (BrdU) labeling was examined. LRC werescattering among renal epithelial tubular cells of normal ratkidneys. During the recovery after renal ischemia, LRC underwentcell division and most of them became positive for proliferatingcell nuclear antigen. In contrast, proliferating cell nuclearantigen–positive but BrdU-negative tubular cells wererarely observed, suggesting that cells proliferating duringtubular regeneration are essentially derived from LRC. At anearly phase of tubular regeneration, descendants of LRC expresseda mesenchymal marker, vimentin, and eventually became positivefor an epithelial marker, E-cadherin, after multiple cell divisions.These findings suggested that LRC function as a source of regeneratingcells to replace injured cells. Collectively, it was concludedthat LRC are renal progenitor-like tubular cells that provideregenerating cells, which actively proliferate and eventuallydifferentiate into epithelial cell, during tubular regeneration.It may be possible to regenerate renal tubules in vivo throughthe activation of LRC.
In general, stem cells have a slow turnover and display minimalphysiologic differentiation. As early descendants of stem cells,transit-amplifying (TA) cells retain significant growth capacitywhile acquiring differentiated functions. TA cells eventuallybecome incapable of proliferation and enter a terminally differentiatedstate (1–4). To conserve growth potential and to preventgenetic injury during mitosis, stem cells are thought to cycleslowly and are recruited only as demanded by tissue turnover.Therefore, much of the increase in cell number in the steadystate occurs in the TA population. One of the most common methodsto identify stem cells is to search for slow-cycling cells bylabeling their DNA. A pulse of bromodeoxyuridine (BrdU) mostlylabels TA cells. Long-term BrdU labeling is thought to markstem cells that retain the label for an extended period as aresult of slow turnover. An adequate labeling intensity anda suitable washout period of the TA and terminal differentiationcompartments thus will result in so-called label-retaining cells(LRC), believed to represent the stem cell compartment. Usingthis method, slow-cycling stem cells have been identified inseveral tissues, including skin (5), cornea (6), intestine (7),lung (8), and prostate (9), and were shown to be involved intissue regeneration.
In the kidney, renal epithelial tubular cells are known to proliferateactively and differentiate to reconstitute tubular epitheliumduring the recovery from a variety of insults (10). To date,several studies have suggested the existence of renal progenitor-liketubular cells (11–13). In response to renal injury, numeroustubular cells proliferate and some express a mesenchymal marker,vimentin, suggesting that they are of an immature phenotype(13). These proliferating cells also express a transcriptionfactor critical for kidney development, Pax-2 (11,12). Consideringthat regeneration processes may recapitulate developmental paradigmsto restore organ or tissue function, it is possible that thesecells are renal progenitor-like tubular cells. However, mostof these cells seem to represent TA cells that replace damagedcells, because they have a high potential to proliferate. Renalstem cells in adult kidney have not been identified definitively.
The present study demonstrated the existence of LRC in tubularcells of normal rat kidneys. During the recovery after renalischemia, LRC underwent cell division, and most of them becamepositive for proliferating cell nuclear antigen (PCNA). At anearly phase of tubular regeneration, descendants of LRC expressedvimentin and eventually became positive for E-cadherin. Thesefindings suggested that descendants of LRC with an immaturephenotype actively proliferate and consequently differentiateinto epithelial tubular cells during tubular regeneration. Thesedata will provide new insights into the mechanism of tubularregeneration after a variety of insults.
Materials
Polyclonal mouse anti-vimentin antibody and polyclonal rabbitanti-cytokeratin antibody were obtained from NEO MARKERS (Fremont,CA) and DAKO (Glostrup, Denmark), respectively. Polyclonal rabbitanti–Tamm Horsfall glycoprotein was obtained from BiomedicalTechnologies (Stoughton, MA). FITC-labeled Lotus tetragonolobusagglutinin and TRITC-labeled Dolichos Biflorus agglutinin werepurchased from Vector Laboratories (Burlingame, CA) and EY Laboratories(San Mateo, CA), respectively. Monoclonal mouse or rat anti-BrdUantibody was obtained from Amersham Bioscience (Tokyo, Japan)and AbCam (Cambridge, UK), respectively. Polyclonal rabbit anti–E-cadherinantibody, goat anti-human PCNA antibody, and goat anti–aquaporin-2antibody were obtained from Santa Cruz Biotechnology (SantaCruz, CA).
BrdU Labeling
Male Wistar rats that weighed 200 to 230 g were obtained fromNihon SLC, (Hamamatsu, Japan). LRC were detected by BrdU labeling.BrdU (100 mg/kg), an analogue of thymidine, was injected intraperitoneallyinto normal rats daily for 1 wk. After 2 wk, rats were killed,and the kidneys were removed and embedded in paraffin. At thisdose, the animals seemed healthy, with normal kidney histologyduring the entire course of the experiments. Four-micrometersections were immunostained using a cell proliferation kit (Amersham,Tokyo, Japan) according to the manufacturer’s instructionsand counterstained with periodic acid-Schiff or hematoxylin.
Ischemia/Reperfusion Injury
Ischemia/reperfusion injury was performed as described previously(14). Briefly, under anesthesia with pentobarbital sodium (30mg/kg body wt), renal ischemia was induced by clamping bothrenal arteries for 45 min using a nontraumatic vascular clamp.After removal of the clamp to allow reperfusion for the indicatedperiods, rats were killed and the kidneys were removed for histologicanalysis. Sham operations were performed in a similar manner,except without clamping the renal arteries.
Immunohistochemistry
Immunohistochemical analysis via an avidin-biotin coupling immunoperoxidasetechnique was performed using a Vectastain Elite ABC kit (VectorLaboratories) according to the manufacturer’s instructions(15). Briefly, kidneys were fixed in 4% formaldehyde and embeddedin paraffin. Four-micrometer sections were deparaffinized andrehydrated in a routine manner. After inactivation of endogenousperoxidase with 1% metaperiodic acid in PBS for 10 min at roomtemperature, sections were preincubated with normal goat/rabbitserum for 1 h. Sections were then incubated with primary antibodyfor 2 h, washed with PBS, and reacted with a biotinylated rabbitanti-goat IgG or a biotinylated goat anti-mouse IgG for 1 h.After washing with PBS, sections were reacted with VectastainElite ABC Reagent. Antibody was detected with diaminobenzidinetetrahydrochloride in PBS, and sections were counterstainedwith hematoxylin. For immunohistochemical controls, the primaryantibody was replaced with normal goat/rabbit serum, which didnot show positive staining, thus confirming specificity. Ina separate experiment, indirect fluorescence immunostainingwas also performed as described previously (12).
Quantification of BrdU and/or PCNA-Positive Cells
Quantitative analysis of BrdU-positive cells was performed bycounting the positive nuclei in tubular cells from five randomlyselected fields of the outer medulla under a light microscopeat x200. The average of the five determinants was calculatedand was recorded as the number of BrdU-positive cells per squaremicrometer. The results were expressed as the proliferationindex. Quantification of PCNA-positive cells was performed ina similar manner. In case of indirect fluorescence staining,quantification of BrdU+/PCNA+, BrdU+/PCNA-, and BrdU-/PCNA+cells was performed by counting positive nuclei from five randomlyselected fields of the kidneys under a fluorescence microscopeat x400. The results were expressed as a percentage of totalcells of five sections per rat. The average of five determinationswas calculated (15).
Statistical Analysis
The differences between means were compared by t test, withP < 0.05 considered significant.
Existence of LRC in Normal Rat Kidneys
To localize slow-cycling cells in normal rat kidneys, we performeda series of kinetic analyses and identified them as the LRC.BrdU was injected intraperitoneally into normal rats daily for7 d, and kidneys were removed immediately after the final administration.As shown in Figure 1A, BrdU-positive cells were observed innormal rat kidney. BrdU-positive cells were localized in tubuli(Figure 1B), glomeruli (Figure 1C), and peritubular capillaries(Figure 1D). For excluding rapidly cycling cells, BrdU was injectedintraperitoneally into normal rats daily for 7 d and kidneyswere removed after a 2-wk chase period. As shown in Figure 1E,LRC were still detectable scattered in proximal and distal tubuli.Of particular interest was that most of these were adjacentto capillary endothelial cells (Figure 1F). LRC were also localizedin collecting ducts but were not detected in glomeruli and capillaryvessels (data not shown). To examine further the distributionof LRC in the kidney, we performed double staining of BrdU andseveral markers for nephron segment (Figure 2). Most of theLRC were co-localized with Lotus tetragonolobus agglutinin,a lectin that labels specifically proximal tubules. However,few LRC were co-localized with Tamm Horsfall glycoprotein, whichis expressed in the thick ascending limb of Henle and distaltubules. Some of the LRC were also present in collecting ductsthat stained positive with Dolichos Biflorus agglutinin butwere undetectable in aquaporin-2–expressing cells. Theseresults indicate that LRC are present in renal epithelial tubularcells of normal rat kidneys.
Figure 1. Localization of label-retaining cells (LRC) in normal rat kidneys. Bromodeoxyuridine (BrdU) was injected intraperitoneally into normal rats once a day for 7 d, and kidneys were removed immediately after the final injection (A to D) or after a 2-wk chase period (E and F). BrdU staining was performed as described in Materials and Methods. Arrowheads in C and D indicate BrdU-positive cells. Arrows in F indicate capillary endothelial cells. PT, proximal tubule; DT, distal tubule. Bars = 50 µm in A and 20 µm in B to F).
Figure 2. Distribution of LRC in nephron segments of normal rat kidneys. BrdU was injected intraperitoneally into normal rats once a day for 7 d, and kidneys were removed after a 2-wk chase period. Localization of Lotus tetragonolobus agglutinin, Tamm Horsfall glycoprotein, Dolichos Biflorus agglutinin, aquaporin-2, and BrdU were examined by indirect fluorescence staining. DAPI (blue). Bars = 50 µm.
Increase in the Number of LRC in the Kidneys after Renal Ischemia
We next examined the involvement of LRC in tubular regeneration.BrdU was injected intraperitoneally into normal rats for 7 d.After a 2-wk chase period, we induced ischemia/reperfusion injuryin these rats, and kidneys were removed at the indicated periods.There was no significant difference in the number of LRC betweennormal (Figure 3A) and sham-operated kidneys (Figure 3B). Asshown in Figure 3C, LRC were abundantly detected in the ischemickidney at 24 h after reperfusion. It should be stressed thatmany BrdU-positive cells were found in clusters of two cells(Figure 3D). Quantitative analysis showed that the number ofLRC increased by twofold at 24 h after reperfusion (Figure 3E).
Figure 3. Increase in the number of LRC in the kidney after renal ischemia. BrdU was injected intraperitoneally into normal rats once a day for 7 d. After a 2-wk chase period, ischemia/reperfusion injury was induced in these rats and the kidneys were removed at the indicated periods after reperfusion. BrdU-positive cells were examined by immunohistochemistry. (A) Normal kidneys. (B) Sham-operated kidneys. (C and D) Ischemic kidneys at 24 h after reperfusion. Arrowheads in D indicate a cluster of BrdU-positive cells. Bars = 50 µm in A to C and 20 µm in D. (E) Quantitative analysis of LRC in the kidney after renal ischemia. Proliferation index was assessed as described in Materials and Methods. •, ischemic kidneys; , sham-operated kidneys. Values are means ± SE (n = 5).
Descendents of LRC Function as TA Cells during Tubular Regeneration
As an alternative approach to detect proliferating cells inthe kidney after renal ischemia, we examined the localizationof tubular cells positive for PCNA, which specifically recognizesthe early G1 and S phases of the cell cycle. There were fewPCNA-positive cells in normal (Figure 4A) and sham-operatedkidneys (Figure 4D). In contrast, a number of PCNA-positivecells were observed among tubular cells in ischemic kidneysat 12 h after reperfusion (Figure 4B). At 24 h after reperfusion,numerous PCNA-positive cells were observed in the outer medullaof ischemic kidneys (Figure 4C). Quantitative analysis showeda significant increase in the number of PCNA-positive cellsin the kidney after renal ischemia (Figure 4E).
Figure 4. Increase in the number of proliferating cell nuclear antigen (PCNA)-positive cells in the kidney after renal ischemia. BrdU was injected intraperitoneally into normal rats once a day for 7 d. After a 2-wk chase period, ischemia/reperfusion injury was induced and rats were then killed at the indicated periods. PCNA-positive cells were detected by immunohistochemistry. (A) Normal kidneys. (B) Ischemic kidneys at 12 h after reperfusion. (C) Ischemic kidneys at 24 h after reperfusion. (D) Sham-operated kidneys. Bars = 50 µm. (E) Quantitative analysis of PCNA-positive cells in the kidney after renal ischemia. Proliferation index was assessed as described in Materials and Methods. Values are means ± SE (n = 5).
We then compared the localization of LRC with that of PCNA-positivecells in the kidney after renal ischemia by double immunostaining.LRC were not positive for PCNA in normal kidneys and ischemickidneys at 12 h after reperfusion (data not shown). However,at 24 h after reperfusion, more than two thirds of LRC werepositive for PCNA (Figure 5, A to E). In contrast, very fewPCNA-positive cells lacking BrdU labeling were observed (Figure 5F).
Figure 5. Co-localization of LRC and PCNA-positive cells in the kidney after renal ischemia. Localization of PCNA-positive cells and BrdU-positive cells in ischemic kidneys at 24 h after reperfusion was examined by indirect fluorescence staining. (A) BrdU. (B) PCNA. (C) Merge image. (D) DAPI. (E) Nomarski image. Bars = 20 µm. (F) Quantitative analysis of PCNA-/BrdU+, PCNA+/BrdU-, and PCNA+/BrdU+ cells in ischemic kidneys at 24 h after reperfusion. Values are the mean ± SE (n = 8).
To investigate further the cell kinetics of LRC after the proliferationphase, we investigated the existence of LRC in ischemic kidneysat 10 d after reperfusion. At this time point, PCNA-positivecells were no longer detectable in ischemic kidneys (data notshown), indicating the completion of the proliferation phase.As shown in Figure 6A, numerous LRC were still present in sham-operatedkidneys. Quantitative analysis revealed that there was no significantdifference in the number of LRC between normal and sham-operatedkidneys (Figure 6C). LRC were also detectable in ischemic kidneysat 10 d after reperfusion, although their numbers were relativelysmall when compared with those of normal and sham-operated kidney(Figure 6, B and C).
Figure 6. Decrease in LRC in ischemic kidneys at 10 d after reperfusion. BrdU was injected intraperitoneally into normal rats once a day for 7 d. After a 2-wk chase period, ischemia/reperfusion injury was performed and kidneys were removed at 10 d after reperfusion. BrdU-positive cells were detected by immunohistochemistry. (A) Sham-operated kidneys. (B) Ischemic kidneys at 10 d after reperfusion. Bars = 50 µm. (C) Quantitative analysis of BrdU-positive cells in normal, sham-operated (sham), and ischemic kidneys at 10 d after reperfusion (I/R). Values are mean ± SE (n = 8); *P < 0.01 versus sham-operated kidneys.
Immature Mesenchymal Phenotype of Descendants of LRC during Tubular Regeneration
To characterize the phenotype of LRC, we examined the expressionof a mesenchymal marker, vimentin, in the kidneys of rats withBrdU labeling after ischemia/reperfusion injury. In normal kidneys,neither LRC nor nonlabeled tubular cells expressed vimentin(data not shown). In contrast, among the pairs of LRC foundin ischemic kidneys at 18 h after reperfusion, one of each pairexpressed vimentin, whereas the other did not (Figure 7, a to d).At 24 h after reperfusion, vimentin was expressed in cellsweakly positive for BrdU but not in those strongly labeled (Figure 7, e to h).At 10 d after reperfusion, vimentin was broadlyexpressed in tubular cells in the outer medulla of ischemickidneys (Figure 7j). Some scattered tubular cells retained BrdUstaining in ischemic kidneys at 10 d after reperfusion (Figure 7i).However, at this time point, LRC barely expressed vimentin,although they were adjacent to vimentin-expressing cells (Figure 7k).
Figure 7. Immature phenotype of descendants of LRC in the kidney after renal ischemia. BrdU was injected intraperitoneally into normal rats once a day for 7 d. After a 2-wk chase period, ischemia/reperfusion injury was induced in these rats and kidneys were removed at 18 h (a to d), 24 h (e to h), and 10 d (i to l, m to p, and q to t) after reperfusion. Localization of BrdU (a, e, i, and m), vimentin (b, f, j, and q), and E-cadherin (n and r) were examined by indirect fluorescence staining. DAPI (blue). (c, g, k, o, and s) Merge images. (d, h, l, p, and t) Nomarski images. Bars = 20 µm.
Differentiation of Descendants of LRC after Proliferation Phase
To investigate further the differentiation of LRC and theirdescendants, we examined the expression of an epithelial marker,E-cadherin, in ischemic kidneys at 10 d after reperfusion. E-cadherinwas expressed in the plasma membrane of some tubular cells inischemic kidneys at 10 d after reperfusion (Figure 7, n and r)but not in normal kidneys (data not shown). Similar to thelocalization of vimentin, E-cadherin was also expressed by cellslying close to LRC (Figure 7, m to p). LRC surrounded by E-cadherin–expressingcells did not express E-cadherin. Moreover, the expression ofE-cadherin and vimentin seems continuous but exclusive (Figure 7, q to t),indicating the various differentiation stages ofregenerating cells.
In the present study, we demonstrated that BrdU was incorporatedinto tubular cells, mesangial cells, and capillary endothelialcells in the kidneys of normal rats after daily injection ofBrdU for 1 wk. In addition, after a 2-wk chase period, BrdU-positivecells were no longer detectable in glomeruli or capillary vesselsin these rats (Figure 1), suggesting that these cells have arapid turnover. However, PCNA-positive cells were not detectedin glomeruli or capillary blood vessels in normal rat kidneys(data not shown), indicating that resident cells constitutingglomeruli or capillary vessels are quiescent. Considering thatproliferating cells are generally detectable by PCNA stainingwhen cell turnover is rapid and PCNA is adequately concentratedinto nuclei, it is possible that BrdU-positive mesangial cellsor capillary endothelial cells are rapidly cycling but the contentof PCNA in their nuclei is not enough to detect them by PCNAstaining. In the present study, we cannot distinguish whetherBrdU-positive mesangial cells or capillary endothelial cellsare resident renal cells or extrarenal cells, but there is somesuggestive evidence about their origin. In the adult, tissueregeneration was thought to occur through the action of tissue-restrictedstem cells. However, it is now believed that stem cells fromone organ system can develop into differentiated cells withinanother organ system (16). Consistent with this notion, it hasbeen reported that bone marrow–derived progenitor cellscould be substituted for mesangial cells (17–19) or vascularendothelial cells (20,21). Therefore, the possibility cannotbe denied that BrdU-positive mesangial cells or capillary endothelialcells are derived from bone marrow. Further study will be neededto clarify this issue. In contrast to mesangial and endothelialcells, LRC are clearly detected in renal tubules (Figures 1 and 2). Recent studies showed that renal tubular cells are substitutedby bone marrow–derived cells under nonphysiologic conditionsincluding renal (22,23) or bone marrow (24) transplantations.Therefore, it is also unknown yet whether LRC represent residenttubular cells or cells derived from bone marrow. Nevertheless,our current study demonstrated for the first time that LRC werepresent in renal tubuli of normal rat kidneys.
Among our results was the novel finding that the number of LRCsignificantly increased in the kidney after renal ischemia (Figure 3).At 24 h after reperfusion, there were numerous PCNA-positivetubular cells in the outer medulla of ischemic kidneys (Figure 4),indicating that renal epithelial tubular cells have thepotential to proliferate in response to ischemic injury. Importantly,most PCNA-positive cells were labeled with BrdU as well (Figure 5).In contrast, PCNA-positive but BrdU-negative cells wererarely observed (Figure 5). Collectively, these results indicatethat the majority of proliferating cells involved in recoveryprocesses in the kidney after renal ischemia are derived fromLRC.
Stem cells are a self-maintaining population. During tissueregeneration, stem cells are considered to divide asymmetricallyinto stem cells and transit-amplifying cells, the latter ofwhich vigorously proliferate, differentiate, and finally reconstitutepart of the tissue (25). At each asymmetric cell division, stemcells are postulated to retain selectively a set of chromosomesthat contain old template DNA strands (26). In the present study,we observed that the BrdU staining frequently displayed unevendistribution (Figure 7). Furthermore, at 10 d after reperfusion,when tubular cells are presumed to have completed multiple cellsdivisions, some LRC were still clearly detectable (Figures 6 and 7). At present, we cannot exclude the possibility that someLRC, which did not divide after renal injury, remain as BrdU-positivecells. However, if this concept of asymmetrical cell divisionmight be applicable to the dynamics of LRC in regeneration processesof the kidney, then the present findings suggest a differencein nuclear BrdU content between daughter cells as a result ofasymmetric cell division and raise the possibility that LRCare programmed to divide asymmetrically during tubular regeneration,thereby retaining the BrdU staining for longer periods. On thebasis of this assumption, we regarded tubular cells that areweakly positive for BrdU and are localized proximal to cellsstrongly positive for BrdU as descendants of LRC. Descendantsof LRC expressed vimentin during the early phases of tubularregeneration (Figure 7). In addition, LRC were surrounded byvimentin-expressing cells after the cell proliferation phasehad subsided (Figure 7). At this time, vimentin-expressing cellswere no longer labeled with BrdU, presumably because multiplecell divisions caused BrdU staining to fade. We also observedthat E-cadherin was expressed in tubular cells localized nearthe LRC in ischemic kidneys at 10 d after reperfusion (Figure 7),suggesting the eventual differentiation of descendants toepithelial cells. Taken together, it is possible that LRC undergoasymmetrical cell division and that their descendants acquireimmature mesenchymal phenotype as well as a high potential toproliferate and differentiate into epithelial tubular cellsand function as TA cells during tubular regeneration.
LRC phenotype remains unknown at present. Morphologically, LRCwere localized in mature tubular cells (Figure 1) and were co-localizedwith some markers for nephron segment (Figure 2), suggestingthat LRC are in a differentiated state. However, these findingscannot totally exclude the possibility that LRC are undifferentiated.Although there might be no morphologic difference between LRCand other tubular cells except the degree of DNA synthesis progression,we consider that there must be some differentiation markersthat express in all epithelial tubular cells but LRC. Such specificmarkers for LRC may be important molecules involved in multiplecellular processes during differentiation such as cytoskeletalreorganization, cell adhesion, and epithelial polarization ormay also be physiologically functional proteins, for example,amino acid or ion transport. Identification of selective markersthat are positive or negative in LRC will be requisite for usto isolate LRC from tubular cells of normal kidneys and to characterizethe mode of cell growth and differentiation of LRC in vitro.Renal epithelial tubular cells are thought to express an epithelialmarker, E-cadherin, in a steady state. However, we could detectE-cadherin in regenerating kidneys after ischemia/reperfusioninjury but not in normal rat kidneys. This may be because itsexpression level is too low to detect in normal kidney or poorsensitivity of antibody used.
In extensively studied tissues such as the epidermis (5,27,28),intestinal epithelium (7,29), and cornea (6,30), LRC residein specialized and generally well-protected niches that arespatially proximal to their more differentiated progeny. Theseniches are believed to be a subset of tissue cells and extracellularsubstrates that can indefinitely house one or more stem cellsand control their self-renewal and progeny production (3,4).We demonstrated here that most LRC are associated with capillaryendothelial cells (Figure 1). Our recent studies demonstratedthat activin A, a member of the TGF- superfamily, inhibited,whereas follistatin, an antagonist of activin A, promoted theproliferation of renal epithelial progenitor-like cells duringtubular regeneration (12,14,31). Because vascular endothelialcells are known to produce activin A (32), it is possible thatactivin A produced by capillary endothelial cells play a rolein maintaining LRC in a quiescent state.
In summary, we demonstrated the presence of LRC in normal ratkidney. During tubular regeneration, LRC act as a source ofregenerating cells that have an immature phenotype, activelyproliferate, and consequently differentiate into epithelialtubular cells. From the aspect of regenerative medicine, identificationof the factors regulating cell growth and differentiation ofLRC is likely to be a very important issue, and such factorsmay become effective therapeutic agents that accelerate tubularregeneration. Transplantation of LRC into damaged kidneys mayalso provide a new therapeutic approach for kidney disease inthe future, provided that a technique for isolating as wellas propagating LRC in vitro is established. At all events, thepresent results will provide new insights into the mechanismof tubular regeneration after injury.
Acknowledgments
This study was supported by a Grant-in-Aid for Exploratory Researchfrom the Ministry of Education, Science, Culture and Sportsof Japan.
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Received for publication March 25, 2003.
Accepted for publication September 3, 2003.
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Top
Abstract
Introduction
). Recent studies showed that renal tubular cells are substituted by bone marrow–derived cells under nonphysiologic conditions including renal (22,23) or bone marrow (24) transplantations. Therefore, it is also unknown yet whether LRC represent resident tubular cells or cells derived from bone marrow. Nevertheless, our current study demonstrated for the first time that LRC were present in renal tubuli of normal rat kidneys. 
superfamily, inhibited, whereas follistatin, an antagonist of activin A, promoted the proliferation of renal epithelial progenitor-like cells during tubular regeneration (12,14,31). Because vascular endothelial cells are known to produce activin A (32), it is possible that activin A produced by capillary endothelial cells play a role in maintaining LRC in a quiescent state. 
, sham-operated kidneys. Values are means ± SE (n = 5).
