Persistent and inducible neogenesis repopulates progenitor renin lineage cells in the kidney
2017; Elsevier BV; Volume: 92; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2017.04.014
ISSN1523-1755
AutoresLinda Hickmann, Anne Steglich, Michael Gerlach, Moath Al-Mekhlafi, Jan Sradnick, Peter J. Lachmann, Maria Luisa S. Sequeira-Lοpez, R. Ariel Gómez, Bernd Hohenstein, Christian Hugo, Vladimir Todorov,
Tópico(s)Birth, Development, and Health
ResumoRenin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow–derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis (de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury. Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow–derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis (de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury. Renin is a limiting enzyme of the circulating endocrine branch of the renin-angiotensin system (RAS).1Castrop H. Hocherl K. Kurtz A. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (195) Google Scholar The RAS is a key player in the control of arterial blood pressure. In adulthood, renin is produced by juxtaglomerular cells in the afferent arterioles (AAs) of the kidney. A small amount of renin mRNA is also expressed by multiple extrarenal cell populations as a part of the tissue RASs, which are believed to modulate the local effects of the systemic RAS.2Paul M. Poyan Mehr A. Kreutz R. Physiology of local renin-angiotensin systems.Physiol Rev. 2006; 86: 747-803Crossref PubMed Scopus (1346) Google Scholar During development, renin is markedly expressed throughout the kidney in precursor cells, which differentiate into mural (vascular smooth muscle cells and pericytes), glomerular, tubular, and interstitial cells.3Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 4Gomez R.A. Chevalier R.L. Sturgill B. et al.Maturation of the intrarenal renin distribution in WistarKyoto rats.J Hypertens. 1986; 4: S31-S33Google Scholar Collectively, these form the renal renin lineage cell (RLC) pool, and the renin-producing cells (or renin-positive cells) in AAs of the adult kidney comprise only a minor portion of this pool (approximately 0.01% of the total kidney cell mass). The RLC pool is central to nephrogenesis because the inactivating renin mutations in both humans and mice can lead to glomerulosclerosis, arterial thickening, and tubular dysgenesis, consequently resulting in the deterioration of renal function and early death.5Gribouval O. Gonzales M. Neuhaus T. et al.Mutations in genes in the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis.Nat Genet. 2005; 37: 964-968Crossref PubMed Scopus (194) Google Scholar, 6Takahashi N. Lopez M.L. Cowhig Jr., J.E. et al.Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type.J Am Soc Nephrol. 2005; 16: 125-132Crossref PubMed Scopus (114) Google Scholar The developmental pattern of renin expression is partially recapitulated in adulthood when after chronic stimulation (e.g., angiotensin II antagonism, arterial hypotension, etc.), the renin gene is switched on in vascular smooth muscle cells of the renin lineage within the upstream portions of AAs.1Castrop H. Hocherl K. Kurtz A. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (195) Google Scholar, 3Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar This process is reversible and is known as metaplastic transformation, which is a confusing term implying pathology, when in fact, it is a normal reenactment of the embryonic pattern by cells previously capable of synthesizing renin. Thus, renin expression in the kidney involves spatiotemporal plasticity, which is preserved in adult life. Importantly, pharmacologic RAS inhibition (RASi), which is the first choice of renoprotective therapy,7Ruggenenti P. Cravedi P. Remuzzi G. Mechanisms and treatment of CKD.J Am Soc Nephrol. 2012; 23: 1917-1928Crossref PubMed Scopus (186) Google Scholar, 8Turner J.M. Bauer C. Abramowitz M.K. et al.Treatment of chronic kidney disease.Kidney Int. 2012; 81: 351-362Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 9Wuhl E. Schaefer F. Managing kidney disease with blood-pressure control.Nat Rev Nephrol. 2011; 7: 434-444Crossref PubMed Scopus (31) Google Scholar potently induces the recruitment of renin-producing cells in AAs,1Castrop H. Hocherl K. Kurtz A. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (195) Google Scholar, 3Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar raising the intriguing possibility that the beneficial effects of RAS inhibitors are at least in part because of the increased number of renin-positive RLCs in the kidney. We recently found that renin-positive RLCs differentiate into renin-negative intraglomerular mesangial cells in a mouse model of mesangial proliferative glomerulonephritis.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar Findings of other studies demonstrated that RLCs are also able to give rise to further glomerular cells after injury, implying that RLCs generally serve as a progenitor cell niche.11Pippin J.W. Kaverina N.V. Eng D.G. et al.Cells of renin lineage are adult pluripotent progenitors in experimental glomerular disease.Am J Physiol Renal Physiol. 2015; 309: F341-F358Crossref PubMed Scopus (45) Google Scholar, 12Pippin J.W. Sparks M.A. Glenn S.T. et al.Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease.Am J Pathol. 2013; 183: 542-557Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Thus, RLCs have important structural and functional roles in the adult kidney, which are not related to classical RAS function (i.e., blood pressure control). Current experimental evidence clearly demonstrates that at least some RLCs retain their differentiating capacity in adult life. However, whether and how the renal RLC niche is replenished after nephrogenesis remains unknown. Here, we attempted to assess 2 mechanisms that might be responsible for maintaining the adult kidney RLC population, namely recruitment of bone marrow (BM) cells and local neogenesis (referred to as de novo differentiation). For this assessment, we generated BM chimeric mice and studied double transgenic mice in which RLC neogenesis could be quantified. We found that BM cells do not differentiate into renal RLCs of the adult kidney. Instead, intrarenal neogenesis of RLCs (defined as switching on of the renin gene in a cell that has never before expressed renin) was detected. RLC neogenesis existed at a relatively constant level, beginning early in nephrogenesis and persisting in adulthood. Importantly, the rate of RLC neogenesis could be stimulated by pharmacologic and damaging factors in adulthood, thus underscoring the regenerative capacity of mature mammalian kidneys. During nephrogenesis, RLCs develop into different cell types of mature kidneys. Double transgenic mice expressing Cre recombinase from the endogenous renin locus (Ren1d) and the mT/mG cassette from the Rosa26 locus (mRenCre-mT/mG) were used to trace RLCs in adult kidneys. The mT/mG construct switches from membrane-directed fluorescent tomato protein (mT, red) to membrane-directed enhanced green fluorescent protein (eGFP; mG) expression after Cre-mediated recombination.13Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2256) Google Scholar Because gene expression from the Rosa26 locus is ubiquitous and gene switch is stably transferred to cell progeny, all RLCs are mG positive, while all non-RLCs remain mT positive. Using this mouse model and confocal laser scanning microscopy, we proved that many kidney cell types develop from RLCs. Renin cells in their classical juxtaglomerular position comprise only a small portion of all RLCs in adult mouse kidneys (Figure 1a and b). In addition to renin cells, RLCs (mG+) include glomerular cells such as some mesangial and parietal epithelial cells (Figure 1c), as well as extraglomerular cells (a subset of tubular cells, NG2-positive interstitial cells, and vascular smooth muscle cells) (Figure 1d). Neither podocytes nor endothelial cells appear to be of RLC origin (Figure 1c and d). These findings are in complete agreement with those previously reported.3Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 14Hu Y. Li M. Gothert J.R. et al.Hemovascular Progenitors in the Kidney Require Sphingosine-1-Phosphate Receptor 1 for Vascular Development.J Am Soc Nephrol. 2016; 27: 1984-1995Crossref PubMed Scopus (27) Google Scholar, 15Sequeira-Lopez M.L. Nagalakshmi V.K. Li M. et al.Vascular versus tubular renin: role in kidney development.Am J Physiol Regul Integr Comp Physiol. 2015; 309: R650-R657Crossref PubMed Scopus (23) Google Scholar Using the mRenCre-mT/mG strain, we assessed whether BM could be a source of RLCs in adult kidneys. This was a plausible assumption because a pool of renin-positive progenitor RLCs was recently identified in BM of adult mice, and they might have the potential to give rise to renal RLCs.16Belyea B.C. Xu F. Pentz E.S. et al.Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.Nat Commun. 2014; 5: 3273Crossref PubMed Scopus (25) Google Scholar According to previous findings, populations of RLCs (mG+) were detected by flow cytometry in BM, peripheral blood, and spleen of mRenCre-mT/mG mice (Figure 2a).16Belyea B.C. Xu F. Pentz E.S. et al.Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.Nat Commun. 2014; 5: 3273Crossref PubMed Scopus (25) Google Scholar BM-derived mG+ cells comprised approximately 1% of BM and 10% of blood and spleen cells (Figure 2b). In agreement with earlier data, most BM-derived RLCs (60%–90%) were positive for B-lymphocyte markers (Supplementary Figure S1).16Belyea B.C. Xu F. Pentz E.S. et al.Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.Nat Commun. 2014; 5: 3273Crossref PubMed Scopus (25) Google Scholar These findings delineate an existing pool of BM-derived RLCs, which we hypothesized to potentially serve as a reservoir for RLCs in the kidney. To test whether BM-derived RLCs may give rise to RLCs in adult kidneys, we transplanted BM from mRenCre-mT/mG mice into irradiated wild-type mice with the same genetic background (C57BL/6, Figure 3a). Two months after BM transplantation (BMTx), BM-derived RLCs were clearly identified in BM, blood, and spleen of the recipient mice, thus confirming that BMTx was efficient (Figure 3b and c). Using fluorescence microscopy, we failed to detect BM-derived RLCs in the kidney of recipient mice despite normal distribution of resident renin-positive cells (Figure 4a). These findings were supported by similar findings 6 months after BMTx and were confirmed by flow cytometry that detected only few BM-derived cells in kidneys. However, these cells were entirely positive for the hematopoietic marker cluster of differentiation (CD)45, were not enriched at the vascular glomerular pole, and were stained negative for renin, indicating low level influx of inflammatory cells but no contribution to the renal RLC niche (Supplementary Figure S2).Figure 4Bone marrow (BM)–derived renin lineage cells (RLCs) are not detected in kidneys of recipient mice after bone marrow transplantation (BMTx). (a) No BM-derived RLCs (mG+) in the kidney sections of control untreated recipient mice 2 months after BMTx (left panel), while the typical juxtaglomerular expression pattern of the renin protein was detectable (right panel). (b,c) No BM-derived RLCs (mG+) in the kidney sections of recipient mice 2 months after BMTx and after enalapril treatment (10 mg/kg for 7 days, b) or mesangial cell injury (10 days, c). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Similarly, pharmacologic RASi (treatment with the angiotensin-converting enzyme [ACE] inhibitor enalapril) or induction of reversible mesangiolysis1Castrop H. Hocherl K. Kurtz A. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (195) Google Scholar, 10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar, 17Desch M. Harlander S. Neubauer B. et al.cAMP target sequences enhCRE and CNRE sense low-salt intake to increase human renin gene expression in vivo.Pflugers Arch. 2011; 461: 567-577Crossref PubMed Scopus (13) Google Scholar did not lead to engraftment of BM-derived RLCs in the kidney or replacement of resident cells (Figure 4b and c, respectively). Thus, we concluded that RLCs in adult mouse kidneys do not originate from BM cells under physiologic or pharmacologic conditions or after kidney damage. Next, we considered the possibility that RLCs develop through intrarenal neogenesis. For comparison and for maintaining consistency, we sought to address this assumption using the same double transgenic mRenCre-mT/mG mice as used previously (Figure 5a). During the Cre-induced transition from mT to mG expression in mRenCre-mT/mG mice, the onset of mG labeling started within the first day of the transcriptional switch, whereas the mT protein was still detectable for at least 1 to 2 days subsequently (Figure 5a).13Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2256) Google Scholar, 18Xiao X. Chen Z. Shiota C. et al.No evidence for beta cell neogenesis in murine adult pancreas.J Clin Invest. 2013; 123: 2207-2217Crossref PubMed Scopus (160) Google Scholar Within this period, cells are mT+mG+ double positive and later become only green (mG+). Newly differentiated RLCs, which express renin for the first time, will express both fluorophores (mT+mG+), whereas long-lived RLC and their descendants will be green (mT−mG+). Using quantitative flow cytometry analysis and the gating provided in Supplementary Figure S3, we detected progressive increases in the relative amount of RLCs (mG+) during nephrogenesis (Figure 5b, left panel and c). RLCs were first observed at embryonic day e14, formed a clearly detectable population in flow cytometric analysis at embryonic day e16, and peaked after postpartum day pp10. After the end of nephrogenesis (pp7–pp10 in mice), the percentage of RLCs remained constant at approximately 10% of all kidney cells until 8 months of age. The time course of RLC neogenesis rate during kidney development fluctuated at 0.25% to 0.5% of all kidney cells until pp5 and peaked from pp7 to pp20, reaching above 1% of all kidney cells (Figure 5b, right panel and c). The peak in the de novo differentiation of RLCs around the end of nephrogenesis timely precedes the steepest increase in the percentage of RLCs, indicating that this process mechanistically represents the essential driver of the maximal RLC pool expansion. Unexpectedly, RLC neogenesis continued in the adult kidney at a remarkable rate of approximately 0.2% to 0.3% of all kidney cells. Within the mG+ RLC pool, neogenesis dominated during the embryonic state and gradually decreased after birth, displaying a marked decrease after pp10 because of RLC pool expansion (Supplementary Figure S4). Apparently, persisting RLC neogenesis in the mature kidney is a central mechanism for compensating for apoptotic cell death occurring in RLCs, as demonstrated in Supplementary Figure S5. Using confocal microscopy, we detected mT+mG+ cells at e16 in vessel-like structures of periglomerular areas (Figure 5d), as well as inside glomeruli (Supplementary Figure S6). In adult mice, mT+mG+ cells were observed at the vascular pole of the glomerulus (Figure 5e). Renin-producing cells in AAs form a distinct subpopulation with established precursor characteristics within the RLC pool.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar, 11Pippin J.W. Kaverina N.V. Eng D.G. et al.Cells of renin lineage are adult pluripotent progenitors in experimental glomerular disease.Am J Physiol Renal Physiol. 2015; 309: F341-F358Crossref PubMed Scopus (45) Google Scholar, 12Pippin J.W. Sparks M.A. Glenn S.T. et al.Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease.Am J Pathol. 2013; 183: 542-557Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Therefore, we particularly tracked their neogenesis. Similar to de novo-differentiated RLCs, renin-positive RLCs formed a steady population during nephrogenesis and in adult kidneys, fluctuating at approximately 0.1% to 0.2% of all renal cells with a sole peak on pp10 (0.4% of all renal cells, Figure 6a). Within the mG+ RLC pool, the percentage of renin-positive cells decreased during nephrogenesis (because of RLC pool expansion), and notably, only <1% of all RLCs in adult kidneys remained renin positive (Supplementary Figure S7; Figure 1a). The same percentage of renin-positive RLCs was previously found in BM, blood, and spleen.16Belyea B.C. Xu F. Pentz E.S. et al.Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.Nat Commun. 2014; 5: 3273Crossref PubMed Scopus (25) Google Scholar The de novo-differentiated subpopulation (mT+mG+) of renin-positive cells decreased during kidney development (e17-pp10, Figure 6b). While during nephrogenesis, up to 9 of 10 renin-positive cells were newly differentiated, in the mature kidney, 1 of 10 renin-positive cells at a given time point differentiated de novo, indicating a low neogenesis rate. Thus, as expected in adulthood, renin-positive cells in AAs appear to be predominantly long lived (mT−mG+) and approximately 10% of them (mT+mG+) are continuously renewed by neogenesis. We addressed the possibility that not only renin expression but also RLC neogenesis could be induced in adult life by pharmacologic RASi (treatment with the ACE inhibitor enalapril). While the overall renal RLC pool did not statistically increase after treatment, the fraction of de novo-differentiated RLCs significantly increased (Figure 7a and b). De novo-differentiated cells were observed in AAs of enalapril-treated animals on confocal microscopy (Figure 7c). The renin-producing cell pool is restricted to AAs in adult wild-type mice after enalapril treatment.1Castrop H. Hocherl K. Kurtz A. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (195) Google Scholar Therefore, we specifically investigated the neogenesis of renin-positive cells because it reflects the changes in RLCs in the AA area. The fraction of renin-positive cells to all RLCs doubled after enalapril treatment, while equivalently, the fraction of newly differentiated RLCs to all RLCs also doubled within the renin-positive cell population (Figure 7d). This indicates that in adults, enalapril mainly expands the renin-positive cell pool in AAs via metaplastic transformation of RLCs that were previously capable of producing renin (at 90%), while 10% of the pool expansion is related to de novo-differentiated RLCs. We further studied the impact of reversible mesangial injury on the rate of RLC neogenesis in a model of mesangiolysis and glomerular damage, which peaks 2 to 3 days after injecting an antimesangial cell serum, followed by a regenerative phase at least up to 10 days.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar We previously reported that RLCs repopulate the injured intraglomerular mesangium in the regenerative phase.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar Considering the abovementioned results, we examined the hypothesis that RLC neogenesis is stimulated to fill up the RLC pool after injury induction. Flow cytometry demonstrated that the total number of RLCs in the kidney did not significantly increase at 2 to 3 days and 10 days after injury (Figure 8a, left panel and b). In contrast, de novo-differentiated mT+mG+ RLCs increased by 3-fold at 2 to 3 days and remained significantly elevated until 10 days after injury (Figure 8a, right panel and b). Three and 10 days after injury, mT+mG+ cells were detected using confocal microscopy, predominantly at the vascular pole of the glomerulus (Figure 8c and d). Some double-positive cells were also detected within the glomerulus during the regenerative stage after mesangial injury (day 10, Supplementary Figure S8). Renin positivity in adult mice is restricted to AAs and is used to label extraglomerular renin-positive RLCs before disease induction.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar Furthermore, renin expression in repopulating RLCs has not been reported to occur at intraglomerular locations.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar Therefore, we investigated the time course of neogenesis of the renin-positive cell fraction of RLCs, which solely reflects the regulation of the renin-positive precursor cell niche in AAs at the vascular pole of the glomerulus. Compared with healthy controls, the fraction of renin-positive cells to RLCs markedly increased in the early injury phase (days 2–3) and was almost indistinguishable from the normal level at the end of the reparative mesangial proliferative phase (10 days; Figure 8e). In contrast, neogenesis of renin-positive cells (mT+mG+) markedly increased (5-fold) on day 2 to 3 and persisted almost at that level up to day 10 (Figure 8e). Thus, during the repair phase of mesangial cell injury, every third renin-positive cell freshly differentiated, demonstrating a shift in the distribution of renin-positive cells from long-lived to de novo-differentiated cells during mesangial regeneration. To further validate our findings in living organisms, we performed intravital microscopy of the mouse kidney cortex by modifying the protocol of Hackl et al.19Hackl M.J. Burford J.L. Villanueva K. et al.Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.Nat Med. 2013; 19: 1661-1666Crossref PubMed Scopus (128) Google Scholar for upright microscopy. Using this technique, we identified double-positive mT+mG+ de novo-differentiated RLCs in the vascular wall of small cortical arteries (Figure 9a, left panel, arrowhead). Z-stack imaging revealed that the vessel shown in the left panel of Figure 9a was preglomerular because it branched before ending at the vascular pole of a deep glomerulus (Z-stack image sequence movie in Supplementary Movie S1). Peritubular de novo-differentiated mT+mG+ double-positive (thus appearing yellow) RLCs were repeatedly observed, although these were difficult to discriminate. These cells were frequently located next to mG+ tubular segments (Figure 9a, middle and right panels, arrowheads). We also monitored single glomeruli for 10 days in healthy and diseased mice using longitudinal intravital microscopy (Figure 9b). There were no marked changes in the glomerular architecture of healthy controls over time (Figure 9b, upper panels). During disease, we observed severe glomerular alterations, including hypertrophy and dilated capillaries, on day 6 (corresponding to the early regeneration phase after the damage peak), which were largely reversed on day 10 (Figure 9b, lower panels). With the ability to repeatedly visualize the very same areas, we could trace cells undergoing transition from mT+mG− (non-RLC, red) over mT+mG+ (de novo-differentiated RLC, yellow) to mT−mG+ (RLC, green) in living animals during mesangial cell injury progression (Figure 9b, lower panels, arrowheads from left to right). We found frequent RLC neogenesis in the walls of afferent and efferent arterioles, as confirmed by Z-stack serial imaging (Figure 9b, lower panels and Supplementary Movies S2 and S3), although it was a generally rare event in diseased mice. RLCs are attracting increasing interest for their role as a progenitor cell niche in the kidney. RLCs act as a precursor pool during nephrogenesis.3Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar Studies in recent years demonstrated that renin-positive RLCs retain their ability to differentiate and contribute to glomerular repair after injury in adult life. We recently found that RLCs replaced damaged mesangial cells in a mouse model of reversible mesangial injury.10Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after injury.J Am Soc Nephrol. 2015; 26: 48-54Crossref PubMed Scopus (59) Google Scholar Other researchers have reported that RLCs might give rise to glomerular epithelial cells such as parietal epithelial cells and podocytes, following the induction of experimental FSGS in mice.12Pippin J.W. Sparks M.A. Glenn S.T. et al.Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease.Am J Pathol. 2013; 183: 542-557Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Lichtnekert J. Kaverina N.V. Eng D.G. et al.Renin-angiotensin-aldosterone system inhibition increases podocyte derivation from cells of renin lineage.J Am Soc Nephrol. 2016; 27: 3611-3627Crossref PubMed Scopus (45) Google Scholar These findings were intriguing because unlike mesangial cells or parietal epithelial cells, podocytes do not belong to the RLC population in the healthy adult mouse kidney (Figure 1). All these findings led us to question whether the RLC pool in the kidney is continuously maintained. We first considered the
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