Biphasic recruitment of microchimeric fetal mesenchymal cells in fibrosis following acute kidney injury
2013; Elsevier BV; Volume: 85; Issue: 3 Linguagem: Inglês
10.1038/ki.2013.459
ISSN1523-1755
AutoresEdwige Roy, Elke Seppanen, Rebecca Ellis, E. S. M. Lee, Kiarash Khosroterani, Nicholas M. Fisk, George Bou‐Gharios,
Tópico(s)Pregnancy and preeclampsia studies
ResumoFetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-β/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis. Fetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-β/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis. Microchimerism, the presence of a rare population of cells within a genetically distinct host, occurs chiefly in pregnancy. Bidirectional trafficking of maternal and fetal cells is a well-described phenomenon occurring during most, if not all, pregnancies and results in long-term persistence of low levels of fetal or maternal cells in the mother and offspring, respectively.1.Bianchi D.W. Zickwolf G.K. Weil G.J. et al.Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.Proc Natl Acad Sci USA. 1996; 93: 705-708Crossref PubMed Scopus (1051) Google Scholar, 2.Krabchi K. Gros-Louis F. Yan J. et al.Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques.Clin Genet. 2001; 60: 145-150Crossref PubMed Scopus (118) Google Scholar, 3.Maloney S. Smith A. Furst D.E. et al.Microchimerism of maternal origin persists into adult life.J Clin Invest. 1999; 104: 41-47Crossref PubMed Scopus (377) Google Scholar Fetal microchimerism has been the subject of intensive research in the past decade, from which it has emerged that fetal microchimeric cells (FMCs) have capacity to home to injured maternal tissues and further differentiate along that tissue's lineage. The identification4.Khosrotehrani K. Johnson K.L. Cha D.H. et al.Transfer of fetal cells with multilineage potential to maternal tissue.JAMA. 2004; 292: 75-80Crossref PubMed Scopus (221) Google Scholar of male FMC bearing epithelial, leukocyte, or hepatocyte markers in various tissue specimens from women with male offspring initiated the concept that fetal cells might have stem cell capabilities with multilineage potential. This was further supported by a range of studies demonstrating FMC plasticity along different lineages such as lymphoid5.Khosrotehrani K. Leduc M. Bachy V. et al.Pregnancy allows the transfer and differentiation of fetal lymphoid progenitors into functional T and B cells in mothers.J Immunol. 2008; 180: 889-897Crossref PubMed Scopus (59) Google Scholar,6.Roy E. Leduc M. Guegan S. et al.Specific maternal microchimeric T cells targeting fetal antigens in beta cells predispose to auto-immune diabetes in the child.J Autoimmun. 2011; 36: 253-262Crossref PubMed Scopus (26) Google Scholar mesenchymal,7.Santos M.A. O'Donoghue K. Wyatt-Ashmead J. et al.Fetal cells in the maternal appendix: a marker of inflammation or fetal tissue repair?.Hum Reprod. 2008; 23: 2319-2325Crossref PubMed Scopus (25) Google Scholar, 8.Dubernard G. Aractingi S. Oster M. et al.Breast cancer stroma frequently recruits fetal derived cells during pregnancy.Breast Cancer Res. 2008; 10: R14Crossref PubMed Scopus (60) Google Scholar, 9.Bou-Gharios G. Amin F. Hill P. et al.Microchimeric fetal cells are recruited to maternal kidney following injury and activate collagen type I transcription.Cells Tissues Organs. 2011; 193: 379-392Crossref PubMed Scopus (12) Google Scholar endothelial,10.Nguyen H.S. Dubernard G. Aractingi S. et al.Feto-maternal cell trafficking: a transfer of pregnancy associated progenitor cells.Stem Cell Rev. 2006; 2: 111-116PubMed Google Scholar, 11.Nguyen K. Eltz S. Drouin S.J. et al.Oncologic outcome after radical prostatectomy in men with PSA values above 20 ng/ml: a monocentric experience.World J Urol. 2009; 27: 653-658Crossref PubMed Scopus (17) Google Scholar, 12.Nassar D. Khosrotehrani K. Aractingi S. Fetal microchimerism in skin wound healing.Chimerism. 2012; 3: 45-47Crossref PubMed Scopus (11) Google Scholar neuronal,13.Zeng X.X. Tan K.H. Yeo A. et al.Pregnancy-associated progenitor cells differentiate and mature into neurons in the maternal brain.Stem Cells Dev. 2010; 19: 1819-1830Crossref PubMed Scopus (59) Google Scholar epithelial, and hepatic.14.Wang Y. Iwatani H. Ito T. et al.Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury.Biochem Biophys Res Commun. 2004; 325: 961-967Crossref PubMed Scopus (102) Google Scholar,15.Nelson J.L. Furst D.E. Maloney S. et al.Microchimerism and HLA-compatible relationships of pregnancy in scleroderma.Lancet. 1998; 351: 559-562Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar This broad differentiation capacity raises the question of the origin of FMC transferred to the mother during pregnancy. Hematopoietic lineage cell types including mature T and B cells, dendritic cells, NK cells as well as more primitive lymphoid precursors1.Bianchi D.W. Zickwolf G.K. Weil G.J. et al.Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.Proc Natl Acad Sci USA. 1996; 93: 705-708Crossref PubMed Scopus (1051) Google Scholar,4.Khosrotehrani K. Johnson K.L. Cha D.H. et al.Transfer of fetal cells with multilineage potential to maternal tissue.JAMA. 2004; 292: 75-80Crossref PubMed Scopus (221) Google Scholar,5.Khosrotehrani K. Leduc M. Bachy V. et al.Pregnancy allows the transfer and differentiation of fetal lymphoid progenitors into functional T and B cells in mothers.J Immunol. 2008; 180: 889-897Crossref PubMed Scopus (59) Google Scholar,16.Guetta E. Gordon D. Simchen M.J. et al.Hematopoietic progenitor cells as targets for non-invasive prenatal diagnosis: detection of fetal CD34+ cells and assessment of post-delivery persistence in the maternal circulation.Blood Cells Mol Dis. 2003; 30: 13-21Crossref PubMed Scopus (61) Google Scholar, 17.Liegeois A. Gaillard M.C. Ouvre E. et al.Microchimerism in pregnant mice.Transplantation proceedings. 1981; 13: 1250-1252PubMed Google Scholar, 18.Walknowska J. Conte F.A. Grumbach M.M. Practical and theoretical implications of fetal-maternal lymphocyte transfer.Lancet. 1969; 1: 1119-1122Abstract PubMed Google Scholar have been documented as transferring from fetus to mother. FMCs implying endothelial nature12.Nassar D. Khosrotehrani K. Aractingi S. Fetal microchimerism in skin wound healing.Chimerism. 2012; 3: 45-47Crossref PubMed Scopus (11) Google Scholar,19.Nguyen H.S. Oster M. Avril M.F. et al.Fetal microchimeric cells participate in tumour angiogenesis in melanomas occurring during pregnancy.Am J Pathol. 2009; 174: 630-637Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,20.Parant O. Dubernard G. Challier J.C. et al.CD34+ cells in maternal placental blood are mainly fetal in origin and express endothelial markers.Lab Invest. 2009; 89: 915-923Crossref PubMed Scopus (39) Google Scholar have been detected in maternal blood and tissues during and after pregnancy as well as FMCs displaying characteristics of terminally differentiated hepatocytes21.Guettier C. Sebagh M. Buard J. et al.Male cell microchimerism in normal and diseased female livers from fetal life to adulthood.Hepatology. 2005; 42: 35-43Crossref PubMed Scopus (53) Google Scholar and epithelial cells.4.Khosrotehrani K. Johnson K.L. Cha D.H. et al.Transfer of fetal cells with multilineage potential to maternal tissue.JAMA. 2004; 292: 75-80Crossref PubMed Scopus (221) Google Scholar,22.Cha D. Khosrotehrani K. Kim Y. et al.Cervical cancer and microchimerism.Obstet Gynecol. 2003; 102: 774-781Crossref PubMed Scopus (85) Google Scholar However, other evidence implicates mesenchymal stem/stromal cells (MSC). FMCs in humans have been identified in trabecular bone, a known MSC niche.23.Chan J. O'Donoghue K. Gavina M. et al.Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration.Stem Cells. 2006; 24: 1879-1891Crossref PubMed Scopus (126) Google Scholar In another study by our group, ex vivo expansion of the adherent cell population isolated from the blood of a mother undergoing first trimester termination, identified FMCs with tri-lineage differentiation capacity, displaying MSC characteristics in vitro.24.O'Donoghue K. Choolani M. Chan J. et al.Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.Mol Hum Reprod. 2003; 9: 497-502Crossref PubMed Google Scholar Limited immunophenotyping of male adherent cells isolated from human female post-reproductive bone marrow was consistent with MSC.24.O'Donoghue K. Choolani M. Chan J. et al.Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.Mol Hum Reprod. 2003; 9: 497-502Crossref PubMed Google Scholar Further studies on human fetal MSC populations suggested increased differentiation potential beyond mesenchymal lineages, rendering them attractive candidates to explain the broad plasticity of FMC.23.Chan J. O'Donoghue K. Gavina M. et al.Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration.Stem Cells. 2006; 24: 1879-1891Crossref PubMed Scopus (126) Google Scholar,25.Kennea N.L. Waddington S.N. Chan J. et al.Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype.Cell Cycle. 2009; 8: 1069-1079Crossref PubMed Scopus (59) Google Scholar However, strictly mesenchymal phenotypes in the context of FMC have rarely been described. In this study, we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis. We used a transgenic approach to track FMCs in mice with aristolochic acid (AA) toxin-induced nephropathy. AA-nephropathy (AAN) was chosen as a model of renal fibrosis because of its simplicity and reproducibility in several mouse strains.26.Sato N. Takahashi D. Chen S.M. et al.Acute nephrotoxicity of aristolochic acids in mice.J Pharmacy Pharmacol. 2004; 56: 221-229Crossref PubMed Scopus (113) Google Scholar,27.Fragiadaki M. Witherden A.S. Kaneko T. et al.Interstitial fibrosis is associated with increased COL1A2 transcription in AA-injured renal tubular epithelial cells in vivo.Matrix Biol. 2011; 30: 396-403Crossref PubMed Scopus (31) Google Scholar AA is a Chinese herbal extract, identified as a kidney toxin capable of inducing progressive interstitial fibrosis in humans and rodents. AAN is characterized by acute tubular injury and interstitial inflammation, and involves distinct long-term outcomes.28.Lebeau C. Debelle F.D. Arlt V.M. et al.Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.Nephrol Dial Transplant. 2005; 20: 2321-2332Crossref PubMed Scopus (112) Google Scholar In this pathology, fibroblasts are involved in the production of excess extracellular matrix deposition resulting in interstitial fibrosis29.Lewis M.P. Norman J.T. Differential response of activated vs. non-activated renal fibroblasts to tubular epithelial cells: a model of initiation and progression of fibrosis?.Exp Nephrol. 1998; 6: 132-143Crossref PubMed Scopus (24) Google Scholar,30.Eddy A.A. Neilson E.G. Chronic kidney disease progression.J Am Soc Nephrol. 2006; 17: 2964-2966Crossref PubMed Scopus (165) Google Scholar involving type I collagen. Using two reporter models, COL1A2 transgenic mice and ROSA-GFP transgenic mice to track mesenchymal, and overall FMC, respectively, in the context of AAN, we describe biphasic recruitment of mesenchymal FMCs to renal injury sites. These two waves of recruitment coincide with the acute and chronic AAN process. Finally, we provide evidence for the active involvement of FMCs in the AAN repair process. C57BL/6 females were crossed with males transgenic for the luciferase gene under the control of Col1a2 promoter (Col1-Luc). After serial pregnancies (at least two), mothers received intraperitoneal injections of AA toxin or saline solution over 5 days (Figure 1a). One group of mice (n=29, 16 AA vs. 13 saline) was killed 60 days after first injection, and analyzed histologically using hematoxylin and eosin and Masson's trichrome staining (Figure 1bi and Supplementary Figure 1 online). Sections revealed patchy tubulointerstitial injury and fibrosis, characterized by tubular dilation or atrophy, interstitial volume expansion, interstitial fibrosis, and massive infiltration of inflammatory cells in AA-treated animals. These observations were quantified and each mouse assigned a histopathological score. Compared with saline-injected controls, AA-treated mice showed a significant increase in pathological manifestations (Figure 1bii) correlating with increased collagen deposition (Figure 1biii). The expression of transforming growth factor-beta (TGF-β), a profibrotic molecule, was increased, but not connective tissue growth factor (CTGF, CCN2; Figure 1c). AA-treated mice had increased plasma urea levels compared with control mice at day 60 (P=0.002), albeit with blood creatinine level remaining normal (Figure 1d). These renal function results, suggesting half the AA-treated animals had measurable renal dysfunction, are consistent with moderate severity AAN at this time point. Download .jpg (.36 MB) Help with files Supplementary Figure 1 Having established that the AA-toxin was capable of driving fibrosis in our model, we addressed the recruitment of collagen-producing FMC at the injury site. C57BL/6 females crossed with Col1-Luc transgenic males were examined for bioluminescence activity over 60 days. In chimeric mice carrying Col1-Luc transgenic fetal cells, bioluminescence was observed and localized at kidney sites (red circles, Figure 2ai). The bioluminescence was significantly increased over time in AA-treated animals (P<0.0001) (Figure 2b). The bioluminescence kinetics in AA-treated mice revealed distinct phases between transgenic virgin mice, which showed a single peak that plateaued (Figure 2c, black triangle) while in wild-type mothers with Col1a2-luc fetal cells, two peaks of luminescence intensity were documented; an early (D16-18) and later time point (D45-52; Figure 2c, black circle). C57BL/6 virgin mice displayed minimal bioluminescence, considered background. Minimal bioluminescence was also observed at D0 in C57BL/6 virgin mice showing no difference between experimental and control groups before experiment (data not shown). The presence of few FMCs was confirmed by fluorescence in situ hybridization studies at day 60 (Supplementary data 4 online). After FMCs were detected at an early time point in the in vivo trafficking experiment, we used another model to investigate FMCs in kidney tissue at a shorter time point. The reason for the different model was the lack of a good luciferase antibody that could be used in wax-embedded samples. To assess FMC participation in tissue harvested during the acute injury stage, virgin C57BL/6 females were crossed with hemizygous GFP+ males, and after two serial pregnancies, females were injected with either AA-toxin or saline. Kidneys were harvested 15 days after first injection and their structure assessed histologically (Figure 3a). Altered renal morphology was confirmed in AA-treated animals (P=0.0002; Figure 3bi and Supplementary Figure 1 online). Unsurprisingly, collagen deposition was also observed (Figure 3bii) but was not different between injured and healthy kidneys. TGF-β expression levels were increased in AA-injected mice (P=0.0008), with the concomitant increase observed in both blood creatinine and urea levels (with P=0.016 and P<0.0001, respectively) reflecting the associated renal dysfunction (Figure 3c and d). We next used anti-enhanced green fluorescent protein (EGFP) immunofluorescence to detect fetal cells in kidney sections from AA-treated versus saline-injected mice with scoring blinded to treatment allocation. Virgin mice injected with AA or saline buffer were used as negative controls. EGFP was detected most readily around vessels, forming clusters of two to three cells (Figure 4a). EGFP+ fetal cells were detected more frequently in kidney sections from AA-treated mice compared with controls (Figure 4b; 16 of 25 AA-treated vs. 3 of 21 saline-treated mice, P=0.0009). Different shapes were observed suggesting the presence of various cell types (Supplementary Figure 2 online). Of note, only a single false-positive EGFP+ cell was found in any of the nine virgin mice sections assessed blindly. The presence and localization of FMCs were confirmed by fluorescence in situ hybridization studies (Supplementary data 4 online). Download .jpg (.11 MB) Help with files Supplementary Figure 2 To assess the phenotype of FMC in maternal kidneys, we performed double immunofluorescence staining with three different markers in combination with EGFP. CD45 was used to identify leukocytes, CD31 for endothelial cells, and vimentin for mesenchymal cells. Most GFP+ cells in these tissues expressed CD45 (40%), indicating a likely hematopoietic origin (Figure 4c). Among these CD45+/GFP+ cells, 23% exhibited a low CD45 staining (Figure 4cii) suggesting a macrophagic profile, which corroborated the cell shapes observed (Supplementary Figure 2 online). To confirm, we performed immunofluorescence staining with the macrophage marker, F4-80, in combination with GFP. More than 5% of FMCs harbored F4-80 staining, confirming the presence of macrophages at the tissue injury site (Supplementary Figure 3 online). A lesser proportion of FMC expressed vimentin (19%) or CD31 (18%). However, 23% of FMCs were not characterized, suggesting other lineage(s). To further analyze their phenotype, we used other markers. More than 3% expressed pan-cytokeratin suggesting FMC contribute to the repair process via mesenchymal–epithelial transition. However, no FMCs exhibited alpha-smooth muscle actin, FSP1, NG2, CD34, or c-kit. The Ki67 marker was not associated with FMCs, suggesting recruitment rather than a proliferation of preexisting FMC located in kidney before injury. Download .jpg (.14 MB) Help with files Supplementary Figure 4 Download .jpg (.07 MB) Help with files Supplementary Figure 3 At day 15, there was a categoric association between the presence of FMCs and an increase in higher TGF-β mRNA levels (P=0.019; Figure 5ai). There was a similar association with elevated plasma urea levels (P=0.002; Figure 5bii), albeit with that for blood creatinine level just falling short of significance (Figure 5bi). Moreover, the pathological score was higher in AA-injected mice previously crossed with GFP males, compared with virgin mice (P=0.0055; Figure 5ci) suggesting an effect on repair post-AA, whereas in the saline-injected group, scores were similar in parous and virgin mice (P=0.0586; Figure 5ci). The collagen deposition score was similar in each group (Figure 5cii). Interestingly, these results may implicate fetal cells in response to injury and fibrosis. At the later time point (days 45–52), AA-injected mice were subdivided into two groups for the analysis in Figure 6a and b, determined according to the median intensity of bioluminescence as an index of the number of participatory FMCs: one group exhibited higher bioluminescence intensity and the other lower. In these results, higher collagen-producing FMCs were associated categorically with higher TGF-β and CTGF expression, and also urea levels (Figure 6a and bii). TGF-β and CTGF expression were increased in the kidneys with higher bioluminescence intensity (CTGF P=0.038, TGF-β P=0.04; Figure 6a), as was serum urea level (P=0.04; Figure 6bii). The severity of renal injury was increased in the parous group injected with AA (Figure 6ci). Both pathological score and collagen deposition were higher in parous compared with virgin AA-injected mice (Figure 6cii; P=0.0035 and 0.005, respectively), but similar in the comparable groups of saline-injected mice (P=0.2 and 0.8, respectively). These results implicate FMCs in the renal injury severity via a contribution to the fibrosis. Publications showed that bone marrow–derived cells are beneficial in nephrotic disease models.31.Togel F. Isaac J. Hu Z. et al.Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury.Kidney Int. 2005; 67: 1772-1784Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,32.Semedo P. Wang P.M. Andreucci T.H. et al.Mesenchymal stem cells ameliorate tissue damages triggered by renal ischemia and reperfusion injury.Transplant Proc. 2007; 39: 421-423Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Whole bone marrow administration in a rodent model also showed improvement in progression of chronic kidney disease.33.Caldas H.C. Fernandes I.M. Gerbi F. et al.Effect of whole bone marrow cell infusion in the progression of experimental chronic renal failure.Transplant Proc. 2008; 40: 853-855Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar FMCs residing in bone marrow have been shown to be recruited in injury models.7.Santos M.A. O'Donoghue K. Wyatt-Ashmead J. et al.Fetal cells in the maternal appendix: a marker of inflammation or fetal tissue repair?.Hum Reprod. 2008; 23: 2319-2325Crossref PubMed Scopus (25) Google Scholar,10.Nguyen H.S. Dubernard G. Aractingi S. et al.Feto-maternal cell trafficking: a transfer of pregnancy associated progenitor cells.Stem Cell Rev. 2006; 2: 111-116PubMed Google Scholar,34.Khosrotehrani K. Bianchi D.W. Multi-lineage potential of fetal cells in maternal tissue: a legacy in reverse.J Cell Science. 2005; 118: 1559-1563Crossref PubMed Scopus (137) Google Scholar,35.Nassar D. Droitcourt C. Mathieu-d'Argent E. et al.Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing.FASEB J. 2012; 26: 149-157Crossref PubMed Scopus (47) Google Scholar In this study, we provide evidence of FMC recruitment in a murine model of maternal kidney fibrosis following tubulointerstitial injury. After characterizing the histological features in our model of AA nephropathy,36.Pozdzik A.A. Salmon I.J. Debelle F.D. et al.Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.Kidney Int. 2008; 73: 595-607Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar we documented both recruitment and functional activity of fetal MSCs at the injury site in affected parous mice. The time course of experimental AAN allowed us to distinguish two distinct yet interconnected phases: (a) an acute phase where recruited fetal MSC produce collagen type I, perhaps as a result of the elevation in TGF-β mRNA that drives this disease37.Zhou L. Fu P. Huang X.R. et al.Mechanism of chronic aristolochic acid nephropathy: role of Smad3.Am J Physiol Renal Physiol. 2010; 298: F1006-F1017Crossref PubMed Scopus (106) Google Scholar and stimulates transcriptional activity of several matrix proteins,38.Massague J. Seoane J. Wotton D. Smad transcription factors.Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1923) Google Scholar,39.Leask A. Abraham D.J. TGF-beta signaling and the fibrotic response.FASEB J. 2004; 18: 816-827Crossref PubMed Scopus (1962) Google Scholar this was then attenuated by day 24; and (b) a late phase indicated by a second wave of bioluminescence at days 45–52 that was also abated by day 60. We suspect that FMCs are recruited as part of the response to injury in the acute phase, as with bone marrow recruitment, since no discernible morphological changes in the kidney were observed at that stage. The evidence for their involvement is based on the fact that fetal MSC are both more frequent and more likely to be found in AA-treated mice compared with saline-injected mice, and that double staining of the FMCs showed that a number of cell types are recruited. Indeed, phenotyping the FMCs revealed a population that was predominantly hematopoietic at the early time point, although mesenchymal and endothelial populations were also detected and that functionally, some of these cells were synthesizing collagen type I, an indicator of their participation in fibrosis following injury.40.Witzgall R. Brown D. Schwarz C. et al.Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.J Clin Invest. 1994; 93: 2175-2188Crossref PubMed Scopus (531) Google Scholar,41.Rumberger B. Vonend O. 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Aractingi S. et al.Feto-maternal cell trafficking: a transfer of pregnancy associated progenitor cells.Stem Cell Rev. 2006; 2: 111-116PubMed Google Scholar,11.Nguyen K. Eltz S. Drouin S.J. et al.Oncologic outcome after radical prostatectomy in men with PSA values above 20 ng/ml: a monocentric experience.World J Urol. 2009; 27: 653-658Crossref PubMed Scopus (17) Google Scholar,42.Lin S.L. Castano A.P. Nowlin B.T. et al.Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations.J Immunol. 2009; 183: 6733-6743Crossref PubMed Scopus (268) Google Scholar Biochemical analysis undertaken to evaluate the severity of the AAN showed increased blood creatinine and urea levels at day 15 in injured mice, but by day 60 only the increase in urea was significant. As creatinine is known to start rising only after a two- to fourfold rise in blood urea, we suggest that the normal creatinine obs
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