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Plasticity of Renal -Producing Cells Governs Fibrosis

† ‡ Tomokazu Souma,* Shun Yamazaki,* Takashi Moriguchi,* Norio Suzuki, Ikuo Hirano,* † † † Xiaoqing Pan,*§ Naoko Minegishi,*§ Michiaki Abe, § Hideyasu Kiyomoto, § Sadayoshi Ito, || and Masayuki Yamamoto*§

*Department of Medical Biochemistry, †Division of Nephrology, Endocrinology, and Vascular Medicine, Department ‡ of Medicine, Division of Interdisciplinary Medical Science, United Centers for Advanced Research and Translational Medicine, and §Tohoku Medical Megabank Organization, Tohoku University Graduate School of Medicine, Sendai, || Miyagi, Japan; and JST, CREST, Sendai, Miyagi, Japan

ABSTRACT CKD progresses with fibrosis and erythropoietin (Epo)-dependent anemia, leading to increased cardio- vascular complications, but the mechanisms linking Epo-dependent anemia and fibrosis remain unclear. Here, we show that the cellular phenotype of renal Epo-producing cells (REPs) alternates between a physiologic Epo-producing state and a pathologic fibrogenic state in response to microenvironmental signals. In a novel mouse model, unilateral ureteral obstruction–induced inflammatory milieu activated NFkB and Smad signaling pathways in REPs, rapidly repressed the Epo-producing potential of REPs, and led to myofibroblast transformation of these cells. Moreover, we developed a unique Cre-based cell-fate tracing method that marked current and/or previous Epo-producing cells and revealed that the majority of myofibroblasts are derived from REPs. Genetic induction of NFkB activity selectively in REPs resulted in myofibroblastic transformation, indicating that NFkB signaling elicits a phenotypic switch. Reversing the unilateral ureteral obstruction–induced inflammatory microenvironment restored the Epo-producing po- tential and the physiologic phenotype of REPs. This phenotypic reversion was accelerated by anti-inflam- matory therapy. These findings demonstrate that REPs possess cellular plasticity, and suggest that the phenotypic transition of REPs to myofibroblasts, modulated by inflammatory molecules, underlies the connection between anemia and renal fibrosis in CKD.

J Am Soc Nephrol 24: 1599–1616, 2013. doi: 10.1681/ASN.2013010030

Renal erythropoietin (Epo)-producing cells (REPs) develops along with the progression of fibrosis, are fibroblast-like cells that express neural markers leading to cardiovascular events.7,9,10 Interest- and produce Epo, an indispensable erythropoietic ingly, REPs in the fibrotic kidney express mark- hormone.1–3 REPs control Epo expression ers of myofibroblasts, such as desmin and a primarily through the prolyl hydroxylases/von smooth muscle actin (aSMA),11,12 suggesting an Hippel-Lindau /hypoxia-inducible factor (HIF) pathway.3,4 Production of Epo at cellular level in REPs is thought to be either “on” or “off,” hence Received January 8, 2013. Accepted April 24, 2013. the total Epo production from kidney is regulated T.S. and S.Y. contributed equally to this work. by the number of Epo-producing REPs, which Published online ahead of print. Publication date available at changes markedly responding to hypoxia and/or www.jasn.org. anemia.2,5,6 fi fi Correspondence: Dr. Masayuki Yamamoto, Department of Renal brosis is considered a nal common Medical Biochemistry, Tohoku University Graduate School of pathway of CKD leading to ESRD.7,8 Because fi- Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, brotic kidneys have a limited Epo-producing ability Japan. Email: [email protected] at any given anemic stimuli,9,10 renal anemia Copyright © 2013 by the American Society of Nephrology

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Figure 1. UUO leads to rapid loss of REP potential. (A) Schematic presentation of ISAM generation. (B) Distribution patterns of G-REPs under anemic (Ht 17%) and normal (Ht 42%) conditions. In the latter case, ISAM are treated with rHuEPO. The inset shows a higher magnification of G-REPs. (C) Epo-GFP mRNA levels after administration of rHuEPO or PBS (n=4 per group). (D) UUO-induced ex- pression changes of Epo-GFP and aSMA in ISAM kidneys. Immunofluorescence is performed for Epo-GFP (green) and aSMA (red)

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Table 1. UUO model did not alter the levels of genetically induced severe anemia UUO Model Erythrocyte Number, 3104/ml Hemoglobin Concentration, g/dl Hematocrit, % Sham 291635 4.660.64 1461.6 UUO day 1 308620 4.860.13 1560.69 UUO day 2 307629 4.760.55 1561.5 UUO day 3 299631 4.660.57 1461.4 UUO day 7 308648 4.760.83 1462.3 UUO day 14 302640 4.860.58 1562.3 All indices were not statistically significant (one-way ANOVA). Data are shown as mean 6 SD. etiological link between renal anemia and fibrosis by dam- inflammatory stresses. These results indicate that a profound aged REPs. cellular plasticity resides in REPs and REPs may be ideal tar- Myofibroblasts are the culprit of renal fibrosis, and their gets for CKD treatment. origin has been debated.8,13 Recent cell-fate mapping studies revealed that myofibroblasts are mainly derived from P0 (my- elin protein 0)-Cre–labeled cells,12 or FoxD1-Cre–labeled per- RESULTS icytes and perivascular fibroblasts.14 However, there remains uncertainty, as these Cre-labeled cells are heterogeneous be- Efficient Monitoring of REPs by a Transgenic Rescue cause of the shared developmental program.14 Moreover, in Approach terms of Epo-producing ability, REPs are only ,10%ofP0- Epo-knockout mice die at around embryonic day 12.5 due to Cre–labeled cells, and approximately 20% of REPs are not anemia.20 Because fetal secretion of Epo from liver is regulated fate-mapped by P0-Cre.6,12 Thus, precise contribution of by its 39 enhancer,21–23 we rescued the embryonic lethality of REPs to renal fibrosis is still unclear. homozygous Epo-knockout/green fluorescent protein (GFP) There are many unanswered important questions related to knock-in mice (EpoGFP/GFP)usingthe39-enhancer-driven 9 9 REPs and renal fibrosis: (1)whetherthemajorityofREPs Epo-expressing transgene (TgEpo3 ). Because TgEpo3 -derived contribute to total myofibroblast population or REPs rather Epo was predominantly expressed in perinatal liver (S. Yama- 9 disappear through cell death upon renal damages, (2)how zaki et al., unpublished observations), this TgEpo3 -rescued REPs change their phenotype to myofibroblasts and lose their EpoGFP/GFP mouse—designated as ISAM— showed severe Epo-producing ability, and (3) whether this phenotypic con- Epo-deficient anemia in adulthood (Figure 1A and Supple- version is reversible. In this regard, because CKD is charac- mental Figure 1). terized by sterile chronic inflammation caused by activated Because GFP was inserted in-frame into the endogenous resident renal cells and infiltrating leukocytes,15–17 we sur- Epo coding exon, the regulatory influences of the Epo gene mised that the phenotypic changes of REPs in diseased kidneys faithfully regulated expressions of Epo-GFP. We refer to these may be attributable to inflammatory signals generated in renal GFP-labeled cells in ISAM kidneys as GFP-labeled renal Epo- microenvironments. producing cells (G-REPs). Although G-REPs did not produce In this study, we utilized two novel mouse lines that manifest Epo, we could monitor their localization and Epo-producing severe anemia (inherited super anemic mice [ISAM]) and that potential by the protein and mRNA expressions of Epo-GFP express Cre enzyme under the regulatory influences of Epo under the reproducible severe anemia. Indeed, G-REPs were gene (Epo-Cre mouse) to monitor phenotypic changes of widely distributed in cortex and outer medulla (Figure 1B, left individual REPs. Utilizing unilateral ureteral obstruction panel). The distribution and Epo-GFP mRNA expression were (UUO), a commonly used inflammatory fibrogenic reduced by recombinant human Epo (rHuEPO) administra- method,18,19 we analyzed how activated inflammatory sig- tion (Figure 1, B right panel and C). Compared with ISAM, naling affects the phenotype of REPs. Through these anal- our previously used models had limitations: P0-Cre labeled all yses, we have discovered that REPs are the major source of glomeruli and almost all interstitial fibroblasts, and a few REPs myofibroblasts and myofibroblast-transformed REPs recover could be GFP-labeled in the juxta-medullary region of kidneys their normal Epo-producing potential upon removal of of EpoGFP/+ mice (Supplemental Figure 2A). Thus, ISAM offer

expressions, showing inverse expression patterns of these . EM staining shows the fibrotic area as blue. (E–H) UUO-induced expression changes of Epo-GFP, Acta2, Tgfb1,andTnfa. Real-time PCR analyses of Epo-GFP, Acta2 (encoding aSMA), Tgfb1 (encoding TGFb1), and Tnfa (encoding TNFa) are performed using UUO-treated and control ISAM kidneys (n.4 per group). Epo-GFP mRNA level is expressed as the relative expression compared with the contralateral kidney. Data of the sham-treated group are used as the starting point (0 day), and are set as 100% (Epo-GFP mRNA) or 1 (the other mRNAs). *P,0.05; **P,0.01. Scale bars, 20 mm (inset) and 200 mm (right) in B; 100 mm in D. Cont, contralateral; EM, Elastica-Masson.

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Figure 2. REPs are the major reservoir of myofibroblasts. (A) Expression of Epo-GFP and aSMA in early stages of UUO-treated ISAM kidneys. Immunofluorescence of Epo-GFP (green) and aSMA (red) is performed along with nuclear staining with DAPI (blue) using UUO-treated kidneys in early stages (day 1 to day 3). Note the overlapping expression of Epo-GFP and aSMA from day 2 after UUO. (B) Quantification of cells expressing both aSMA and Epo-GFP. Numbers of aSMA-positive G-REPs are compared with either those of total

1602 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1599–1616, 2013 www.jasn.org BASIC RESEARCH significant advantages for efficient monitoring of REPs in the characteristic juxta-medullary distribution of REPs, which adult mice. increased with anemia, and expressed CD73, a commonly used marker of REPs (Supplemental Figure 5).1,2,25,26 To max- Loss of Epo-Producing Potential under Fibrogenic imally label and fate-track REPs by full induction of Epo-Cre, Conditions because its induction is dependent on the level of anemia Totest how inflammatory and fibrogenic insults affect G-REPs, (Supplemental Figure 5G), we crossbred Epo-Cre::R26T mice we applied UUO to ISAM. UUO treatment led to progressive with ISAM to generate ISAM::Epo-Cre::R26T. Almost all cor- renal fibrosis (Figure 1, D and F–H). GFP intensities and the tical and outer medullary fibroblast-like cells were labeled by number of G-REPs decreased at day 7, and a scarce number of tdTomato in sham-treated kidneys of ISAM::Epo-Cre::R26T. G-REPs in the juxta-medullary region was detected at day 14 The tdTomato-positive cells were much more abundant than (Figure 1D and Supplemental Figure 2, B–D). The Epo-GFP G-REPs (Figure 2E, sham), presumably due to labeling of cells transcript level was rapidly reduced by 80% at day 2 despite the that were previously and/or are currently producing Epo-GFP persistent severe anemia (Figure 1E and Table 1). Epo-GFP (i.e., “history of Epo production”). mRNA level is suitable for real-time monitoring of Epo-pro- Notably, the vast majority of cortical and outer medullary ducing potential, whereas Epo-GFP protein allowed us to trace interstitial cells were tdTomato-positive in the UUO-treated the cell-fate for a couple of days longer by virtue of its long t1/2. kidney, and they lost the Epo-GFP fluorescence despite severe These findings demonstrate that Epo-producing potentials are anemia (Figure 2E, UUO day 14). Importantly, around 80% of lost in the majority of myofibroblasts even in the severe ane- aSMA-positive cells (including myofibroblasts and vascular mic conditions. smooth muscle cells) at day 14 after UUO were tdTomato- positive (Figure 2, F and G). These results demonstrate that REPs as the Major Reservoir of Myofibroblasts almost all myofibroblasts are derived from cells with a history Closer examinations revealed that aSMA-positive myofibro- of Epo production. blasts appeared and increased from day 2 after UUO (Figure 2A). Most G-REPs changed into myofibroblasts, as aSMA was Active Contribution of REPs to Renal Inflammation and expressed in 50% and 80% of G-REPs at day 2 and day 3 after Fibrosis UUO, respectively. Thirty to forty percent of aSMA-positive To address mechanisms underlying the phenotypic transition, cells were Epo-GFP positive (Figure 2, A and B). Con- we focused on the inflammatory and fibrogenic signals. TNFa comitantly, G-REPs entered into the cell cycle and started and TGFb1 mRNA levels were upregulated at day 1 (Figure 1, proliferation (Supplemental Figure 3). The myofibroblast- G and H), and downstream transcriptional factors, phosphor- transformed G-REPs were also observed in other models of ylated p65 (p-p65) and Smad2/3 (p-Smad2/3), accumulated kidney disease, indicating that the myofibroblastic transfor- in the nuclei of G-REPs (Figure 3, A and B), suggesting that mation of G-REPs is a common pathologic outcome (Supple- TGFb1-Smad2/3 and TNFa-NFkB signaling contribute to the mental Figure 4). myofibroblastic transformation. To ascertain that the G-REP–derived myofibroblasts con- Next, we isolated G-REPs by FACS using Epo-GFP fluo- tribute to the total population of myofibroblasts in later stages rescence and analyzed transcriptional changes (Figure 3 and of fibrosis, we generated bacterial artificial Supplemental Figure 3F). We found that G-REPs lost Epo-GFP (BAC)-transgenic mouse lines that expressed Cre recombi- expression by 90% at day 2. In contrast, mRNA expression of nase under the control of a 180-kb Epo gene regulatory region HIF factors (Hif1a, Hif2a,andArnt) was maintained, suggest- (Epo-Cre mice; Figure 2C). We performed cell-fate tracing of ing that the reduction of Epo-GFP was not due to the impaired REPs by crossing Epo-Cre mice with the tdTomato reporter transcription of HIF factors. Expression of microtubule-asso- line (Rosa26-tdTomato or R26T).24 We refer to Epo-Cre line- ciated protein 2 (Map2), a neural marker, was significantly age-labeled cells as Epo-Cre cells. Epo-Cre cells recapitulated decreased, whereas target of Smad2/3 (Acta2, Col1a1,

G-REPs (upper panel) or those of total aSMA-positive cells (lower panel) in five independent fields. **P,0.01 (n=3 per group). (C) Schematic presentation of the strategy for cell-fate analysis of REPs. A 180-kb BAC clone containing the Epo gene is used to generate an Epo-Cre mouse line. Crossing of the Epo-Cre mice with Rosa26-tdTomato (R26T) reporter mice generates Epo-Cre::R26T mice. (D) Cell- fate analysis of REPs in later stage of UUO kidneys. Native fluorescence of tdTomato (red; Epo-Cre cells) and immunofluorescence of aSMA (green) overlaps in UUO-treated kidneys at day 14, but not in sham-treated kidneys. (E) Distribution pattern of Epo-GFP and tdTomato in ISAM::Epo-Cre::R26T. Expressions of native Epo-GFP (green) and tdTomato (red) are analyzed using kidneys that underwent UUO or sham procedures (day 14). Note that tdTomato fluorescence, which represents previous and/or current expression of the Epo gene, is widely distributed in the UUO-treated kidney, but GFP fluorescence is diminished at this time point. (F) Myofibroblast trans- formation of Epo-Cre cells. Immunofluorescence for aSMA and tdTomato is performed using UUO kidneys at day 14. (G) Quantification of cells expressing both aSMA and tdTomato. Number of aSMA-positive Epo-Cre cells is compared with either those of total Epo-Cre cells or those of total aSMA-positive cells. (n=4). DAPI, 4’,6-diamidino-2-phenylindole. Scale bars, 100 mm.

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Figure 3. REPs contribute to renal fibrosis and inflammation. (A and B) TGFb and NFkB signaling in G-REPs of UUO-treated ISAM kidney. Immunofluorescence of Epo-GFP (green), p-Smad2/3 or p-p65 (red), and nucleus (blue) is performed using UUO-treated kidneys at day 1 after UUO. White arrows indicate cells that show nuclear accumulation of p-p65 or p-Smad2/3 in G-REPs. (C) Schematic presentation of the FACS protocol for isolation of G-REPs from UUO-treated (day 2) and normal ISAM kidneys. (D)

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Col3a1,andSerpine1)andNFkB(Il6 and Ccl2)wereup- Reversal of Epo-Producing Potential by Removing regulated upon UUO (Figure 3E). Collectively, these results Microenvironmental Cues demonstrate that G-REP–derived myofibroblasts actively To elucidate whether this phenotypic transition of G-REPs is contribute to renal fibrosis and inflammation by aberrantly reversible, we utilized a reversible UUO model (Clip-ClipR producing extracellular matrix proteins, an inhibitor of extra- treatment). Because most G-REPs were primed for the cellular matrix degradation, and inflammatory cytokines and myofibroblastic transformation and lost their Epo-producing chemokines. potential at day 2 after UUO, we reopened the obstructed ureter at this time point (Figure 5A). Notably, the Epo-GFP transcript Repression of Epo-Producing Potential by NFkB level was restored to normal level (Figure 5B) and production Signaling of collagens are returned to the baseline at 12 days after the To explore roles that NFkB and Smad signals play in pheno- reopening (Figure 5C). We then analyzed the distribution and typic changes of G-REPs, we administered LPS, an NFkB population of currently Epo-GFP producing REPs (ON-REPs signal activator, or TGFb1, and analyzed at 6 hours after or G-REPs) and total population of REPs (Epo-Cre cells) using the injection. LPS administration rapidly diminished the ISAM::Epo-Cre::R26T mice that underwent Clip-ClipR treat- Epo-GFP expression in ISAM (Figure 4A). Coadministration ment (Figure 5D and Supplemental Figure 7). Population of of , which disrupts the NFkB activity, atten- ON-REPs were comparable to those of contralateral kidneys, uated this repression, indicating the presence of an NFkB- but the number of total REPs was increased by 2-fold (Figure mediated repressive regulation. However, injection of TGFb1 5, E–G), indicating that individual REPs restored normal Epo- did not affect the Epo-GFP expression, despite substantial producing machinery after transient proliferation through inductions of its target genes (Figure 4A). Interestingly, the UUO-induced myofibroblastic transformation. These results LPS-induced Epo-GFP repression was transient and revers- thus demonstrate that G-REP–derived myofibroblasts retain ible (Supplemental Figure 6). These results suggest that the functional reversibility in response to the improvement of mi- rapid repression of Epo-GFP after UUO is dependent on the croenvironmental cues. NFkB signaling, and imply that this repression could be re- versible. Evidence for the Plasticity of Damaged REPs Toelucidate consequences of sustained activation of NFkB We used immunoelectron microscopy to evaluate the mor- signals in REPs, we selectively activated canonical NFkBsig- phologic changes of G-REPs resulting from the Clip-ClipR nals in REPs by crossing Epo-Cre mice with constitutively treatment (Figure 6A). Normal G-REPs were observed as os- active inhibitor of IkB kinase 2 (IKK2) knock-in mice mium-positive angular cells that bridged capillaries and prox- (R26-IKK2ca;Figure4B).27 Forced expression of IKK2ca re- imal tubules. At 2 days postobstruction, all G-REPs gained sults in phosphorylation and degradation of IkB, thereby the characteristic features of myofibroblasts, as indicated by a stel- NFkB complex activates its target genes. REPs in Epo-Cre:: late cell shape with round nuclei and widened processes. At 12 R26-IKK2ca/+ mice were visualized by the GFP expression days after the release, such changes completely disappeared. from the IRES-EGFP cassette (Figure 4B). We found that These results indicate that G-REPs possess cellular plasticity. approximately 20% of the GFP-positive cells expressed To prove the plasticity of G-REPs, we performed two aSMA (Figure 4, C and D). Because Epo-Cre cells without additional cell-fate tracking experiments. First, we tracked the the R26-IKK2ca allele in Epo-Cre::R26T mice were aSMA cell-fate of G-REPs by detecting residual aSMA (Supplemental negative (Figure 2D), the observed aSMA expression could Figure 8, A and B). Most G-REPs retained aSMA immunore- be attributable to the induced NFkBactivation.Although activity at 5 and 12 days after the release of UUO (ClipR-7 and some REPs transformed into myofibroblasts, Epo production ClipR-14, respectively; Supplemental Figure 8, C and D), in- of kidney was not attenuated, presumably due to insufficient dicating that the recovering G-REPs originated from myofi- expression of IKK2ca, and/or high reserve capacity of kidneys broblasts. to produce Epo (Figure 4E). Taken together, these data sug- Second, we pulse labeled G-REPs with bromodeoxyuridine gest that NFkB signal is important not only for repressing (BrdU), which is incorporated into the DNA of cells in S-phase Epo production but also for transforming REPs to myofibro- and can be detected in their daughter cells,28 and determined blasts. whether BrdU-labeled myofibroblasts returned to functional

Appearance of FACS-isolated G-REPs. Immunofluorescence of FACS-isolated G-REPs is performed for Epo-GFP (green) and aSMA (red), and nucleus was stained by DAPI (blue). (E) Transcriptional profiles in G-REPs upon UUO. Real-time PCR analyses are performed with FACS-isolated G-REPs to quantify Epo-GFP, Hif1a (HIF-1a), Hif2a (HIF-2a), Arnt (HIF-1b), Acta2, Col1a1 (type I collagen a1), Col3a1 (type III collagen a1), Serpine1 (plasminogen activator inhibitor 1), Rela (p65), Ccl2 (MCP1), Il6 (IL6), and Map2 (MAP2); names in parentheses indicate encoded proteins. *P,0.05; **P,0.01 (n=3, per group; one sample comprises 100,000 G-REPs collected from 5–10 mice). Scale bars, 20 mminB.

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Figure 4. Essential contribution of NFkB signaling for the dysfunction of REPs. (A) Pharmacologic activations of NFkB and Smad signals in ISAM. Real-time PCR analyses are performed using ISAM kidneys 6 hours after single intraperitoneal injection of LPS, TGFb1, or PBS. For antagonizing NFkB signaling, dexamethasone is administered simultaneously with LPS (LPS+Dex). *P,0.05 and **P,0.01 versus PBS-injected group; #P,0.05 versus LPS-injected group (n.3 per group). (B) Schematic presentation of the strategy to selectively

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G-REPs (Figure 6B). Normal G-REPs did not incorporate renal cells (Figure 8C). Whereas Tnfa, Tgfb1,andMmp9 were BrdU, indicating that the G-REPs are normally in quiescence enriched in the CD45-positive fraction, others were not en- (Figure 6C). Upon application of UUO, G-REPs incorporated riched in either fraction (Figure 8D), suggesting that the BrdU, and BrdU-incorporated G-REPs were found through- infiltrating leukocytes and other resident renal cells collabo- out the observation period (Figure 6, C and D). These results ratively created the deleterious inflammatory microenviron- support the notion that the UUO insults transform G-REPs ments. into myofibroblasts, but G-REPs return to functional upon We surmised that interventions to the inflammatory signals removal of UUO, and contribute to the restoration of the might exerttherapeutic potentialsby facilitating therecoveryof Epo-producing potential. myofibroblast-transformed REPs. Administration of dexa- methasone after the Clip-ClipR treatment accelerated the Induced DNA Methyltransferases in Myofibroblast- recovery of Epo-producing potential of G-REPs (Figure 8, E Transformed REPs and F), demonstrating the importance of inflammatory sig- To further explore the molecular basis underlying the re- nals for the phenotypic transition and the feasibility of the versibility of damaged REPs, we examined expression profiles therapeutic strategy (Figure 9). of DNA methyl transferases (DNMTs) (Dnmt1, Dnmt3a, and Dnmt3b) in UUO-treated ISAM kidneys. Expression levels of Dnmt1 and Dnmt3b were increased from early stages (from DISCUSSION day 2), whereas Dnmt3a level was increased in later stages (from day 7; Figure 7A). Notably, expressions of Dnmt1 and In this study, we demonstrate that REPs possess functional and Dnmt3b in UUO-treated G-REPs were increased at day 2 after cellular plasticity in response to microenvironmental changes. UUO (Figure 7B). Clip removal reversed the expression levels Damaged REPs transform into myofibroblasts, lose their Epo- of DNMTs to normal levels, suggesting that the epigenetic producing potentials, and become the major contributor of modifying cues were halted by this procedure (Figure 7C). fibrosis. However, they maintain the inherent potentials to These results indicate that epigenetic alteration may play a revert to their original state when environmental cues subside. role in progression of CKD by changing the epigenetic codes We discovered that most myofibroblasts are derived from of renal microenvironments as well as those of G-REPs. REPs, whereas others showed that FoxD1-labeled were a major source.14,29 These data raise a question of Comprehensive Analyses of Microenvironmental Cues whether these different Cre driver lines, which are based on To delineate the microenvironmental cues, we performed either cellular function or developmental origin, labeled the genome-wide transcriptome analyses utilizing sham-, Clip-, same or different cells. On the basis of our findings that REPs and ClipR-14-treated kidneys. Pairwise comparisons of these share distinctive cellular markers, such as CD73 and groups revealed 1191 transcripts specifically increased in PDGFRb,26 with renal pericytes and that REPs have intimate UUO-treated kidneys (Figure 8A). These were then subjected interactions with endothelium, we consider that there is a sig- to a pathway analysis with Ingenuity Pathways Analysis (IPA), nificant crossover between REPs and renal pericytes. demonstrating that atherosclerosis signal and acute phase re- Inflammatory cytokines, such as TNFa, have been shown to sponse signal were the top two key-regulated pathways (Figure repress hypoxia-responsive Epo-producing ability,30,31 and 8B and Table 2). These results indicate that inflammatory cues are assumed to be key pathogenic regulators of anemia of were responsible for the UUO-induced phenotypic transition chronic disease32 and UUO-induced renal fibrosis.18,19,33 In of G-REPs. turn, TGFb/Smad signaling is the central pathway for myofi- broblast activation and transformation.34,35 In progressive fi- Accelerated Recovery of Damaged REPs by Anti- brogenesis, these two pathways amplify each other to drive Inflammatory Therapy inflammation and fibrosis.36–39 Here, we show that both To clarify cellular sources responsible for the inflammatory NFkB and Smad signaling were induced in damaged REPs. signals, we fractionated UUO-treated kidneys into CD45 By using pharmacologic activators of each signaling type, we (leukocyte common antigen)–positive leukocytes and residual clarified differential roles of these two signaling pathways.

activate NFkB signaling in REPs. Epo-Cre mice are crossed with R26-IKK2ca mice. Expression of EGFP from the IRES cassette allows the identification of Epo-Cre cells, and indicates recombination of the R26-IKK2ca locus. (C) NFkB signals as a phenotypic switch in REPs. Immunofluorescence is performed for EGFP (green) and aSMA (red), and nucleus is stained by DAPI (blue) using kidneys from Epo-Cre:: R26-IKK2ca/+ mice. White arrows indicate aSMA-positive Epo-Cre cells, indicating myofibroblastic transformation. (D) Quantification of cells expressing GFP and aSMA. The number of aSMA-positive Epo-Cre cells (GFP-positive cells) is compared with either that of total Epo- Cre cells or total aSMA-positive cells (n=4). (E) Epo mRNA expression of Epo-Cre::R26-IKK2ca/+ mice. Real-time PCR analyses are performed for quantifying Epo mRNA levels using whole kidneys. EGFP, enhanced GFP; IRES, internal ribosome entry site; Dex, dexa- methasone. Scale bar, 20 mminC.

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Figure 5. Elimination of UUO stress restores the normal Epo-producing potential. (A) Representative photographs of the UUO reversal model using vascular clips (Clip-ClipR treatment). We obstruct the left ureter by a vascular clip for 2 days (Clip). The vascular clip is removed 2 days after the obstruction, and then mice are euthanized 12 days after the removal (ClipR-14). (B) Recovery of Epo-producing potential of G-REPs in UUO-treated kidneys after the clip removal. Real-time PCR analysis is performed to quantify Epo-GFP mRNA

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NFkB signaling is responsible for the rapid repression of Epo- renal microenvironments, and attenuates and even reverses producing potential, whereas Smad signaling is more related fibrosis.50–52 Together with our findings that damaged REPs to the fibrogenic activity. Furthermore, we revealed that the produce proinflammatory factors and an anti-inflammatory sustained activation of NFkB signals in REPs results in a phe- drug accelerates the recovery of physiologic phenotype of notypic transition. This finding is supported by the recent REPs, the pathogenic significance of a deleterious inflam- finding that inhibition of NFkB signaling in renal fibro- matory cycle by REPs and surrounding cells is implied. We blasts/myofibroblasts prevents the UUO-induced fibrosis.19 consider that the therapeutic treatments that control the However, the fact that not all of the IKK2ca-overexpressed proinflammatory activity of tubular epithelial cells, infiltrating REPs changed into myofibroblasts implies the requirement leukocytes, and REPs would halt the inflammatory cycle, pro- of other coinsulting signals that might synergize with the tect REPs, and even reverse their transformation. NFkB pathway.34–39 In summary, our study demonstrates that REPs possess There are growing numbers of clinical and experimental profound cellular plasticity and that the damaged REPs directly reports suggesting that structural damage of the kidney, link fibrosis and anemia. Future studies are needed to elucidate including fibrosis, can be reversible.40–44 Therefore, elucidat- the exact mechanism underlying the plasticity of REPs, which ing whether diseased REPs in ESRD can be reversed to their will open up new avenues for treating CKD by protecting and physiologically normal state is an important lingering issue. regenerating REPs. Considering our findings that normal mature REPs are quies- cent, but damaged REPs proliferate with a notable regen- erative capacity, we assume that damaged REPs might be CONCISE METHODS replenished throughout life by self-renewal under physiologic insults, such as aging, or short-term pathologic insults. More- Mice over, clinical observations that some patients on dialysis with Mice were maintained in a specific pathogen-free facility. All ESRD become anemia-free and rHuEPO-independent during experimental procedures were approved by the Animal Care Com- the course of treatment suggest that some REPs retain a func- mittee at Tohoku University. Mice aged 8–14 weeks were used. We tional reversibility, even in ESRD.45–47 used age- and sex-matched mice for each experiment. Cre-negative TGFb1-mediated activation of Dnmt1 is reported to be littermates were used as a control when phenotypic differences were fundamental for perpetuating fibrosis.44 We found that examined with mice groups of different genotypes. ISAM were gen- DNMTs are increased in myofibroblast-transformed REPs. erated by transgenic complementary rescue of homozygous Epo Interestingly, the Epo gene locus is known to be highly meth- GFP-knock-in mice (EpoGFP/GFP)(S.Yamazakiet al., unpublished ylated in cell lines lacking Epo-producing abilities,48,49 indi- observations). The 8-kb DNA construct containing the Epo gene 9 cating the importance of DNA methylation on epigenetic was used (TgEpo3 ) for this purpose. ISAM showed adult-onset 9 silencing of Epo . This evidence raises a pos- Epo-deficient anemia because TgEpo3 -derived Epo in serum de- sibility that sustained activation of inflammatory signaling creased to almost undetectable level from weaning age. Epo-Cre might alter the epigenetic code of REPs and lead to difficulty mice were generated using a BAC clone containing the 180-kb frag- in restoring myofibroblast-transformed REPs to their original ment sufficient for recapitulating endogenous Epo gene regulation.2 state. However, considering that only a small number of REPs To prepare the Epo-Cre construct, Cre cDNA was integrated into the are required to maintain basal erythropoiesis, we surmise that a second exon of Epo-60K/BAC (S. Yamazaki et al., unpublished ob- reversion of Epo-producing ability of a small number of myofi- servations). Of the multiple transgenic mouse lines established, we broblasts or functionally repressed REPs would be sufficient to chose one line (line #44) for further study, which showed the char- support basal erythropoiesis of patients with ESRD. acteristic renal interstitial expression pattern of REPs and the anemic The pathway analysis revealed the series of inflammatory induction, when examined by crossing with R26Tmice (from Jackson signaling cascades as the most significant canonical signaling Laboratory)24 and EpoGFP/+ mice.2,23 To generate Epo-Cre::R26- pathways responsible for the phenotypic transition. Recent IKK2ca/+ mice, we crossed Epo-Cre mice with homozygous reports have shown that genetic inactivation of tubule- R26-IKK2ca mice (from Jackson Laboratory).27 Genotyping was per- activating signals or the depletion of macrophages improves formed by PCR using primers described in Supplemental Table 1.

levels, which are expressed as the relative expression compared with the contralateral (Cont) kidney. **P,0.01 versus sham and ClipR-14 (n=5 per group). (C) Changes of fibrogenic markers during Clip-ClipR treatment. Real-time PCR analyses are performed to quantify Acta2, Col1a1,andCol3a1 mRNA expressions using kidneys of sham, Clip, and ClipR-14 groups. ***P,0.01 versus sham and contralateral kidneys (n=5 per group). (D) Schematic presentation for the cell-fate analyses of REPs upon Clip-ClipR treatment. (E) Distribution of ON- REPs (Epo-GFP+ cells) and total REPs (tdTomato+ cells) upon Clip-ClipR treatment. Immunohistochemical analysis performed for Epo-GFP and tdTomato using kidneys of ISAM::Epo-Cre::R26T. (F and G) Changes in cell number of ON-REPs and total REPs upon Clip-ClipR treatment. FACS analyses are performed to count the number of Epo-GFP+ cells and tdTomato+ cells in ClipR-14 kidneys and contralateral (Cont) kidneys (n=5). Cont, contralateral; IHC, immunohistochemical analysis; NS, not significant. Scale bar in E, 200 mm.

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Figure 6. REPs possess cellular plasticity that responds to environmental signals. (A) Morphologic changes of G-REPs upon Clip-ClipR treatment determined by immunoelectron microscopic analyses. Uninjured G-REPs appear as osmium-positive (black) cells that adhere to proximal tubular cells and capillaries (sham). In contrast, UUO alters the morphology of G-REPs to show characteristic myofibroblastic features (Clip). The morphologic features of G-REPs regain normal features 12 days after the clip release (ClipR-14). Green arrows and red arrowheads indicate G-REPs and their processes, respectively. Lower panels are higher magnification of dotted boxes in upper panels. (B) Schematic presentation for the cell-fate analyses of G-REPs. Cell-fate of G-REPs is tracked by pulse labeling using single injection of BrdU, and then mice are euthanized at day 7 (ClipR-7) and day 14 (ClipR-14). (C) BrdU incorporation into G-REPs upon Clip-ClipR treatment. Immunofluorescence of Epo-GFP (green) and BrdU (red) are performed, and nucleus is stained with DAPI (blue) using kidneys treated as depicted in B. Right panels are higher magnifications of dotted boxes in left panels. (D) Efficacy of the cell tracking using BrdU incorporation. The BrdU-incorporated G- REPs are counted in five independent fields (n$3 per group). Approximately 7%–15% (yellow) of total G-REPs (green and yellow) are labeled with BrdU after UUO. Some tubular and GFP-negative interstitial cells also incorporate BrdU (red). PT, proximal tubular cell; C, capillary; DAPI, 4’,6-diamidino-2-phenylindole. Scale bars, 10 mm (upper panels) and 0.1 mm (lower panels) in A; 20 mminC.

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Figure 7. DNMTs are increased in myofibroblast-transformed REPs. (A) UUO-induced expression changes of DNMTs. Real-time PCR analyses of Dnmt1, Dnmt3a,andDnmt3b are performed using UUO-treated and control ISAM kidneys (n$3 per group). Data of the sham-treated group are used as the starting point (0 day), and set as 1. *P,0.05 and **P,0.01 versus sham. (B) Transcriptional profiles of DNMTs in G-REPs upon UUO. Real-time PCR analyses are performed with FACS-isolated G-REPs to quantify Dnmt1, Dnmt3a,and Dnmt3b. #P,0.05 and ##P,0.01 (n=3 per group; see Figure 3, C and D, for details). (C) Recovery of expression profiles of DNMTs after † †† the clip removal. Real-time PCR analyses are performed to quantify Dnmt1, Dnmt3a,andDnmt3b. P,0.05 and P,0.01 versus sham and ClipR-14 (n=5 per group). Cont, contralateral.

UUO Model expression of the contralateral kidney was used to normalize the For the ligature method, left ureter was exposed by a flank incision and anemic stimuli due to individual differences of anemic severity. ligated twice by 4-0 polysorb (Syneture) at the level of the lower pole of Sham operation was performed in the same way except for ligating the kidney. The ureter was cut between ligatures. For the reversible or clipping the ureter. UUO model (Clip-ClipR treatment), a vascular clip (Bear Medical Corp.) was used instead of the suture. After 48 hours of the ureter rHuEPO Treatment clipping, the vascular clip was removed and mice were euthanized 5 rHuEPO (3000 U/kg, a generous gift from Chugai Pharmaceutical Co. and 12 days after releasing the clips (ClipR-7 and ClipR-14, re- Ltd.) was intraperitoneally administered every other day for 12 days to spectively). Sham-operated uninjured kidneys were used as controls. reverse Epo-deficient anemia. A PBS-treated group was used as a For anti-inflammatory treatment, mice were intraperitoneally in- control. jected with dexamethasone (10 mg/g body weight) daily after the re- moval of vascular clip, and euthanized at 3 days after clip removal. LPS, hTGFb1, and Dexamethasone Treatment The appropriate vehicle treatment group was used as a control. To Mice were intraperitoneally injected with 0.1 and 1 mg/g body weight evaluate the Epo-producing potential, the Epo-GFP mRNA LPS (less than one tenth of the dose for proteinuria induction, and

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Figure 8. Phenotypic reversions of damaged REPs are accelerated by anti-inflammatory therapy. (A) A Venn diagram summarizing the differential expression of transcripts in the kidneys with Clip-ClipR treatment. The sum of 1191 differentially regulated transcripts is identified by the microarray analyses using kidneys of sham, Clip, and ClipR-14 groups (n=3 per group, per array). (B) Top signaling pathway responsible for the myofibroblastic transformation of REPs. IPA reveals that the atherosclerosis signaling is the most significant

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Table 2. Top 10 canonical signaling pathways responsible buffer (pH 6.0) was used for antigen retrieval. For p-p65 staining, for myofibroblastic transformation of REPs tyramide amplification was used (Invitrogen). Fluorescent images Ingenuity Canonical Pathway -Log (P Value) were obtained using an LSM510 confocal imaging system (Carl Atherosclerosis signaling 1.55E01 Zeiss). Acute phase response signaling 1.46E01 Dendritic cell maturation 1.33E01 Immunoelectron Microscopic Analyses Hepatic fibrosis/ hepatic stellate cell activation 1.29E01 Frozen sections (10 mm) were used for immunoelectron microscopic LXR/RXR activation 1.2E01 analysis. Sections were incubated with an anti-GFPantibody (Medical & Leukocyte extravasation signaling 1.02E01 Biological Laboratories) to detect G-REPs. Then, 3,3-diaminobenzidine Cell cycle control of chromosomal replication 9.6E00 staining was performed using appropriate secondary antibody and a fi Role of macrophages, broblasts, and endothelial 9.51E00 3,3-diaminobenzidine staining (DAKO). After osmium staining, sec- cells in rheumatoid arthritis tions were embedded in resin. Embedded sections were processed to Role of BRCA1 in DNA damage response 9.02E00 ultrathin sections for electron microscopic analysis. Cell cycle: G2/M DNA damage checkpoint regulation 8.84E00

Isolation of G-REPs by FACS one fiftieth of the dose for the sepsis model; Sigma) and with 10, 20, Kidneys were dissected after intracardiac perfusion with ice-cold PBS. and 40 ng/g body weight of human TGFb1 (R&D Systems). To an- Then, kidneys were decapsulated and minced. The minced whole tagonize the NFkB signaling, dexamethasone (10 mg/g body weight; kidney was digested with Liberase TM (Roche) with hyaluronidase Sigma) was coadministered with LPS (0.1 mg/g body weight). At the (Sigma) and DNase I (Roche) for 2.5 hours at 37°C. The cell suspen- time indicated, mice were euthanized and their kidneys were dis- sion was sieved through 40-mm nylon meshes. The resulting single- sected out for RNA isolation. Appropriate vehicle treatment groups cell suspension was washed with 5% FBS in PBS. Dead cells were were used as controls. stained with 1 mg/ml propidium iodide and excluded from collec- tion. FACS was performed using a FACSAria2 (BD Bioscience). GFP- Tissue Preparation for Histologic Analysis positive cells from 5–10 kidneys were sorted and combined to collect To prepare frozen sections, tissues were fixed with 4% paraformal- 100,000 cells for RNA extraction. dehyde for 2 hours, washed with 20% sucrose in PBS overnight, and then embedded in optimal cutting temperature compound (Sakura Isolation of Leukocytes from Kidneys Using Magnetic fi fi FineTek). Tissues for paraf n-embedded sections were xed with 4% Beads paraformaldehyde for 48 hours, and then processed by conventional Kidneys were digested into single-cell suspensions, and incubated procedures. with biotinylated anti-CD45 antibody (eBioscience) and with strep- tavidin-conjugated magnetic beads (Invitrogen). Fractionated cells Immunohistochemistry were resuspended into Isogen (Nippon gene). For fluorescence labeling, 10-mmfrozenorparaffin-embedded sec- tions were used. Sections were blocked and incubated with primary Real-Time PCR Analyses antibodies. Primary antibodies against GFP (Medical & Biological Isogen was used to prepare total RNA. Total RNA (3 mg) was used for Laboratories and Abcam), aSMA (Sigma), BrdU (BD Pharmingen), reverse transcription using random hexamers and a Superscript III Ki67 (DAKO), CD73 (eBioscience), p-Smad2/3 (Santa Cruz Bio- polymerase kit (Invitrogen). Real-time PCR was then performed with technology), and p-p65 (Cell Signaling Technology) were used. Ap- appropriate primers (Supplemental Table 1) using an ABI prism 7300 propriate Alexa 488- or 555-conjugated secondary antibodies were (Applied Biosystems). Gene expression levels were calculated based used for fluorescent detection, and nuclei were stained with 4’,6- on the threshold cycle (Ct) values and the efficiency of each primer diamidino-2-phenylindole. For paraffin-embedded sections, citrate set, and expressed as relative to 18S rRNA.

canonical signaling pathway and a heat-map representation of genes involved in this pathway is shown. Genes marked with red asterisks are examined in D. (C) Schematic presentation of cell isolation method from UUO-treated kidneys. Infiltrating and resident leukocytes are collected from single-cell suspensions of kidneys by using anti-CD45 antibody at UUO day 2. Fractionated cells were subjected for real-time PCR analyses. (D) Infiltrated leukocytes and residual renal cells collaboratively create inflammatory microenvironments. Real-time PCR analyses are performed to quantify mRNA expression levels for Pdgfb (encoding PDGFb), Tgfb1, Il6, Tnfa, Mmp3 (encoding matrix metalloproteinase 3), Mmp9, and Itgam (also known as CD11b, a surface marker for monocyte/macrophage). Relative mRNA levels in – CD45+ fraction, CD45 fraction, and whole UUO kidneys are expressed as fold changes compared with those of contralateral kidneys (set as 1). *P,0.05 and **P,0.01 versus UUO-treated kidneys (n=5 per group). (E) Schematic presentation of dexamethasone treatment. Dex is injected daily after the clip removal for 4 days, and mice are then euthanized for the subsequent analyses. (F) Effects of dexamethasone on the recovery of G-REPs. Real-time PCR analyses are performed for quantifying mRNA levels of Epo-GFP, Col1a1,andCol3a1 using vehicle-treated and dexamethasone-treated ISAM kidneys. Epo-GFP level is expressed as the relative expression compared with the contralateral kidney. #P,0.05 and ##P,0.01 (n$3 per group). Cont, contralateral; Dex, dexamethasone.

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Figure 9. REPs show the dynamic phenotypic changes in health and disease. In healthy conditions, most REPs are in “OFF” state and do not produce Epo. In response to anemic and/or hypoxic stimuli, REPs turn to “ON” state and start to produce Epo. In injured kidneys, microinflammation and other stresses transform REPs into myofibroblasts, possibly due to the NFkB and Smad signals. Myofibroblast-transformed REPs lose Epo-producing potential and actively contribute to renal fibrosis and inflammation. By elimi- nating the inflammatory signals, myofibroblast-transformed REPs restart producing Epo, regaining their physiologic phenotypes. Persisting inflammatory signals augment expression levels of DNMTs in myofibroblast-transformed REPs and might change their epigenetic codes (“epigenetic hits”). Modulation of inflammatory signals halts the inflammatory cycles and negates the deleterious link between fibrosis and anemia. We speculate that drugs targeting epigenetic modification would be an adjunct therapeutic strategy to bring back irreversibly transformed REPs to physiologic states. In summary, REPs possess profound cellular and functional plasticity in response to microenvironmental changes. Regulating the cellular status of REPs is important for treating renal fibrosis and anemia.

Microarray Analyses ClipR-14. The top canonical signaling pathways were identified using Total RNA was prepared from whole kidneys using Isogen. The RNA IPA software (http;//www.ingenuity.com; Ingenuity Systems). Heat quantity and quality were determined using an Agilent Bioanalyzer maps were generated using Cluster3.0 and JAVATreeview software. 2100 (Agilent Technologies). Gene expression data were obtained by hybridizing a total of nine ISAM kidney samples: sham, Clip, and Cell-Fate Tracking and Proliferation Analyses by BrdU ClipR-14 (n=3 per group) to a 4344K whole-mouse genome array To label proliferating cells, we pulse labeled cells with BrdU (50 mg/g (Agilent Technologies). Data were analyzed by Gene Spring software body weight; BD Pharmingen) 2 days after clipping the left ureter. (Agilent Technologies), normalized to the 75th percentile shift and Three groups were prepared: Clip (mice were euthanized at 3 hours the baseline transformed to the median of all samples to identify after BrdU injection without releasing the clip), and ClipR-7 and differentially expressed genes (P,0.01, one-way ANOVA). The data ClipR-14 (mice were euthanized 5 and 12 days after the releasing were submitted to the Gene Expression Omnibus (GSE45304). Toiden- the clip, respectively). Releasing of the clip was performed 3 hours tify factors responsible for myofibroblastic transformation of REPs, we after BrdU injection. For control experiments, uninjured mice were chose .2-fold upregulated genes by comparing Clip with either sham or euthanized 3 hours and 12 days after BrdU injections.

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Statistical Analyses 11. Maxwell PH, Ferguson DJ, Nicholls LG, Johnson MH, Ratcliffe PJ: The Data were expressed as means 6 SD. Comparisons between groups interstitial response to renal injury: Fibroblast-like cells show pheno- were performed using one-way ANOVA or an unpaired t test as ap- typic changes and have reduced potential for erythropoietin gene – propriate. For multiple comparisons, the Tukey–Kramer test was expression. Kidney Int 52: 715 724, 1997 fi , 12. Asada N, Takase M, Nakamura J, Oguchi A, Asada M, Suzuki N, used. Data were considered statistically signi cant at P 0.05. Yamamura K, Nagoshi N, Shibata S, Rao TN, Fehling HJ, Fukatsu A, Minegishi N, Kita T, Kimura T, Okano H, Yamamoto M, Yanagita M: Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis ACKNOWLEDGMENTS and renal anemia in mice. JClinInvest121: 3981–3990, 2011 13. Kriz W, Kaissling B, Le Hir M: Epithelial-mesenchymal transition (EMT) in kidney fibrosis: Fact or fantasy? JClinInvest121: 468–474, 2011 The authors thank Professors Hozumi Motohashi and Mitchell 14. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre Halperin for their invaluable advice, Ms. Maggie Patient for editing JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the our manuscript, and Ms. Kiyomi Kisu for helping with im- and not epithelial origin of myofibroblasts in kidney fibrosis. munoelectron microscopy. The authors also thank Biomedical Re- Am J Pathol 176: 85–97, 2010 search Core and Center for Laboratory Animal Research of Tohoku 15. Mezzano SA, Barría M, Droguett MA, Burgos ME, Ardiles LG, Flores C, Egido J: Tubular NF-kappaB and AP-1 activation in human proteinuric University Graduate School of Medicine for technical support, and renal disease. Kidney Int 60: 1366–1377, 2001 Chugai Pharmaceutical Co Ltd for comments. 16. Schmid H, Boucherot A, Yasuda Y, Henger A, Brunner B, Eichinger F, This work was supported in part by JST, CREST (to M.Y.); Grants- Nitsche A, Kiss E, Bleich M, Gröne HJ, Nelson PJ, Schlöndorff D, Cohen in-Aid for Creative Scientific Research (to M.Y.), for ScientificRe- CD, Kretzler M; European Renal cDNA Bank (ERCB) Consortium: search (to M.Y.), and for JSPS fellows (to T.S. and S.Y.) from the JSPS; Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy. Diabetes 55: 2993–3003, 2006 Grants-in-Aid for Scientific Research on Priority Areas (to M.Y.) from 17. Woroniecka KI, Park AS, Mohtat D, Thomas DB, Pullman JM, Susztak K: the MEXT; and a research grant from the Kidney Foundation, Japan Transcriptome analysis of human diabetic kidney disease. Diabetes 60: (JKFB12-1 to T.S.). 2354–2369, 2011 18. 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