Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 2;9(1):11254.
doi: 10.1038/s41598-019-47766-5.

An immortalized cell line derived from renal erythropoietin-producing (REP) cells demonstrates their potential to transform into myofibroblasts

Koji Sato  1   2 , Ikuo Hirano  1   3 , Hiroki Sekine  1   4 , Kenichiro Miyauchi  1   2 , Taku Nakai  1 , Koichiro Kato  1 , Sadayoshi Ito  2 , Masayuki Yamamoto  5 , Norio Suzuki  6
Affiliations

An immortalized cell line derived from renal erythropoietin-producing (REP) cells demonstrates their potential to transform into myofibroblasts

Koji Sato et al. Sci Rep. .

Abstract

The erythroid growth factor erythropoietin (Epo) is produced by renal interstitial fibroblasts, called REP (renal Epo-producing) cells, in a hypoxia-inducible manner. In chronic kidney disease (CKD), REP cells lose their Epo-production ability, leading to renal anaemia. Concurrently, REP cells are suggested to be transformed into myofibroblasts, which are the major player of renal fibrosis. Although establishment of cultured cell lines derived from REP cells has been a long-term challenge, we here successfully established a REP-cell-derived immortalized and cultivable cell line (Replic cells) by using a genetically modified mouse line. Replic cells exhibited myofibroblastic phenotypes and lost their Epo-production ability, reflecting the situation in renal fibrosis. Additionally, we found that cell-autonomous TGFβ signalling contributes to maintenance of the myofibroblastic features of Replic cells. Furthermore, the promoters of genes for Epo and HIF2α, a major activator of Epo gene expression, were highly methylated in Replic cells. Thus, these results strongly support our contention that REP cells are the origin of myofibroblasts in fibrotic kidneys and demonstrate that cell-autonomous TGFβ signalling and epigenetic silencing are involved in renal fibrosis and renal anaemia, respectively, in CKD. The Replic cell line is a useful tool to further investigate the molecular mechanisms underlying renal fibrosis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Establishment of a Replic cell line. (a) A representative image of cells cultured in MSCM for one week after isolation from ISAM-REC kidneys. The mixed cell culture contains cells positive (red) and negative (asterisk) for tdTomato fluorescence. (b) A colony of tdTomato-positive cells was generated after immortalization and isolation of tdTomato-positive REP cell lineage cells from the mixed renal cell culture. (c) Phase-contrast (left) and tdTomato fluorescent (right) images of Replic cells cultured with MSCM (upper) or DMEM (lower). (d) Growth curves of Replic cells cultured with DMEM (blue) or MSCM (red). On Day 0, 1.0 × 104 cells were seeded onto 3.5-cm dishes with 2.5 mL medium, and cell numbers were counted daily. The data are shown as the means ± standard errors. n = 3 for each group. **p < 0.01 by two-tailed and unpaired Student’s t tests. (e) Genomic PCR of Replic cells compared with that of organs from wild-type C57BL/6 (WT) and ISAM-REC mice. N, a non-template negative control.
Figure 2
Figure 2
Fibroblastic phenotype of Replic cells. (a) Flow cytometry of Replic cells (upper) and MEFs (lower) cultured with MSCM. Every Replic cell expressed high-level tdTomato fluorescence. The mean fluorescent intensity of CD73 in Replic cells was higher than that in MEFs (dotted red lines). (b,c) The mRNA expression levels of marker genes for fibroblasts (b) and non-fibroblastic renal cells (c) in Replic cells were compared to those in wild-type (WT) kidneys and MEFs. Arrows indicate undetectable levels. Cell culture was conducted with MSCM. Ten biologically independent samples for each cell line were analysed. The average expression level of WT kidneys (n = 3) was set at 1.0, and error bars indicate standard errors. **p < 0.01 by multiple comparisons using one-way ANOVA with Tukey-Kramer tests.
Figure 3
Figure 3
Hypoxic response of Replic cells. (a,b) mRNA expression levels of genes related to the hypoxic response in Replic cells exposed to hypoxia (a) or GSK360A (b) for 24 hours were compared to those in ISAM-REC kidneys. Twelve biologically independent samples for each group of Replic cells were analysed. The average expression level of ISAM-REC kidneys (n = 3) was set at 1.0, and error bars indicate standard errors. Arrows indicate undetectable levels. *p < 0.05 and **p < 0.01 by multiple comparisons using one-way ANOVA with Tukey-Kramer tests. (c) Immunoblots for HIF1α and HIF2α in Replic cells cultured under normoxic (N) or hypoxic (H, 1% oxygen) conditions for 24 hours (upper) and in Replic cells cultured with or without GSK360A (G and V, respectively) for 24 hours (lower). The HIF1α- and HIF2α-derived band intensities normalized with βTubulin-derived band intensities were quantified and are shown in the right panels. Hep3B cells was used as positive controls for the hypoxia-inducible accumulation of HIF1α and HIF2α. βTubulin was used as an internal control. Cell culture was conducted with MSCM (a–c).
Figure 4
Figure 4
Epigenetic suppression of the Epo-production ability in Replic cells. (a) Summary of results from bisulfite sequencing in the Epo (upper) and Epas1 (lower) gene promoters of Replic cells and ISAM-REC livers. Genomic regions between red arrows were sequenced after cloning 8 clones for each promoter from bisulfite-converted genomic DNAs. Each row represents a single clone. Vertical bars indicate CpG sites in the tested regions, and white and black dots represent unmethylated and methylated CpG sites, respectively. TSS, transcription start site. *p < 0.05 and **p < 0.01 between the methylated ratio of each CpG site in Replic cells and ISAM-REC livers, using χ2 tests. (b) Immunoblots for HIF2α in Replic cells transiently transfected with plasmids expressing GFP or constitutively active HIF2α. βTubulin was used as an internal control for immunoblots. (c) EpoGFP mRNA expression levels in Replic cells transiently transfected with plasmids expressing GFP or constitutively active HIF2α. ISAM-REC kidneys were used as positive controls for EpoGFP mRNA expression. Six biologically independent samples for each group of Replic cells were analysed. The average expression level of ISAM-REC kidneys (n = 3) was set at 1.0, and error bars indicate standard errors. Arrows indicate undetectable levels. **p < 0.01 by multiple comparisons using one-way ANOVA with Tukey-Kramer tests. Cell culture was conducted with MSCM (a–c).
Figure 5
Figure 5
Myofibroblastic property of Replic cells. (a) mRNA expression levels of genes related to myofibroblasts in Replic cells cultured with MSCM. In addition to MEFs cultured with DMEM, injured and contralateral kidneys (n = 3 for each) of UUO-treated normal mice were compared to Replic cells with respect to mRNA expression. Twelve biologically independent samples for each cell line were analysed. (b) mRNA expression levels of genes for TGFβ superfamily receptors in Replic cells cultured with MSCM were compared to those in mouse kidneys (n = 3 for ISAM-REC kidneys and WT kidneys) and MEFs. Six biologically independent samples for each cell line were analysed. (c) Flow cytometry of TGFβR2 expression on the cell surface of Replic cells and MEFs cultured with DMEM. Negative controls (NC) were stained only with APC-conjugated streptavidin. The mean fluorescent intensities are shown (red dotted lines). (d) mRNA expression levels of genes related to myofibroblasts in Replic cells cultured with DMEM or MSCM. Six biologically independent samples for each cell line were analysed. The average expression levels of injured kidneys (a), ISAM-REC kidneys (b), or Replic cells cultured with DMEM (d) were set at 1.0. Error bars are indicated standard errors, and arrows indicate undetectable levels (a,b). *p < 0.05 and **p < 0.01 by multiple comparisons using one-way ANOVA with Tukey-Kramer tests (a,b) or by two-tailed, unpaired Student’s t-tests (d).
Figure 6
Figure 6
Upregulation of profibrotic TGFβ-signalling in Replic cells. (a) Immunoblots for p-Smad2/3 and total Smad2/3 in Replic cells cultured with DMEM were compared to those in MEFs cultured with DMEM (left) or Replic cells cultured with MSCM (right). βTubulin was used as an internal control. (b) TGFβ concentrations in serum-free DMEM incubated with Replic cells or MEFs for 24 hours. Three biologically independent samples for each cell line were analysed. (c) Relative amounts of the listed factors in culture supernatants of Replic cells and MEFs, both of which were cultured with serum-free DMEM for 24 hours, were determined by the antibody array based on a double antibody sandwich assay. The data indicate the chemiluminescent intensities of each spot (duplicate for each antibody, A and B). Undetectable cytokines, both spots of which showed signal intensities lower than 50, are listed in the right column. The scanned image of the antibody array is shown in Supplementary Fig. 3.
Figure 7
Figure 7
TGFβ-dependent myofibroblastic property of Replic cells. (a) mRNA expression levels of genes related to myofibroblasts in Replic cells incubated with or without SB431542 in DMEM for 24 hours. EpoGFP mRNA levels were also examined. ISAM-REC kidneys (n = 3) were used as controls. Ten biologically independent samples for each treatment of Replic cells were analysed. (b) The Acta2 mRNA expression levels in Replic cells incubated with vehicle or neutralizing antibody against TGFβ (N-Ab) in DMEM for 24 hours. Six biologically independent samples for each treatment of Replic cells were analysed. (c) Immunoblots for p-Smad2/3 and total Smad2/3 in Replic cells cultured with or without SB431542 in DMEM for 1 hour. βTubulin was used as an internal control. (d) Relative mRNA expression levels of genes related to hypoxic response were measured in Replic cells cultured with DMEM in the presence or absence (Vehicle) of the inhibitor cocktail. ISAM-REC kidneys (n = 3) were used as controls. As the inhibitor cocktail, SB431542, 5-Aza, and GSK360A were added to the cell culture at 24 hours, 5 days, and 24 hours before sampling, respectively. Five biologically independent samples for each treatment of Replic cells were analysed. The average expression level of ISAM-REC kidneys (a,d) or vehicle-treated Replic cells (b) was set at 1.0, and the error bars represent standard errors for each experimental group. (a,b,d) Arrows indicate undetectable levels. *p < 0.05 and **p < 0.01 by multiple comparisons using one-way ANOVA with Tukey-Kramer tests (a,d) or by two-tailed, unpaired Student’s t-tests (b).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nangaku M, Eckardt KU. Pathogenesis of renal anemia. Semin Nephrol. 2006;26:261–268. doi: 10.1016/j.semnephrol.2006.06.001. - DOI - PubMed
    1. Friedman SL, Sheppard D, Duffled JS, Violette S. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med. 2013;5:167sr1. doi: 10.1126/scitranslmed.3004700. - DOI - PubMed
    1. Iwano M, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002;110:341–350. doi: 10.1172/JCI0215518. - DOI - PMC - PubMed
    1. Zeisberg EM, Potenta SE, Sugimoto H, Zeisberg M, Kalluri R. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J Am Soc Nephrol. 2008;19:2282–2287. doi: 10.1681/ASN.2008050513. - DOI - PMC - PubMed
    1. Humphreys BD, et al. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol. 2010;176:85–97. doi: 10.2353/ajpath.2010.090517. - DOI - PMC - PubMed

Publication types

MeSH terms