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Musculin/MyoR is expressed in kidney side population cells and can regulate their function

Musculin/MyoR is expressed in kidney side population cells and can regulate their function JCB: ARTICLE Musculin/MyoR is expressed in kidney side population cells and can regulate their function 1,2 1,2 1 1 4,9 1 Keiichi Hishikawa, Takeshi Marumo, Shigeki Miura, Asato Nakanishi, Yumi Matsuzaki, Katsunori Shibata, 1 4,9 4,9 6 5 5 Tomoko Ichiyanagi, Hiroko Kohike, Takuya Komori, Ichiro Takahashi, Osamu Takase, Naohiko Imai, 1,2 1,3 5 7 8 Masahiro Yoshikawa, Toshihiko Inowa, Matsuhiko Hayashi, Toshio Nakaki, Hiromitsu Nakauchi, 4,9 1,2 Hideyuki Okano, and Toshiro Fujita 1 2 Department of Clinical Renal Regeneration, Graduate School of Medicine, Department of Internal Medicine, Division of Nephrology and Endocrinology, and Department of Urology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan 4 5 Department of Physiology and Department of Internal Medicine, Keio University School of Medicine, Shionanomachi 35, Shinjuku-ku, Tokyo 160, Japan 6 7 Central Electron Microscopic Laboratory and Department of Pharmacology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo 173, Japan The Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108, Japan Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo 102-8666, Japan usculin/MyoR is a new member of basic helix- positive cells and improved renal function. Kidney SP loop-helix transcription factors, and its expres- cells in reversible acute renal failure expressed a high sion is limited to skeletal muscle precursors. level of renoprotective factors and leukemia inhibitory Here, we report that musculin/MyoR is expressed in adult factor (LIF), but not in irreversible chronic renal failure. In kidney side population (SP) cells and can regulate their cultured kidney SP cells, LIF stimulated gene expression of function. SP phenotype can be used to purify stem cell– renoprotective factors, and down-regulation of musculin/ rich fractions. Microarray analysis clarified that musculin/ MyoR augmented LIF-induced gene expression. Our re- MyoR was exclusively expressed in kidney SP cells, and sults suggest that musculin/MyoR may play important the cells resided in the renal interstitial space. Musculin/ roles not only in developmental processes but also in re- MyoR-positive cells were decreased in acute renal failure, generative processes in adult tissue. but infusion of kidney SP cells increased musculin/MyoR- Introduction In 1996, Goodell et al. (1996) reported a new method of obtain- BCRP1/ABCG2 prevents hematopoietic differentiation (Zhou ing an enriched population of hematopoietic stem cells from et al., 2001). However, the physiological role of this ATP- adult mouse bone marrow in a single step by using Hoechst binding cassette (ABC) transporter (Gottesman et al., 2002) in 33342 dye staining and FACS. The isolated cells were called SP cells is still unclear. side population (SP) cells, and the SP phenotype can be used to In this study, we first tried to clarify the gene expression purify a stem cell-rich fraction (Zhou et al., 2001; Matsuzaki et profile of kidney SP cells in healthy control and renal failure al., 2004). Bone marrow–derived SP cells differentiate into car- models by microarray analysis. The results of microarray and diomyocytes, endothelial cells (Jackson et al., 2001), and os- quantitative PCR analysis clarified that musculin/MyoR is ex- teoblast precursors (Olmsted-Davis et al., 2003), and skeletal clusively expressed in kidney SP cells but not in non-SP cells, muscle SP cells (Asakura et al., 2002) may differentiate into and we further examined the functional role of musulin/MyoR endothelial cells (Majka et al., 2003). The SP phenotype is de- in kidney SP cells. Musculin/MyoR is a newly discovered basic termined by BCRP1/ABCG2, and enforced expression of helix-loop-helix (bHLH) protein that represses myogenesis (Robb et al., 1998; Lu et al., 1999) and controls facial muscle development with capsulin (Lu et al., 2002). Musculin/MyoR Correspondence to Keiichi Hishikawa: [email protected] mRNA is first expressed at 9.8 days postcoitum in the mouse Abbreviations used in this paper: ABC, ATP-binding cassette; ARF, acute renal failure; bHLH, basic helix-loop-helix; BM, bone marrow-derived mononuclear; embryo, and is almost exclusively restricted to cells of skeletal BMP, bone morphologic protein; BUN, blood urea nitrogen; CRF, chronic renal muscle lineage, with peak expression at 14–15 days postcoitum. failure; HGF, hepatocyte growth factor; LIF, leukemia inhibitory factor; RA, To our knowledge, no study has confirmed the expression of retinoic acid; SP, side population; TSA, trichostatin A. The online version of this article includes supplemental material. musculin/MyoR in adult kidney tissue. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 6, June 20, 2005 921–928 http://www.jcb.org/cgi/doi/10.1083/jcb.200412167 JCB 921 THE JOURNAL OF CELL BIOLOGY Figure 1. FACS and microarray analysis of kidney SP cells. (A) Representative FACS profile of SP cells isolated from the kidney of normal (ddY and ICR) and renal failure (HIGA and ICGN) mice (total events:1.0  10 to 3.0 10 ). (B) Kidney SP cells isolated from ddY, ICR, HIGA, and ICGN mice stained with Hoechst dye (white bars). Total event of all mice were set 1.0  10 , and uptake of Hoechst dye was significantly blocked in the presence of reserpine (res) in all samples (black bars). Results are presented as the mean  SEM, n  15. *, P  0.05 versus control (without reserpine). (C) Scatter-plot analyses of gene expression (3,800 genes) in SP cells versus kidney tissue prepared from normal (ddY and ICR) and renal failure (HIGA and ICGN) model mice. Average expression levels of each gene were calculated from three independent hybridizations. We then aimed to determine the functional role of 3.1  0.9%, respectively) than did bone marrow (1%; not de- BCRP1 and musculin/MyoR in the kidney. We found that kid- picted). In both renal failure models (HIGA and ICGN), the ney SP cells, but not non-SP cells, are protected from cell population of reserpine-sensitive SP cells in the kidney was re- death by BCRP1. Leukemia inhibitory factor (LIF) is up-regu- duced to 30% of control (1.5  0.1 and 1.1  0.2%, respec- lated in reversible cisplatin-induced acute renal failure (ARF), tively). In our preparation, kidney SP cells were Sca-1 positive and is considered to play a role for regeneration process (70% on average; n  20) but CD45 negative (2% on aver- (Yoshino et al., 2003). In this study, we also examined the ex- age; n  20) in all mice, indicating that they were not part of pression of renoprotective factors such as hepatocyte growth the hematopoietic system and were not likely to be contami- factor (HGF), VEGF, bone morphologic protein (BMP) 7, and nated with peripheral blood SP cells. To characterize kidney SP LIF in kidney SP cells. In an ARF model, gene expression of cells, gene expression profiling was performed using freshly renoprotective factors and LIF was up-regulated in kidney SP isolated kidney SP cells from control (ddY and ICR) and renal cells but not in non-SP cells. On the other hand, there was no failure models (HIGA and ICGN), as well as cortical tissue of up-regulation of renoprotective factors and LIF in irreversible the kidney from control and renal failure mice. Among the chronic renal failure (CRF) models. In cultured kidney SP 3,800 genes analyzed, musculin/MyoR (Robb et al., 1998, cells, LIF augmented gene expression of renoprotective fac- 1999) was the only gene that was commonly and highly ex- tors. Although inhibition of BCRP1 showed no effect on LIF- pressed in SP cells of all mice (greater than sixfold higher com- induced gene expression of renoprotective factors, down-regu- pared with cortical tissue; Fig. 1 C, red square with arrow). We lation of musculin/MyoR augmented it. These results provide also performed gene expression profiling using kidney SP cells evidence that expression of musculin/MyoR is not limited to and non-SP cells, and found that musculin/MyoR was also ex- the skeletal muscle lineage and the production of renoprotective pressed greater than sixfold higher in SP cells compared with factors in kidney SP cells is regulated by LIF and musculin/ non-SP cells in all mice (unpublished data). MyoR, but nor by BCRP1. Immunohistochemical staining of musculin/MyoR and BCRP1 Results To confirm the protein expression and localization of musulin/ Gene expression profile of kidney SP cells MyoR-positive cells, we performed immunohistochemical We found a significant increase in blood urea nitrogen (BUN) and IgA in IgA nephropathy (HIGA) mice compared with con- Table I. Biochemical features in control (ddY and ICR) and CRF trol mice (ddY; Table I). In nephrotic syndrome mice (ICGN), (HIGA and ICGN) mice BUN and total cholesterol were significantly increased as com- BUN IgA Total cholesterol pared with control (ICR). We isolated whole kidney cells from mg/dl mg/dl mg/dl these mice and stained them with Hoechst 33342 dye (Zhou et ddY 20  2 98  12 ND al., 2001). The isolated cells were subjected to FACS analysis a a HIGA 33  3 396  26 ND in the presence or absence of reserpine to quantitate the number ICR 36  4 ND 108  13 of cells with a SP and non-SP phenotype (Fig. 1, A and B). As ICGN 96  4 ND 188  12 previously reported (Asakura and Rudnicki, 2002), kidney tis- BUN, IgA, and total cholesterol levels in serum. Results are presented as the sue from control mice (ddY and ICR) contained a much mean  SEM (n  12). higher number of reserpine-sensitive SP cells (5.1  0.1 and P  0.05 versus normal mice. 922 JCB • VOLUME 169 • NUMBER 6 • 2005 Figure 2. Immunohistochemical analysis of kidney SP cells. (A and B) Representative photomicrographs of fluorescent images of musculin, BCRP1, and nuclear staining (TO-PRO3) in cytospin of kidney SP cells (C57BL/6) just after FACS sorting. Overlay images are shown in the right Figure 3. TNF-  induced cell death in kidney SP cells. (A) Kidney SP and panels. (C and D) Representative photomicrographs of fluorescent images non-SP cells just after FACS sorting were incubated with TNF- at the indi- of musculin and nuclear staining in renal tissue (C57BL/6). Musculin-positive cated concentrations for 24 h. TNF- induced cell death in SP cells (black cells (arrows) reside in the interstitial space of the kidney. Overlay images of bars) in a concentration-dependent manner, but not in non-SP cells (white musculin and nuclear (yellow) images also showed that the cells reside in bars). Cell survival was determined by trypan blue exclusion assay. (B and C) the interstitial space of the kidney. Kidney SP cells just after FACS sorting were incubated with 50 M reser- pine (res), 8 ng/ml TNF-, or both for 24 h. Compared with control (no treatment), TNF- alone significantly increased DNA fragmentation and apoptosis, and cotreatment with reserpine significantly augmented it. analysis. Kidney SP cells were very small, but strongly ex- DNA fragmentation assay was performed using an ELISA kit, and percent- pressed both musculin/MyoR and BCRP1 protein (Fig. 2, A age of apoptosis was evaluated by nuclear staining with Hoechst 33258. and B). Although the SP population is a heterogeneous popu- Results are presented as means  SEM of four independent experiments. *, P  0.05 compared with control; **, P  0.05 compared with TNF-. lation, musculin/MyoR staining clarified that kidney SP cells were musulin/MyoR positive (95%), but non-SP cells were negative (5%; Fig. S1, available at http://www.jcb.org/cgi/ content/full/jcb.200412167/DC1). In other words, the purity apoptosis, and cotreatment with TNF- and reserpine signifi- of kidney SP cells was 95% with respect to musculin/ cantly increased it (Fig. 3, B and C). MyoR staining. As shown in Fig. 2 (C and D), musculin/ Kidney SP cells contribute to regeneration MyoR-positive cells were localized in the interstitial space of the kidney. To clarify the functional role of kidney SP cells in vivo, we in- jected isolated kidney SP cells systemically via the tail vain BCRP protects SP cells, but not non-SP into mice with ARF induced by cisplatin administration cells, from cell death (Ramesh and Reeves, 2002). In this model, i.p. injection of 12 BCRP1/ABCG2 is a member of ABC transporters, which gen- mg/kg cisplatin induced a peaked BUN level on day 7 (Fig. 4 A). erally protect cells from toxic agents (Gottesman et al., 2002). On day 7, musculin/MyoR-positive cells in the interstitial To clarify the reason why kidney SP cells were decreased in re- space were significantly reduced by cisplatin treatment alone, nal failure models (Fig. 1 B) and to examine the protective role but kidney SP cell infusion on day 1 significantly increased of BCRP1 in kidney SP cells, SP and non-SP cells were treated these cells on day 7 (Fig. 4, B–E). We also compared the effect with TNF-. Treatment with TNF- (at up to 16 ng/ml) did kidney SP cell infusion on renal function to those of infusion of not reduce cell viability of non-SP cells, but significantly re- non-SP cells or bone marrow-derived mononuclear (BM) cells. duced cell viability of SP cells in a concentration-dependent Infusion of kidney SP cells significantly suppressed the peak manner (Fig. 3 A). We further examined the effect of an inhibi- BUN and creatinine level on day 7 (Fig. 5, A and B), whereas tor of BCRP1/ABCG2 on TNF-–induced cell death in kidney infusion of non-SP and BM cells had no effect. Tubular necro- SP cells. Compared with control, reserpine alone showed no ef- sis scores were also significantly improved by SP cell infusion fect (Fig. 3, B and C). TNF- alone significantly increased (Fig. 5, C and D). MYOR IN KIDNEY SP CELLS • HISHIKAWA ET AL. 923 that contribute to the kidney regeneration process, such as HGF (Mizuno et al., 1998), VEGF (Ostendorf et al., 1999), and BMP7 (Wang and Hirschberg, 2003), in a reversible cisplatin- induced ARF model and two irreversible CRF models (IgA nephropathy, HIGA; nephritic syndrome, ICGN). In ARF mice, kidney SP cells expressed significantly higher levels of HGF, VEGF, and BMP7 compared with the cells in control mice (Fig. 6 A). In contrast, in the CRF models, there was no significant difference between disease mice and control mice (Fig. 6, B and C). Recently, LIF was reported to be up-regu- lated and to contribute to regeneration processes (Yoshino et al., 2003), so we examined the expression of LIF in kidney SP cells. We also confirmed up-regulation of LIF in the ARF model but not in the CRF models (Fig. 6 A). On the other hand, we could not find significant changes of these factors in kidney non-SP cells of all disease mice as compared with their control mice (unpublished data). Musculin/MyoR but not BCRP1 regulates function of kidney SP cells As infusion of kidney SP cells improved renal function in an ARF model and LIF is also up-regulated in this model, we ex- amined the effect of LIF on gene expression of renoproptec- Figure 4. Systemic kidney SP cell infusion increases musculin/MyoR- tive factors and musculin/MyoR in kidney SP and non-SP positive cells in renal interstitial space in ARF model. (A) Time course of plasma BUN level in cisplatin-induced ARF model. Values are the mean  cells. Compared with control (without LIF), treatment with SEM (n  8). *, P  0.05 versus day 0. (B–D) Representative fluorescence LIF significantly up-regulated gene expression of HGF, photographs and overlay images (fluorescent and phase contrast) of renal VEGF, and BMP7 in SP but not in non-SP cells (Fig. 7, A and tissues of control, cisplatin alone (cis), and infusion of kidney SP cells (cisSP) on day 7. Green spots with red arrowheads are musculin/MyoR- B). To clarify the role of BCRP1 in LIF-induced gene expres- positive cells. (E) Musculin/MyoR-positive cells were significantly reduced sion of renoprotective genes, we treated cells with LIF and re- in the cisplatin-induced ARF model (cis) but were markedly increased by serpine, but reserpine showed no effect. Retinoic acid (RA) is systemic kidney SP cell infusion (cisSP). Values are the average number of musculin-positive cells in 10 randomly selected low power fields reported to down-regulate musculin/MyoR (Yu et al., 2003), (200) in four independent experiments. Values are the mean  SEM. and cotreatment with LIF and RA significantly augmented the *, P  0.05 versus control; **, P  0.05 versus cis. LIF-induced gene expression in kidney SP cells but not in non-SP cells. Finally, we confirmed the effect of LIF, reser- Kidney SP cells express growth factors pine, and RA on musculin/MyoR expression in SP and non- that contribute to tissue regeneration SP cells by RT-PCR and real-time PCR. LIF and reserpine To clarify the role of kidney SP cells in renal failure, we per- alone showed no effect on basal musculin/MyoR gene expres- formed quantitative real-time PCR analysis of several factors sion, and cotreatment with reserpine also showed no effect Figure 5. Systemic kidney SP cell infusion augments recovery of renal function in ARF model. (A and B) Compared with control (cont), cisplatin alone (cis) significantly increased the BUN and creatinine levels, but infusion of kidney SP cells (cisSP) on day 1 (day after cisplatin injection) significantly reduced the BUN and creatinine levels on day 7. On the other hand, infusion of non-SP cells (cisNSP) and BM cells (cisBM) showed no effect. (C and D) Compared with con- trol (cont), cisplatin alone (cis) significantly increased the tu- bular necrosis scores, but infusion of kidney SP cells (cisSP) on day 1 (day after cisplatin injection) significantly reduced it on day 7. Values are the mean  SEM. *, P  0.05 versus control; **, P  0.05 versus cis. 924 JCB • VOLUME 169 • NUMBER 6 • 2005 Discussion Members of the bHLH family of transcription factors are in- volved in cell fate determination and tissue differentiation in several developmental processes including hematopoiesis, myogenesis, and neurogenesis. Musculin/MyoR is a newly dis- covered bHLH protein that acts as a lineage-restricted repres- sor of embryonic skeletal muscle development. In this study, we clarified that musculin/MyoR is expressed in adult kidney SP cells and regulates LIF-induced gene expression of renopro- tective factors such as HGF, VEGF, and BMP7. Robb et al. (1998) failed to detect expression of muscu- lin/MyoR in adult tissue by Northern blotting but demonstrated expression in the adult brain, spleen, and muscle but not in the kidney, liver, lung, or thymus by RNase protection assay. Yu et al. (2003) detected expression of musculin/MyoR in the adult brain, heart, liver, lung, small intestine, spleen, and thymus by Northern blotting. This discrepancy may be explained by the sensitivity of their assay, but our results also suggest that mus- culin/MyoR expression is not lineage restricted as has been re- ported, but is expressed in a relatively broad spectrum of adult tissue. Musculin/MyoR was originally identified as a transcrip- tional repressor in embryonic skeletal muscle precursors, but its function in adult tissue is not known. In fact, musculin/ MyoR knockout mice were not embryonic lethal, and analysis of kidney function in these mice has not been completed (un- Figure 6. Quantitative real-time PCR analysis of HGF, VEGF, BMP7, and LIF in kidney SP cells. (A) Cisplatin-induced gg model. White bars show published data). In the present study, we demonstrated that control (no treatment) and black bars show cisplatin treatment. Compared down-regulation of musculin/MyoR augmented LIF-induced with control, gene expression of HGF, VEGF, BMP7, and LIF was signifi- gene expression of HGF, VEGF, and BMP7 in kidney SP cells. cantly up-regulated in kidney SP cells. (B) IgA nephropathy mice. White bars show control (ddY) and black bars show IgA nephropathy (HIGA). These results showed that musculin/MyoR plays a role as a (C) Nephritic syndrome mice. White bars show control (ICR) and black transcriptional repressor in adult kidney SP cells as reported in bars show nephritic syndrome (ICGN). Expression was normalized to embryonic skeletal muscle. The physiological role of kidney GAPDH and is shown as the ration to the value of control. Values repre- sent means  SEM (n  6). *, P  0.05 versus control. SP cells is still unclear, but systemic administration of RA, which down-regulates musculin/MyoR (Yu et al., 2003), aug- (Fig. 7, C and D). On the other hand, RA significantly down- mented the recovery of ARF (Perez et al., 2004). To down-regu- regulated musculin/MyoR in the presence of LIF (Fig. 7, C late musculin/MyoR in vivo, we injected histone deacetylase in- and D). In non-SP cells, basal musculin/MyoR expression was hibitor trichostatin A (TSA), which down-regulates musculin/ almost absent (Fig. 6 E). MyoR (Yu et al., 2003), in control and CRF model (nephrotoxic Figure 7. LIF-induced gene expression is regulated by musculin/MyoR. (A and B) Kidney SP cells (A) or non-SP cells (B) just after FACS sorting were incubated with vehicle (white bars), LIF (10 ng/ml; black bars), LIF with reserpine (10 ng/ml and 50 M, respectively; dotted bars), and LIF with RA (10 ng/ml and 5  10 mol/l, respectively; hatched bars) for 12 h, and then gene expression of HGF, VEGF, and BMP7 was examined by TaqMan real-time PCR. (C and D) Gene expression of musculin/MyoR in kidney SP cells. An aliquot of RNA in panel A was examined by RT-PCR (C) and Taq- Man real-time PCR (D). (E) Gene expression of musculin/ MyoR in non-SP cells. Basal expression of musculin/MyoR was almost absent. Values represent means  SEM (n  6). *, P  0.05 versus control; **, P  0.05 versus LIF. MYOR IN KIDNEY SP CELLS • HISHIKAWA ET AL. 925 serum model, NTN model). As in the case with HIGA and BMP7 in cultured kidney SP cells. Moreover, the number of SP ICGN, kidney SP cells analyzed by FACS were reduced in cells was decreased in the irreversible CRF models (Fig. 1 B). NTN model. Surprisingly, daily injection (for 21 d) of TSA al- We also examined the effect of kidney SP cell infusion on CRF most completely abolished musculin/MyoR-positive cells in model (unilateral ureteral obstruction model) but found no sig- the kidney of control and NTN model and completely pre- nificant improvement (Fig. S3, available at http://www.jcb.org/ vented progression of CRF in NTN model. Moreover, regener- cgi/content/full/jcb.200412167/DC1). Recently, we also re- ative factors were also up-regulated by treatment with TSA ported that LIF induces multilineage differentiation of kidney (unpublished data). Together, these results suggest that muscu- SP cells in culture (Hishikawa et al., 2005), and some other lin/MyoR-positive cells could be new targets for regeneration pathways can be involved in the restoration of renal function. in renal failure, and we are currently trying to clarify its role us- Although the precise mechanism regarding the regenerative role ing a renal failure model in musculin/MyoR knockout mice. of kidney SP cells remains to be clarified, our results suggest Theoretically, an activated transcription factor should be that loss of musulin/MyoR-positive kidney SP cells and inade- located within the nucleus, but musculin/MyoR was expressed quate expression of key factors such as LIF and BMP7 in CRF throughout kidney SP cells. Inactive sterol regulatory element may determine the fate of CRF. binding proteins, another member of the bHLH transcription BCRP1/ABCG2 is reported to determine the SP pheno- factor family, are synthesized as precursors in the cytoplasm type (Zhou et al., 2001). Enforced expression of BCRP1/ and bind to the endoplasmic reticulum membrane. Upon acti- ABCG2 inhibits hematopoietic development (Zhou et al., vation, the active forms of sterol regulatory element binding 2001), but its mechanism is still unclear. BCRP1/ABCG2 is a proteins enter the nucleus and activate several genes that con- member of ABC transporters (Gottesman et al., 2002) and is trol the synthesis and uptake of cholesterol and unsaturated known to transport doxorubicin, daunorubicin, mitoxantrone, fatty acids (Nagoshi and Yoneda, 2001). Pod1, which is also a topotecan, and prazocin. Although many ABC transporters bHLH transcription factor expressed in kidney stromal cells have been identified as drug-resistance proteins, they are all and is required for glomerulogenesis, is distributed throughout expressed in normal tissues. Consistent with their wide distri- cells located in peritubular interstitial space in the kidney (Cui bution, it is becoming clear that in addition to exogenously ad- et al., 2003). These results suggest that localization of a tran- ministered drugs, ABC proteins transport numerous endoge- scription factor such as musculin/MyoR throughout kidney SP nous substrates. It is well know that ABC transporters have cells is not unique, but further characterization may be needed an important role in protecting the central nervous system to fully understand its mechanism of translocation. through the blood brain barrier (Schinkel et al., 1996), and Infusion of kidney SP cells significantly augmented re- BCRP1/ABCG2 in the placenta may play a role in protect- covery of ARF and increased musculin/MyoR-positive cells in ing fetal blood from toxic organic anions and excreting the interstitial space. Therefore, we speculate that homing of glutathione/glucuronide metabolites into maternal circulation kidney SP cells to the interstitial space is necessary for regener- (Gottesman et al., 2002). Considering these results, we exam- ation in the ARF model induced by cisplatin. Recently, cardiac ined the protective role of BCRP1 in kidney SP cells. The role stem cells were found in the interstitium between well-differen- for TNF- in cisplatin-induced nephrotoxicity has been well tiated myocytes (Beltrami et al., 2003), and myogenic-endothe- established (Ramesh and Reeves, 2002), and important roles of lial progenitor cells with the SP phenotype were also identified TNF- are also reported in IgA nephropathy (Tuglular et al., in the interstitial spaces of skeletal muscle (Tamaki et al., 2002), 2003; Chan et al., 2005) and nephrotic syndrome (Suranyi et suggesting that SP cells commonly reside in the interstitial al., 1993; Kim et al., 2004; Raveh et al., 2004). As shown in space. We also systemically injected kidney SP cells from GFP Fig. 3, kidney SP cells were more sensitive to TNF- than non- mice and DiI-labeled kidney SP cells, and found that these cells SP cells, and BCRP1 protected kidney SP cells from cell death. were located in the interstitial spaces of kidney (Fig. S2, avail- The precise mechanism of TNF-–induced cell death in kidney able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1). SP cells and direct in vivo evidence linking these effects to pro- As the interstitial space of the kidney has a fine microcircula- gression of renal failure remain to be determined, but our re- tion system, musculin/MyoR-positive kidney SP cells in the in- sults may explain why the number of musculin/MyoR-positive terstitial space may provide paracrine growth support for neigh- kidney SP cells was decreased in renal failure in which inflam- boring cells in vivo. Several growth factors such as HGF, matory cytokines were overexpressed. VEGF, and BMP7 have been reported to contribute to kidney In summary, our study provides evidence for a new role regeneration. Especially, BMP7 is the only factor that reverses of musculin/MyoR and a protective role of BCRP1 in kidney chronic renal injury (Zeisberg et al., 2003) and Lin et al. (2005) SP cells. Our results suggest that members of the bHLH family recently reported the important role of BMP7 enhancer in CRF. of transcription factors such as musculin/MyoR may play im- Administration of exogenous LIF ameliorates glomerulonephri- portant roles not only in embryonic developmental processes tis (Tang et al., 1996), and LIF is up-regulated in ARF (Yoshino but also in regenerative processes in adult tissue. et al., 2003). Therefore, we examined the expression of these Materials and methods factors in kidney SP cells in renal failure. As shown in Fig. 6 and 7, all of these factors were significantly up-regulated in the Mice reversible ARF model, but not in the irreversible CRF models, HIGA mice (Muso et al., 1996) were donated by E. Muso (Kitano Hospital, Osaka, Japan). ICGN mice (Ogura et al., 1989) were donated by J. and LIF increased the gene expression of HGF, VEGF, and 926 JCB • VOLUME 169 • NUMBER 6 • 2005 Matsuda (National Institute of Infectious Diseases, Tokyo, Japan). ICR, Cisplatin-induced ARF and SP cell infusion ddY, and C57/B6 mice were purchased from Clea Japan. HIGA mice Mice (C57BL/6; 8 wk old) were given a single i.p. injection of either ve- and their control ddY mice were used for analysis at 14 wk of age. ICGN hicle (saline) or cisplatin (12 mg/kg of body weight). Several doses of mice and their control ICR mice were used for analysis at 30 wk of age. In cisplatin were tested (5–20 mg/kg of body weight), and 12 mg/kg was Figs. 2, 3, 4, 6 A, and 7, kidney SP and non-SP cells were prepared from chosen to produce mild renal failure with the ability for partial or full recov- C57B/6 (8 wk old). All the procedures described here were approved by ery. Bone marrow mononuclear cells were isolated from the femurs and tib- the Animal Committee of the University of Tokyo. ias of mice by density gradient centrifugation with Ficoll-Paque Plus (Amer- sham Biosciences). Approximately 10,000 freshly isolated bone marrow Clinical biochemistry mononuclear, kidney SP, and non-SP cells prepared from C57BL/6 mice Blood samples were measured using an automated analyzer (model Fuji were injected via the tail vein 24 h after cisplatin injection (Table S1, avail- Dri-Chem 3500V; Fuji Film Co.). able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1). SP cell analysis and cell sorting RT-PCR and real-time PCR Mice were anesthetized and were perfused via the abdominal aorta with The sequences of primers for RT-PCR were as follows. GAPDH forward 5 - normal saline. Kidneys were harvested and the tissue was minced with a TGCTGAGTATGTCGTGGA-3 and reverse 5 -AGTTGCTGTTGAAGT- razor blade and digested by collagenase. The cell suspensions were fil- CGC-3 ; musculin/MyoR forward 5 -GGAGGACCGCTACGAGGACA- tered through a c ell strainer (Falcon 2350; Becton Dickinson) to remove 3 and reverse 5 -ACCCACAGAAGGCTATGCT-3 . RT-PCR conditions debris. The filtrates were analyzed as previously described (Matsuzaki et for musculin/MyoR and GAPDH were 35 cycles of 94 C for 30 s, 59 al., 2004). In brief, after filtration by cell strainer, the kidney cells were for 1 min, and 72 C for 1 min. Quantitative real-time PCR was performed cells/ml in Hank’s balanced salt solution (sup- resuspended at 1  10 using commercially available TaqMan probes (for HGF, VEGF, BMP7, plemented with 2% FCS and 10 mM Hepes) and then incubated with 5 and musculin) and analyzed on a sequence detector system (model ABI g/ml Hoechst33342 (Sigma-Aldrich) for 60 min for 37 C. A parallel PRISM 7000; BD Biosciences). Quantitative values were obtained from aliquot was stained with Hoechst33342 in the presence of 50 M of the threshold PCR cycle number at which an increase in signal associ- reserpine (Sigma-Aldrich). As the batch of the Hoechst33342 some- ated with exponential growth of the PCR product started to be detected. times affected FACS profile, we screened several batches and ultimately The relative mRNA levels in each sample were normalized to its GAPDH chose lot number 31K4028 for use in the experiments. Cell analysis and content. sorting were performed on a triple laser MoFlo (DakoCytomation). Hoechst33342 was excited at 350 nm, and fluorescence emission was Online supplemental material detected by using a 405/BP30 and 570/BP20 optical filter for Hoechst Fig. S1 shows immunohistochemical analysis of kidney non-SP cells. Fig. S2 blue and Hoechst red, respectively, and a 550-nm long-pass dichroic mir- shows engraftment of kidney SP cells in interstitial spaces by systemic kidney ror to separate the emission wavelengths. Both Hoechst blue and red fluo- SP cell infusion. Fig. S3 shows the effect of SP cell injection on CRF model rescence are shown on a linear scale. PI fluorescence was measured (unilateral ureteral obstruction model). Online supplemental material is avail- through 630BP30 after excitation at 499 nm with an argon laser, and able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1. live cell gate was defined as that which excluded the cells positive for PI. We thank Dr. Barry S. Oemar for his help with the preparation of this manuscript. events, the SP population was defined as reported After collecting 1  10 This study was supported by Mochida Pharmaceutical Co., Ltd., Me- previously (Matsuzaki et al., 2004). biol Inc., and Health and Labor Science Research Grants for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare. Microarray analysis DNA microarray hybridization experiments were performed using Atlas Submitted: 29 December 2004 glass Mouse3.8 I microarray (CLONTECH Laboratories, Inc.) according to Accepted: 16 May 2005 the manufacturer’s protocol. The protocol and the complete list of genes can be viewed at the manufacturer’s website. The DNA arrays were scanned using GenePix4000A (Hishikawa et al., 2001). References Histological analysis Sections were blocked with 1% skimmed milk (Morinaga) in PBS for 60 Asakura, A., and M.A. Rudnicki. 2002. Side population cells from diverse adult min at RT, and then incubated with 2 g/ml of goat anti–mouse musculin tissues are capable of in vitro hematopoietic differentiation. Exp. Hematol. polyclonal antibody (Santa Cruz Biotechnology, Inc.) or rabbit anti-BCRP1 30:1339–1345. antibody (PC-138; Kamiya Biomedical Company) for overnight at 4 C. Af- Asakura, A., P. Seale, A. Girgis-Gabardo, and M.A. Rudnicki. 2002. Myogenic ter washing three times in PBS for 5 min at RT, sections were incubated specification of side population cells in skeletal muscle. J. Cell Biol. with Alexa Fluor 488 or 546 donkey anti–goat IgG (Molecular Probes) or 159:123–134. Alexa Fluor 594 goat anti–rabbit IgG (Molecular Probes) at a dilution of Beltrami, A.P., L. Barlucchi, D. Torella, M. Baker, F. Limana, S. Chimenti, H. 1:200 for 30 min at RT. All sections were washed three times in PBS for 5 Kasahara, M. Rota, E. Musso, K. Urbanek, et al. 2003. 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Robb, L., T. Brodnicki, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, and R.P. Harvey. 1999. Assignment of the human helix-loop-helix transcription factor gene musculin/activated B-cell factor-1 (MSC) to chromosome 8q21 and its mouse homologue (Msc) to the proximal region of chromo- some 1. Genomics. 57:318–319. Schinkel, A.H., E. Wagenaar, C.A. Mol, and L. van Deemter. 1996. P-glycopro- tein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J. Clin. Invest. 97:2517–2524. Suranyi, M.G., A. Guasch, B.M. Hall, and B.D. Myers. 1993. Elevated levels of tumor necrosis factor-alpha in the nephrotic syndrome in humans. Am. J. Kidney Dis. 21:251–259. Tamaki, T., A. Akatsuka, K. Ando, Y. Nakamura, H. Matsuzawa, T. Hotta, R.R. Roy, and V.R. Edgerton. 2002. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle. J. Cell Biol. 157:571–577. Tang, W.W., M. Qi, G.Y. Van, G.P. Wariner, and B. Samal. 1996. Leukemia in- hibitory factor ameliorates experimental anti-GBM Ab glomerulonephritis. Kidney Int. 50:1922–1927. Tuglular, S., P. Berthoux, and F. Berthoux. 2003. Polymorphisms of the tumour necrosis factor alpha gene at position -308 and TNFd microsatellite in primary IgA nephropathy. Nephrol. Dial. Transplant. 18:724–731. 928 JCB • VOLUME 169 • NUMBER 6 • 2005 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Musculin/MyoR is expressed in kidney side population cells and can regulate their function

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Abstract

JCB: ARTICLE Musculin/MyoR is expressed in kidney side population cells and can regulate their function 1,2 1,2 1 1 4,9 1 Keiichi Hishikawa, Takeshi Marumo, Shigeki Miura, Asato Nakanishi, Yumi Matsuzaki, Katsunori Shibata, 1 4,9 4,9 6 5 5 Tomoko Ichiyanagi, Hiroko Kohike, Takuya Komori, Ichiro Takahashi, Osamu Takase, Naohiko Imai, 1,2 1,3 5 7 8 Masahiro Yoshikawa, Toshihiko Inowa, Matsuhiko Hayashi, Toshio Nakaki, Hiromitsu Nakauchi, 4,9 1,2 Hideyuki Okano, and Toshiro Fujita 1 2 Department of Clinical Renal Regeneration, Graduate School of Medicine, Department of Internal Medicine, Division of Nephrology and Endocrinology, and Department of Urology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan 4 5 Department of Physiology and Department of Internal Medicine, Keio University School of Medicine, Shionanomachi 35, Shinjuku-ku, Tokyo 160, Japan 6 7 Central Electron Microscopic Laboratory and Department of Pharmacology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo 173, Japan The Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108, Japan Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo 102-8666, Japan usculin/MyoR is a new member of basic helix- positive cells and improved renal function. Kidney SP loop-helix transcription factors, and its expres- cells in reversible acute renal failure expressed a high sion is limited to skeletal muscle precursors. level of renoprotective factors and leukemia inhibitory Here, we report that musculin/MyoR is expressed in adult factor (LIF), but not in irreversible chronic renal failure. In kidney side population (SP) cells and can regulate their cultured kidney SP cells, LIF stimulated gene expression of function. SP phenotype can be used to purify stem cell– renoprotective factors, and down-regulation of musculin/ rich fractions. Microarray analysis clarified that musculin/ MyoR augmented LIF-induced gene expression. Our re- MyoR was exclusively expressed in kidney SP cells, and sults suggest that musculin/MyoR may play important the cells resided in the renal interstitial space. Musculin/ roles not only in developmental processes but also in re- MyoR-positive cells were decreased in acute renal failure, generative processes in adult tissue. but infusion of kidney SP cells increased musculin/MyoR- Introduction In 1996, Goodell et al. (1996) reported a new method of obtain- BCRP1/ABCG2 prevents hematopoietic differentiation (Zhou ing an enriched population of hematopoietic stem cells from et al., 2001). However, the physiological role of this ATP- adult mouse bone marrow in a single step by using Hoechst binding cassette (ABC) transporter (Gottesman et al., 2002) in 33342 dye staining and FACS. The isolated cells were called SP cells is still unclear. side population (SP) cells, and the SP phenotype can be used to In this study, we first tried to clarify the gene expression purify a stem cell-rich fraction (Zhou et al., 2001; Matsuzaki et profile of kidney SP cells in healthy control and renal failure al., 2004). Bone marrow–derived SP cells differentiate into car- models by microarray analysis. The results of microarray and diomyocytes, endothelial cells (Jackson et al., 2001), and os- quantitative PCR analysis clarified that musculin/MyoR is ex- teoblast precursors (Olmsted-Davis et al., 2003), and skeletal clusively expressed in kidney SP cells but not in non-SP cells, muscle SP cells (Asakura et al., 2002) may differentiate into and we further examined the functional role of musulin/MyoR endothelial cells (Majka et al., 2003). The SP phenotype is de- in kidney SP cells. Musculin/MyoR is a newly discovered basic termined by BCRP1/ABCG2, and enforced expression of helix-loop-helix (bHLH) protein that represses myogenesis (Robb et al., 1998; Lu et al., 1999) and controls facial muscle development with capsulin (Lu et al., 2002). Musculin/MyoR Correspondence to Keiichi Hishikawa: [email protected] mRNA is first expressed at 9.8 days postcoitum in the mouse Abbreviations used in this paper: ABC, ATP-binding cassette; ARF, acute renal failure; bHLH, basic helix-loop-helix; BM, bone marrow-derived mononuclear; embryo, and is almost exclusively restricted to cells of skeletal BMP, bone morphologic protein; BUN, blood urea nitrogen; CRF, chronic renal muscle lineage, with peak expression at 14–15 days postcoitum. failure; HGF, hepatocyte growth factor; LIF, leukemia inhibitory factor; RA, To our knowledge, no study has confirmed the expression of retinoic acid; SP, side population; TSA, trichostatin A. The online version of this article includes supplemental material. musculin/MyoR in adult kidney tissue. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 6, June 20, 2005 921–928 http://www.jcb.org/cgi/doi/10.1083/jcb.200412167 JCB 921 THE JOURNAL OF CELL BIOLOGY Figure 1. FACS and microarray analysis of kidney SP cells. (A) Representative FACS profile of SP cells isolated from the kidney of normal (ddY and ICR) and renal failure (HIGA and ICGN) mice (total events:1.0  10 to 3.0 10 ). (B) Kidney SP cells isolated from ddY, ICR, HIGA, and ICGN mice stained with Hoechst dye (white bars). Total event of all mice were set 1.0  10 , and uptake of Hoechst dye was significantly blocked in the presence of reserpine (res) in all samples (black bars). Results are presented as the mean  SEM, n  15. *, P  0.05 versus control (without reserpine). (C) Scatter-plot analyses of gene expression (3,800 genes) in SP cells versus kidney tissue prepared from normal (ddY and ICR) and renal failure (HIGA and ICGN) model mice. Average expression levels of each gene were calculated from three independent hybridizations. We then aimed to determine the functional role of 3.1  0.9%, respectively) than did bone marrow (1%; not de- BCRP1 and musculin/MyoR in the kidney. We found that kid- picted). In both renal failure models (HIGA and ICGN), the ney SP cells, but not non-SP cells, are protected from cell population of reserpine-sensitive SP cells in the kidney was re- death by BCRP1. Leukemia inhibitory factor (LIF) is up-regu- duced to 30% of control (1.5  0.1 and 1.1  0.2%, respec- lated in reversible cisplatin-induced acute renal failure (ARF), tively). In our preparation, kidney SP cells were Sca-1 positive and is considered to play a role for regeneration process (70% on average; n  20) but CD45 negative (2% on aver- (Yoshino et al., 2003). In this study, we also examined the ex- age; n  20) in all mice, indicating that they were not part of pression of renoprotective factors such as hepatocyte growth the hematopoietic system and were not likely to be contami- factor (HGF), VEGF, bone morphologic protein (BMP) 7, and nated with peripheral blood SP cells. To characterize kidney SP LIF in kidney SP cells. In an ARF model, gene expression of cells, gene expression profiling was performed using freshly renoprotective factors and LIF was up-regulated in kidney SP isolated kidney SP cells from control (ddY and ICR) and renal cells but not in non-SP cells. On the other hand, there was no failure models (HIGA and ICGN), as well as cortical tissue of up-regulation of renoprotective factors and LIF in irreversible the kidney from control and renal failure mice. Among the chronic renal failure (CRF) models. In cultured kidney SP 3,800 genes analyzed, musculin/MyoR (Robb et al., 1998, cells, LIF augmented gene expression of renoprotective fac- 1999) was the only gene that was commonly and highly ex- tors. Although inhibition of BCRP1 showed no effect on LIF- pressed in SP cells of all mice (greater than sixfold higher com- induced gene expression of renoprotective factors, down-regu- pared with cortical tissue; Fig. 1 C, red square with arrow). We lation of musculin/MyoR augmented it. These results provide also performed gene expression profiling using kidney SP cells evidence that expression of musculin/MyoR is not limited to and non-SP cells, and found that musculin/MyoR was also ex- the skeletal muscle lineage and the production of renoprotective pressed greater than sixfold higher in SP cells compared with factors in kidney SP cells is regulated by LIF and musculin/ non-SP cells in all mice (unpublished data). MyoR, but nor by BCRP1. Immunohistochemical staining of musculin/MyoR and BCRP1 Results To confirm the protein expression and localization of musulin/ Gene expression profile of kidney SP cells MyoR-positive cells, we performed immunohistochemical We found a significant increase in blood urea nitrogen (BUN) and IgA in IgA nephropathy (HIGA) mice compared with con- Table I. Biochemical features in control (ddY and ICR) and CRF trol mice (ddY; Table I). In nephrotic syndrome mice (ICGN), (HIGA and ICGN) mice BUN and total cholesterol were significantly increased as com- BUN IgA Total cholesterol pared with control (ICR). We isolated whole kidney cells from mg/dl mg/dl mg/dl these mice and stained them with Hoechst 33342 dye (Zhou et ddY 20  2 98  12 ND al., 2001). The isolated cells were subjected to FACS analysis a a HIGA 33  3 396  26 ND in the presence or absence of reserpine to quantitate the number ICR 36  4 ND 108  13 of cells with a SP and non-SP phenotype (Fig. 1, A and B). As ICGN 96  4 ND 188  12 previously reported (Asakura and Rudnicki, 2002), kidney tis- BUN, IgA, and total cholesterol levels in serum. Results are presented as the sue from control mice (ddY and ICR) contained a much mean  SEM (n  12). higher number of reserpine-sensitive SP cells (5.1  0.1 and P  0.05 versus normal mice. 922 JCB • VOLUME 169 • NUMBER 6 • 2005 Figure 2. Immunohistochemical analysis of kidney SP cells. (A and B) Representative photomicrographs of fluorescent images of musculin, BCRP1, and nuclear staining (TO-PRO3) in cytospin of kidney SP cells (C57BL/6) just after FACS sorting. Overlay images are shown in the right Figure 3. TNF-  induced cell death in kidney SP cells. (A) Kidney SP and panels. (C and D) Representative photomicrographs of fluorescent images non-SP cells just after FACS sorting were incubated with TNF- at the indi- of musculin and nuclear staining in renal tissue (C57BL/6). Musculin-positive cated concentrations for 24 h. TNF- induced cell death in SP cells (black cells (arrows) reside in the interstitial space of the kidney. Overlay images of bars) in a concentration-dependent manner, but not in non-SP cells (white musculin and nuclear (yellow) images also showed that the cells reside in bars). Cell survival was determined by trypan blue exclusion assay. (B and C) the interstitial space of the kidney. Kidney SP cells just after FACS sorting were incubated with 50 M reser- pine (res), 8 ng/ml TNF-, or both for 24 h. Compared with control (no treatment), TNF- alone significantly increased DNA fragmentation and apoptosis, and cotreatment with reserpine significantly augmented it. analysis. Kidney SP cells were very small, but strongly ex- DNA fragmentation assay was performed using an ELISA kit, and percent- pressed both musculin/MyoR and BCRP1 protein (Fig. 2, A age of apoptosis was evaluated by nuclear staining with Hoechst 33258. and B). Although the SP population is a heterogeneous popu- Results are presented as means  SEM of four independent experiments. *, P  0.05 compared with control; **, P  0.05 compared with TNF-. lation, musculin/MyoR staining clarified that kidney SP cells were musulin/MyoR positive (95%), but non-SP cells were negative (5%; Fig. S1, available at http://www.jcb.org/cgi/ content/full/jcb.200412167/DC1). In other words, the purity apoptosis, and cotreatment with TNF- and reserpine signifi- of kidney SP cells was 95% with respect to musculin/ cantly increased it (Fig. 3, B and C). MyoR staining. As shown in Fig. 2 (C and D), musculin/ Kidney SP cells contribute to regeneration MyoR-positive cells were localized in the interstitial space of the kidney. To clarify the functional role of kidney SP cells in vivo, we in- jected isolated kidney SP cells systemically via the tail vain BCRP protects SP cells, but not non-SP into mice with ARF induced by cisplatin administration cells, from cell death (Ramesh and Reeves, 2002). In this model, i.p. injection of 12 BCRP1/ABCG2 is a member of ABC transporters, which gen- mg/kg cisplatin induced a peaked BUN level on day 7 (Fig. 4 A). erally protect cells from toxic agents (Gottesman et al., 2002). On day 7, musculin/MyoR-positive cells in the interstitial To clarify the reason why kidney SP cells were decreased in re- space were significantly reduced by cisplatin treatment alone, nal failure models (Fig. 1 B) and to examine the protective role but kidney SP cell infusion on day 1 significantly increased of BCRP1 in kidney SP cells, SP and non-SP cells were treated these cells on day 7 (Fig. 4, B–E). We also compared the effect with TNF-. Treatment with TNF- (at up to 16 ng/ml) did kidney SP cell infusion on renal function to those of infusion of not reduce cell viability of non-SP cells, but significantly re- non-SP cells or bone marrow-derived mononuclear (BM) cells. duced cell viability of SP cells in a concentration-dependent Infusion of kidney SP cells significantly suppressed the peak manner (Fig. 3 A). We further examined the effect of an inhibi- BUN and creatinine level on day 7 (Fig. 5, A and B), whereas tor of BCRP1/ABCG2 on TNF-–induced cell death in kidney infusion of non-SP and BM cells had no effect. Tubular necro- SP cells. Compared with control, reserpine alone showed no ef- sis scores were also significantly improved by SP cell infusion fect (Fig. 3, B and C). TNF- alone significantly increased (Fig. 5, C and D). MYOR IN KIDNEY SP CELLS • HISHIKAWA ET AL. 923 that contribute to the kidney regeneration process, such as HGF (Mizuno et al., 1998), VEGF (Ostendorf et al., 1999), and BMP7 (Wang and Hirschberg, 2003), in a reversible cisplatin- induced ARF model and two irreversible CRF models (IgA nephropathy, HIGA; nephritic syndrome, ICGN). In ARF mice, kidney SP cells expressed significantly higher levels of HGF, VEGF, and BMP7 compared with the cells in control mice (Fig. 6 A). In contrast, in the CRF models, there was no significant difference between disease mice and control mice (Fig. 6, B and C). Recently, LIF was reported to be up-regu- lated and to contribute to regeneration processes (Yoshino et al., 2003), so we examined the expression of LIF in kidney SP cells. We also confirmed up-regulation of LIF in the ARF model but not in the CRF models (Fig. 6 A). On the other hand, we could not find significant changes of these factors in kidney non-SP cells of all disease mice as compared with their control mice (unpublished data). Musculin/MyoR but not BCRP1 regulates function of kidney SP cells As infusion of kidney SP cells improved renal function in an ARF model and LIF is also up-regulated in this model, we ex- amined the effect of LIF on gene expression of renoproptec- Figure 4. Systemic kidney SP cell infusion increases musculin/MyoR- tive factors and musculin/MyoR in kidney SP and non-SP positive cells in renal interstitial space in ARF model. (A) Time course of plasma BUN level in cisplatin-induced ARF model. Values are the mean  cells. Compared with control (without LIF), treatment with SEM (n  8). *, P  0.05 versus day 0. (B–D) Representative fluorescence LIF significantly up-regulated gene expression of HGF, photographs and overlay images (fluorescent and phase contrast) of renal VEGF, and BMP7 in SP but not in non-SP cells (Fig. 7, A and tissues of control, cisplatin alone (cis), and infusion of kidney SP cells (cisSP) on day 7. Green spots with red arrowheads are musculin/MyoR- B). To clarify the role of BCRP1 in LIF-induced gene expres- positive cells. (E) Musculin/MyoR-positive cells were significantly reduced sion of renoprotective genes, we treated cells with LIF and re- in the cisplatin-induced ARF model (cis) but were markedly increased by serpine, but reserpine showed no effect. Retinoic acid (RA) is systemic kidney SP cell infusion (cisSP). Values are the average number of musculin-positive cells in 10 randomly selected low power fields reported to down-regulate musculin/MyoR (Yu et al., 2003), (200) in four independent experiments. Values are the mean  SEM. and cotreatment with LIF and RA significantly augmented the *, P  0.05 versus control; **, P  0.05 versus cis. LIF-induced gene expression in kidney SP cells but not in non-SP cells. Finally, we confirmed the effect of LIF, reser- Kidney SP cells express growth factors pine, and RA on musculin/MyoR expression in SP and non- that contribute to tissue regeneration SP cells by RT-PCR and real-time PCR. LIF and reserpine To clarify the role of kidney SP cells in renal failure, we per- alone showed no effect on basal musculin/MyoR gene expres- formed quantitative real-time PCR analysis of several factors sion, and cotreatment with reserpine also showed no effect Figure 5. Systemic kidney SP cell infusion augments recovery of renal function in ARF model. (A and B) Compared with control (cont), cisplatin alone (cis) significantly increased the BUN and creatinine levels, but infusion of kidney SP cells (cisSP) on day 1 (day after cisplatin injection) significantly reduced the BUN and creatinine levels on day 7. On the other hand, infusion of non-SP cells (cisNSP) and BM cells (cisBM) showed no effect. (C and D) Compared with con- trol (cont), cisplatin alone (cis) significantly increased the tu- bular necrosis scores, but infusion of kidney SP cells (cisSP) on day 1 (day after cisplatin injection) significantly reduced it on day 7. Values are the mean  SEM. *, P  0.05 versus control; **, P  0.05 versus cis. 924 JCB • VOLUME 169 • NUMBER 6 • 2005 Discussion Members of the bHLH family of transcription factors are in- volved in cell fate determination and tissue differentiation in several developmental processes including hematopoiesis, myogenesis, and neurogenesis. Musculin/MyoR is a newly dis- covered bHLH protein that acts as a lineage-restricted repres- sor of embryonic skeletal muscle development. In this study, we clarified that musculin/MyoR is expressed in adult kidney SP cells and regulates LIF-induced gene expression of renopro- tective factors such as HGF, VEGF, and BMP7. Robb et al. (1998) failed to detect expression of muscu- lin/MyoR in adult tissue by Northern blotting but demonstrated expression in the adult brain, spleen, and muscle but not in the kidney, liver, lung, or thymus by RNase protection assay. Yu et al. (2003) detected expression of musculin/MyoR in the adult brain, heart, liver, lung, small intestine, spleen, and thymus by Northern blotting. This discrepancy may be explained by the sensitivity of their assay, but our results also suggest that mus- culin/MyoR expression is not lineage restricted as has been re- ported, but is expressed in a relatively broad spectrum of adult tissue. Musculin/MyoR was originally identified as a transcrip- tional repressor in embryonic skeletal muscle precursors, but its function in adult tissue is not known. In fact, musculin/ MyoR knockout mice were not embryonic lethal, and analysis of kidney function in these mice has not been completed (un- Figure 6. Quantitative real-time PCR analysis of HGF, VEGF, BMP7, and LIF in kidney SP cells. (A) Cisplatin-induced gg model. White bars show published data). In the present study, we demonstrated that control (no treatment) and black bars show cisplatin treatment. Compared down-regulation of musculin/MyoR augmented LIF-induced with control, gene expression of HGF, VEGF, BMP7, and LIF was signifi- gene expression of HGF, VEGF, and BMP7 in kidney SP cells. cantly up-regulated in kidney SP cells. (B) IgA nephropathy mice. White bars show control (ddY) and black bars show IgA nephropathy (HIGA). These results showed that musculin/MyoR plays a role as a (C) Nephritic syndrome mice. White bars show control (ICR) and black transcriptional repressor in adult kidney SP cells as reported in bars show nephritic syndrome (ICGN). Expression was normalized to embryonic skeletal muscle. The physiological role of kidney GAPDH and is shown as the ration to the value of control. Values repre- sent means  SEM (n  6). *, P  0.05 versus control. SP cells is still unclear, but systemic administration of RA, which down-regulates musculin/MyoR (Yu et al., 2003), aug- (Fig. 7, C and D). On the other hand, RA significantly down- mented the recovery of ARF (Perez et al., 2004). To down-regu- regulated musculin/MyoR in the presence of LIF (Fig. 7, C late musculin/MyoR in vivo, we injected histone deacetylase in- and D). In non-SP cells, basal musculin/MyoR expression was hibitor trichostatin A (TSA), which down-regulates musculin/ almost absent (Fig. 6 E). MyoR (Yu et al., 2003), in control and CRF model (nephrotoxic Figure 7. LIF-induced gene expression is regulated by musculin/MyoR. (A and B) Kidney SP cells (A) or non-SP cells (B) just after FACS sorting were incubated with vehicle (white bars), LIF (10 ng/ml; black bars), LIF with reserpine (10 ng/ml and 50 M, respectively; dotted bars), and LIF with RA (10 ng/ml and 5  10 mol/l, respectively; hatched bars) for 12 h, and then gene expression of HGF, VEGF, and BMP7 was examined by TaqMan real-time PCR. (C and D) Gene expression of musculin/MyoR in kidney SP cells. An aliquot of RNA in panel A was examined by RT-PCR (C) and Taq- Man real-time PCR (D). (E) Gene expression of musculin/ MyoR in non-SP cells. Basal expression of musculin/MyoR was almost absent. Values represent means  SEM (n  6). *, P  0.05 versus control; **, P  0.05 versus LIF. MYOR IN KIDNEY SP CELLS • HISHIKAWA ET AL. 925 serum model, NTN model). As in the case with HIGA and BMP7 in cultured kidney SP cells. Moreover, the number of SP ICGN, kidney SP cells analyzed by FACS were reduced in cells was decreased in the irreversible CRF models (Fig. 1 B). NTN model. Surprisingly, daily injection (for 21 d) of TSA al- We also examined the effect of kidney SP cell infusion on CRF most completely abolished musculin/MyoR-positive cells in model (unilateral ureteral obstruction model) but found no sig- the kidney of control and NTN model and completely pre- nificant improvement (Fig. S3, available at http://www.jcb.org/ vented progression of CRF in NTN model. Moreover, regener- cgi/content/full/jcb.200412167/DC1). Recently, we also re- ative factors were also up-regulated by treatment with TSA ported that LIF induces multilineage differentiation of kidney (unpublished data). Together, these results suggest that muscu- SP cells in culture (Hishikawa et al., 2005), and some other lin/MyoR-positive cells could be new targets for regeneration pathways can be involved in the restoration of renal function. in renal failure, and we are currently trying to clarify its role us- Although the precise mechanism regarding the regenerative role ing a renal failure model in musculin/MyoR knockout mice. of kidney SP cells remains to be clarified, our results suggest Theoretically, an activated transcription factor should be that loss of musulin/MyoR-positive kidney SP cells and inade- located within the nucleus, but musculin/MyoR was expressed quate expression of key factors such as LIF and BMP7 in CRF throughout kidney SP cells. Inactive sterol regulatory element may determine the fate of CRF. binding proteins, another member of the bHLH transcription BCRP1/ABCG2 is reported to determine the SP pheno- factor family, are synthesized as precursors in the cytoplasm type (Zhou et al., 2001). Enforced expression of BCRP1/ and bind to the endoplasmic reticulum membrane. Upon acti- ABCG2 inhibits hematopoietic development (Zhou et al., vation, the active forms of sterol regulatory element binding 2001), but its mechanism is still unclear. BCRP1/ABCG2 is a proteins enter the nucleus and activate several genes that con- member of ABC transporters (Gottesman et al., 2002) and is trol the synthesis and uptake of cholesterol and unsaturated known to transport doxorubicin, daunorubicin, mitoxantrone, fatty acids (Nagoshi and Yoneda, 2001). Pod1, which is also a topotecan, and prazocin. Although many ABC transporters bHLH transcription factor expressed in kidney stromal cells have been identified as drug-resistance proteins, they are all and is required for glomerulogenesis, is distributed throughout expressed in normal tissues. Consistent with their wide distri- cells located in peritubular interstitial space in the kidney (Cui bution, it is becoming clear that in addition to exogenously ad- et al., 2003). These results suggest that localization of a tran- ministered drugs, ABC proteins transport numerous endoge- scription factor such as musculin/MyoR throughout kidney SP nous substrates. It is well know that ABC transporters have cells is not unique, but further characterization may be needed an important role in protecting the central nervous system to fully understand its mechanism of translocation. through the blood brain barrier (Schinkel et al., 1996), and Infusion of kidney SP cells significantly augmented re- BCRP1/ABCG2 in the placenta may play a role in protect- covery of ARF and increased musculin/MyoR-positive cells in ing fetal blood from toxic organic anions and excreting the interstitial space. Therefore, we speculate that homing of glutathione/glucuronide metabolites into maternal circulation kidney SP cells to the interstitial space is necessary for regener- (Gottesman et al., 2002). Considering these results, we exam- ation in the ARF model induced by cisplatin. Recently, cardiac ined the protective role of BCRP1 in kidney SP cells. The role stem cells were found in the interstitium between well-differen- for TNF- in cisplatin-induced nephrotoxicity has been well tiated myocytes (Beltrami et al., 2003), and myogenic-endothe- established (Ramesh and Reeves, 2002), and important roles of lial progenitor cells with the SP phenotype were also identified TNF- are also reported in IgA nephropathy (Tuglular et al., in the interstitial spaces of skeletal muscle (Tamaki et al., 2002), 2003; Chan et al., 2005) and nephrotic syndrome (Suranyi et suggesting that SP cells commonly reside in the interstitial al., 1993; Kim et al., 2004; Raveh et al., 2004). As shown in space. We also systemically injected kidney SP cells from GFP Fig. 3, kidney SP cells were more sensitive to TNF- than non- mice and DiI-labeled kidney SP cells, and found that these cells SP cells, and BCRP1 protected kidney SP cells from cell death. were located in the interstitial spaces of kidney (Fig. S2, avail- The precise mechanism of TNF-–induced cell death in kidney able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1). SP cells and direct in vivo evidence linking these effects to pro- As the interstitial space of the kidney has a fine microcircula- gression of renal failure remain to be determined, but our re- tion system, musculin/MyoR-positive kidney SP cells in the in- sults may explain why the number of musculin/MyoR-positive terstitial space may provide paracrine growth support for neigh- kidney SP cells was decreased in renal failure in which inflam- boring cells in vivo. Several growth factors such as HGF, matory cytokines were overexpressed. VEGF, and BMP7 have been reported to contribute to kidney In summary, our study provides evidence for a new role regeneration. Especially, BMP7 is the only factor that reverses of musculin/MyoR and a protective role of BCRP1 in kidney chronic renal injury (Zeisberg et al., 2003) and Lin et al. (2005) SP cells. Our results suggest that members of the bHLH family recently reported the important role of BMP7 enhancer in CRF. of transcription factors such as musculin/MyoR may play im- Administration of exogenous LIF ameliorates glomerulonephri- portant roles not only in embryonic developmental processes tis (Tang et al., 1996), and LIF is up-regulated in ARF (Yoshino but also in regenerative processes in adult tissue. et al., 2003). Therefore, we examined the expression of these Materials and methods factors in kidney SP cells in renal failure. As shown in Fig. 6 and 7, all of these factors were significantly up-regulated in the Mice reversible ARF model, but not in the irreversible CRF models, HIGA mice (Muso et al., 1996) were donated by E. Muso (Kitano Hospital, Osaka, Japan). ICGN mice (Ogura et al., 1989) were donated by J. and LIF increased the gene expression of HGF, VEGF, and 926 JCB • VOLUME 169 • NUMBER 6 • 2005 Matsuda (National Institute of Infectious Diseases, Tokyo, Japan). ICR, Cisplatin-induced ARF and SP cell infusion ddY, and C57/B6 mice were purchased from Clea Japan. HIGA mice Mice (C57BL/6; 8 wk old) were given a single i.p. injection of either ve- and their control ddY mice were used for analysis at 14 wk of age. ICGN hicle (saline) or cisplatin (12 mg/kg of body weight). Several doses of mice and their control ICR mice were used for analysis at 30 wk of age. In cisplatin were tested (5–20 mg/kg of body weight), and 12 mg/kg was Figs. 2, 3, 4, 6 A, and 7, kidney SP and non-SP cells were prepared from chosen to produce mild renal failure with the ability for partial or full recov- C57B/6 (8 wk old). All the procedures described here were approved by ery. Bone marrow mononuclear cells were isolated from the femurs and tib- the Animal Committee of the University of Tokyo. ias of mice by density gradient centrifugation with Ficoll-Paque Plus (Amer- sham Biosciences). Approximately 10,000 freshly isolated bone marrow Clinical biochemistry mononuclear, kidney SP, and non-SP cells prepared from C57BL/6 mice Blood samples were measured using an automated analyzer (model Fuji were injected via the tail vein 24 h after cisplatin injection (Table S1, avail- Dri-Chem 3500V; Fuji Film Co.). able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1). SP cell analysis and cell sorting RT-PCR and real-time PCR Mice were anesthetized and were perfused via the abdominal aorta with The sequences of primers for RT-PCR were as follows. GAPDH forward 5 - normal saline. Kidneys were harvested and the tissue was minced with a TGCTGAGTATGTCGTGGA-3 and reverse 5 -AGTTGCTGTTGAAGT- razor blade and digested by collagenase. The cell suspensions were fil- CGC-3 ; musculin/MyoR forward 5 -GGAGGACCGCTACGAGGACA- tered through a c ell strainer (Falcon 2350; Becton Dickinson) to remove 3 and reverse 5 -ACCCACAGAAGGCTATGCT-3 . RT-PCR conditions debris. The filtrates were analyzed as previously described (Matsuzaki et for musculin/MyoR and GAPDH were 35 cycles of 94 C for 30 s, 59 al., 2004). In brief, after filtration by cell strainer, the kidney cells were for 1 min, and 72 C for 1 min. Quantitative real-time PCR was performed cells/ml in Hank’s balanced salt solution (sup- resuspended at 1  10 using commercially available TaqMan probes (for HGF, VEGF, BMP7, plemented with 2% FCS and 10 mM Hepes) and then incubated with 5 and musculin) and analyzed on a sequence detector system (model ABI g/ml Hoechst33342 (Sigma-Aldrich) for 60 min for 37 C. A parallel PRISM 7000; BD Biosciences). Quantitative values were obtained from aliquot was stained with Hoechst33342 in the presence of 50 M of the threshold PCR cycle number at which an increase in signal associ- reserpine (Sigma-Aldrich). As the batch of the Hoechst33342 some- ated with exponential growth of the PCR product started to be detected. times affected FACS profile, we screened several batches and ultimately The relative mRNA levels in each sample were normalized to its GAPDH chose lot number 31K4028 for use in the experiments. Cell analysis and content. sorting were performed on a triple laser MoFlo (DakoCytomation). Hoechst33342 was excited at 350 nm, and fluorescence emission was Online supplemental material detected by using a 405/BP30 and 570/BP20 optical filter for Hoechst Fig. S1 shows immunohistochemical analysis of kidney non-SP cells. Fig. S2 blue and Hoechst red, respectively, and a 550-nm long-pass dichroic mir- shows engraftment of kidney SP cells in interstitial spaces by systemic kidney ror to separate the emission wavelengths. Both Hoechst blue and red fluo- SP cell infusion. Fig. S3 shows the effect of SP cell injection on CRF model rescence are shown on a linear scale. PI fluorescence was measured (unilateral ureteral obstruction model). Online supplemental material is avail- through 630BP30 after excitation at 499 nm with an argon laser, and able at http://www.jcb.org/cgi/content/full/jcb.200412167/DC1. live cell gate was defined as that which excluded the cells positive for PI. We thank Dr. Barry S. Oemar for his help with the preparation of this manuscript. events, the SP population was defined as reported After collecting 1  10 This study was supported by Mochida Pharmaceutical Co., Ltd., Me- previously (Matsuzaki et al., 2004). biol Inc., and Health and Labor Science Research Grants for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare. Microarray analysis DNA microarray hybridization experiments were performed using Atlas Submitted: 29 December 2004 glass Mouse3.8 I microarray (CLONTECH Laboratories, Inc.) according to Accepted: 16 May 2005 the manufacturer’s protocol. The protocol and the complete list of genes can be viewed at the manufacturer’s website. The DNA arrays were scanned using GenePix4000A (Hishikawa et al., 2001). References Histological analysis Sections were blocked with 1% skimmed milk (Morinaga) in PBS for 60 Asakura, A., and M.A. Rudnicki. 2002. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: Jun 20, 2005

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