TY - JOUR
AU1 - Imai, Naohiko
AU2 - Hishikawa, Keiichi
AU3 - Marumo, Takeshi
AU4 - Hirahashi, Junichi
AU5 - Inowa, Toshihiko
AU6 - Matsuzaki, Yumi
AU7 - Okano, Hideyuki
AU8 - Kitamura, Tadaichi
AU9 - Salant, David
AU1 - Fujita, Toshiro
AB - Abstract Bone morphogenic protein (BMP)-7 is expressed in the adult kidney and reverses chronic renal injury when given exogenously. Here, we report that a histone deacetylase inhibitor, trichostatin A (TSA), attenuates chronic renal injury, in part, by augmenting the expression of BMP-7 in kidney side population (SP) cells. We induced accelerated nephrotoxic serum nephritis (NTN) in C57BL/6 mice and treated them with TSA for 3 weeks. Compared with vehicle-treated NTN mice, treatment with TSA prevented the progression of proteinuria, glomerulosclerosis, interstitial fibrosis, and loss of kidney SP cells. Basal gene expression of renoprotective factors such as BMP-7, vascular endothelial growth factor, and hepatocyte growth factor was significantly higher in kidney SP cells as compared with non-SP cells. Treatment with TSA significantly upregulated the expression of BMP-7 in SP cells but not in non-SP cells. Moreover, initiation of treatment with TSA after 3 weeks of NTN (for 3 weeks, until 6 weeks) partially but significantly reversed renal dysfunction. Our results indicate an important role of SP cells in the kidney as one of the possible generator cells of BMP-7 and TSA as a stimulator of the cells in reversing chronic renal disease. Disclosure of potential conflicts of interest is found at the end of this article. Stem cell, Side population, Transcription regulation, Chronic renal failure, Anti-glomerular basement membrane disease Introduction Bone morphogenic protein (BMP)-7 [1] is a member of the transforming growth factor (TGF)-β superfamily and plays pivotal roles during embryogenic renal development. On the other hand, BMP-7 expression is retained in only a few adult tissues, most prominently in the kidney [2–4], but its physiologic functions in the adult kidney are still unclear. BMP-7 is an antifibrotic agent, and administration of recombinant BMP-7 has shown marked efficacy in the reduction of glomerular and interstitial fibrosis in nephrotoxic serum nephritis (NTN) mice [5]. In addition, kielin/chordin-like protein, a novel enhancer of BMP, attenuates renal fibrotic disease [6]. Although chronic renal disease is considered to be clinically irreversible, these findings suggest that BMP-7 may play a key role in clinical renal regeneration. However, regulation of BMP-7 in the adult kidney and the cells that produce it are not known. In 1996, Goodell et al. reported a new method of obtaining an enriched population of hematopoietic stem cells in a single step by using Hoechst 33342 dye and fluorescence-activated cell sorting (FACS) [7]. The isolated cells were called side population (SP) cells, and the SP phenotype can be used to purify a stem cell-rich fraction [8–11]. Recently, we reported that kidney SP cells differentiate into multiple lineages [12], and the function of these cells is regulated by MyoR [13–15]. Infusion of kidney SP cells improved renal function in acute renal failure [13], and downregulation of MyoR by retinoic acid augmented the gene expression of renoprotective factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and BMP-7 [13], suggesting that kidney SP cells may play a role as a source of renoprotective factors in the kidney. Challen et al. also reported that the kidney SP cell reveals multilineage potential and suggested a humoral role for SP cells in renal repair [16]. Trichostatin A (TSA) is a specific and reversible inhibitor of histone deacetylases (HDACs) in vitro and in vivo that is active at low nanomolar concentrations [17]. TSA and a structurally related compound, suberoylanilide hydroxamic acid (SAHA), have been used effectively and without toxicity in mouse models of cancer, and SAHA is currently in phase I clinical trials as anticancer therapy [18]. TSA also has a potential role in autoimmune diseases, and Mishra et al. reported that HDACs significantly ameliorated glomerulonephritis in MRL-lpr/lpr mice [19]. Moreover, Yu et al. reported that TSA downregulates MyoR expression [20]. To further investigate the role of BMP-7 and kidney SP cells in reversing chronic renal disease, we assessed the effect of TSA in a NTN model [21] in vivo and FACS sorted kidney SP cells in vitro. Our results showed that TSA not only prevents the progression of chronic renal failure but also reverses it, in part, by augmenting the expression of BMP-7 in kidney SP cells. Materials and Methods Mice C57BL/6 mice 6–8-weeks old were purchased from Oriental Yeast (Tokyo, http://www.oyc.co.jp/e). The mice were bred and maintained in a pathogen-free animal facility at the University of Tokyo. All the procedures described here were approved by the Animal Committee of the University of Tokyo. Drug TSA was purchased from Wako Pharmaceutical Co., Ltd. (Tokyo, http://www.wako-chem.co.jp/). For in vitro studies, a 300-nM solution of TSA in dimethyl sulfoxide (DMSO) was prepared and stored at −20°C until use. Induction of Accelerated NTN Nephrotoxic serum was prepared as described previously [21]. Nephrotoxic serum and nonimmune sheep control serum were filter-sterilized. C57BL/6 mice were preimmunized by subcutaneous injection of 200 μg of normal sheep IgG in Freud's complete adjuvant (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Five days later, the mice were injected intravenously with 50 μl of nephrotoxic serum on 3 consecutive days. Control mice were injected with 50 μl of normal sheep serum on the same schedule. In Vivo Treatment NTN mice were treated with subcutaneous injection of TSA (0.5 mg/kg body weight) in 40 μl of DMSO daily for 3 weeks. The dose of TSA was based on published results demonstrating significant inhibition of tumor progression in mice. Control mice were injected with 40 μl of DMSO alone. There were no differences between TSA-treated mice and vehicle-treated mice in age, weight, or food and water consumption. Urine Protein Excretion Total protein and creatinine were measured at intervals from 1–21 days in 24-hour urine collection samples from mice housed in individual metabolic cages. During urine collection, mice were allowed free access to food and water. Total protein and creatinine in urine were measured by enzymatic assay (SRL, Tokyo, http://www.srl-group.co.jp/en). Blood Urea Nitrogen At the end of the studies (3 weeks or 6 weeks), serum was obtained by cardiac puncture. Blood urea nitrogen (BUN) was measured in serum 21 and 42 days after the initial challenge with nephrotoxic serum. Blood urea nitrogen level was measured by enzymatic assay (SRL). Histological Studies To assess the light microscopic appearance, 5-μm paraffin sections were stained with periodic acid-Schiff (PAS). Percentages of glomeruli with crescents and those with more than 50% sclerotic area positive for PAS were calculated [22]. For the detection of α-smooth muscle actin (SMA), actin were stained with mouse anti-α-SMA monoclonal antibody (1A4; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com; 1:50 dilution) according to a previously described method [23] with minor modification. Morphometric analysis of α-SMA-positive interstitial area was calculated by image analysis using Nikon (Tokyo, http://www.nikon.com) ACT-1 version 220, Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, http://www.adobe.com), and NIH Image [24]. Fluorescence-Activated Cell Sorting Mice were anesthetized and perfused via the abdominal aorta with normal saline. Kidney tissue was minced and digested at 37°C in 0.1 mg/ml collagenase type I-AS (Sigma). The incubation time for mouse tissue was 60 minutes and for human tissue was 15–20 minutes, to avoid cell death. The cell suspensions were filtered through a cell strainer (Falcon 2350; BD Biosciences, San Diego, http://www.bdbiosciences.com) to remove debris. The filtrates were analyzed as previously described [13]. In brief, after filtration by the cell strainer, the kidney cells were resuspended at 1 × 106 cells per milliliter in Hanks' balanced saline solution (supplemented with 2% fetal calf serum [FCS] and 10 mM HEPES) and then incubated with 5 μg/ml Hoechst 33342 (Sigma) for 60 minutes at 37°C. The cells were kept on ice just before FACS analysis. A parallel aliquot was stained with Hoechst 33342 in the presence of 50 μM reserpine (Sigma). As the batch of the Hoechst 33342 sometimes affected the FACS profile, we screened several batches and ultimately chose lot number 31K4028 for use in the experiments. Cell analysis and sorting were performed on a triple laser MoFlo (DakoCytomation, Fort Collins, CO, http://www.dakocytomation.com). After collecting 1 × 105 events, the SP population was defined as described in a previous report. For cell surface antigen determination, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD45 monoclonal antibody (1553079; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) and phycoerythrin (PE)-conjugated anti-mouse Sca-1 antibody (MSCA04; Caltag Laboratories, Burlingame, CA, http://www.caltag.com) or FITC and PE-conjugated rat IgG monoclonal immunoglobulin isotype control (10 μg/ml; BD Biosciences) at 4°C. After staining with 2 μg/ml propidium iodide (Sigma) to discriminate dead cells, the cells were analyzed using a triple laser LSR II (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Immunostaining Frozen sections of kidney tissue from NTN mice were cut into 5-μm-thick sections. Sections were blocked with 1% skimmed milk in phosphate-buffered saline for 60 minutes at room temperature and then incubated with 2 mg/ml goat anti-mouse musculin/MyoR polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) overnight at 4°C. Tissues and cells were also stained with TO-PRO-3 (T3605; Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com) for nuclear staining. Microscopic images were acquired with an Olympus (Tokyo, http://www.olympus-global.com) microscopic workstation equipped with a laser-scanning confocal unit and 15-m krypton and argon lasers and a ×63 Plan Apochromat/1.4 NA or ×40 Plan Neofluar/1.3 NA oil-immersion objective. Western Blotting of Acetylated Histone H3 Renal tissue was immediately snap frozen in liquid nitrogen and stored at −70°C for further processing. The antibody for acetylated histone H3 was purchased from Upstate (Charlottesville, VA, http://www.upstate.com). The signals were visualized with an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com), and the signal intensity was measured by NIH Image. Real-Time Polymerase Chain Reaction For real-time polymerase chain reaction (PCR) analysis, kidney SP cells just after FACS were incubated in the presence and absence of TSA (300 nM) for 6 hours in Dulbecco's modified Eagle's medium/F12 with 10% FCS. Total RNA was extracted from the supernatant following treatment with DNase using an RNeasy midi kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Quantitative PCR was performed using an ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) according to manufacturer's instructions. TaqMan Gene Expression Assays (Applied Biosystems), namely Hs00708019_s1 for BCL2L11 (BMP7), Hs00236329_m1 for BCL2L1 (HGF), and Hs00187848_m1 for BCL2L2 (VEGF) were used following the manufacturer's protocol. To correct for variability in RNA recovery and efficiency of reverse transcription, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified and quantitated in each cDNA preparation. The mRNA levels were normalized to GAPDH. Statistical Analysis Statistical significance was determined using paired Student's t test, Mann-Whitney U rank-sum test, or analysis of variance. A p value less than .05 was considered to be significant. Mean values (± SEM) are used throughout the text. Analysis was performed using the StatView program (Abacus Concepts Inc., Berkeley, CA, http://www.abacus.com). Results TSA Prevents Progression of Glomerular Injury and Interstitial Fibrosis To investigate the effect of TSA on chronic kidney disease, we induced accelerated NTN in C57/B6 mice. We found extensive glomerulosclerosis and crescent formation 21 days after the induction of NTN (Fig. 1A). NTN mice treated with TSA developed less glomerulosclerosis and fewer crescents than those treated with NTN mice. We also calculated the area of SMA staining. On day 21, NTN mice showed a marked increase in interstitial area compared with control, but treatment with TSA significantly decreased it (Fig. 1B). The increase in interstitial area was also confirmed by another analysis software (MetaMorpho analysis program) [6]. Figure 1. Open in new tabDownload slide Treatment with TSA prevented progression of chronic renal injury in NTN mice. (A): Upper panels are histological appearance on day 21 (periodic acid-Schiff staining). Bar graphs are quantitative assessment of glomerulosclerosis and crescent formation in control and NTN mice. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Each group contained six mice, and four sections were made per mouse. Ten glomeruli per section were evaluated in a blind fashion. (B): Representative histological appearance and quantitation of α-smooth muscle actin (SMA) staining. Upper panels are low-power (×40) photographs of histological appearance on day 21 (α-SMA staining), and lower panels are high-power (×200). White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Each group contained six mice, and four sections were made per mouse. Averages of ten randomly selected fields per section are shown. Abbreviations: NTN, nephrotoxic serum nephritis; TSA, trichostatin A. Figure 1. Open in new tabDownload slide Treatment with TSA prevented progression of chronic renal injury in NTN mice. (A): Upper panels are histological appearance on day 21 (periodic acid-Schiff staining). Bar graphs are quantitative assessment of glomerulosclerosis and crescent formation in control and NTN mice. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Each group contained six mice, and four sections were made per mouse. Ten glomeruli per section were evaluated in a blind fashion. (B): Representative histological appearance and quantitation of α-smooth muscle actin (SMA) staining. Upper panels are low-power (×40) photographs of histological appearance on day 21 (α-SMA staining), and lower panels are high-power (×200). White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Each group contained six mice, and four sections were made per mouse. Averages of ten randomly selected fields per section are shown. Abbreviations: NTN, nephrotoxic serum nephritis; TSA, trichostatin A. TSA Prevents Progression of Renal Dysfunction To clarify whether TSA alters the progression of renal dysfunction, we quantified urinary protein excretion and BUN. NTN mice in the TSA treated group showed less urinary protein excretion as compared with control on day 8, 14, and 21 (Fig. 2A). BUN level measured on day 21 after the induction of NTN was significantly elevated, but treatment with TSA prevented the elevation (Fig. 2B). Figure 2. Open in new tabDownload slide Treatment with TSA prevented progression of renal dysfunction. (A): Urinary protein/creatinine ratio. (B): BUN on day 21. Data are mean ± SEM (n = 6); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Abbreviations: BUN, blood urea nitrogen; NTN, nephrotoxic serum nephritis; TSA, trichostatin A. Figure 2. Open in new tabDownload slide Treatment with TSA prevented progression of renal dysfunction. (A): Urinary protein/creatinine ratio. (B): BUN on day 21. Data are mean ± SEM (n = 6); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Abbreviations: BUN, blood urea nitrogen; NTN, nephrotoxic serum nephritis; TSA, trichostatin A. Kidney SP Cells Are Decreased in NTN Mice and TSA Prevents SP Cell Decrease. We have recently reported that kidney SP cells were decreased in chronic renal failure models such as IgA nephropathy mice and nephritic syndrome mice [13]. Moreover, infusion of kidney SP cells improved renal function [13]. So we next tried to clarify the role of kidney SP cells in the prevention of renal dysfunction by TSA. We isolated whole kidney cells from C57/B6 mice and stained them with Hoechst 33342 dye. The isolated cells were subjected to FACS analysis, and the SP population was confirmed by cotreatment with reserpine, an inhibitor of the ATP-binding cassette transporter that determines the SP phenotype (Fig. 3A) [25]. In control mice, kidney SP cells were almost negative (less than 1% positive) for a hematopoietic cell marker, CD45, and positive (over 70%) for a stem cell antigen, Sca-1 (Fig. 2B, 2D, 2E). On the other hand, anti-CD45 labeled a higher proportion of non-SP cells, and anti-Sca-1 labeled a lower proportion of them. As in the case with IgA nephropathy mice and nephritic syndrome mice [13], kidney SP cells were significantly reduced in NTN mice (Fig. 3C), but the proportion of SP and non-SP cells positive for CD45 and Sca-1 was not changed as compared with control mice (Fig. 3C–3E). Although TSA showed no effect on SP population of TSA injected control mice without NTN (data not shown), treatment with TSA significantly prevented the reduction of SP cells in NTN mice (Fig. 3C), and the proportion of SP and non-SP cells positive for CD45 and Sca-1 was not changed as compared with control mice (Fig. 3C–3E). Figure 3. Open in new tabDownload slide Fluorescence-activated cell sorting (FACS) analysis of kidney SP and non-SP cells in NTN model. (A): Representative FACS dot plot showing the presence of kidney SP cells (left panel: P2) and their disappearance in the presence of reserpine (right panel: P2) in control mice. Reserpine blocks the activity of drug transport proteins, preventing them from effluxing the dye. (B): Representative FACS dot plot showing the phenotype of kidney SP cells in control mice. CD45 positive in Q1–3 and Sca-1 positive in Q4–1. (C): Population of kidney SP cells in NTN mice. The values are expressed as a ration relative to the mean SP fraction of the control mice. Data are mean ± SEM (n = 12); * p < .05 versus control mice. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. (D, E): Characterization of kidney SP and non-SP cells in NTN mice. Data are mean ± SEM (n = 6); ** p < .05 versus non-SP cells. White bars represent SP cells in control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Dotted white bars are non-SP cells in control mice, dotted black bars in NTN mice, and dotted gray bars in NTN mice treated with TSA. Abbreviations: cont, control; NTN, nephrotoxic serum nephritis; SP, side population; TSA, trichostatin A. Figure 3. Open in new tabDownload slide Fluorescence-activated cell sorting (FACS) analysis of kidney SP and non-SP cells in NTN model. (A): Representative FACS dot plot showing the presence of kidney SP cells (left panel: P2) and their disappearance in the presence of reserpine (right panel: P2) in control mice. Reserpine blocks the activity of drug transport proteins, preventing them from effluxing the dye. (B): Representative FACS dot plot showing the phenotype of kidney SP cells in control mice. CD45 positive in Q1–3 and Sca-1 positive in Q4–1. (C): Population of kidney SP cells in NTN mice. The values are expressed as a ration relative to the mean SP fraction of the control mice. Data are mean ± SEM (n = 12); * p < .05 versus control mice. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. (D, E): Characterization of kidney SP and non-SP cells in NTN mice. Data are mean ± SEM (n = 6); ** p < .05 versus non-SP cells. White bars represent SP cells in control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Dotted white bars are non-SP cells in control mice, dotted black bars in NTN mice, and dotted gray bars in NTN mice treated with TSA. Abbreviations: cont, control; NTN, nephrotoxic serum nephritis; SP, side population; TSA, trichostatin A. TSA Downregulates MyoR and Augments Histone Acetylation TSA is a histone deacetylase inhibitor and downregulates MyoR, which determines the function of kidney SP cells [20]. Next, we confirmed the effect of TSA on MyoR and histone acetylation in vivo. The population of kidney SP cells was reduced in NTN mice, and kidney SP cells were positive for MyoR. As expected, MyoR-positive cells were significantly reduced in NTN mice (Fig. 4A, right upper panel). Although the reduction of SP cells was prevented by treatment with TSA, MyoR-positive cells were abolished in NTN mice treated with TSA (Fig. 4A, left lower panel). Treatment with TSA also abolished MyoR-positive cells in control C57/B6 mice without NTN (data not shown), suggesting that TSA strongly downregulates MyoR in vivo. Next, we examined histone acetylation by Western blot. Compared with control, histone acetylation was not altered in NTN mice, but treatment with TSA significantly augmented it (Fig. 4B). Figure 4. Open in new tabDownload slide Immunohistochemical analysis of kidney side population cells. (A): Representative merged (yellow) photomicrographs of fluorescent images of MyoR (green) and nuclear staining (red) in kidney tissue. Data are mean ± SEM (n = 6); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. (B): Western immunoblot analysis of acetylated histone H3 in kidney tissue. Data are mean ± SEM (n = 4); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Abbreviations: cont, control; LPF, low-power field; NTN, nephrotoxic serum nephritis; TSA, trichostatin A. Figure 4. Open in new tabDownload slide Immunohistochemical analysis of kidney side population cells. (A): Representative merged (yellow) photomicrographs of fluorescent images of MyoR (green) and nuclear staining (red) in kidney tissue. Data are mean ± SEM (n = 6); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. (B): Western immunoblot analysis of acetylated histone H3 in kidney tissue. Data are mean ± SEM (n = 4); * p < .05 versus control; ** p < .05 versus NTN. White bars represent control mice, black bars NTN mice, and gray bars NTN mice treated with TSA. Abbreviations: cont, control; LPF, low-power field; NTN, nephrotoxic serum nephritis; TSA, trichostatin A. TSA Augments Expression of BMP-7 in Kidney SP Cells To clarify the mechanism by which TSA prevents progression of renal dysfunction, we examined the effect of TSA on regulation of renoprotective factors such as BMP-7, VEGF, and HGF in kidney cells. First, we examined whether TSA downregulates MyoR in kidney SP cells as reported by Yu et al. [20]. TSA abolished gene expression of MyoR in kidney SP cells, but MyoR was not detected in non-SP cells (Fig. 5A). Compared with non-SP cells, basal expression of all renoprotective factors was significantly higher in SP cells (Fig. 5B–5D). TSA significantly upregulated BMP-7 in kidney SP cells but not in non-SP cells (Fig. 5B–5D). However, TSA showed no effect on VEGF and HGF in both SP and non-SP cells. Figure 5. Open in new tabDownload slide Quantitative real-time polymerase chain reaction analysis of (A) MyoR, (B) BMP-7, (C) VEGF, and (D) HGF in kidney SP and non-SP cells. White bars represent SP cells and black bars non-SP cells. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. Values represent mean ± SEM (n = 6); * p < .05 versus non-SP cells; ** p < .05 versus SP cells. Abbreviations: BMP, bone morphogenic protein; HGF, hepatocyte growth factor; SP, side population; TSA, trichostatin A; VEGF, vascular endothelial growth factor. Figure 5. Open in new tabDownload slide Quantitative real-time polymerase chain reaction analysis of (A) MyoR, (B) BMP-7, (C) VEGF, and (D) HGF in kidney SP and non-SP cells. White bars represent SP cells and black bars non-SP cells. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. Values represent mean ± SEM (n = 6); * p < .05 versus non-SP cells; ** p < .05 versus SP cells. Abbreviations: BMP, bone morphogenic protein; HGF, hepatocyte growth factor; SP, side population; TSA, trichostatin A; VEGF, vascular endothelial growth factor. TSA Partially Reverses Chronic Renal Dysfunction Administration of recombinant BMP-7 has shown marked efficacy in the reduction of glomerular and interstitial fibrosis in NTN mice [5]. As TSA augments the expression of BMP-7 in kidney SP cells, we tried to examine the effect of TSA to reverse chronic dysfunction. Initiation of treatment with TSA after 3 weeks of NTN (for 3 weeks, until 6 weeks) significantly prevented the progression of proteinuria (Fig. 6A). Moreover, mice treated with TSA starting 3 weeks after NTN injection showed significant improvement in renal function at week 6, as measured by BUN, when compared with the week-3 measurement before TSA treatment was initiated (Fig. 6B). We also performed histological analysis but could not find significant improvement, unlike the case with recombinant BMP-7 (data not shown), suggesting partial reverse of chronic renal dysfunction. Figure 6. Open in new tabDownload slide Treatment with TSA reversed chronic renal dysfunction. (A): Urinary protein/creatinine ratio. White squares represent NTN-alone mice, and black circles represent NTN mice treated with TSA. Data are mean ± SEM; * p < .05 versus NTN alone at 3 weeks; ** p < .05 versus NTN alone at 5 weeks and 6 weeks. (B): BUN on days 21 (3W NTN) and 42 (6W NTN and 6W NTN+TSA). White bars represent NTN-alone mice, and black bars NTN mice treated with TSA. Data are mean ± SEM (n = 6); * p < .05 versus 3W NTN; ** p < .05 versus 6W NTN. Abbreviations: BUN, blood urea nitrogen; NS, not significant; NTN, nephrotoxic serum nephritis; TSA, trichostatin A; W, week. Figure 6. Open in new tabDownload slide Treatment with TSA reversed chronic renal dysfunction. (A): Urinary protein/creatinine ratio. White squares represent NTN-alone mice, and black circles represent NTN mice treated with TSA. Data are mean ± SEM; * p < .05 versus NTN alone at 3 weeks; ** p < .05 versus NTN alone at 5 weeks and 6 weeks. (B): BUN on days 21 (3W NTN) and 42 (6W NTN and 6W NTN+TSA). White bars represent NTN-alone mice, and black bars NTN mice treated with TSA. Data are mean ± SEM (n = 6); * p < .05 versus 3W NTN; ** p < .05 versus 6W NTN. Abbreviations: BUN, blood urea nitrogen; NS, not significant; NTN, nephrotoxic serum nephritis; TSA, trichostatin A; W, week. Discussion To determine core genes comprising a stem cell genetic program, several comprehensive microarray studies have been performed [26, 27]. However, a limited number of genes overlapped among the reports; ABCG2/BCRP-1, which determines the SP phenotype [28], was the only gene expressed in embryonic stem cells, hematopoietic stem cells, and neurospheres [29]. Recently, we reported that kidney SP cells differentiate into multiple lineages, and transplantation of these cells augmented recovery of acute renal failure. Recently, Challen et al. also confirmed the multilineage potential of kidney SP cells and a humoral role of the cells in renal repair [16]. As the next step, we focused on the activation of kidney SP cells in situ and recovery of chronic renal injury through this. We found that: (a) treatment with TSA prevented the progression of chronic injury and reduction of kidney SP cells; (b) kidney SP cells were not derived from bone marrow, and TSA augmented gene expression of BMP-7 in these cells; and (c) treatment with TSA partially reversed chronic renal injury. Although dialysis treatment is well advanced, end-stage renal failure is a leading cause of morbidity and mortality throughout the world. Recently, Zeisberg et al. reported reversal of chronic renal injury by recombinant human BMP-7 [5], and BMP-7 is also thought to be involved in regeneration after ischemic [30] or toxic renal injury. These results strongly suggest that BMP-7 could be a potential therapeutic agent for chronic renal disease, but its gene expression is retained in only a few types of adult tissue, most prominently in the kidney, and its physiologic functions and regulation in the adult kidney are unknown at present. In these studies, we showed that TSA augmented the expression of BMP-7 in kidney SP cells, suggesting that kidney SP cells are one of the promising generator cells and TSA a stimulator for endogenous BMP-7 production. However, since SP cells were sorted by dye efflux function but not by histological definition, we should await further histological studies to define a predominant source of BMP-7. TSA is a histone deacetylase inhibitor and can modulate gene expression either positively or negatively in a gene-specific manner. Although TSA increased histone acetylation in whole kidney tissue, TSA augmented BMP-7 expression only in kidney SP cells but not in non-SP cells. This different response to TSA suggests that augmentation of BMP-7 was not simply mediated by nonspecific HDAC inhibition. Recently, we reported that MyoR, a basic-helix-loop-helix transcriptional factor, is exclusively expressed in kidney SP cells [13]. MyoR was originally identified as a transcriptional repressor [14] that antagonizes the action of MyoD, and MyoD enhances the effect of BMP-7 [31]. Recently, Yu et al. reported that MyoR is expressed in nonmyogenic cells, and TSA downregulates MyoR [20]. As shown in Figure 6, MyoR was expressed in kidney SP cells but was absent in non-SP cells. Moreover, treatment with TSA abolished MyoR expression in vivo and in vitro (Figs. 4A, 5A). Taken together, these findings indicate that TSA may downregulate the transcriptional repressor MyoR, and this may lead to augmentation of BMP-7 expression in kidney SP cells. Recently, kielin/chordin-like protein (KCP) was reported as a novel enhancer of BMP-7 [6], but KCP does not augment BMP-7 production. Although TSA showed limited reversal of chronic histological changes as compared with recombinant BMP-7, combination therapy of TSA and KCP may effectively reverse it by augmenting production of BMP-7 and enhancing its effect. Kidney SP cells can differentiate into multiple lineages, but our results showed that the cells, both in control and NTN models, were positive for a stem cell marker, Sca-1 (over 75%), and were not bone marrow derived (CD45 negative: less than 2% positive). Treatment with TSA prevented the reduction of SP cells, but the preserved cells were also Sca-1 positive and CD45 negative. Kidney SP cells are more sensitive to inflammatory cytokines than non-SP cells [13], and Mishra et al. demonstrated that TSA downregulates cytokines in the MRL-lpr/lpr mouse. These results suggest that TSA did not induce infiltration of bone marrow-derived cells into the kidney but prevented damage of kidney SP cells. Recently, we reported that TSA suppressed TGF-β induced epithelial to mesenchymal transition (EMT) in cultured human renal proximal tubular epithelial cells [32]. Taken together, we speculate that TSA prevented and partially reversed chronic renal injury by both augmenting BMP-7 expression in kidney SP cells and protecting the cells from inflammatory damage and also, in part, by direct inhibition of EMT in renal epithelial cells. Label-retaining cells and slow cycling cells in kidney are reported to be kidney progenitor or stem cells [33, 34]. Interestingly, kidney SP cells and slow cycling cells have some common characteristics. Kidney SP cells are localized to the interstitial space [13], and slow cycling cells are both interstitial cells and well differentiated epithelial cells [34]. When kidney SP cells are infused in an acute renal failure model, they localize to the interstitial space [13]. Challen et al. also showed that injected kidney SP cells are localized to the renal interstitium [16]. When slow cycling cells are injected into the subcapsular space of the kidney, they are located in both the interstitial space and inside tubules [34], and our results showed that TSA strongly prevented interstitial fibrosis where SP cells and slow cycling cells exist. TSA can directly inhibit the transdifferentiation of myofibroblasts [35], and we should think about the possibility that TSA directly decreased α-SMA positive fibroblasts and attenuated interstitial fibrosis independent of SP cells. However, these results suggest that the kidney interstitial space may play a key role in renal regeneration as a niche for multipotent cells such as slow cycling cells and SP cells. Recent studies have demonstrated that stem cells and multipotent cells contribute to the regenerative process by producing protective and regenerative factors rather than direct replacement of damaged cells by differentiation [24, 36, 37]. Our results showed that TSA activated multipotent kidney SP cells and augmented the expression of a regenerative factor, BMP-7. For clinical regenerative medicine, we believe the focus of stem cell biology may shift from multiple differentiations of cells to multiple functions of cells, such as the production of regenerative factors. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. Acknowledgements This study was supported by Mochida Pharmaceutical Co., Ltd., Takeda Science Foundation, Health and the Open Competition for the Development of Innovative Technology, and Research Grant DK30932 from National Institutes of Health (to D.S.). References 1 Dudley AT , Lyons KM, Robertson EJ. 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Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2007 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Inhibition of Histone Deacetylase Activates Side Population Cells in Kidney and Partially Reverses Chronic Renal Injury
JF - Stem Cells
DO - 10.1634/stemcells.2007-0049
DA - 2007-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/inhibition-of-histone-deacetylase-activates-side-population-cells-in-UU7V9TbpZi
SP - 2469
EP - 2475
VL - 25
IS - 10
DP - DeepDyve
ER -