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

Abstract

Musculin/MyoR is a new member of basic helix-loop-helix transcription factors, and its expression is limited to skeletal muscle precursors. Here, we report that musculin/MyoR is expressed in adult kidney side population (SP) cells and can regulate their function. SP phenotype can be used to purify stem cell-rich fractions. Microarray analysis clarified that musculin/MyoR was exclusively expressed in kidney SP cells, and the cells resided in the renal interstitial space. Musculin/MyoR-positive cells were decreased in acute renal failure, but infusion of kidney SP cells increased musculin/MyoR-positive cells and improved renal function. Kidney SP cells in reversible acute renal failure expressed a high level of renoprotective factors and leukemia inhibitory factor (LIF), but not in irreversible chronic renal failure. In cultured kidney SP cells, LIF stimulated gene expression of renoprotective factors, and down-regulation of musculin/MyoR augmented LIF-induced gene expression. Our results suggest that musculin/MyoR may play important roles not only in developmental processes but also in regenerative processes in adult tissue.

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 × 10⁵ to 3.0 × 10⁵). (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 × 10⁵, 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.

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 panels. (C and D) Representative photomicrographs of fluorescent images of musculin and nuclear staining in renal tissue (C57BL/6). Musculin-positive cells (arrows) reside in the interstitial space of the kidney. Overlay images of musculin and nuclear (yellow) images also showed that the cells reside in the interstitial space of the kidney.

Figure 3.

TNF- α induced cell death in kidney SP cells. (A) Kidney SP and non-SP cells just after FACS sorting were incubated with TNF-α at the indicated concentrations for 24 h. TNF-α induced cell death in SP cells (black bars) in a concentration-dependent manner, but not in non-SP cells (white bars). Cell survival was determined by trypan blue exclusion assay. (B and C) Kidney SP cells just after FACS sorting were incubated with 50 μM reserpine (res), 8 ng/ml TNF-α, or both for 24 h. Compared with control (no treatment), TNF-α alone significantly increased DNA fragmentation and apoptosis, and cotreatment with reserpine significantly augmented it. DNA fragmentation assay was performed using an ELISA kit, and percentage of apoptosis was evaluated by nuclear staining with Hoechst 33258. Results are presented as means ± SEM of four independent experiments. *, P < 0.05 compared with control; **, P < 0.05 compared with TNF-α.

Figure 4.

Systemic kidney SP cell infusion increases musculin/MyoR-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 ± SEM (n = 8). *, P < 0.05 versus day 0. (B–D) Representative fluorescence photographs and overlay images (fluorescent and phase contrast) of renal tissues of control, cisplatin alone (cis), and infusion of kidney SP cells (cis+SP) on day 7. Green spots with red arrowheads are musculin/MyoR-positive cells. (E) Musculin/MyoR-positive cells were significantly reduced in the cisplatin-induced ARF model (cis) but were markedly increased by systemic kidney SP cell infusion (cis+SP). Values are the average number of musculin-positive cells in 10 randomly selected low power fields (×200) in four independent experiments. Values are the mean ± SEM. *, P < 0.05 versus control; **, P < 0.05 versus cis.

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 (cis+SP) 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 (cis+NSP) and BM cells (cis+BM) showed no effect. (C and D) Compared with control (cont), cisplatin alone (cis) significantly increased the tubular necrosis scores, but infusion of kidney SP cells (cis+SP) 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.

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 control (no treatment) and black bars show cisplatin treatment. Compared with control, gene expression of HGF, VEGF, BMP7, and LIF was significantly up-regulated in kidney SP cells. (B) IgA nephropathy mice. White bars show control (ddY) and black bars show IgA nephropathy (HIGA). (C) Nephritic syndrome mice. White bars show control (ICR) and black bars show nephritic syndrome (ICGN). Expression was normalized to GAPDH and is shown as the ration to the value of control. Values represent means ± SEM (n = 6). *, P < 0.05 versus control.

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 μM, respectively; dotted bars), and LIF with RA (10 ng/ml and 5 × 10⁻⁷ 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 TaqMan 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.