Mesenchymal stem cells differentiate into renin-producing juxtaglomerular (JG)-like cells under the control of liver X receptor-alpha

Abstract

Renin is a key enzyme for cardiovascular and renal homeostasis and is produced by highly specialized endocrine cells in the kidney, known as juxtaglomerular (JG) cells. The nature and origin of these cells remain as mysteries. Previously, we have shown that the nuclear hormone receptor liver X receptor-alpha (LXRalpha) is a major transcriptional regulator of the expression of renin, c-myc, and other genes involved with growth/differentiation. In this study we test the hypothesis that LXRalpha plays an important role not only in renin expression but also in renin-containing cell differentiation, specifically from the mesenchymal stem cell (MSC), which may be the origin of the JG cell. Indeed, our data demonstrated that LXRalpha activation by its ligands or cAMP stimulated renin gene expression in both murine and human MSCs. Furthermore, sustained cAMP stimulation of murine MSCs overexpressing LXRalpha led to their differentiation into JG-like cells expressing renin and alpha-smooth muscle actin. These MSC-derived JG-like cells contained renin in secretory granules and released active renin in response to cAMP. In conclusion, the activation of LXRalpha stimulates renin expression and induces MSCs differentiation into renin-secreting, JG-like cells. Our results suggest that the MSC may be the origin of the juxtaglomerular cell and provide insight into novel understanding of pathophysiology of the renin-angiotensin system.

FIGURE 1.

Characteristics of murine MSCs. Murine MSCs were analyzed for the expression of the cell surface markers CD44 (A–C), CD105 (D–F), CD34 (G–I), and CD 45 (J–L) by FACS (A, D, G, and J) and immunofluorescent staining (C, F, I, and L). Negative controls for antibody type were performed (red area in A, D, G, and J) by using isotype control immunoglobulin G (for CD 44 and CD45) or by omitting the primary antibody (for CD 105 and CD 34). B, C, E, F, H, I, K, and L, original magnification, ×400.

FIGURE 2.

Effects of LXRα ligands on the expression of Ren1c- and LXRα-related genes in murine MSCs. One μmol/liter LXRα synthetic ligand T0901317, 0.1 μmol/liter LXRα natural ligand 22-hydroxycholesterol (22OH-C), or 1 mmol/liter 8-bromocyclic AMP (cAMP) was added to media after 16 h of serum depletion. Murine MSCs were harvested after 6 h of pharmacological treatment, and total RNA was analyzed by quantitative RT-PCR for mRNA expression of Ren1c (A, E, and F), Abca1 (B), LXRα (C), and LXRβ (D). -Fold changes versus without treatment group (Without Tx). Data are the mean ± S.E.; n = 4; *, p < 0.05 versus without treatment group. n.s., statistically non-significant.

FIGURE 3.

Effect of cAMP or 22-hydroxycholesterol on the expression of Renin, LXRα, and LXRβ in human MSCs. One mmol/liter 8-bromo-cyclic AMP (cAMP) or 0.1 μmol/liter LXRα natural ligand 22-hydroxycholesterol (22OH-C) was added to media after 16 h of serum depletion. Human MSCs were harvested after 6 h of pharmacological treatment, and total RNA was analyzed by quantitative RT-PCR for mRNA expression of renin (A and B), LXRα (C and D), and LXRβ (E and F). Data are -fold changes versus without the treatment group (Without Tx). Data are the mean ± S.E.; n = 4; *, p < 0.05 versus without treatment group. n.s., statistically non significant.

FIGURE 4.

Effect of LXRα siRNA in cAMP- or 22-hydroxycholesterol-induced up-regulation of renin expression in human MSCs. Human MSCs were transfected with LXRα siRNA or control siRNA. 1 mmol/liter 8-bromo-cyclic AMP (cAMP) (A) or 0.1 μmol/liter LXRα (B) natural ligand 22-hydroxycholesterol (22OH-C) was added to media after 16 h of serum depletion. Cells were harvested after 6 h of pharmacological treatment, and total RNA was analyzed by quantitative RT-PCR for mRNA expression of LXRα and Renin. Data are -fold changes versus the control siRNA group. Data are the mean ± S.E.; n = 5 in each group; *, p < 0.05 versus control siRNA group.

FIGURE 5.

Effect of cAMP on the expression of Ren1c gene in mMSC/GFP and mMSC/LXRα/GFP. One mmol/liter 8-bromo-cyclic AMP (cAMP) was added to media after 16 h of serum depletion. Murine MSCs overexpressing GFP (mMSC/GFP) or GFP-LXRα (mMSC/LXRα/GFP) with or without cAMP treatment were harvested after 6 h of pharmacological treatment, and total RNA was analyzed by quantitative RT-PCR for mRNA expression of Ren1c. Without Tx, without the treatment group. Data are -fold changes versus mMSC/GFP without Tx. Data are the mean ± S.E.; n = 3. *, p < 0.05 versus mMSC/GFP without Tx. #, p < 0.05 versus mMSC/GFP, cAMP. +, p < 0.05 versus mMSC/LXRα/GFP without Tx.

FIGURE 6.

Phenotypic changes of mMSC/LXRα/GFP induced by the prolonged treatment with cAMP or 22-hydroxycholesterol. Prolonged treatment of mMSC/LXRα/GFP with cAMP (A, B, and E) or 22-hydroxycholesterol (C and D) was performed. The arrows show granules in cytoplasm (A, cAMP treatment for 3 weeks; C, 22-hydroxycholesterol treatment for 6 weeks). Immunocytochemistry for renin antibody exhibited that granules were positive for renin (B, cAMP treatment for 2 weeks; D, 22-hydroxycholesterol treatment for 6 weeks). E, negative control for renin staining using isotype control immunoglobulin G. Original magnification, ×400.

FIGURE 7.

Immunofluorescent staining for α-smooth muscle actin and immunoblotting for renin. A–C, murine MSCs overexpressing GFP-LXRα (mMSC/LXRα/GFP) were treated with cAMP for 5 weeks. A, microscopic findings showed granules in cytoplasm. B, cells induced from mMSC/LXRα/GFP exhibited green fluorescence. C, granulated cells were positive for α-smooth muscle actin. Original magnification, ×400. D, murine MSCs overexpressing GFP alone (mMSC/GFP) and murine MSCs overexpressing GFP-LXRα (mMSC/LXRα/GFP) were treated with or without cAMP daily for 8 weeks. Cell lysates were isolated and subjected to immunoblotting analysis using antibodies for renin and GAPDH. Lane 1, mMSC/GFP without treatment (Tx); lane 2, mMSC/GFP with cAMP Tx; lane 3, mMSC/LXRα/GFP without Tx; lane 4, mMSC/LXRα/GFP with cAMP Tx; lane 5, positive control (human renin recombinant protein).

FIGURE 8.

Renin activity in conditioned media. Cyclic AMP-LXRα-activated mMSCs (mMSC/LXRα/GFP with cAMP treatment), 22-hydroxycholesterol-LXRα-activated mMSCs (mMSC/LXRα/GFP with 22-hydroxycholesterol treatment), and control MSCs (mMSC/GFP without treatment) were cultured for 8 weeks. Conditioned media from cells were collected, and active free renin activity in conditioned media was measured by angiotensin I (Ang I) generation. Data are the mean ± S.E.; n = 6; *, p < 0.05 versus control mMSC.

FIGURE 9.

Hypotheses of the lineage of renin-containing cell. A, shown is a traditional hypothesis. The JG cell (Renin-containing cell) is hypothesized to be derived from the vascular smooth muscle cell (VSMC) through metaplastic transformation. B, shown is a new hypothesis. Renin progenitor cell may exist, and the renin-containing cell can give rise to vascular smooth muscle cells. The MSC may be the origin of renin-containing cell.