Essential roles of oncostatin M receptor β signaling in renal crystal formation in mice

Abstract

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ^-/- mice). There were fewer renal crystal deposits in OSMRβ^-/- mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ^-/- mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.

Figure 1

Expressions of OSM and OSMRβ in the kidney of a mouse model of renal crystal formation. C57BL/6 J mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for three, six, or nine days. (a,b) Gene expression of OSM (a) and OSMRβ (b) in the kidney on days 0 (without GOx injection), 3, 6, or 9. (c) Protein expression of OSM and OSMRβ in the kidney on days 0, 3, 6, or 9. The apparent molecular weights are indicated on the right. (d,e) Quantitative analysis of the protein expressions of OSM and OSMRβ. The band intensities of OSM and OSMRβ were normalized to β-actin and represented as the fold induction relative to the intensities on day 0. The full-length blots are presented in Supplementary Fig. S4. Data are expressed as mean ± SEM; n = 4–7 per group. *P < 0.05 compared with day 0.

Figure 2

Renal crystal deposits in WT and OSMRβ^−/− mice. WT and OSMRβ^−/− mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for three or six days. (a) Pizzolato staining in the kidney of WT and OSMRβ^−/− mice on days 0 (without GOx injection), 3, or 6. The boxed regions are shown at a higher magnification in the insets. Scale bars = 100 μm; 50 μm (insets). (b) The ratio of areas with renal crystal deposits. The ratio was quantified by calculating the percentage of the area containing crystal deposits to the total kidney area. Data are expressed as mean ± SEM; n = 6 per group. *P < 0.05 compared with WT mice.

Figure 3

Gene expressions in the kidney of WT and OSMRβ^−/− mice after GOx injection. WT and OSMRβ^−/− mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for three or six days. (a–c) Gene expressions of crystal-binding molecules, OPN (a), ANXA1 (b), and ANXA2 (c), in the kidney of WT and OSMRβ^−/− mice on days 0 (without GOx injection), 3, or 6. (d–g) Expressions of genes related to inflammation, TNF-α (d), IL-1β (e), F4/80 (f), and MCP-1 (g), in the kidney of WT and OSMRβ^−/− mice at days 0, 3, or 6. (h–l) Expressions of genes related to kidney injury, KIM-1 (h), and fibrosis, TGF-β (i), Col1a2 (j), αSMA (k), and Timp2 (l), in the kidneys of WT and OSMRβ^−/− mice on days 0, 3, or 6. Data are expressed as mean ± SEM; n = 5–7 per group. *P < 0.05 compared with WT mice.

Figure 4

Protein expressions in the kidney of WT and OSMRβ^−/− mice after GOx injection. WT and OSMRβ^−/− mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for three or six days. (a,c) Western blot analysis of crystal-binding molecules, OPN, ANXA1, and ANXA2, in the kidney of WT and OSMRβ^−/− mice on day 3 (a) and day 6 (c). The apparent molecular weights are indicated on the right. (b,d) Quantitative analysis of the protein expressions of OPN, ANXA1, and ANXA2 on day 3 (b) and day 6 (d). The band intensities of OPN, ANXA1, and ANXA2 were normalized to β-actin and are represented as the fold induction relative to the intensities of WT mice in the bar graph. (e,g) Western blot analysis of inflammation-related molecules, TNF-α (e,g), IL-1β (e,g), and MCP-1 (g), in the kidney of WT and OSMRβ^−/− mice on day 3 (e) day 6 (g). The apparent molecular weights are indicated on the right. (f,h) Quantitative analysis of the protein expressions of TNF-α (f,h), IL-1β (f,h), and MCP-1 (h) on day 3 (f) and day 6 (h). The band intensities of TNF-α, IL-1β, and MCP-1 were normalized to β-actin and are represented as the fold induction relative to the intensities of WT mice in the bar graph. (i) Western blot analysis of fibrosis-related molecules, TGF-β and Col1, in the kidney of WT and OSMRβ^−/− mice on day 3. The apparent molecular weights are indicated on the right. (j) Quantitative analysis of the protein expressions of TGF-β and Col1. The band intensities of TGF-β and Col1 were normalized to β-actin and are represented as the fold induction relative to the intensities of WT mice in the bar graph. The full-length blots are presented in Supplementary Fig. S5. Data are expressed as mean ± SEM; n = 4 per group. *P < 0.05 compared with WT mice.

Figure 5

Flow cytometric analysis of renal macrophages in the kidney of WT and OSMRβ^−/− mice. WT and OSMRβ^−/− mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for three or six days. (a) Dot plots of total macrophages (upper panels) and histogram plots of M1 (middle panels) and M2 (lower panels) macrophages in the kidney of WT and OSMRβ^−/− mice on day 3 and day 6. The isotype controls are shown as red dots (upper panels) and red areas (middle and lower panels). (b–d) The number of CD45⁺/F4/80⁺/CD11b⁺ cells (total macrophages; b), CD45⁺/F4/80⁺/CD11b⁺/Ly6C⁺ cells (M1 macrophages; c), and CD45⁺/F4/80⁺/CD11b⁺/CD206⁺ cells (M2 macrophages; d) in the kidney of WT and OSMRβ^−/− mice was evaluated by flow cytometric analysis. Data are expressed as mean ± SEM; n = 4–5 per group. *P < 0.05 compared with WT mice.

Figure 6

Localization of OSM and OSMRβ protein in the kidney. WT mice were given intraperitoneal injection of GOx (80 mg/kg) once daily for 3 days. Immunofluorescence staining for OSM (a) or OSMRβ (d, g, j, m) is shown in the left line (red). Immunofluorescence staining of EpCAM (b, e), OPN (h), PDGFRβ (k), and F4/80 (n) is shown in the middle line (green). The figures in the right line (c, f, i, l, o) show the merged images of panels in the left and middle lines. Nuclei were counterstained with DAPI (blue in c, f, i, l, o). Arrowheads indicate double-positive cells. Scale bars = 100 μm.

Figure 7

Direct effects of OSM on gene expressions in the RTECs and renal fibroblasts. RTECs and renal fibroblasts were isolated from the kidney of C57BL/6 J mice on day 3. Isolated cells were treated with OSM (50 ng/ml) for 1 or 2 h. (a) Western blot analysis of EpCAM and PDGFRβ in the RTECs and fibroblasts. The apparent molecular weights are indicated on the right. The full-length blots are presented in Supplementary Fig. S6. (b) Effects of OSM on the expressions of crystal-binding molecules (OPN, ANXA1, and ANXA2), inflammation-related genes (TNF-α and MCP-1), and fibrosis-related genes (TGF-β) in RTECs. (c) Effects of OSM on the expressions of crystal-binding molecules (OPN, ANXA1, and ANXA2), inflammation-related genes (TNF-α and MCP-1), and fibrosis-related genes (Col1a2 and αSMA) in renal fibroblasts. n = 4–6 per group. *P < 0.05 compared with controls.

Figure 8

Direct effects of OSM on protein expressions in the RTECs and renal fibroblasts. RTECs and renal fibroblasts were isolated from the kidney of C57BL/6 J mice on day 3. Isolated cells were treated with OSM (50 ng/ml) for 2 or 4 h. (a) Western blot analysis of crystal-binding molecules (OPN, ANXA1, and ANXA2) and fibrosis-related molecules (TGF-β) in the OSM-stimulated RTECs. The apparent molecular weights are indicated on the right. (b) Quantitative analysis of the protein expressions of OPN, ANXA1, ANXA2, and TGF-β in the RTECs. The band intensities of OPN, ANXA1, ANXA2, and TGF-β were normalized to β-actin and are represented as the fold induction relative to the intensities of the controls (white bar) in the bar graph. (c) Western blot analysis of crystal-binding molecules (OPN, ANXA1, and ANXA2), inflammation-related molecule (TNF-α), and fibrosis-related molecules (Col1) in the OSM-stimulated renal fibroblasts. The apparent molecular weights are indicated on the right. (d) Quantitative analysis of the protein expression of OPN, ANXA1, ANXA2, TNF-α, and Col1 in the renal fibroblasts. The band intensities of OPN, ANXA1, ANXA2, TNF-α, and Col1 were normalized to β-actin and are represented as the fold induction relative to the intensities of the controls (white bar) in the bar graph. The full-length blots are presented in Supplementary Fig. S7. Data are expressed as mean ± SEM; n = 4 per group. *P < 0.05 compared with controls.