Fig 1.
Expression of IL-34 and its two receptors on CP-stimulated MRPTEpiC.
Scheme for the in vitro analysis using cultured MRPTEpiC stimulated with CP (A). MRPTEpiC and the culture supernatants were harvested after stimulation with CP (2 μg/mL) for 0 to 24 h. Real-time RT-PCR for Kim-1 (B), IL-34 (C), and CSF-1 (D) in MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h. IL-34 and CSF-1 protein levels evaluated by ELISA in the culture supernatants of MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h (E and F). Real-time RT-PCR for cFMS (G) and PTP-ζ (H) in MRPTEpiC after stimulation with CP or TNF-α for 0 to 24 h. In the analysis of real-time RT-PCR, the values were normalized to the β-actin transcript, and are expressed as a relative ratio. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 2.
Mø proliferation induced by the culture supernatants of MRPTEpiC stimulated with CP.
Scheme for the analysis. Mouse Møs (RAW 264) were cultured with medium or the culture supernatant of MRPTEpiC stimulated with CP (2 μg/mL) for 24 h. The medium for culturing RAW 264 contained vehicle, CP, rIL-34 (500 pg/mL), or anti-IL-34 Ab (1000 pg/mL). CP-stimulated MRPTEpiC were treated with vehicle or anti-IL-34 Ab (1000 pg/mL), followed by the collection of the supernatant to culture RAW 264 (A). MTT assay for Mø proliferation among the study groups (B). Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 3.
Intra-renal expression of IL-34 and its two receptors in CP-N mice.
Representative photos of IL-34 expression on TECs identified by immunostaining among the NC (A), CP+V (B), and CP+anti-IL-34 Ab (C) groups of mice. Original magnification, 400×. Graph of the IL-34 positivity among the study groups (D). IL-34 protein levels in homogenate kidney tissues analyzed by ELISA among the study groups (E). Representative WB analysis for cFMS, PTP-ζ, and GAPDH (F). Densitometric analysis of WB for cFMS (G) and PTP-ζ (H). The values shown are the values after normalization to GAPDH expression, and they are depicted as the relative ratio of cFMS or PTP-ζ to GAPDH. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 4.
Biochemical parameters and renal histological findings in CP-N mice.
The serum IL-34 levels as evaluated by ELISA (A), the body weight (BW) (B), and the ratios of the kidney weight (KW) to the BW (C) among the study groups. The serum Cr levels determined by the Cr assay kit (D) among the study groups. The mRNA levels for Kim-1 among the study groups (E). Values were normalized to the GAPDH transcript, and are expressed as the relative ratio. Representative photos of kidney tissues stained with PAS in a NC mouse (F), CP+V mouse (G), and CP+anti-IL-34 Ab mouse (H). Original magnification, 200×. Quantification of the tubulointerstitial damage score per field (I), and the number of casts per field (J) among the study groups. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 5.
Intra-renal Mø proliferation and TEC apoptosis in CP-N mice.
Representative photos of F4/80-positive Mø infiltration in the tubular interstitium, TUNEL-positive apoptotic cells in TECs, and caspase-3-positive cells in TECs identified by immunostaining among the NC (A, E, and I), CP+V (B, F and J), and CP+anti-IL-34 Ab (C, G, and K) groups of mice. Original magnification, 200×. Quantitative evaluation of the IHC for F4/80 (D), TUNEL (H), and caspase-3 (L) among the study groups. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 6.
Intra-renal expression of chemokines, apoptosis-regulatory molecules, and proinflammatory cytokines in CP-N mice.
Real-time RT-PCR for genes encoding MCP-1/CCL2 (A), MIP-1α/CCL3 (B), Bax (C), Bcl-2 (D), TNF-α (E), IL-6 (F), IL-1β (G), and IL-10 (H) among the NC, CP+V, and CP+anti-IL-34 Ab groups of mice. Values were normalized to the GAPDH transcript, and are expressed as the relative ratio. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 7.
Intra-renal cyto-destructive (M1-like) and cyto-protective (M2-like) Mø accumulation in CP-N mice.
Single-cell suspensions from both kidneys of each mouse were stained. The CD11b+F4/80+ cell population was gated first, then the F4/80+TNFα+ or IL-10+ cell population was gated. Representative FACS plots with gating for F4/80+TNFα+ or IL-10+ cells in the CP+V (A) and CP+anti-IL-34 Ab mouse (B) groups. Graphs of the numbers of cyto-destructive Møs (F4/80+TNFα+) (C) and cyto-protective Møs (F4/80+IL-10+) (D) in the kidneys of the mouse groups. Data are expressed as the mean ± SEM. Broken lines indicate the data for NC mice (n = 3). The Mann-Whitney U test was used for statistical analysis.
Fig 8.
Cellular injury and apoptosis in CP-stimulated MRPTEpiC.
Scheme for the in vitro analysis. Cultured MRPTEpiC were stimulated with or without CP (2 μg/mL), followed by treatment with vehicle or anti-IL-34 Ab (1000 pg/mL) for 12 or 24 h (A). The IL-34 (B) and Kim-1 (C) mRNA expression levels in TECs, as assessed by real-time RT-PCR, among the study groups. Real-time RT-PCR for Bax (D) and Bcl-2 (E) in CP-stimulated MRPTEpiC with or without anti-IL-34 Ab treatment for 0 to 24 h. Values were normalized to the GAPDH transcript, and are expressed as the relative ratio. Representative WB analysis for Bax and GAPDH (F). Densitometric analysis of WB for Bax. The values shown are the values after normalization to GAPDH expression, and they are depicted as the relative ratio of Bax to GAPDH (G). Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 9.
Expression of CSF-1 in CP-N mice and CP-stimulated MRPTEpiC.
Representative photos of the intra-renal CSF-1 expression on TECs identified by immunostaining among the NC (A), CP+V (B), and CP+anti-IL-34 Ab (C) groups of mice. Original magnification, 400×. Graph of CSF-1 positivity per field among the study groups (D). The serum CSF-1 levels evaluated by ELISA among the study groups (E). Real-time RT-PCR for CSF-1 in CP-stimulated MRPTEpiC with or without anti-IL-34 Ab treatment for 0 to 24 h (F). Values were normalized to the GAPDH transcript, and are expressed as the relative ratio. The CSF-1 protein levels evaluated by ELISA in the culture supernatants of MRPTEpiC after CP stimulation with or without anti-IL-34 Ab treatment for 0 to 24 h (G). Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.
Fig 10.
CP-induced ERK1/2 phosphorylation in CP-N mice and CP-stimulated MRPTEpiC.
Representative WB analysis for p-ERK1/2 and t-ERK1/2 in renal cortical tissues after stimulation with CP for 72 h among the NC, CP+V, and CP+anti-IL-34 Ab groups of mice (A). Densitometric analysis of WB for p-ERK1/2 among the study groups (B). Representative WB analysis for p-ERK1/2 and t-ERK1/2 in MRPTEpiC after stimulation with CP for 6 h among the NC, CP+V, and CP+anti-IL-34 Ab groups (C). Densitometric analysis of WB for p-ERK1/2 and t-ERK1/2 in MRPTEpiC among the study groups (D). The values shown are the values after normalization to the t-ERK1/2 expression, and they are depicted as the relative ratio of p-ERK1/2 to t-ERK1/2. Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analysis.