1. Volume 88, Issue 5, Pages 1013-1029 (November 2015)
Excess 25-hydroxyvitamin D3 exacerbates tubulointerstitial injury in mice by modulating macrophage phenotype 
Yasuo Kusunoki, Isao Matsui, Takayuki Hamano, Akihiro Shimomura, Daisuke Mori, Sayoko Yonemoto, Yoshitsugu Takabatake, Yoshiharu Tsubakihara, René St-Arnaud, Yoshitaka Isaka, Hiromi Rakugi 
Kidney International 
Volume 88, Issue 5, Pages (November 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Excess 25-hydroxyvitamin D3 (25(OH)D3) exacerbated tubulointerstitial injury in unilateral ureteral obstructed (UUO) mice. (a) Seven-week-old 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) knockout mice were randomly divided into three groups: vehicle, sufficiency 25(OH)D3 (6.25ng/g body weight (BW) per injection), and excess 25(OH)D3 (100ng/g BW per injection). Mice were injected subcutaneously with vehicle or 25(OH)D3 three times before undergoing the UUO operation. The analyses were performed on UUO day 7. (b) Real-time PCR analyses showed that excess 25(OH)D3, but not sufficiency 25(OH)D3, upregulated mRNA levels for α-smooth muscle actin (α-SMA) and extracellular matrix genes in the obstructed kidneys. (c) Representative micrographs of (A) periodic acid–Schiff (PAS)-, (B) α-SMA-, and (C) collagen I–stained kidney sections are shown (bars=50μm for PAS staining and 100μm for α-SMA and collagen I staining). (d) Quantitative analyses of these histological sections—tubular injury index, interstitial volume index, α-SMA-positive area, and collagen I–positive area—revealed that excess 25(OH)D3 exacerbated tubulointerstitial injury in the UUO kidney but not in the contralateral kidney. All results are presented as means±s.d. Values of the vehicle group served as references. Values of contralateral kidneys and UUO kidneys were separately analyzed with Dunnett’s test (N=12–18 in each group: *P<0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Excess 25-hydroxyvitamin D3 (25(OH)D3) aggravated oxidative stress in the obstructed kidneys. Kidney tissues of the mice in Figure 1 were analyzed. (a) Kidney sections were immunohistochemically stained with γH2AX in green and nuclei in blue (bar=20μm). Both single-stained (γH2AX) and double-stained (γH2AX+DAPI (4′,6-diamidino-2-phenylindole)) micrographs are shown. Mice in the excess 25(OH)D3 group had greater numbers of γH2AX–positive cells in the obstructed kidney (N=12–18 in each group: *P<0.05, Dunnett’s test). (b) Western blot analysis demonstrated that excess 25(OH)D3 increased nitrotyrosine (nitro-Tyr)-containing proteins in the obstructed kidneys. No difference was seen between the vehicle and sufficiency 25(OH)D3 groups. Values of the vehicle group served as references. Values of contralateral kidneys and UUO kidneys were separately analyzed with Dunnett’s test (N=4 in each group: *P<0.05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; UUO, unilateral ureteral obstructed; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
The extent of tubulointerstitial injury was not affected by 24,25(OH)2D3 (24,25-dihydroxyvitamin D3). (a) CYP27B1 knockout mice were randomly divided into two groups: vehicle and 24,25(OH)2D3. Mice in the 24,25(OH)2D3 group received 24,25(OH)2D3 at a dose of 100ng/g body weight (BW) per injection at the indicated time points. No parameters—(b) mRNA levels for α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, (c) (A) periodic acid–Schiff (PAS)-, (B) α-SMA-, and (C) collagen I–stained kidney sections, and (d) quantitative analyses of the kidney sections—were different between the vehicle and 24,25(OH)2D3 groups. All results are presented as means±s.d. Values of contralateral kidneys and unilateral ureteral obstructed (UUO) kidneys were separately analyzed with the Student’s t-test (N=12–16 in each group: P<0.05) (bars=50μm for PAS staining and 100μm for α-SMA and collagen I staining). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
The levels of oxidative stress were not different between the vehicle and 24,25(OH)2D3 (24,25-dihydroxyvitamin D3) groups. Kidney tissues of the mice in Figure 3 were analyzed. (a) Kidney sections were immunohistochemically stained with γH2AX in green and nuclei in blue (bar=20μm). (b) Western blot analysis was performed using anti-nitrotyrosine (nitro-Tyr) antibody. Both (a) γH2AX staining (N=10–13 in each group: P<0.05) and (b) western blotting for nitro-Tyr (N=4 in each group: P<0.05) showed that 24,25(OH)2D3 did not affect the levels of oxidative stress. Values of contralateral kidneys and UUO kidneys were separately analyzed with the Student’s t-test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
In 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1)/vitamin D receptor (VDR) double knockout (KO) mice, excess 25-hydroxyvitamin D3 (25(OH)D3) did not exacerbate tubulointerstitial injury. (a) Seven-week-old CYP27B1/VDR double KO mice were randomly divided into two groups: vehicle and excess 25(OH)D3. Mice in the excess 25(OH)D3 group received 25(OH)D3 at a dose of 100ng/g body weight (BW) per injection. Determination of all parameters—(b) mRNA levels for α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, (c) (A) periodic acid–Schiff (PAS)-, (B) α-SMA-, and (C) collagen I–stained kidney sections, and (d) quantitative analyses of the kidney sections—demonstrated that excess 25(OH)D3 did not exacerbate tubulointerstitial injury in CYP27B1/VDR double KO mice. All results are presented as means±s.d. Values of contralateral kidneys and unilateral ureteral obstructed (UUO) kidneys were separately analyzed with the Student’s t-test (N=8–12 in each group: P<0.05) (bars=50μm for periodic acid–Schiff (PAS) staining and 100μm for α-SMA and collagen I staining). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
In 25-Hydroxyvitamin D 1α-hydroxylase (CYP27B1)/vitamin D receptor (VDR) double knockout mice, oxidative stress was not aggravated by excess 25-hydroxyvitamin D3 (25(OH)D3). Kidney tissues of the mice in Figure 5 were analyzed. Both (a) γH2AX staining of the kidney in green (bar=20μm, N=8–12 in each group: P<0.05) and (b) western blot analysis for nitrotyrosine (nitro-Tyr) (N=4 in each group: P<0.05) demonstrated that excess 25(OH)D3 did not affect the levels of oxidative stress in CYP27B1/VDR double knockout mice. Values of contralateral kidneys and UUO kidneys were separately analyzed with the Student’s t-test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

8. Figure 7
Macrophage depletion abrogated the exacerbating effect of 25-hydroxyvitamin D3 (25(OH)D3). (a) To deplete macrophages, clodronate liposomes (200μl on day 0 and 100μl on days 2 and 5) were injected intraperitoneally. 25-Hydroxyvitamin D 1α-hydroxylase (CYP27B1) knockout (KO) mice were randomly divided into four groups: vehicle+empty liposome, excess 25(OH)D3+empty liposome, vehicle+clodronate liposome, and excess 25(OH)D3+clodronate liposome. Data were obtained from obstructed kidneys but not from contralateral kidneys. All parameters obtained from the obstructed kidneys—(b) mRNA levels for α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, (c) (A) periodic acid–Schiff (PAS)-, (B) α-SMA-, and (C) collagen I–stained kidney sections, and (d) quantitative analyses of the kidney sections—showed that excess 25(OH)D3 did not exacerbate tubulointerstitial injury in macrophage-depleted CYP27B1 KO mice. All results are presented as means±s.d. Values of empty liposome–treated unilateral ureteral obstructed (UUO) kidneys and clodronate liposome–treated UUO kidneys were separately analyzed with the Student’s t-test (N=5–6 in each group: *P<0.05) (bars=50μm for periodic acid–Schiff (PAS) staining and 100μm for α-SMA and collagen I staining). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

9. Figure 8
Excess 25-hydroxyvitamin D3 (25(OH)D3) did not aggravate oxidative stress in macrophage-depleted kidneys. Obstructed kidney tissues of the mice in Figure 7 were analyzed. Both (a) γH2AX staining of the obstructed kidney in green (bar=20μm, N=5 in each group: *P<0.05) and (b) western blot analysis for nitrotyrosine (nitro-Tyr) (N=4 in each group: *P<0.05) revealed that excess 25(OH)D3 did not affect the levels of oxidative stress in macrophage-depleted UUO kidneys. Values of empty liposome–treated UUO kidneys and clodronate liposome–treated UUO kidneys were separately analyzed with the Student’s t-test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

10. Figure 9
Excess 25-hydroxyvitamin D3 (25(OH)D3) changed the phenotype of kidney-infiltrating macrophages. Kidney tissues of the mice in Figure 1 were analyzed. (a) F4/80-stained kidney sections are shown. The extent of the F4/80-positive area was not different between the vehicle and excess 25(OH)D3 groups (bar=100μm). Values of contralateral kidneys and unilateral ureteral obstructed (UUO) kidneys were separately analyzed with the Student’s t-test (N=16 in each group). (b) Real-time PCR analyses of whole kidney samples revealed that both macrophage M1 marker (tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS)) and M2 marker genes (transforming growth factor-β1 (TGF-β1) and mannose receptor C type 1 (MRC)) were upregulated in the obstructed kidneys of the excess 25(OH)D3 group. Values of contralateral kidneys and UUO kidneys were separately analyzed with the Student’s t-test (N=15–17 in each group: *P<0.05). (c) Sections of obstructed kidneys were immunohistochemically stained with (A) TNF-α, (B) iNOS, (C) TGF-β1, or (D) MRC in green, F4/80 in red, and nuclei in blue (bar=10μm). Marker-positive macrophages were quantitatively analyzed (N=7 in each group: *P<0.05 with the Student’s t-test). (d) Obstructed kidney sections stained with (A) TNF-α, (B) iNOS, (C) TGF-β1, or (D) MRC in green were counterstained with Lotus tetragonolobus lectin in red and nuclei in blue (bar=10μm). Proximal tubular cells were identified as L. tetragonolobus lectin-positive cells. (A) TNF-α-, (B) iNOS–, (C) TGF-β1-, or (D) MRC–positive proximal tubular cells were quantified (N=7 in each group: *P<0.05 with the Student’s t-test). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, nonsignificant.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

11. Figure 10
In vitro analyses revealed that 25-hydroxyvitamin D3 (25(OH)D3) upregulates mRNA levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in CYP27B1 knockout macrophages, but not in proximal tubular cells, in a vitamin D receptor (VDR)-dependent manner. Effects of 25(OH)D3 on cultured (a) macrophages and (b) proximal tubular cells were analyzed. Both cell types were isolated from CYP27B1 knockout mice. (a) Cultured macrophages were treated with increasing doses of 25(OH)D3. The concentrations of 25(OH)D3 were: 0mol/l (column 1), 1.0×10−8mol/l (column 2), 1.0×10− 7mol/l (column 3), or 1.0×10−6mol/l (column 4). Real-time PCR analyses demonstrated that 25(OH)D3 upregulates TNF-α and TGF-β1 in cultured CYP27B1 knockout macrophages. Inducible nitric oxide synthase (iNOS) was not detected. Macrophages stimulated with interferon-γ (100 U/ml) served as a positive control for iNOS analysis (N=6 in each group: *P<0.05, Dunnett’s test). (b) Cultured CYP27B1 knockout proximal tubular cells were treated with increasing doses of 25(OH)D3 in the absence (columns 1–4) or presence (columns 5–8) of TNF-α at a dose of 10ng/ml. The concentrations of 25(OH)D3 were: 0mol/l (columns 1 and 5), 1.0×10−8mol/l (columns 2 and 6), 1.0×10−7mol/l (columns 3 and 7), or 1.0×10−6mol/l (columns 4 and 8). TNF-α upregulated mRNA levels of TNF-α itself in cultured proximal tubular cells, whereas 25(OH)D3 had no effect on TNF-α levels. Neither TNF-α nor 25(OH)D3 affected mRNA levels of TGF-β1. TNF-α increased iNOS levels in cultured proximal tubular cells. In the presence of TNF-α, 25(OH)D3 (1.0×10−6mol/l) upregulated mRNA levels for iNOS (N=6 in each group: *P<0.05, Dunnett’s test). (c) Cultured macrophages from CYP27B1/vitamin D receptor (VDR) double knockout mice were treated with increasing doses of 25(OH)D3 in the same way as in a. Real-time PCR analyses demonstrated that CYP27B1/VDR double knockout macrophages did not respond to 25(OH)D3. CYP27B1/VDR double knockout macrophages stimulated with interferon-γ (100 U/ml) served as a positive control for iNOS analysis (N=5 in each group: *P<0.05, Dunnett’s test).
Kidney International  , DOI: ( /ki )
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