Figure 1.
CYP24A1 is increased 2–3 fold in renal proximal tubules of diabetic mice.
mRNA expression levels in renal proximal tubules of mice 20 weeks old were semi-quantified by RT-PCR/PCR, wild type compared to db/db (A) CYP24A1 (a) gel scan, (B) CYP27B1(b) gel scan, (C) VDR (c) gel scan. (*p<0,05; **p<0.01; ***p<0.001). (D) CYP24A1 immuno-staining of paraffin kidney sections (a,c) C57/BL6 wild type 24 weeks old, (b,d) C57/BL6-J db/db 24 weeks old (magnification X200 (a,b), X400 (c,d)).
Figure 2.
CYP24A1 increase in high glucose conditions is reactive oxygen species production and Protein kinase C activation-dependent.
(A) Time course of CYP24A1 protein expression in 25 mM D-Glucose , transfected or not with pCMV-CYP24A1, (B) (A) Time course of pro-caspase-3 protein expression in 25 mM D-Glucose , transfected or not with pCMV-CYP24A1, (C) CYP24A1 protein expression levels in hPRPTCs cultured for 4 days with H2O2 or with 25 mM D-Glucose plus Tiron. (D) CYP24A1 protein variation in hPRPTCs cultured for 4 days with 25 mM D-Glucose plus PD98059, SB3050208, Calcineurin C or Genistein. (*p<0.05; **p<0.01; ***p<0.001)
Figure 3.
High Glucose and CYP24A1 over-expression induce G1 arrest by themselves, but synergistically induce apoptosis in hPRPTCs.
(A) FACS cell cycle profiles of propidium iodide stained cells over a 8 day time course in high glucose, (a) hPRPTCs, (b) hPRPTCs transfected with pCMV-CYP24A1. (B) % of cells in a specific cell cycle phase over 10 days time course in high glucose, (a) SubG1, (b) G1. (*p<0.05; **p<0.01; ***p<0.001)
Figure 4.
G1 arrest-induction in high glucose of hPRPTCs is partly CYP24A1-dependent, but apoptosis induction is entirely CYP24A1-dependent.
CYP24A1 expression and Caspase-3 activation profiles in hPRPTCs cultured for 4 days under NG or HG and transfected or not with pCMV-CYP24A1 (A). hPRPTCs in NG, hPRPTCs in HG, hPRPTC in HG with 10−9 M Calcitriol, hPRPTCs transfected with pCMV-CYP24A1 in NG, hPRPTCs transfected with pCMV-CYP24A1 in HG, and hPRPTCs transfected with pCMV-CYP24A1 in HG with 10–6 M Genistein. % of cells in SubG1after 6 days incubation (B), and G1 (C). (*p<0.01; **p<0.05; ***p<0.001)
Figure 5.
VDR expression is CYP24A1-dependent and vice-versa CYP24A1 expression is VDR dependent.
(A, B and C) CYP24A1 and VDR protein expression analysis by western blotting 4 days after transfection with siRNAs scrambled (control), siCYP24A1, siVDR or pCMV-CYP24A1. (*p<0.05; **p<0.01; ***p<0.001)
Figure 6.
High Glucose G1 arrest and senescence induction is CYP24A1-dependent.
(A) Senescence analysis by SA-βGalactosidase assay after 6 days (a) hPRPTCs in NG, (b) hPRPTCs in HG, (c) hPRPTC after transfection with pCMV-CYP24A1 in NG (d) hPRPTC after transfection with pCMV-CYP24A1 in HG. (magnification X400). (B) Senescence analysis by SA-βGalactosidase assay after 6 days hPRPTCs after transfection with siRNAs (a) scrambled (control) in NG (b) scrambled (control) in HG and (c) siCYP24A1 in HG. (magnification X200). (C) % of senescent cells after 6 days incubation. (D) FACS cell cycle profiles of propidium iodide cell-stained, % of cells in a specific cell cycle phase after 4 days incubation in HG. (*p<0.05; **p<0.01; ***p<0.001)
Figure 7.
Control of CYP24A1 high fat fed animals prevents increase of pro-caspase-3 protein expression.
(A) Cyp24A1 immuno-staining, proteins (a) C57/BL6 wild type on normo-diet, (b) C57/BL6 wild type on high fat diet, (c) C57/BL6-Cyp24A1 −/− on normo-diet, (d) C57/BL6-Cyp24A1 −/− on high fat diet. (magnification X400). (B) Caspase-3 immuno-staining, proteins (a) C57/BL6 wild type on normo-diet, (b) C57/BL6 wild type on high fat diet, (c) C57/BL6-Cyp24A1 −/− on normo-diet, (d) C57/BL6-Cyp24A1 −/− on high fat diet. (magnification X400). (C) SA-βGalactosidase activity appears in tubular structures of high fat fed animals, but not in C57/BL6-Cyp24a1 −/− animals, as shown by the blue staining. (a) C57/BL6 wild type on normo-diet, (b and e) C57/BL6 wild type on high fat diet, (c) C57/BL6-Cyp24a1 −/− on normo-diet, (d) C57/BL6-Cyp24a1 −/− on high fat diet. (magnification X200 (a,b,c,d) and X400 (e)), (D) Circulating levels of 1,25(OH)2D3 in C57/BL6 wild type and C57/BL6-Cyp24a1 −/− , on either normo or high fat diet, at day of sacrifice (16 weeks) . (Each group with N = 4 animals and *p<0.05; **p<0.01; ***p<0.001)
Figure 8.
CYP24A1, pro-caspase-3, VDR and p27 rise in high fat fed animals, but not in C57/BL6-Cyp24a1 −/− animals high fat fed.
(A) Rise and disappearance of Cyp24A1, pro-caspase-3, VDR and p27, in animals after 8 weeks of high fat diet. (B) semi-quantification of CYP24A1 protein expression, (C) semi-quantification of pro-caspase-3 protein expression, (D) semi-quantification of p27 protein expression, (E) semi-quantification of VDR protein expression. (Each group with N = 6–8 animals and *p<0.05; **p<0.01; ***p<0.001)