CYP24A1 Exacerbated Activity during Diabetes Contributes to Kidney Tubular Apoptosis via Caspase-3 Increased Expression and Activation

<div><p>Decreases in circulating 25,hydroxyl-vitamin D3 (25 OH D3) and 1,25,dihydroxyl-vitamin D3 (1,25 (OH)2 D3) have been extensively documented in patients with type 2 diabetes. Nevertheless, the molecular reasons behind this drop, and whether it is a cause or an effect of disease progression is still poorly understood. With the skin and the liver, the kidney is one of the most important sites for vitamin D metabolism. Previous studies have also shown that CYP24A1 (an enzyme implicated in vitamin D metabolism), might play an important role in furthering the progression of kidney lesions during diabetic nephropathy. In this study we show a link between CYP24A1 increase and senescence followed by apoptosis induction in the renal proximal tubules of diabetic kidneys. We show that CYP24A1 expression was increased during diabetic nephropathy progression. This increase derived from protein kinase C activation and increased H₂O₂ cellular production. CYP24A1 increase had a major impact on cellular phenotype, by pushing cells into senescence, and later into apoptosis. Our data suggest that control of CYP24A1 increase during diabetes has a beneficial effect on senescence induction and caspase-3 increased expression. We concluded that diabetes induces an increase in CYP24A1 expression, destabilizing vitamin D metabolism in the renal proximal tubules, leading to cellular instability and apoptosis, and thereby accelerating tubular injury progression during diabetic nephropathy.</p> </div

VDR expression is CYP24A1-dependent and <i>vice-versa</i> CYP24A1 expression is VDR dependent.

<p>(A, B and C) CYP24A1 and VDR protein expression analysis by western blotting 4 days after transfection with siRNAs scrambled (control), si<i>CYP24A1</i>, si<i>VDR</i> or pCMV-<i>CYP24A1</i>. (*p<0.05; **p<0.01; ***p<0.001)</p

CYP24A1 is increased 2–3 fold in renal proximal tubules of diabetic mice.

<p>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 <i>db/db</i> 24 weeks old (magnification X200 (a,b), X400 (c,d)).</p

CYP24A1, pro-caspase-3, VDR and p27 rise in high fat fed animals, but not in C57/BL6-<i>Cyp24a1 −/−</i> animals high fat fed.

<p>(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)</p

G1 arrest-induction in high glucose of hPRPTCs is partly CYP24A1-dependent, but apoptosis induction is entirely CYP24A1-dependent.

<p>CYP24A1 expression and Caspase-3 activation profiles in hPRPTCs cultured for 4 days under NG or HG and transfected or not with pCMV-<i>CYP24A1</i> (A). hPRPTCs in NG, hPRPTCs in HG, hPRPTC in HG with 10⁻⁹ M Calcitriol, hPRPTCs transfected with pCMV-<i>CYP24A1</i> in NG, hPRPTCs transfected with pCMV-<i>CYP24A1</i> 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)</p

High Glucose G1 arrest and senescence induction is CYP24A1-dependent.

<p>(A) Senescence analysis by SA-βGalactosidase assay after 6 days (a) hPRPTCs in NG, (b) hPRPTCs in HG, (c) hPRPTC after transfection with pCMV-<i>CYP24A1</i> in NG (d) hPRPTC after transfection with pCMV-<i>CYP24A1</i> 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) si<i>CYP24A1</i> 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)</p

Control of CYP24A1 high fat fed animals prevents increase of pro-caspase-3 protein expression.

<p>(A) Cyp24A1 immuno-staining, proteins (a) C57/BL6 wild type on normo-diet, (b) C57/BL6 wild type on high fat diet, (<b>c</b>) C57/BL6-<i>Cyp24A1 −/−</i> on normo-diet, (d) C57/BL6-<i>Cyp24A1 −/−</i> 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, (<b>c</b>) C57/BL6-<i>Cyp24A1 −/−</i> on normo-diet, (d) C57/BL6-<i>Cyp24A1 −/−</i> on high fat diet. (magnification X400). (C) SA-βGalactosidase activity appears in tubular structures of high fat fed animals, but not in C57/BL6-<i>Cyp24a1 −/−</i> 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-<i>Cyp24a1 −/−</i> on normo-diet, (d) C57/BL6-<i>Cyp24a1 −/−</i> 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-<i>Cyp24a1 −/−</i> , 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)</p