Vitamin D receptor agonist doxercalciferol modulates dietary fat-induced renal disease and renal lipid metabolism

Abstract

Diet-induced obesity (DIO) and insulin resistance in mice are associated with proteinuria, renal mesangial expansion, accumulation of extracellular matrix proteins, and activation of oxidative stress, proinflammatory cytokines, profibrotic growth factors, and the sterol regulatory element binding proteins, SREBP-1 and SREBP-2, that mediate increases in fatty acid and cholesterol synthesis. The purpose of the present study was to determine whether treatment of DIO mice with the vitamin D receptor (VDR) agonist doxercalciferol (1α-hydroxyvitamin D2) prevents renal disease. Our results indicate that treatment of DIO mice with the VDR agonist decreases proteinuria, podocyte injury, mesangial expansion, and extracellular matrix protein accumulation. The VDR agonist also decreases macrophage infiltration, oxidative stress, proinflammatory cytokines, and profibrotic growth factors. Furthermore, the VDR agonist also prevents the activation of the renin-angiotensin-aldosterone system including the angiotensin II type 1 receptor and the mineralocorticoid receptor. An additional novel finding of our study is that activation of VDR results in decreased accumulation of neutral lipids (triglycerides and cholesterol) and expression of adipophilin in the kidney by decreasing SREBP-1 and SREBP-2 expression and target enzymes that mediate fatty acid and cholesterol synthesis and increasing expression of the farnesoid X receptor. This study therefore demonstrates multiple novel effects of VDR activation in the kidney which prevent renal manifestations of DIO in the kidney.

Fig. 1.

Treatment of NON/LtJ (NON) mice with doxercalciferol regulates renal vitamin D receptor (VDR) and vitamin D (VitD)-metabolizing enzymes. The mRNA expression level of VDR, 1,25-dihydroxyvitamin D3 24-hydroxylase, 25-hydroxyvitamin D3–1α-hydroxylase, and TRPV5 was determined by quantitative real-time PCR. *P < 0.05 vs. NON mice on low-fat (LF) diet with vehicle (Veh). **P < 0.05 vs. NON mice on high-fat (HF) diet with Veh. #P < 0.05 vs. NON mice on LF diet with doxercalciferol (n = 6 mice per group).

Fig. 2.

A: treatment of NON mice with doxercalciferol significantly decreases albuminuria in NON mice fed a HF diet. Urinary albumin excretion is expressed as urinary albumin-to-creatinine ratio. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group). B: representative periodic acid Schiff (PAS) staining of kidney sections (×400) shows that treatment of NON mice with doxercalciferol decreases mesangial expansion and matrix accumulation in NON mice fed a HF diet.

Fig. 3.

Treatment with doxercalciferol decreases mRNA abundance of renin (A), angiotensin II type 1 receptor (AT1R; B), mineralocorticoid receptor (MR; C), and hydroxysteroid 11-β-dehydrogenase isozyme 2 (Hsd11b2; D) in NON mice. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 4.

Treatment of NON mice fed a HF diet with doxercalciferol prevents macrophage infiltration, decreases NF-κB activity, and prevents expression of proinflammatory cytokines. A: immunofluorescence staining of kidney sections for CD68. B: NF-κB DNA binding activity. C–F: analysis of mRNA expression of renal inflammatory markers by quantitative real-time PCR for Cox-2 (C), receptor for advanced glycation products (RAGE; D), toll-like receptor-4 (TLR-4; E), and monocyte chemotactic protein-1 (MCP-1; F). *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 5.

Treatment of NON mice fed a HF diet with doxercalciferol prevents renal oxidative stress. A–C: expression of oxidative stress markers: Nox-4 (A), p47-phox (B), and Nox2 (C). D: ELISA quantification of oxidized proteins in kidney homogenate. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 6.

Treatment of NON mice fed a HF diet with doxercalciferol prevents the increase in renal lipid accumulation. A: oil red O staining of kidney frozen sections. B: mRNA expression of adipophilin. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 7.

Treatment with doxercalciferol downregulates mRNA expression of sterol regulatory element binding protein 1c (SREBP-1c; A), ACC (B), FAS (C), and ACL (D), which all mediate fatty acid synthesis. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group). Treatment with doxercalciferol increases mRNA expression of PPAR-α (E), mediator of fatty acid oxidation, and decreases expression of CD36 (F), mediator of fatty acid uptake. *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group). Treatment with doxercalciferol regulates kidney cholesterol synthesis and uptake pathways by decreasing mRNA expression of SREBP-2 (G), LDLR (H), and HMG CoA R (I). *P < 0.05 vs. NON mice on LF diet with Veh. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 8.

Treatment with doxercalciferol increases farnesoid X receptor (FXR) expression in NON mice fed with HF diet. **P < 0.05 vs. NON mice on HF diet with Veh (n = 6 mice per group).

Fig. 9.

Summary of effects of VDR activation on the prevention of diet-induced obesity (DIO)-induced nephropathy in the NON mice.