Zhongyi Zhang

1,25-Dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter

We have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from –2725 to –2647 (distal fragment) and from –117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)2D3 inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)2D3-induced suppression. EMSA revealed that 1,25(OH)2D3 markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)2D3 suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

Combination therapy with AT1 blocker and vitamin D analog markedly ameliorates diabetic nephropathy: Blockade of compensatory renin increase

The renin–angiotensin system (RAS) plays a critical role in the development of diabetic nephropathy, and blockade of the RAS is currently used for treatment of diabetic nephropathy. One major problem for the current RAS inhibitors is the compensatory renin increase, which reduces the efficacy of RAS inhibition. We have shown that vitamin D exerts renoprotective actions by transcriptionally suppressing renin. Here we demonstrated that combination therapy with an AT1 receptor blocker and a vitamin D analog markedly ameliorated renal injury in the streptozotocin (STZ)-induced diabetes model due to the blockade of the compensatory renin rise by the vitamin D analog, leading to more effective RAS inhibition. STZ-treated diabetic DBA/2J mice developed progressive albuminuria and glomerulosclerosis within 13 weeks, accompanied by increased intrarenal production of angiotensin (Ang) II, fibronection, TGF-β, and MCP-1 and decreased expression of slit diaphragm proteins. Treatment of the diabetic mice with losartan or paricalcitol (19-nor-1,25-dihydroxyvitamin D2, an activated vitamin D analog) alone moderately ameliorated kidney injury; however, combined treatment with losartan and paricalcitol completely prevented albuminuria, restored glomerular filtration barrier structure, and markedly reduced glomerulosclerosis. The combined treatment suppressed the induction of fibronection, TGF-β, and MCP-1 and reversed the decline of slit diaphragm proteins nephrin, Neph-1, ZO-1, and α-actinin-4. These were accompanied by blockade of intrarenal renin and Ang II accumulation induced by hyperglycemia and losartan. These data demonstrate that inhibition of the RAS with combination of vitamin D analogs and RAS inhibitors effectively prevents renal injury in diabetic nephropathy.

Suppression of renin–angiotensin gene expression in the kidney by paricalcitol

The renal renin-angiotensin system plays a major role in determining the rate of chronic renal disease progression. Treatment with activators of the vitamin D receptor retards the progression of experimental chronic renal disease, and vitamin D is known to suppress the renin-angiotensin system in other organs. Here we determined if the beneficial effects of paricalcitol (19-nor 1,25-dihydroxyvitamin D(2)) were associated with suppression of renin-angiotensin gene expression in the kidney. Rats with the remnant kidney model of chronic renal failure (5/6 nephrectomy) were given two different doses of paricalcitol thrice weekly for 8 weeks. Paricalcitol was found to decrease angiotensinogen, renin, renin receptor, and vascular endothelial growth factor mRNA levels in the remnant kidney by 30-50 percent compared to untreated animals. Similarly, the protein expression of renin, renin receptor, the angiotensin type 1 receptor, and vascular endothelial growth factor were all significantly decreased. Glomerular and tubulointerstitial damage, hypertension, proteinuria, and the deterioration of renal function resulting from renal ablation were all similarly and significantly improved with both treatment doses. These studies suggest that the beneficial effects of vitamin D receptor activators in experimental chronic renal failure are due, at least in part, to down-regulation of the renal renin-angiotensin system.

Involvement of the vitamin D receptor in energy metabolism: regulation of uncoupling proteins

Recent studies have established that vitamin D plays multiple biological roles beyond calcium metabolism; however, whether vitamin D is involved in energy metabolism is unknown. To address this question, we characterized the metabolic phenotypes of vitamin D receptor (VDR)-null mutant mice. Under a normocalcemic condition, VDR-null mice displayed less body fat mass and lower plasma triglyceride and cholesterol levels compared with wild-type (WT) mice; when placed on a high-fat diet, VDR-null mice showed a slower growth rate and accumulated less fat mass globally than WT mice, even though their food intake and intestinal lipid transport capacity were the same as WT mice. Consistent with the lower adipose mass, plasma leptin levels were lower and white adipocytes were histologically smaller in VDR-null mice than WT mice. The rate of fatty acid beta-oxidation in the white adipose tissue was higher, and the expression of uncoupling protein (UCP) 1, UCP2 and UCP3 was markedly upregulated in VDR-null mice, suggesting a higher energy expenditure in the mutant mice. Experiments using primary brown fat culture confirmed that 1,25-dihydroxyvitamin D3 directly suppressed the expression of the UCPs. Consistently, the energy expenditure, oxygen consumption, and CO2 production in VDR-null mice were markedly higher than in WT mice. These data indicate that vitamin D is involved in energy metabolism and adipocyte biology in vivo in part through regulation of beta-oxidation and UCP expression.

Combined vitamin D analog and AT1 receptor antagonist synergistically block the development of kidney disease in a model of type 2 diabetes

We recently showed that losartan and paricalcitol are synergistic in the treatment of diabetic nephropathy in a model of type 1 diabetes. To test this strategy in a model of type 2 diabetes, we treated 2-month-old diabetic Lprdb/db mice with losartan, paricalcitol, or a combination of losartan and paricalcitol for 3 months. Vehicle-treated diabetic mice developed progressive albuminuria and glomerular abnormalities with mesangial expansion and glomerulosclerosis compared to their non-diabetic littermate control mice. Accompanying damage of the glomerular filtration barrier was a marked reduction in podocyte number as well as reduced expression of slit diaphragm proteins. Further, there was increased glomerular expression of extracellular matrix proteins, monocyte chemoattractant protein-1 and transforming growth factor-beta. Losartan or paricalcitol each alone moderately ameliorated albuminuria and glomerular damage. However, their combined use showed a dramatic therapeutic synergism, manifested by prevention of progressive albuminuria, restoration of the glomerular filtration barrier, reversal of the decline in slit diaphragm proteins, reduced synthesis of extracellular matrix proteins, and reduction of glomerulosclerosis. These effects were accompanied by blockade of the compensatory increase of renin production and angiotensin I/II accumulation in the kidney. Thus, our study further shows that vitamin D analogs can increase the efficacy of AT1 receptor blockade, leading to a more effective prevention of kidney disease in type 2 diabetes.

1,25-Dihydroxyvitamin D3 suppresses high glucose-induced angiotensinogen expression in kidney cells by blocking the NF-κB pathway

The renin-angiotensin system (RAS) is a major mediator of renal injury in diabetic nephropathy. Our previous studies demonstrated that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a renoprotective role by suppressing the RAS, with renin and angiotensinogen (AGT) as the main targets. The mechanism whereby 1,25(OH)(2)D(3) transcriptionally suppresses renin gene expression has been elucidated; however, how vitamin D regulates AGT remains unknown. Exposure of mesangial cells or podocytes to high glucose (HG; 30 mM) markedly stimulated AGT expression. In mesangial cells, the stimulation was inhibited by 1,25(OH)(2)D(3) (20 nM) or NF-kappaB inhibitor BAY 11-7082, suggesting the involvement of NF- kappaB in HG-induced AGT expression and the interaction between 1,25(OH)(2)D(3) and NF-kappaB in the regulation. Plasmid pNF-kappaB-Luc luciferase reporter assays showed that 1,25(OH)(2)D(3) blocked HG-induced NF-kappaB activity. EMSA and ChIP assays demonstrated increased p65/p50 binding to a NF-kappaB binding site at -1734 in the AGT gene promoter upon high glucose stimulation, and the binding was disrupted by 1,25(OH)(2)D(3) treatment. Overexpression of p65/p50 overcame 1,25(OH)(2)D(3) suppression, and mutation of this NF-kappaB binding site blunted 1,25(OH)(2)D(3) suppression of the promoter activity. In mice lacking the vitamin D receptor, AGT mRNA expression in the kidney was markedly increased compared with wild-type mice, and AGT induction in diabetic mice was suppressed by treatment with a vitamin D analog. These data indicate that 1,25(OH)(2)D(3) suppresses hyperglycemia-induced AGT expression by blocking NF-kappaB-mediated pathway.

Targeted vitamin D receptor expression in juxtaglomerular cells suppresses renin expression independent of parathyroid hormone and calcium

Previously, we showed that vitamin D receptor gene knockout leads to hyperreninemia independent of calcium metabolism; however, the contribution of parathyroid hormone to renin upregulation remained unclear. Here we separated the role of vitamin D and parathyroid hormone in the regulation of renin expression in vivo by generating transgenic mice that overexpressed the human vitamin D receptor in renin-producing cells using the 4.1 kb Ren-1c gene promoter. Targeting of human vitamin D receptor to the juxtaglomerular cells of the mice was confirmed by immunohistochemistry. Renal renin mRNA levels and plasma renin activity were decreased in these transgenic mice by about 50% and 30%, respectively, with no significant change in blood pressure, calcium, or parathyroid hormone levels. Moreover using vitamin D receptor knockout mice, we found that expression of the human receptor in their juxtaglomerular cells reduced renin expression in these mice without affecting calcium or parathyroid hormone status. Our study shows that suppression of renin expression by 1,25-dihydroxyvitamin D in vivo is independent of parathyroid hormone and calcium.

Long-term therapeutic effect of vitamin D analog doxercalciferol on diabetic nephropathy: strong synergism with AT1 receptor antagonist

The intrarenal renin-angiotensin system (RAS) plays a key role in the development of diabetic nephropathy. Recently, we showed that combination therapy with an AT(1) receptor blocker (ARB) and an activated vitamin D analog produced excellent synergistic effects against diabetic nephropathy, as a result of blockade of the ARB-induced compensatory renin increase. Given the diversity of vitamin D analogs, here we used a pro-drug vitamin D analog, doxercalciferol (1alpha-hydroxyvitamin D(2)), to further test the efficacy of the combination strategy in long-term treatment. Streptozotocin-induced diabetic DBA/2J mice were treated with vehicle, losartan, doxercalciferol (0.4 and 0.6 microg/kg), or losartan and doxercalciferol combinations for 20 wk. Vehicle-treated diabetic mice developed progressive albuminuria and glomerulosclerosis. Losartan alone moderately ameliorated kidney injury, with renin being drastically upregulated. A similar therapeutic effect was seen with doxercalciferol alone, which markedly suppressed renin and angiotensinogen expression. The losartan and doxercalciferol combination most effectively prevented albuminuria, restored glomerular filtration barrier structure, and dramatically reduced glomerulosclerosis in a dose-dependent manner. These effects were accompanied by blockade of intrarenal renin upregulation and ANG II accumulation. These data demonstrate an excellent therapeutic potential for doxercalciferol in diabetic renal disease and confirm the concept that blockade of the compensatory renin increase enhances the efficacy of RAS inhibition and produces synergistic therapeutic effects in combination therapy.

Vitamin D Receptor Signaling in Podocytes Protects against Diabetic Nephropathy

Vitamin D and its analogs have antiproteinuric activity and podocytes express the vitamin D receptor, but whether vitamin D signaling in podocytes accounts for this renoprotection is unknown. To investigate this question, we used the 2.5 kb podocin promoter to target Flag-tagged human vitamin D receptor (hVDR) to podocytes in DBA/2J mice. After the induction of diabetes with streptozotocin, transgenic mice had less albuminuria than wild-type controls. In transgenic mice, a low dose of the vitamin D analog doxercalciferol prevented albuminuria, markedly attenuated podocyte loss and apoptosis, and reduced glomerular fibrosis, but it had little effect on the progression of diabetic nephropathy in wild-type mice. Moreover, reconstitution of VDR-null mice with the hVDR transgene in podocytes rescued VDR-null mice from severe diabetes-related renal damage. In culture, 1,25-dihydroxyvitamin D suppressed high-glucose-induced apoptosis of podocytes by blocking p38- and ERK-mediated proapoptotic pathways. Taken together, these data provide strong evidence that vitamin D/VDR signaling in podocytes plays a critical role in the protection of the kidney from diabetic injury.

Inactivation of the vitamin D receptor in APCmin/+ mice reveals a critical role for the vitamin D receptor in intestinal tumor growth

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/β‐catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/β‐catenin pathways in intestinal tumor development. APCmin/+ mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APCmin/+ and APCmin/+VDR−/− mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APCmin/+ and APCmin/+VDR−/− mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APCmin/+VDR−/− mice in all age groups. Immunostaining showed significant increases in β‐catenin, cyclin D1, phosphorylated Stat‐3 and MSH‐2 levels and decreases in Stat‐1 in APCmin/+VDR−/− tumors compared to APCmin/+ tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APCmin/+VDR−/− mice is likely due to the loss of the growth‐inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and β‐catenin signaling pathways in intestinal tumorigenesis.