1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system

Abstract

Inappropriate activation of the renin-angiotensin system, which plays a central role in the regulation of blood pressure, electrolyte, and volume homeostasis, may represent a major risk factor for hypertension, heart attack, and stroke. Mounting evidence from clinical studies has demonstrated an inverse relationship between circulating vitamin D levels and the blood pressure and/or plasma renin activity, but the mechanism is not understood. We show here that renin expression and plasma angiotensin II production were increased severalfold in vitamin D receptor-null (VDR-null) mice, leading to hypertension, cardiac hypertrophy, and increased water intake. However, the salt- and volume-sensing mechanisms that control renin synthesis are still intact in the mutant mice. In wild-type mice, inhibition of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] synthesis also led to an increase in renin expression, whereas 1,25(OH)(2)D(3) injection led to renin suppression. We found that vitamin D regulation of renin expression was independent of calcium metabolism and that 1,25(OH)(2)D(3) markedly suppressed renin transcription by a VDR-mediated mechanism in cell cultures. Hence, 1,25(OH)(2)D(3) is a novel negative endocrine regulator of the renin-angiotensin system. Its apparent critical role in electrolytes, volume, and blood pressure homeostasis suggests that vitamin D analogues could help prevent or ameliorate hypertension.

Figure 1

Effect of VDR inactivation on renin expression and plasma Ang II production. (a) Renin mRNA expression in the kidney. Kidney total RNAs were isolated from wild-type (+/+) and VDR^–/– (–/–) mice and analyzed by Northern blot. The same membrane was sequentially hybridized with mouse renin and 36B4 cDNA probes. Each lane represents an individual animal. (b) Quantitative results of the Northern blot analyses shown in a. Values represent the ratio of renin mRNA to 36B4 mRNA. *P < 0.001 vs. wild-type mice. (c) Immunohistochemical staining of the kidney cortex from wild-type and VDR^–/– mice with anti-renin antiserum. Arrows indicate the afferent glomerular arterioles in the JG region. Scale bar, 25 μm. (d) Plasma Ang II concentrations in wild-type and VDR^–/– mice. *P < 0.001 vs. wild-type mice; n = 15 in each group. (e) Liver angiotensinogen mRNA expression in wild-type and VDR^–/– mice, determined by Northern blot. The membrane was sequentially hybridized with mouse angiotensinogen and 36B4 cDNA probes. Each lane represents an individual mouse.

Figure 2

Effect of VDR inactivation on blood pressure and heart weight/body weight ratio. (a) Systolic and diastolic blood pressures of wild-type (white bars) and VDR^–/– (black bars) mice. *P < 0.01 vs. corresponding wild-type mice; n = 9 for wild-type mice; n = 8 for VDR^–/– mice. (b) Ratio of heart weight to body weight of wild-type and VDR^–/– mice. *P < 0.05 vs. wild-type mice; n = 9 in each genotype. (c) Mean blood pressure (BP) of wild-type (white bars) and VDR^–/– (black bars) mice untreated or treated with captopril for 5 days. *P < 0.05 vs. corresponding untreated wild-type mice; n = 4 in each genotype in each group.

Figure 3

Effect of high sodium load and volume depletion on renin mRNA expression and plasma Ang II production in wild-type and VDR^–/– mice. (a) Northern blot analysis of renal renin mRNA from mice treated, for different numbers of days as indicated, with the normal rodent diet supplemented with 8% NaCl. Each lane represents an individual mouse. Control mice were untreated. (b) Plasma Ang II concentrations in the 8% NaCl diet–treated animals. White bars, wild-type mice; black bars, VDR^–/– mice. *P < 0.01 vs. corresponding wild-type mice at the same time point; **P < 0.05 vs. untreated control wild-type mice; n = 3 in each genotype at each time point. (c) Northern blot analysis of renal renin mRNA expression in mice dehydrated for 24 hours (24 h). Each lane represents an individual mouse. Control mice were untreated. (d) Plasma Ang II levels in untreated control and dehydrated (24 h) mice. White bars, wild-type mice; black bars, VDR^–/– mice. *P < 0.01 vs. corresponding wild-type mice; **P < 0.01 vs. untreated control wild-type mice; n = 3 in each genotype in each group.

Figure 4

Elevation of renin expression in strontium-treated wild-type mice. Two-month-old wild-type mice were fed the normal diet supplemented with 2.5% strontium chloride for 7 weeks before sacrifice. (a) Northern blot analysis of renin mRNA expression in the kidney from untreated and strontium-treated wild-type mice. Each lane represents an individual animal. (b) Quantitative results of the Northern analysis. (c) Blood ionized calcium concentration determined at the end of the treatment (n = 5 in each group). *P < 0.01 vs. untreated value (in b and c). NT, not treated; STR, strontium-treated.

Figure 5

1,25-Dihydoxyvitamin D₃ suppresses renin expression in wild-type mice. (a) Wild-type mice (3 months old) were injected intraperitoneally with two or five doses of 30 pmol of 1,25(OH)₂D₃ (VD) dissolved in propylene glycol or vehicle (V). The two doses were given in 2 consecutive days at 9 am. The five doses were given in 3 consecutive days at 9 am and 7 pm on the first 2 days and 9 am on the third day. Total renal RNA was isolated 6 hours after the last injection. Renin, calbindin-D9k (CaBP-D9k), and 36B4 mRNA levels were determined by Northern blot analysis. (b) Quantitation of renin mRNA levels. Black bars, vehicle treatment; white bars, 1,25(OH)₂D₃ treatment. *P < 0.05 vs. vehicle treatment; n ≥ 3 in each group.

Figure 6

Renin upregulation is independent of the calcium status. (a and b) Blood ionized calcium levels (a) and serum iPTH concentrations (b) in wild-type (white bars) and VDR^–/– (black bars) mice at 20 days of age, at 3 months of age, or treated with the HCa-Lac diet for 5 weeks (5 wks Ca). *P < 0.01 vs. corresponding wild-type value; n ≥ 5 in each group. Note that the white bars are barely visible in b. (c) Northern blot analysis of renal renin mRNA from 20-day-old wild-type and VDR^–/– mice. (d) Quantitative data of the Northern blot analyses from 20-day-old mice. *P < 0.001 vs. wild-type mice. (e) Northern blot analysis of renin mRNA expression in the kidney of wild-type and VDR^–/– mice treated with the HCa-Lac diet for 5 weeks. (f) Quantitative result of the Northern blot analysis in shown in e. *P < 0.001 vs. wild-type mice. (g) Plasma Ang II concentrations of the mice analyzed in e. *P < 0.01 vs. wild-type mice; n = 5 in each genotype.

Figure 7

Suppression of renin mRNA expression by 1,25(OH)₂D₃ in As4.1 cells. (a) As4.1 cells were transiently transfected with (+) or without (–) pcDNA-hVDR plasmid containing the full-length human VDR cDNA and then treated with 5 × 10^–8 M 1,25(OH)₂D₃ (+) or ethanol (–) for 24 hours. Total cellular RNA was isolated and analyzed by Northern blot with renin and 36B4 cDNA probes. (b) Quantitative results of Northern blot analyses obtained from three independent experiments. C, ethanol-treated; VD, 1,25(OH)₂D₃-treated; T, transfected with pcDNA-hVDR and treated with ethanol; T/VD, transfected with pcDNA-hVDR and treated with 1,25(OH)₂D₃. *P < 0.001 vs. C, VD, or T value. (c) Renin mRNA expression in As4.1 cells (left panel), and in As4.1 cells transfected with pcDNA-PTH/PTHrPR containing the rat PTH/PTHrP receptor cDNA (right panel) and treated with bovine PTH(1–34) as indicated. –, untreated.

Figure 8

1,25(OH)₂D₃ suppresses renin gene transcription. (a) Expression of hVDR mRNA in stable As4.1 clones. P, parental As4.1 cells; #57, As4.1 clone 57 stably transfected with pcDNA-hVDR; V, As4.1 clone stably transfected with the empty vector pcDNA3.1. (b) Renin mRNA expression in As4.1 clone V (Vector) and #57 treated with ethanol (E) or different doses of 1,25(OH)₂D₃ as indicated. (c) As4.1-hVDR cells (clone #57) were transfected with pGL3-control, pGL-4.1kb, or pGL-117bp luciferase reporter plasmid and then treated with ethanol (black bars) or 10^–8 M 1,25(OH)₂D₃ (white bars). Luciferase activity was determined 48 hours after transfection. Similar results were obtained in other stable clones (data not shown).