CALCITRIOL AS A NEGATIVE REGULATOR OF THE RENIN-ANGIOTENSIN SYSTEM

Yan Chun Li

Department of Medicine, University of Chicago.
Chicago, USA.

cyan@medicine.bsd.uchicago.edu

Introduction

The renin-angiotensin system (RAS) plays a crucial role in the regulation of electrolyte, extracellular volume and blood pressure homeostasis. The primary physiological role of the vitamin D endocrine system is to regulate calcium homeostasis.

As the most active metabolite of vitamin D, calcitriol [1,25-dihydroxyvitamin D₃, 1,25(OH)₂D₃] is responsible for most of the biological actions of the vitamin D endocrine system, which are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily ¹. Studies using genetically mutant mice have demonstrated that the vitamin D endocrine system has additional physiological functions beyond calcium homeostasis, which include immune responses, reproduction, mammary gland development and hair growth ^2-6. Our recent works show that calcitriol also functions as a negative endocrine regulator of the RAS and thus plays an important role in the regulation of the renocardiovascular functions ^{7, 8}.

Clinical and epidemiological evidence linking vitamin D-deficiency with hypertension

Accumulating evidence from epidemiological and clinical studies in the last decades has suggested a connection between vitamin D and blood pressure. As ultraviolet (UV) irradiation is essential for the cutaneous production of vitamin D, circulating vitamin D levels are greatly influenced by geographic locations, seasonal changes and skin pigmentations. Data obtained from the INTERSALT study centers reveal a linear correlation between the rise in blood pressure or the prevalence of hypertension and the latitudes north or south of the equator ⁹. Data from a national survey in China also demonstrate that the prevalence of hypertension and stroke incidence display a high-to-low gradient from the north to the south of the country ¹⁰. Seasonal variations in blood pressure have been reported in temperate climates, with blood pressure higher in the winter (low UV irradiation) than in the summer (high UV irradiation) ^{11, 12}. Dark skin pigmentation, which affects an efficient UV light penetration ¹³, has also been reported to be associated with higher blood pressure ^{14, 15}. Indeed, UV irradiation has been reported to lower blood pressure in patients with mild essential hypertension ¹⁶.

Numerous studies have shown that the serum level of 1,25(OH)₂D₃ is inversely associated with blood pressure in normotensive and hypertensive subjects ^17-19. More interestingly, such inverse relationship has also been reported between circulating 1,25(OH)₂D₃ levels and plasma renin activity in patients with essential hypertension ²⁰. Vitamin D supplement has been reported to be beneficial to the heart. In clinical trials, vitamin D treatment has been reported to reduce blood pressure in hypertensive or elderly patients ^{21, 22}. In several cases, 1,25(OH)₂D₃ treatment has been shown to reduce the blood pressure, plasma renin activity, Ang II level and myocardial hypertrophy ^{23, 24}.

Vitamin D as a negative endocrine regulator of the renin-angiotensin system

The clinical and epidemiological evidence suggests that vitamin D may regulate blood pressure via regulating the RAS. Based on the inverse relationship between serum 1,25(OH)₂D₃ levels and plasma renin activity found previously, we speculated that 1,25(OH)₂D₃ might be a negative endocrine regulator of renin production in vivo. If this hypothesis is correct, disruption of the vitamin D signaling pathway should lead to a deregulated stimulation of renin synthesis, whereas an increase in serum 1,25(OH)₂D₃ levels should lead to renin suppression.

To test this hypothesis, we examined VDR-null mutant mice, reasoning that renin expression should be increased in these mice due to the lack of VDR-mediated vitamin D signaling. Indeed, we found that both renin mRNA and protein levels in the kidney, as well as the plasma Ang II production, were drastically increased in VDR(-/-) mice, whereas the expression of angiotensinogen in the liver was the same as in wildtype mice. Thus, the increase in plasma Ang II appeared to be mainly due to an increase in renin activity. As a consequence of the aberrant RAS over-stimulation, VDR(-/-) mice developed hypertension, cardiac hypertrophy and an over-drinking behavior. Plasma and urinary aldosterone levels were also increased in VDR(-/-) mice. Cardiac hypertrophy, likely induced by Ang II, was reflected by a higher heart to body weight ratio and increased cardiac myocyte size in the ventricle revealed by histological analyses. Accompanying the cardiac hypertrophy, both cardiac mRNA expression and plasma concentration of atrial natriuretic peptide (ANP) were found to be increased in VDR(-/-) mice, presumably as a compensatory mechanism.

The high blood pressure, cardiac hypertrophy and increased water intake seen in VDR(-/-) mice could be corrected by treatment with captopril, an ACE inhibitor, or losartan, an Ang II AT₁ receptor antagonist, confirming that over-stimulation of the RAS is indeed responsible for the abnormalities. Interestingly, captopril or losartan treatment resulted in a drastic up-regulation of renin expression in both wild-type and VDR(-/-) mice. We also showed that renin expression in both wild-type and VDR(-/-) mice was stimulated by dehydration, and suppressed by a high sodium diet. In all cases VDR(-/-) mice still maintained a significantly higher renin expression than wild-type mice. Therefore, despite a high basal renin synthesis, the regulatory mechanisms that control renin production, including the Ang II feedback inhibition and the volume- and salt-sensing mechanisms, are still intact in VDR(-/-) mice. Thus, the sustained renin up-regulation is mediated by other mechanisms.

VDR(-/-) mice develop hypocalcemia and secondary hyperparathyroidism ⁴, which may influence renin production and secretion. To address the contribution of serum calcium or parathyroid hormone (PTH) to renin up-regulation in VDR(-/-) mice, we found that renin up-regulation was already evident in 20-day old VDR(-/-) mice, before the hypocalcemia developed, and persistent in normocalcemic adult VDR(-/-) mice after their blood calcium was normalized through dietary intervention. The plasma Ang II level remained elevated in the normocalcemic state. On the other hand, renin expression was normal in Gcm2(-/-) mice ²⁵, even though these mutant mice were as hypocalcemic as VDR(-/-) mice. In addition, renin expression was still elevated in VDR(-/-) mice whose alopecia was rescued by targeted expression of human VDR in the skin ²⁶. These date strongly suggest that regulation of renin expression by 1,25(OH)₂D₃ is independent of calcium metabolism or alopecia.

However, the contribution of PTH to the renin up-regulation in VDR(-/-) is less certain yet, because serum PTH starts to rise early in life before hypocalcemia develops and can not be completely normalized by dietary treatment, due to the lack of the VDR-mediated vitamin D inhibition of PTH biosynthesis ²⁷.

We also tested the hypothesis using wild-type mice. We showed that in wild-type mice rendered vitamin D-deficient by dietary strontium treatment, which inhibits 1,25(OH)₂D₃ biosynthesis ²⁸, renin expression in the kidney was up-regulated as in VDR(-/-) mice. On the other hand, in wild-type mice receiving several doses of 1,25(OH)₂D₃ injection renin expression was suppressed. Thus, the inhibitory role of 1,25(OH)₂D₃ in renin biosynthesis was confirmed in normal mice.

We further tested the hypothesis using As4.1 cells, a JG cell-like cell line that was derived from kidney tumors in SV40 T antigen transgenic mice and maintains a high level of renin synthesis ²⁹. We found that 1,25(OH)₂D₃ drastically reduced renin mRNA expression in As4.1 cells transiently or stably transfected with human VDR cDNA. To elucidate the molecular mechanism whereby 1,25(OH)₂D₃ suppresses renin gene expression, the stably transfected cells were used to analyze the renin gene promoter by luciferase reporter assays. When the cells were transfected with a luciferase reporter plasmid containing a 4.1kb 5'-flanking sequence of the murine Ren-1^c gene, 1,25(OH)₂D₃ treatment markedly reduced the promoter activity, confirming that 1,25(OH)₂D₃ directly and negatively regulates renin gene transcription by a VDR-mediated mechanism. Deletion analysis of the Ren-1^c gene promoter has identified two short fragments, from -2720 to -2642 and -117 to +1, that are required to mediate the repression by 1,25(OH)₂D₃.

Conclusion

The discovery that 1,25(OH)₂D₃ suppresses renin gene expression helps explain, at least in part, the relationship between sunlight exposure, vitamin D and blood pressure observed previously. Figure 1 outlines the interaction between the vitamin D endocrine system and the RAS in the regulation of calcium, electrolytes, volume and blood pressure homeostasis. It is speculated that, under normal physiological conditions, in addition to maintaining the blood calcium concentration, 1,25(OH)₂D₃ may be one of the general "gate-keepers" to maintain an appropriate level of renin in the body, and/or may act as a counter-balance regulator to antagonize other renin-stimulating factors. The counter-balance of 1,25(OH)₂D₃ may be crucial to prevent the detrimental over-activation of the RAS. Therefore, vitamin D-deficiency may increase the risk of hypertension, and vitamin D supplement may be beneficial to the cardiovascular system.

This finding also raises a possibility to develop vitamin D analogs into renin inhibitors for therapeutic purposes. With a large number of low calcemic vitamin D analogs available, and some of them already approved for clinical uses such as treatment of secondary hyperparathyroidism ^{30, 31}, such possibility is not unrealistic. As high-renin hypertension accounts for 10-20% of the patient population with essential hypertension, specific inhibitors for renin production are of significant therapeutic values. Low calcemic vitamin D analogs might open a new era for the long-sought therapeutic renin inhibitors and potentially offer a new class of anti-hypertensive drugs.

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