Losartan and vitamin D inhibit colonic tumor development in a conditional Apc-deleted mouse model of sporadic colon cancer

Abstract

Colorectal cancer (CRC) is a leading cause of cancer-deaths. The renin-angiotensin system (RAS) is up-regulated in CRC and epidemiological studies suggest RAS inhibitors reduce cancer risk. Since vitamin D receptor negatively regulates renin, we examined anti-cancer efficacy of vitamin D (VD) and losartan (L), an angiotensin receptor blocker. Control Apc^+/LoxP mice, and tumor-forming Apc^+/LoxP Cdx2P-Cre mice were randomized to unsupplemented Western diet (UN), or diets supplemented with vitamin D (VD), losartan (L) or VD+L, the latter to assess additive or synergistic effects. At 6-mo mice were killed. Plasma Ca²⁺, 25(OH)D3, 1α,25 (OH)2D3, renin and Ang II were quantified. Colonic transcripts were assessed by qPCR and proteins by immunostaining and blotting. Cancer incidence and tumor burden were significantly lower in Cre+ VD and Cre+ L, but not in Cre+ VD+L group. In Apc^+/LoxP mice, VD increased plasma 1,25(OH)2D3 and colonic VDR. In Apc^+/LoxP-Cdx2P-Cre mice, plasma renin and Ang II, and colonic tumor AT1, AT2 and Cyp27B1 were increased and VDR down-regulated. Losartan increased, whereas VD decreased plasma renin and Ang II in Cre+ mice. VD or L inhibited tumor development, while exerting differential effects on plasma VD metabolites and RAS components. We speculate that AT1 is critical for tumor development, whereas RAS suppression plays a key role in VD chemoprevention. When combined with L, VD no longer increases active VD and colonic VDR in Cre- mice nor suppresses renin and Ang II in Cre+ mice, likely contributing to lack of chemopreventive efficacy of the combination.

INTRODUCTION

Colorectal cancer (CRC) remains a major health problem in the US, with more than 50,000 deaths expected in 2018 (1). For patients with distant metastatic disease the prognosis is poor, with less than 15% surviving at 5 years. For these reasons, efforts to prevent colon cancer are a high priority. Aspirin is the only agent shown clinically to inhibit colon cancer development, but serious GI toxicities have limited its widespread use (2). Vitamin D (VD) is another promising agent for chemoprevention. VD is metabolically activated in vivo to 1α,25-dihydroxyvitamin D3, which binds to vitamin D receptor (VDR), a transcription factor that regulates diverse cellular processes. VD has epidemiological support and strong pre-clinical evidence for colon cancer chemoprevention (3–7). However, results from a recent randomized clinical trial to assess VD efficacy to prevent colonic adenoma recurrences were negative (8). Several problems were noted in the study design, especially the low dose of VD intervention (9). Subsequent genotype analysis of trial participants suggested that specific SNPs in the vitamin D receptor (VDR) gene were associated with VD efficacy to prevent advanced adenoma progression (10). This finding suggests that vitamin D might exert a chemopreventive effect in some populations.

Western diets (WDs) are associated with increased CRC incidence (11). These diets, rich in saturated fats and red meat and low in fiber, are relatively deficient in vitamin D and calcium. Numerous animal studies, using both carcinogen-induced and spontaneous/genetic models of colon cancer, have demonstrated that WDs promote colon cancer (12–18). Factors implicated in WD-related tumor promotion include changes in the colonic microbiome and colonic milieu (19); and altered host factors including up-regulated growth factors, increased pro-inflammatory enzymes and suppressed immune responses (15, 20, 21). The renin angiotensin system [RAS], which includes circulating and colonic components, is implicated in tumor development (22, 23). The RAS includes angiotensinogen, which is converted to angiotensin I (Ang I) by renin. Angiotensin converting enzyme (ACE) in turn converts Ang I to Ang II, which binds to Ang II receptors, AT1 and AT2 to modulate numerous cellular functions. We showed that VDR, when bound to 1α,25-dihydroxyvitamin D3, inhibits renin transcription and thus suppresses RAS signals (24). Renin is up-regulated in many colon tumors (25–27). Furthermore, we showed that vitamin D suppressed colonic renin levels in mice with wild type Vdr fed a WD and treated with AOM/DSS (6). Intriguingly, renin is required for diet induced obesity and the metabolic syndrome (28).

Most experimental animal studies showing protective effects of vitamin D against colon cancer have employed 1α, 25-dihydroxyvitamin D3, or an active vitamin D analogue (5, 29). Supplemented dietary vitamin D (precursor of active vitamin D) has not been widely studied for chemoprevention in sporadic models of colon cancer, and results have been inconsistent (25, 30, 31). Some of these differences may reflect differences in species (rat vs. mouse), different amounts of dietary fat (standard vs. high fat) and different vitamin D doses. Our laboratory showed that global deletion of the Vdr gene increased inflammation-associated colon cancer and other groups showed that dietary vitamin D could suppress such tumors (6, 32, 33). Colitis-associated tumors, however, constitute only a small fraction of colon cancers found in humans. Since vitamin D efficacy in a sporadic colon cancer model has only been shown in rats, our goal was to directly test the efficacy of VD in the setting of a WD-fed GEM model that targets tumors to the colon. To also directly test the role of RAS, we examined the chemopreventive effects of losartan, an angiotensin receptor (AgtR1) blocker. Since vitamin D not only suppresses RAS signals via renin inhibition, but also exerts RAS-independent chemopreventive effects (34), we also investigated whether the combination of vitamin D and L could exert additive or synergistic effects.

For a colon cancer model, we employed a genetically engineered mouse with a conditional allele for the Apc gene (LoxP-exon14-LoxP). The conditional Apc gene, when deleted in colonic epithelial cells using a constitutively active Cre-recombinase transgene under the control of colonocyte-specific Cdx2 promoter, yields colonocytes with Apc+/Δ genotype. The remaining wild type Apc allele is subsequently mutated or deleted through loss of heterozygosity. This model phenocopies sporadic human colon cancer in that 85% of human tumors possess APC mutations (35). The model allowed us to assess effects of VD and L on sporadic colonic tumorigenesis. We also explored their effects on ADAM17 and Notch signals that are implicated in tumorigenesis. We demonstrate that as single agents, vitamin D or L suppress tumor development, but surprisingly the combination is not effective.

MATERIALS AND METHODS

Materials

Defined diets, enriched in Western fat (WD, 20% fat) and relatively low in vitamin D (100 IU/kg chow) or supplemented with vitamin D (20,000 IU/kg chow) were obtained from Harlan Teklad (Madison, WI) (14). Rabbit polyclonal anti-ADAM17 antibodies (catalog ab2051) were purchased from Abcam (Cambridge, MA). Mouse monoclonal antibodies to pERK (SC-7383) and rabbit polyclonal antibodies to Jagged 1 (sc-390177) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-pAKT antibodies (#9271) and NICD antibodies (4147T) were obtained from Cell Signaling Technology (Beverly, MA). Monoclonal β-actin antibodies (#A-5441) were purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal β-catenin antibodies (#13–8400) were obtained from Invitrogen. Polyclonal anti-Hes1 antibodies (AB5702) were obtained from Millipore. Anti-AT1 (PA5–18587) and -AT2 (PA3–210) antibodies were obtained from Thermo-Fisher Scientific. Plasma renin was measured using RayBiotech Mouse Renin1 ELISA (#ELM-Renin1, RayBiotech, Norcross, GA 30092). Plasma Ang II was measured using Ang II ELISA (ADI-900–204, Enzo Life Sciences, Farmingdale, NY 11735). Plasma 25-hydroxyvitamin D3 was measured using Mouse 25-OH Vitamin D ELISA (SKU:VID21-K01, Eagle Biosciences, Inc. Nashua, NH 03063). 1α,25-dihydroxyvitamin D3 was measured using EIA (AC-62F1, Immunodiagnostic Systems, Gaithersburg, MD 20878). See supplemental Materials and Methods for additional details.

Animals

Mice were used under an approved animal protocol (ACUP 71350). Transgenic mice, expressing constitutively active Cre recombinase controlled by a modified cdx2 promoter (Cdx2-P-Cre), with expression restricted to distal small intestine and colon, were obtained from Jackson Laboratories (stock #00935). Genetically engineered mice with a conditional Apc gene (Apc^{LoxP-exon14-LoxP}) were obtained from NCI (strain 01XAA). Both genetically engineered mouse strains were on a C57Bl6/J background. Cdx2-P-Cre and Apc^LoxP/LoxP mice were intercrossed. The experimental tumor-forming groups were Apc^+/LoxP ; Cdx2P-Cre (Cre+) (to generate colonocyte Apc+/Δ) and the control groups were Apc^+/LoxP (Cre-). Cre+ and Cre- mouse colonies were expanded and control and tumor-forming mice randomized at 6 wks of age to receive unsupplemented Western diet alone (UN) as described (15), or vitamin D supplemented diet (VD, 500 μg/kg chow), losartan supplemented diet (L, 160 mg/L drinking water) or VD + L supplemented diet. The experimental protocol, including diets and supplemented agents, are summarized in Fig 1A. The WD included 100 IU vitamin D/kg chow that reflects the murine equivalent of vitamin D in the American diet (see supplemental Table 1 NWD in reference #12). The vitamin D supplemented diet contained 20,000 IU vitamin D/kg chow (500 μg/kg chow). We previously showed that this dose of vitamin D was well tolerated and did not induce hypercalcemia, but suppressed AOM/DSS-induced colonic renin in Vdr wild type mice fed a WD (6). This vitamin D dose was well tolerated in other rodent studies and shown to increase circulating 1α,25-dihydroxyvitamin D3 (32, 36).

Figure 1. Experimental protocol and effects of vitamin D and losartan on mouse weight gain and on plasma 25-hydroxyvitamin D3, renin and Ang II.

A. Experimental protocol. B. Left panel Body weight gain in control (Cre-) mice on the indicated diets; (§p<0.005 compared to Cre- UN mice). Right panel body weight gain in Cre+ tumor-bearing mice. Weights for Cre+ L or Cre+ VD+L were significantly lower than Cre- UN mice for time points > 3 mo age and for Cre+ UN or Cre+ VD tumor bearing mice for time points > 4.5 mo age (*p<0.01, ‡p<0.005; †p<0.001, compared to Cre- UN mice). C. Plasma 25-hydroxyvitamin D3 (ng/ml; *p<0.001, †p<0.005, compared to Cre- UN mice. D. Plasma renin; (pg/ml, *p<0.00005, †p<0.005, compared to Cre- UN mice, ‡p<0.01 compared to Cre+ UN mice). E. Plasma Ang II levels (pg/ml, *p<0.001, compared to treatment-matched Cre- UN mice; ‡p<0.01, †p<0.001 compared to Cre+ UN mice).

We estimated that the reduction in tumor multiplicity would be approximately 50% based on prior studies in DMH-treated rats fed high fat diet alone or with vitamin D (30) and that such a reduction would be biologically meaningful. Based on this estimate we determined that 10 animals per group would be sufficient to provide 80% power to discriminate differences in tumor multiplicity. We included additional animals to compensate for unexpected losses. There were 5 mice in each Cre- control group (Cre- UN, Cre- VD, Cre- L and Cre- VD+L group). In the Cre+ tumor-bearing groups there were 15 mice on unsupplemented group (Cre+ UN), 18 mice in the Cre+ VD group, 17 mice in the Cre+ L group, and 16 mice in the Cre+ VD + L group.

To assess for emergence of overt tumors, colonoscopy was performed in a subset of mice beginning at 5 months of age using Karl Storz-Endoskope under an approved animal protocol (ACUP71350) as described (37). After sedation, the rectal area was cleaned with saline-saturated gauze and mice secured by holding the tail with the left hand and inserting and advancing the scope with the right hand to allow for flexibility and gauge resistance better and allow for active and responsive positioning of the mouse to avoid scope trauma. The scope, lubricated with KY jelly, was inserted into the rectum and 500 μl air slowly introduced to insufflate the colon and images of tumors captured. After the procedure, the air was evacuated. Mice were killed at 6 months age and tumors harvested and blood collected for plasma. Tissue aliquots were preserved in RNA later, or flash frozen or fixed in 10% buffered formalin. Tumor stage was confirmed on H&E stained sections by a GI pathologist (JH). Subsequent tissue analyses included Western blotting, immunostaining and real time PCR.

Human tissue

Fresh human colon cancers and adjacent normal-appearing colonic mucosa were obtained under IRB 10–209-A approved by the University of Chicago. Stromal cells and colonocytes were prepared by differential centrifugation in EDTA-EGTA buffer (13). See supplemental Materials and Methods for further details.

Cell proliferation

HCT116 and HT29 human colon cancer cells and CCD-18Co colonic fibroblasts were obtained from ATCC and authenticated using short tandem repeat DNA fingerprinting by IDEXX (Elmhurst, IL). Cells were cultured in 96 well plates at 37°C in a humidified atmosphere of 5%CO₂-95% air as described (13). Pre-confluent cells were treated with Ang II (100 ng/ml) or vehicle (PBS). Where indicated, cells were pretreated with AT1 inhibitor losartan (10 μM) and/or AT2 inhibitor PD123319 (10 μM) for 2 hrs before adding Ang II. Proliferation was quantified at 48 hrs by MTT assay and expressed as fold vehicle-treated.

Real-time PCR

Real-time PCR was performed as described (14). Primer sequences are provided below:

Gene	Forward primer	Reverse primer
Cyp27B1	F: 5’-AGA ATG CAC TCC ACT CTG AGA TCA CA-3’	R: 5’-GAT TCC TAC ACG GAT GTC TCT GTC TGG-3’
Jag1	F: 5’- GAC AAC TGG TAT CGG TGC GA-3’	R: 5’-TGG AGG GCA GAT ACA CTG GT- 3’
Jag2	F: 5’-TGT AAT TTG CTC CAC GGG GG −3’	R: 5’-CAC CCC AGT TGG TCT CAC AG - 3’
Notch1	F: 5’-GCT TCA GTG GCC CTA ATT GC −3’	R: 5’- TGC ATA CCC CGC TGT TTT TG- 3’
Notch2	F: 5’-AGC AGA CTG GAT GAA CCG TG −3’	R: 5’-AAG TCA CGA TGG GAG GCA AG - 3’
Hes1	F: 5’- ATA GCT CCC GGC ATT CCA AG-3’	R: 5’- TAT TTC CCC AAC ACG CTC GG- 3’
Ren1	F: 5’-GAG GCC TTC CTT GAC CAA TC-3’	R: 5’-TGT GAA TCC CAC AAG CAA GG-3’
Agtr1a	F: 5’-CTG CTC TCC CGG ACT TAA CA-3’	R: 5’-CTG GGT TGA GTT GGT CTC AGA-3’
Agtr2	F: 5’-GGT CTG CTG GGA TTG CCT TA-3’	R: 5’- TCA GGA CTT GGT CAC GGG TA-3’
Vdr	F: GAT GCC CAC CAC AAG ACC TA-3’	R: 5’-CGG TTC CAT CAT GTC CAG TG-3’
Actb	5’-GAA GCT GTG CTA TGT TGC TCT A-3’	5’-GGA GGA AGA GGA TGC GGCA-3’

For real time PCR,we analyzed colonic mRNA from 3–4 mice per group. Reactions were run in triplicate, and Ct values averaged. Relative abundance, expressed as 2exp(-ΔΔC_T), was calculated by exponentiating differences in C_T between target gene and β-actin and normalized to fold of Cre- control (6). There were no detectable amplifications in negative control reactions (reactions omitting reverse transcriptase or reactions lacking template). Fold of control was compared among groups using one-way ANOVA with effects of genotype, treatment conditions, and tissue type (tumor or normal mucosa) (14). For gene expression changes that were significant among the groups by ANOVA, two-group comparisons were made by two-sided unpaired Student’s t-test and the Bonferroni or Tukey’s HSD correction was applied as indicated for multiple comparisons.

Immunohistochemistry

Formalin fixed paraffin-embedded blocks were stained as described (6, 13). Primary antibody concentrations included: 1:500 dilution for anti Ki67 antibodies, 1:150 dilution for anti-β-catenin, 1:200 dilution for anti-AT1 antibodies and 1:250 dilution for anti-Jagged1 antibodies. Tumors of comparable stage from each group were used for immunostaining comparisons. For negative controls, primary antibodies were omitted, or sections were incubated with isotype-matched non-immune antibodies. Control sections showed no specific staining. The Image J plug in immunoRatio was used to quantify the number of tumor nuclei with positive Ki67 or β-catenin staining as described (13). Sections from five tumors randomly selected from each Cre+ group and coded to blind investigators to treatment conditions, were used for quantification. After stained images were scanned and quantified identifiers were unmasked to compile mean ± SD for each group. See Supplemental Materials and Methods for additional details.

Western Blotting

Western blotting was carried out as previously described with primary antibodies (dilutions) to ADAM17 (1:250), AT1 (1:500), AT2 (1:500), NICD (1:200), pERK (1:1000), pAKT (1:400), Hes1 (1:500), Jagged1 (1:500), renin (1:500), VDR (1:200), β-actin (1:3000), CK20 (1:100) and VIM (1:500) as noted in Materials (13). Proteins were measured using RC-DC assay (Bio-Rad) and lysates adjusted to the same concentration. Separate aliquots were run to blot for β-actin to confirm equal loading.

Statistical Methods

Continuous data (plasma levels of calcium, 25-hydroxyvitamin D3, 1α,25 hydroxyvitamin D3, renin and Ang II, animal weights, tumor sizes and Western blotting densitometry units) were shown to be normally distributed using the Shapiro-Wilk normality test and summarized as means ± SD (38). All analyses of normally distributed variables involving more than two groups were calculated by one-way ANOVA and subsequent two group comparisons made using two-sided un-paired Student’s t-test. The Bonferroni correction, or where indicated, Tukey’s HSD correction, was applied to control for multiple comparisons for independent univariable tests (e.g., VD alone). For those significant univariables, we evaluated “multivariable models” (e.g., VD+L) and the nominal p value was used to determine statistical significance.

Cancer incidence was defined as the percentage of mice with at least one cancer and significance was calculated by Chi-square test adjusted with the Bonferroni correction. Tumor multiplicity (TM) was defined as the average number of tumors and includes both cancers and adenomas per tumor bearing mouse. Tumor multiplicities were compared among groups using the non-parametric Kruskal-Wallis (KW) test and significance determined by p-values adjusted with the Bonferroni correction.

RESULTS

Effects of vitamin D and losartan on mouse weights, and on plasma calcium, 25-hydroxyvitamin D3 and RAS components

VD did not alter mouse weight gain in the Cre- group (Fig 1B). In preliminary studies, we determined that L, added to the drinking water at a dose of 160 mg /L, was well tolerated. This was equivalent to a dose we had used in prior short term mouse studies (39). Cre- mice (Fig 1B, left panel) showed similar weight gains except for the Cre- L group that showed delayed weight gain (§p<0.005, compared to unsupplemented Cre- mice). By 6 months, however, weights of Cre- L mice were comparable to other Cre- groups (Fig 1B, left panel). Weight gain in the Cre+ groups (all Cre+ mice developed tumors, see below), lagged significantly behind treatment-matched Cre- mice (Fig 1B right panel *p<0.01, ‡p<0.005, †p<0.001, compared to Cre- UN mice). The Cre+ L group showed the slowest weight gain. For the Cre+ L group, the average weight at 6 mo was 21.5±1.6 g, which was significantly less than other Cre+ groups: 25.0±1.8 g (Cre+ UN, p<0.01); 25.3±1.1 g (Cre+ VD, p<0.005); and 24.6±2.1 g, (Cre+ VD+L, p<0.01). Interestingly, VD appeared to mitigate the effects of L on delayed weight gain in both Cre- and Cre+ mice. While plasma 25-hydroxyvitamin D3 levels were 4–5-fold higher in the vitamin D supplemented groups, plasma calcium levels were normal in all mice (Fig. 1C and Supplemental Fig S1). Losartan was shown to increase plasma levels of Ang II and renin (40). As shown in Fig 1D–E, Cre+ UN mice had significantly higher plasma renin and Ang II levels compared to Cre- UN mice, indicating that tumor development up-regulates plasma renin and Ang II. Compared to Cre+ UN mice, Cre+ VD group had significantly lower plasma renin and Ang II levels, whereas Cre+ L group had significantly higher levels. Renin and Ang II were also increased in Cre+ VD+L group compared to diet matched Cre- mice. In contrast to Cre+ VD and Cre+ L, renin and Ang II in the Cre+ VD+L were comparable to Cre+ UN mice.

Effects of vitamin D and losartan on tumor development

There were no tumors in the Cre- mice. In contrast, in the Cre+ groups tumors were detected on colonoscopy at 5 mo age (Fig 2A). By 6 mo, tumors were present in all Cre+ mice (all Cre+ mice had adenomas and/or carcinomas). As shown in Fig 2B however, cancer incidence was significantly different among the groups (p<0.015, Chi-square test). Among Cre+ mice, cancer incidence in the unsupplemented group was 66.7%, compared to 22.2% in the Cre+ VD group (p<0.01) and 17.6% in the Cre+ L group (p<0.01). In contrast, the difference in cancer incidence between the Cre+ VD+L group (31.3%) and the Cre+ UN group (66.7%) did not reach statistical significance after applying the Bonferroni correction (p=0.05).

Figure 2. Vitamin D and losartan suppress tumor progression.

A. upper panel: Colonoscopy image of tumor at 5 mo; lower panel: tumors in situ at 6 mo. B. Cancer incidence (p<0.015, Chi-square). C. Tumor multiplicity (p<0.0005, KW test). There were 5 mice in each control group (Cre- UN, Cre- VD, Cre- L and Cre- VD+L). In the Cre+ tumor-forming groups there were 15 unsupplemented (UN) mice, 18 Cre+ VD mice, 17 Cre+ L mice and 16 Cre+ VD+L mice. D. Tumor size, (p*<0.005, compared to tumors in Cre+ UN mice. E. Ki67 staining in tumors from the indicated Cre+ groups. Images are representative of tumors from each group. (20x magnification; scale = 100 microns). F. Ki67 quantitation. (*p<0.001, compared to Cre- mice matched to treatment; †p<0.01 compared to tumors from Cre+ UN mice (n= 4 mice from each group).

Tumor multiplicities (adenomas plus carcinomas) were also significantly different among the Cre+ groups as assessed by KW test (p<0.0005). As shown in Fig 2C, compared to the Cre+ UN group (tumor multiplicity 4.3±2.1), the Cre+ VD group was 1.7±0.7 (VD, p<0.0005); and Cre+ L group was 1.6±0.6 (L, p<0.0005). As in the case of cancer incidence, tumor multiplicity was numerically less in the Cre+ VD+L group (2.8±1.2) compared to Cre+ UN mice, but the difference was not significant (p=0.07). Tumor sizes were significantly lower in all Cre+ supplemented groups compared to the Cre+ UN group (Fig 2D). Ki67 was increased in tumors compared to mucosa from diet-matched Cre- mice (Fig 2E, F, *p<0.0001). Ki67 was significantly lower in tumors from the Cre+ VD group compared to the Cre+ UN group (Fig 2F, †p<0.01).

Effects of VD and losartan on colonic Cyp27B1 and VDR and on plasma 1α,25-dihydroxyvitamin D3

Conversion of circulating 25-hydroxyvitamin D3 to active 1α,25-dihydroxyvitamin D3 requires Cyp27B1 and mostly occurs in the proximal renal tubule. Cyp27B1 was shown to be increased in human colon tumors (data accessible at NCBI Geo database, accession GSE10950) (41). To assess the potential role of VDR signals, we measured plasma 1α,25-dihydroxyvitamin D3 and colonic Cyp27B1 and VDR. As shown in Fig 3A, Cyp27B1 transcripts were significantly increased in tumors from all groups except Cre VD+L. Compared to the Cre+ UN group, Cre+ L group showed reduced colonic tumor Cyp27B1. Plasma 1α,25-dihydroxyvitamin D3 was significantly increased only in the Cre- VD group (Fig 3B). As shown in Fig 3C, colonic Vdr transcripts were increased in Cre- VD group, and down-regulated in tumors in agreement with prior studies (data accessible at NCBI Geo database, accession GDS2947 and GDS389) (25, 42). Vdr transcripts were higher in Cre+ VD and Cre+ L, but not Cre+ VD+L in adjacent (normal-appearing) mucosa compared to Cre+ UN group. VDR receptors were significantly elevated in the Cre- VD group and significantly decreased in tumors in the Cre+ VD+L group (Fig 3D).

Fig 3. Effects of vitamin D and losartan on colonic Cyp27B1, plasma 1α,25 dihydroxyvitamin D3 and colonic VDR.

A. Cyp27B1 is increased in colonic tumors (*p<0.005 compared to Cre- UN group. †p<0.003 compared to Cre+ UN group. B. Plasma 1α,25 hydroxyvitamin D3 is increased in Cre- VD group. (*p<0.001 compared to Cre- UN group; †p< 0.005 compared to Cre- VD group (Tukey’s HSD correction). C. Colonic Vdr mRNA is up-regulated in Cre- VD group and down-regulated in colonic tumors. (*p<0.0001, compared to Cre- UN group; †p<0.005, compared to adjacent mucosa in Cre- UN group; D. Colonic VDR protein expression. (*p<0.001, compared to Cre- UN group; †p<0.005, compared to tumors in Cre+ VD group.

Effects of VD and losartan on renin and Ang II receptors, AT1 and AT2

Renin transcripts and protein were increased in tumors from the Cre+ UN group. Compared to the Cre+ UN tumors, renin was down-regulated in Cre+ VD and Cre+ VD+L tumors (Fig 4A–B). It was of interest to examine the effects of VD and L on colonic AT1 and AT2 that mediate Ang II signals. As shown in Fig 4C, Agtr1 transcripts were increased in tumors from Cre+ UN group and Cre+ L groups. VD significantly reduced Agtr1 up-regulation in tumors from Cre+ VD group compared to the Cre+ UN group. AT1 receptors were lower in tumors in Cre+ VD groups compared to Cre+ UN group (Fig 4D). As shown in Fig 4E Agtr2 transcripts were also increased in tumors from Cre+ UN group and Cre+ L group. As shown in Fig 4F, AT2 receptors were increased in Cre+ UN group and significantly reduced in tumors from Cre+ VD+L group compared to tumors from Cre+ UN group.

Figure 4. Renin, and angiotensin II receptors, AT1 and AT2 are up-regulated in colonic tumors.

RNA was extracted from colonic mucosa from Cre- groups and from tumors from Cre+ mice in the indicated groups. Transcripts were measured by real time PCR. (n = 8 treatment-matched controls and n=16 tumors per group). Control mucosal and tumor lysates were probed for indicated proteins by Western blotting. A. Ren1 transcripts are suppressed in Cre+ VD and Cre+ VD+L colonic tumors. (*p<0.0001, †p<0.02, compared to mucosa from diet matched Cre- mice; ‡p<0.00001, §p<0.001 compared to tumors from Cre+ UN group). B. VD suppresses up-regulated renin protein in colonic tumors. Inset renin WB of Cre- UN control mucosa and tumors from indicated Cre+ groups. Blot is representative of 4 tumors in each group (*p<0.01, compared to Cre- UN group; †p<0.01 compared to tumors from Cre+ UN group). There were no differences in colonic mucosal renin levels among dietary groups in Cre- groups. C. Agtr1 mRNA; *p<0.00005, †p<0.01, compared to mucosa from treatment matched Cre- groups; §p<0.0005 compared to tumors in Cre+ UN group. D. AT1 receptors. Inset AT1 WB of Cre- UN mucosa and tumors from indicated groups. Mouse liver is included as WB positive control for AT1. Blot is representative of 4 tumors in each group (*p<0.005, **p<0.01 compared to treatment matched Cre- mucosa, †p<0.01 compared to tumors in Cre+ UN group). E. Agtr2 mRNA (*p<0.0005, §p<0.05, compared to treatment-matched Cre- mucosa. †p<0.005, compared to tumors in Cre+ UN group. F. AT2 receptors Inset AT2 WB of Cre- UN mucosa and tumors from indicated groups. HT29 cells are included as WB positive control for AT2. Blot is representative of 4 tumors in each group (*p<0.0005, compared to mucosa from Cre- UN group; †p<0.005, compared to tumors in Cre+ UN group.

Effects of VD and losartan on ADAM17, pAKT and pERK

We showed that ADAM17-EGFR signals play important roles in genetic and carcinogen-induced colon cancer (13, 15, 43). In other tissues, Ang II was shown to transactivate EGFR by an ADAM17-dependent mechanism (44). We, therefore, examined colon tumors for ADAM17 and EGFR effectors. While there were no differences in Cre- control groups, ADAM17 was significantly increased in tumors in Cre+ UN group compared to Cre- groups (Fig 5A,B). ADAM17 up-regulation was significantly reduced in tumors in the Cre+ VD group, but not in the Cre+ VD+L group (Fig 5A,B). We also investigated AKT and ERK, down-stream effectors of ADAM17-EGFR signals. AKT and ERK are activated in Apc mutant Min adenomas (45). In agreement with this, phospho-AKT (pAKT) and phospho-ERK (pERK) were increased in tumors in the Cre+ UN group. Compared to tumors in the Cre+ UN group, pAKT was significantly reduced in tumors by VD and/or L, whereas pERK was significantly reduced only in tumors from the Cre+ losartan-treated groups.

Figure 5. Effects of VD and losartan on ADAM17 signals and expression of β-catenin and Notch components.

A. Western blots of ADAM17, pAKT and pERK. B. Densitometry ADAM17 (y1-axis), pAKT and pERK. (y2-axis) (^ap<0.01, ^bp<0.005 compared to colonic mucosa in Cre- UN fed mice. ^cp<0.005, ^dp<0.01 compared to tumors in Cre+ UN mice; n=3 tumors in each group). C. β-catenin immunostaining. Images are representative of 5 tumors in each group. Note that β-catenin is predominantly expressed in colonocytes and is both cytoplasmic and nuclear (20x magnification; scale: 100 microns). D. ImmunoRatio quantitation of β-catenin staining. (^ap<0.0005, ^bp<0.001, compared to mucosa from Cre- UN mice. ^cp<0.001, compared to tumors from Cre+ UN group); E. β-catenin Western blotting; Protein lysates from the indicated groups were probed for β-catenin by Western blotting. F. β-catenin densitometry. (^ap<0.01 compared to control mucosa from Cre- UN mice; ^bp<0.01 compared to tumors from Cre+ UN mice, n=3 per group). G. Notch signaling components. NICD, notch intracellular domain. (^ap<0.01, ^bp<0.005 compared to colonic mucosa in Cre- UN mice. ^cp<0.005, ^dp<0.01 compared to tumors from Cre+ UN mice (n=3 tumors in each group). There were no differences in expression of these proteins among the Cre- groups.

Effects of VD and losartan on β-catenin expression

Since β-catenin plays a key oncogenic role in this model and EGFR is required for β-catenin activation (46, 47), we examined the effects of VD and L on β-catenin. As shown in Fig 5C, both nuclear and cytoplasmic β-catenin stained strongly in tumors from Cre+ UN group. Quantitation of nuclear β-catenin is shown in Fig 5D. As shown in Fig 5E–F, Western blotting confirmed this up-regulation and showed that β-catenin expression was significantly reduced (3.9±0.5 vs. 2.0±0.6, p<0.01) in tumors from the Cre+ VD group, but not in the Cre+ L or Cre+ VD+L group compared to Cre+ UN tumors.

Effects of VD and L on Notch Signaling

Notch signaling is regulated by ADAM17 and β-catenin and implicated in colonic tumorigenesis (48–50). The Notch receptor, when ligand bound, is cleaved by gamma secretase, releasing the Notch intracellular domain (NICD) that migrates to the nucleus as an active transcription factor. Since ADAM17 and β-catenin were altered by VD and/or L in this model, we examined VD and L effects on transcript levels of notch components: Notch 1 and Notch 2 receptors; Notch ligands, Jagged1 and Jagged2; and Notch effector, Hes1. As shown in Supplemental Table S1, transcripts for Jagged1 and Jagged2 and Notch1 and Notch2 were significantly up-regulated in colonic tumors in the Cre+ UN group. L and/or VD inhibited up-regulations of Jagged2 and Notch1 and Notch2 transcripts. Jagged1 up-regulation was reduced by these agents, but did not reach significance. Similarly, Notch target Hes1 was greater in tumors in the Cre+ UN group (p=0.14), and decreased in the Cre+ VD, Cre+ L and Cre+ VD+L, but differences were not significant after correction for multiple comparisons.

We next examined the protein expression levels of several Notch signaling components. As shown in Supplemental Fig S2A–B, Jagged1 and NICD were increased in tumors in the Cre+ UN group. Interestingly, NICD was shown to activate renin promoter in rat (51). Jagged1 was reduced in tumors from the Cre+ L and Cre+ VD+L group. Levels of NICD and Hes1 were significantly reduced in tumors in all supplemented groups, compared to Cre+ UN group. We next examined the cellular localization of AT1 and Jagged1 by immunostaining. As shown in supplemental Fig S2C, AT1 was expressed predominantly in stromal cells of tumors. Jagged1 was expressed in a heterogenous cytoplasmic pattern in tumor cells and appeared more restricted in the Cre+ supplemented groups.

AT1 and AT2 in human colon cancers and cell lines

To assess AT1 and AT2 expression in human colon cancer, we isolated stromal cells and colonocytes from tumors and adjacent normal-appearing colonic mucosa. As shown in Fig 6A–B and Supplemental Figure S3, AT1 was expressed in stromal cells (VIM positive) and colonocytes (CK20 positive) in normal colon and increased in stroma cells from tumors. AT2 was expressed at low levels in normal colon, but was significantly up-regulated in stromal cells and colonocytes from colon cancers (see also Supplemental Fig S3).

Figure 6. AT1 and AT2 in human colon cancers and colonic cells.

Isolated stromal cells (S) and colonocytes (C) were prepared from colon cancers and adjacent normal-appearing colonic mucosa by EDTA incubation and differential centrifugation as described in Methods. Protein lysates were probed for AT1 and AT2 by Western blotting. Samples are representative of 8 controls and 8 tumors. A. Western blot of AT1 and AT2. VIM, vimentin (stromal cell marker), CK20, cytokeratin 20 (epithelial cell marker). B. AT1 and AT2 densitometry. ^ap<0.05, compared to AT1 in control stroma; ^bp<0.05, ^cp<0.005, compared to AT2 in matched control cells. C. AT1 and AT2 regulate Ang II-induced proliferation in human colonic cells. Losartan (inhibitor of AT1) and PD123319 (inhibitor of AT2) were used to assess the contributions of AT1 and AT2 to Ang II stimulated proliferation. HT29 and HCT116 colon cancer cells and CCD-18Co colonic fibroblasts were pretreated with 10 μM losartan (L) or 10 μM PD123319 (PD) or DMSO (control) for 2 hrs followed by treatment with 100 ng/ml Ang II (Ang) for 24 hrs. Cell proliferation was measured by WST-1 assay. Cells were plated in replicates of 12 and values are representative of two independent platings. Values are normalized to unstimulated cells and expressed as % control. ^ap<0.05, ^bp<0.00005, compared to control. ^cp<0.0005, ^dp<0.005, ^ep<0.00005, compared to Ang II alone (Tukey’s HSD correction). D. VDR and RAS signals modulate colonic tumor development. Colonocyte Apc deletion initiates colon tumors and up-regulates RAS components (64). Renin is required for Western diet-induced metabolic syndrome and insulin resistance and suppressed by dietary vitamin D3 (6, 28). Active vitamin D inhibits renin induction and antagonizes β-catenin signals, whereas losartan blocks AT1 signals that can transactivate EGFR and induce Notch signals that drive tumorigenesis (49, 51). Renin can also bind Atp6ap2 [(pro)renin receptor] that signals via Wnt/β-catenin pathway (68) Cholecalciferol, the precursor to active vitamin D3, can suppress smoothened (SMO) to inhibit hedgehog signaling that is also implicated in colon cancer development (69, 70).

To begin to assess the roles of AT1 and AT2 in tumor growth, we examined the effects of Ang II and losartan (AT1 inhibitor) and PD123319 (AT2 inhibitor) on proliferation of colon cancer cells and colonic fibroblasts. As shown in Fig 6C, Ang II induced nearly a 50% increase in proliferation of colon cancer cells and 100% increase in proliferation of colonic fibroblasts. Colon cancer cell proliferation in the Ang II + L or Ang II + PD treated cells was significantly lower than cells treated with Ang II alone. While separately, L or PD had less effect on Ang II-induced colonic fibroblast proliferation, the combination of L+PD significantly reduced Ang II-induced proliferation of these cells. These results suggest that both AT1 and AT2 might drive proliferation in transformed colonocytes and colonic fibroblasts.

Discussion

Vitamin D is a pro-hormone synthesized in the skin from 7-dehydrocholesterol by solar UV radiation, and metabolically transformed in the liver by 25-hydroxylation followed by 1α-hydroxylation in the kidney catalyzed by Cyp27B1 to yield active 1α,25 dihydroxyvitamin D3. VDR is ubiquitous and regulates numerous processes, including cellular proliferation, differentiation, apoptosis and immunity. Multiple mechanisms have been proposed for its putative chemopreventative actions and several VDR SNPs suggested as protective (34, 52). AA genotype in rs7968585, or CC genotype in rs731236 decreased the risk of advanced adenomas in the presence of VD supplementation (10). In contrast, other studies have not supported VDR SNPs as modulators of VD effects on CRC risk (53). Interestingly, the TT genotype in rs731236, in association with an HLA-DRB1*15 allele, was associated with 7.9-fold higher VDR expression compared to the putative protective CC genotype (54).

A Western diet was shown to induce spontaneous colon tumors in mice that were suppressed by increasing calcium and vitamin D (12). However, the low penetrance and long latency make this model challenging to test chemopreventative agents. Pre-clinical studies have demonstrated that 1α,25-dihydroxyvitamin D3, or its active analogues can inhibit tumor development in several models of colon cancer, including the azoxymethane (AOM) model, the AOM/DSS model of colitis-associated colon cancer and the Apc mutant Min mouse model (5, 29, 55). A major question remains whether the pro-hormone vitamin D, when supplemented in the diet, can inhibit tumorigenesis in a sporadic model of colon cancer. Studies using carcinogenic and genetic models have provided conflicting results that have not resolved the controversy (25, 30, 31). The study by Pence et al., in DMH-treated rats compared a standard fat and high fat diet (30). The latter was similar to our Western diet and vitamin D supplementation inhibited tumor development in the high fat diet, but interestingly not the standard fat diet (30). In contrast, in a study in Apc mutant Min mice by Giardina et al., vitamin D did not alter tumor development in the setting of standard or high fat diet, but the dose of vitamin D in that study was 4-fold lower than the current study. Moreover, colon tumors are infrequent in the Apc mutant Min mouse model (25). In a study by Irving et al., using the Apc mutant Pirc rat and the Min mouse, dietary supplemented 25-hydroxyvitamin D3 was also not chemopreventive, but that study only examined rodents fed a standard 5% fat diet (31).

To address the clinically important question of colon cancer prevention in mice on Western diet, we employed the Apc^+/LoxP; Cdx2P-Cre mouse model with a conditional Apc exon 14 deletion that closely mimics sporadic human colon cancer (35). We used a Western diet, rich in saturated and omega-6 fatty acids and relatively deficient in vitamin D and calcium that mimics a high-risk Western diet (15). This diet is similar in fat composition and concentrations of vitamin D and calcium used by Yang et al., to induce spontaneous colon tumors in mice (12). In the current study, vitamin D increased 1α,25-dihydroxyvitamin D3 and colonic VDR in the Cre- VD group and inhibited colon cancer development in Cre+ VD group. The vitamin D dose of 3000 IU/kg body wt per day compares to 20 IU/kg body weight per day in humans supplemented with 10,000 IU vitamin D weekly, a standard repletion regiment. While this dose was well tolerated, with mice showing normal plasma calciums and weight gain, a comparable dose in humans will require further study.

We previously showed that active vitamin D is a negative regulator of renin gene transcription (24). Renin drives the renin-angiotensin system (RAS), which is implicated in tumor development (22, 56). We postulated that renin suppression might mediate some chemopreventive effects of vitamin D. Our prior studies in the AOM/DSS model showed that global Vdr deletion up-regulated renin and other RAS components in the colon and increased tumor development (6). Studies by other investigators showed increased renin in human colonic adenomas (data accessible at NCBI Geo database, accession GDS2947) (25). We also showed that supplemented vitamin D suppressed colonic renin expression in Vdr wild type mice treated with AOM/DSS (6). In those studies, however, we did not directly test the anti-tumor effects of RAS blockade. Furthermore, for the AOM/DSS tumor studies we employed a Vdr null mouse to dissect the role of VDR signals. Results of the global Vdr KO and the AOM/DSS model are not readily translatable to humans.

In the current study, in addition to VD supplementation, we blocked the RAS signals with losartan, a specific inhibitor of angiotensin II type 1A receptors (AT1) to directly address the role of RAS in colon cancer. In prior studies we showed that L suppressed tumor development in the AOM model (57). We chose a L dose that was well tolerated in preliminary experiments (comparable weight gain at 2 wks). In our longer-term studies, however, Cre- L and Cre+ L groups both showed delayed weight gain. This was unexpected as we found that a higher L dose of 10 mg/ml in the drinking water was protective in a colitis model (58). While L blocked RAS as assessed by increased plasma renin and Ang II and concomitantly reduced tumor development, its effect on weight gain confounds a firm conclusion that losartan is chemopreventive in this model. In this regard, reduced weight gain by caloric restriction suppressed tumor burden in the small intestine of the Apc+/Min mouse, but interestingly not in the colon (59). Future studies with lower L doses will be needed to separate its effects on AT1 blockade from effects on growth. To assess renin-independent anti-tumor effects of vitamin D, we also included the VD+L group. Unexpectedly, the combination was not chemopreventative, though intriguingly the addition of VD partially mitigated the inhibitory effects of L on weight gain.

To explore potential RAS- and VD-dependent mechanisms that might contribute to chemoprevention, we examined several plasma and colon markers implicated in VDR and RAS signaling. With respect to VDR signaling, we showed that plasma 1α,25-dihydroxyvitamin D3 and colonic VDR were increased in the Cre- VD group, but not in the Cre- VD+L group. The change in plasma active vitamin D level was in agreement with prior studies (36). Megalin (LRP2) is a multivalent ligand required for uptake of 25 hydroxyvitamin D3 into the proximal renal tubule cell, a step required for 1α,25 dihydroxyvitamin D3 synthesis (60). Colonic megalin is decreased in colon cancer (data accessible at NCBI Geo database, accession GSE89076) (61), which could theoretically suppress colonic 1α,25-dihydroxyvitamin D3 synthesis. Interestingly, Ang II is also taken up from the renal tubule by a megalin-dependent mechanism, which is blocked by L (62). We speculate that L might inhibit 25-hydroxyvitamin D3 endocytosis by a steric mechanism involving blockade of Ang II uptake, or perhaps block AT1 signals required for uptake or hydroxylation of 25-hydroxyvitamin D3. While Cyp27B1 was increased in adenomas in agreement with studies in human colonic adenomas (data accessible at NCBI Geo database, accession GDS2947) (25), plasma 1α,25 dihydroxyvitamin D3 was not increased in Cre+ VD group, in contrast to Cre- VD group. This might reflect tumor-related effects on synthesis or catabolism of active vitamin D. In this regard, Cyp24A1, the enzyme that catabolizes active vitamin D, is frequently increased in colon cancer (61, 63).

The VDR was down-regulated in tumors from all Cre+ groups in agreement with other studies (25), suggesting that VD might be less effective if given after tumor initiation. Vdr transcripts were higher in adjacent normal-appearing mucosa in Cre+ VD and Cre+ L group, but not in Cre+ VD+L group. We speculate that failure to increase plasma 1α,25-dihydroxyvitamin D3 and colonic VDR in the Cre- VD+L group in contrast to effects of VD in the Cre- VD group, contributed to the lack of chemopreventative efficacy of VD+L in the Cre+ group.

With respect to RAS signaling, we showed that plasma RAS markers, renin and Ang II, and colonic tumor renin were increased in Cre+ UN group. Renin is also increased in human and mouse colonic adenomas (data accessible at NCBI Geo database, accession GDS2947, GSE8671, GSE5261) (25–27). Up-regulation of the RAS in colon cancer is likely linked to increased Wnt/β-catenin signaling driving expression of RAS components (64). L further increased RAS components, uncovering an inhibitory AT1-dependent feedback mechanism. Plasma renin and Ang II were decreased in the Cre+ VD group, presumably via inhibited renin gene transcription. Interestingly, in Cre+ VD+L group renin and Ang II levels were comparable to the Cre+ UN group. VD, by inhibiting renin expression and thereby reducing Ang II, and L by blocking AT1, leading to counter regulatory feed-back increases in plasma renin and Ang II, are predicted to differentially modulate AT1 and AT2 signals. Both AT1 and AT2 expression levels were increased in our model in all Cre+ groups. Increased AT1 and AT2 were previously noted in stromal cells from human colonic tumors (data accessible at NCBI Geo databases, accession GSE39397 and GSE70468) (65, 66). In the AOM model both AT1 and AT2 appear to promote tumor development (57, 67). In primary human colon cancers, we showed that AT1 and AT2 were up-regulated and in cell culture these receptors mediated Ang II-induced colonic cell proliferation. Based on the observations that plasma 1α,25-dihydroxyvitamin D3 and colonic VDR did not increase in the Cre- VD + L group, together with failure of VD+L in Cre+ mice to suppress plasma renin and Ang II suggests that in Cre + VD+L group VDR signals will be lower, whereas RAS signals will be enhanced compared to the Cre+ VD group that is expected to contribute to the lack of chemopreventive efficacy of VD+L in Cre+ mice.

ADAM17, an upstream activator of EGR, is required for robust colon tumor development in this model (13). EGFR in turn is required for β-catenin activation and both ADAM17 and β-catenin were increased in our model (47). Decreases in β-catenin expression in tumors from the Cre+ VD group, compared to the Cre+ VD+L group are consistent with increased 1α,25 dihydroxyvitamin D3 in Cre- VD group, but not the Cre- VD+L group. This is noteworthy given the role of β-catenin in regulating both oncogenesis and transcription of RAS signaling components (64). Notch signals, while variably decreased in all supplemented groups, did not provide further insights to distinguish preventative (VD or L monotherapies) versus ineffective therapies (combination VD+L therapy).

Renin can also bind to the (pro)renin receptor (Atp6ap2) that is increased in colon tumors and promotes Wnt/β-catenin signals independent of RAS (68). Given the observed differences in plasma renin between Cre+ VD group and Cr+ VD+L group, increased renin signaling through (pro)renin receptor could also contribute to lack of chemopreventive efficacy seen in the Cre+ VD+L group. Hedgehog signaling is also implicated in colon cancer development and the active VD precursor, cholecalciferol appears to directly suppress smoothened (SMO) in this pathway (69, 70). Circulating cholecalciferol is expected to be high in the Cre+ VD groups, compared to Cre+ UN and Cre+ L group, but the role of this pathway in this model will require further study.

In summary, we showed for the first time that RAS is up-regulated in the Apc^+/LoxP Cdx2P-Cre sporadic model of colon cancer. These studies have highlighted the potential importance of systemic and colonic renin angiotensin systems in tumor development. Furthermore, VD or L inhibited development of colon tumors in this model, though the effect of L on weight gain complicates the interpretation of chemopreventive efficacy. These agents exert differential effects on circulating 1α,25-dihydroxyvitamin D3 in Cre- mice, and on plasma renin and Ang II in Cre+ mice. While L inhibits increases in 1α,25 dihydroxyvitamin D3 and increases pro-inflammatory plasma renin and Ang II, systemic and/or colonic AT1 blockade appears to play an essential role in losartan’s chemopreventive effects. Since in the Cre+ VD group, Ang II and renin were significantly down-regulated, this suggests RAS blockade plays an important role in VD chemopreventive effects. A schema summarizing Western diet-promoted RAS signals promoting Apc mutant colonic tumors, and tumor suppression by VD or L is shown in Fig 6D. Additional studies will be needed to dissect the potential roles of (pro)renin receptors and hedgehog signaling in colon cancer.

Future studies will be directed to uncover the underlying tumor-associated mechanisms that up-regulate RAS signals and dissect the respective roles of AT1 and AT2 in colon cancer development. While prior studies in humans failed to show that low dose vitamin D could prevent adenoma recurrence, this study suggests that higher vitamin D doses, perhaps in individuals with vitamin D responsive VDR SNPs, might be required to protect against colon cancer (8, 10). Moreover, VD and L are widely available and well tolerated. These agents could potentially be rapidly advanced to clinical trials for colon cancer prevention, especially in high risk individuals such as patients with a history of colon cancer or advanced adenomas. Further studies in humans would be needed to assess whether combination therapy might be antagonistic as suggested by our mouse studies.

Abbreviations used in this paper

ADAM17

a disintegrin and metalloproteinase17

Ang II

angiotensin II

Agtr1

angiotensin receptor 1 (AT1)

Agtr2

angiotensin receptor 2 (AT2)

CRC

colorectal cancer

EGFR

epidermal growth factor receptor

GPCR

G-protein coupled receptor

GDS

Geo dataset

GSE

gene data series

L

Losartan

NICD

Notch intracellular domain

PD

PD123319

PBS

phosphate buffered saline

RAS

renin-angiotensin system

VDR

vitamin D receptor

VD

vitamin D

1,25(OH)2D3, 1α

25 dihydroxyvitamin D3