Vitamin D receptor agonist VS-105 improves cardiac function in the presence of enalapril in 5/6 nephrectomized rats

Abstract

Vitamin D receptor (VDR) agonists (VDRAs) are commonly used to manage hyperparathyroidism secondary to chronic kidney disease (CKD). Patients with CKD experience extremely high risks of cardiovascular morbidity and mortality. Clinical observations show that VDRA therapy may be associated with cardio-renal protective and survival benefits in patients with CKD. The 5/6 nephrectomized (NX) Sprague-Dawley rat with established uremia exhibits elevated serum parathyroid hormone (PTH), hypertension, and abnormal cardiac function. Treatment of 5/6 NX rats with VS-105, a novel VDRA (0.05 and 0.5 μg/kg po by gavage), once daily for 8 wk in the presence or absence of enalapril (30 mg/kg po via drinking water) effectively suppressed serum PTH without raising serum calcium. VS-105 alone reduced systolic blood pressure (from 174 ± 6 to 145 ± 9 mmHg, P < 0.05) as effectively as enalapril (from 174 ± 6 to 144 ± 7 mmHg, P < 0.05). VS-105 improved cardiac functional parameters such as E/A ratio, ejection fraction, and fractional shortening with or without enalapril. Enalapril or VS-105 alone significantly reduced left ventricular hypertrophy (LVH); VS-105 plus enalapril did not further reduce LVH. VS-105 significantly reduced both cardiac and renal fibrosis. The lack of hypercalcemic toxicity of VS-105 is due to its lack of effects on stimulating intestinal calcium transport and inducing the expression of intestinal calcium transporter genes such as Calb3 and TRPV6. These studies demonstrate that VS-105 is a novel VDRA that may provide cardiovascular benefits via VDR activation. Clinical studies are required to confirm the cardiovascular benefits of VS-105 in CKD.

Keywords: PTH; cardiac function; chronic kidney disease; vitamin D analog; vitamin D receptor.

Fig. 1.

Left ventricular hypertrophy in 5/6 NX rats. Sham and 5/6 nephrectomized (NX) rats were given vehicle or VS-105 at indicated doses (once daily by oral gavage) with or without enalapril (E, 30 mg/kg po via drinking water) for 8 wk as described in methods. Hearts were collected and weighed and left ventricles were then dissected and weighed. A: left ventricle weight was first normalized by body wt and then expressed as % of control (sham). B: heart weight was first normalized by body wt and then expressed as % of control (sham). Group means ± SE are presented. One-way ANOVA Dunnett's test with 95% confidence intervals of difference was performed for statistical comparisons. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. sham. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NX.

Fig. 2.

Cardiac function. Sham and 5/6 NX rats were given vehicle or VS-105 at indicated doses (once daily by oral gavage) with or without enalapril (30 mg/kg po via drinking water) for 8 wk as described in methods. Cardiac function was determined as described in methods. A: E/A ratio. B: fractional shortening. C: ejection fraction. D: septum relative wall thickness ratio [(IVSd + LVPWd)/LVIDd]. Group means ± SE are presented. A t-test was used to assess differences between two groups. #P < 0.05, ##P < 0.01 vs. sham rats. *P < 0.05, **P < 0.01 vs. 5/6 NX rats. IVSd: interventricular septum thickness in diastole; LVPWd: left ventricular posterior wall thickness in diastole; LVIDd: left ventricular internal diameter in diastole.

Fig. 3.

Systolic blood pressure. Sham and 5/6 NX rats were given vehicle or VS-105 (0.5 μg/kg once daily by oral gavage) with or without enalapril (30 mg/kg po via drinking water) for 8 wk as described in methods. Blood pressure was determined as described in methods. Group means ± SE are presented. A t-test was used to assess differences between two groups. #P < 0.05, ###P < 0.001 vs. sham rats. *P < 0.05 vs. 5/6 NX rats.

Fig. 4.

Left ventricular fibrosis. 5/6 NX rats were treated as described in Fig. 1. Left ventricle (LV) tissue sections were prepared and stained with Masson trichrome as described in methods. Randomly selected areas were examined. Photographs are representative of four fields per section per rat, four rats per treatment group. A: sham rat; B: 5/6 NX rat treated with vehicle; C: 5/6 NX rat treated with enalapril (30 mg/kg po via drinking water) alone; D: 5/6 NX rat treated with VS-105 at 0.05 μg/kg alone; E: 5/6 NX rat treated with VS-105 at 0.5 μg/kg alone; F: 5/6 NX rat treated with VS-105 at 0.5 μg/kg plus enalapril; G: quantitative determination of tissue collagen abundance. Group means ± SE are presented. One-way ANOVA Dunnett's test with 95% confidence intervals of difference was performed for statistical comparisons. #P < 0.05, ###P < 0.001 vs. sham rats. *** P < 0.001 vs. 5/6 NX rats. §P < 0.05 vs. 5/6 NX rats receiving enalapril treatment.

Fig. 5.

Remnant kidney fibrosis. 5/6 NX rats were treated as described in Fig. 1 with VS-105 at 0.05 μg/kg with or without enalapril (30 mg/kg po via drinking water) for 8 wk. The kidney tissue sections were prepared and stained with Masson trichrome as described in methods. Randomly selected areas were examined. Photographs are representative of 10 fields per section per rat, four rats per treatment group. A: sham rat; B: 5/6 NX rat treated with vehicle; C: 5/6 NX rat treated with enalapril (30 mg/kg po via drinking water) alone; D: 5/6 NX rat treated with VS-105 at 0.05 μg/kg alone; E: quantitative determination of tissue collagen abundance. Group means ± SE are presented. One-way ANOVA Dunnett's test with 95% confidence intervals of difference was performed for statistical comparisons. ###P < 0.001 vs. sham rat. ***P < 0.001 vs. 5/6 NX rat.

Fig. 6.

Urinary albumin in 5/6 NX rats. Rats were treated as described in Fig. 1 with VS-105 at 0.05 μg/kg with or without enalapril (30 mg/kg po via drinking water) for 8 wk. Urine samples were collected for albumin determination as described in methods. Means ± SE were calculated for each group. Statistical comparisons between two groups were performed by unpaired t-test with 95% confidence intervals of difference. #P < 0.05, ##P < 0.01 vs. predosing. *P < 0.05, **P < 0.01 vs. 5/6 NX rat receiving vehicle at week 8.

Fig. 7.

Effects of VS-105 or paricalcitol on expression of NPPB, CYP24A1, or vitamin D receptor (VDR). A: neonatal rat cardiomyocytes were treated with or without VS-105 or paricalcitol (concentrations as indicated) in the presence or absence of angiotensin II (1 μM) for 24 h. Cells were harvested, RNA was isolated, and NPPB mRNA (A), CYP24A1 mRNA (B), or VDR mRNA (C) levels were analyzed by real-time RT-PCR. The NPPB level was first normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and then expressed as % of control (no treatment). The CYP24A1 level was expressed as a ratio of GAPDH because there was no detectable CYP24A1 expression in the untreated samples (control) that could be used for normalization. The VDR level was also expressed as a ratio of GAPDH. Statistical analysis was performed by unpaired t-test. #P < 0.05, ###P < 0.001 compared with control (no treatment); *P < 0.05 compared with angiotensin II alone; n = 3 per condition. Results shown are representative of two independent experiments. D: 5/6 NX rats at week 6 after surgery were treated with vehicle (20% hydroxypropyl-β-cyclodextrin, 1.65 ml/kg once daily by oral gavage) or VS-105 (0.01 or 0.1 μg/kg in vehicle) for 12 days. The left ventricle from each rat was harvested, RNA was isolated, and NPPB mRNA level was analyzed by real-time RT-PCR. The NPPB level was first normalized with GAPDH and then expressed as % of sham (kidneys intact). Statistical analysis was performed by unpaired t-test. ##P < 0.05 compared with sham rats; *P < 0.05 compared with 5/6 NX rats; n = 6 per condition.

Fig. 8.

Calb3 and TRPV6 gene expression, and intestinal calcium transport. Sham and 5/6 NX rats were given vehicle or VS-105 at indicated doses (once daily by oral gavage) for 8 wk as described in methods. RNA samples were prepared from small intestines using a standard RNA isolation procedure. Real-time RT-PCR was performed according to the description in methods. Calb3 (A) or TRPV6 (B) values were first normalized with GAPDH, and then expressed as % of control (sham). C: segments of proximal small intestine were processed and calcium transport was determined in the mucosal-to-serosal direction according to the description in methods. Calcium levels were determined in both serosal and mucosal compartments and the serosal/mucosal ratio was calculated. The data were expressed as % of control (sham rats). A t-test was used to assess differences between two groups.

Fig. 9.

VS-105 vs. paricalcitol on intestinal calcium transport parameters. 5/6 NX rats at week 6 after surgery were treated with vehicle (5% ethanol + 95% propylene glycol, 0.4 ml/kg), VS-105, or paricalcitol (0.1 μg/kg in vehicle) ip 3×/wk for 12 days. Calb3 (A) or TRPV6 (B) gene expression was determined as described in Fig. 8. C: intestinal calcium transport was determined as described in methods and Fig. 8. A t-test was used to assess differences between two groups. *P < 0.05, **P < 0.01 vs. sham rats.