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Body composition, Vitamin D, Calcium, Rats, What&,rsquo s Known Paricalcitol has been proposed for the treatment of secondary hyperparathyroidism in patients with renal failure and vitamin D deficiency Few studies have evaluated the effects of paricalcitol on vitamin D-deficient rats. What&,rsquo s New Paricalcitol reduced global fat mass and fat percentage in our rat model with vitamin D deficiency. Paricalcitol reduced tibia and spine fat mass in our rat model. IntroductionIn the past, calcitriol therapy was prescribed to treat vitamin D deficiency (VDD) and secondary hyperparathyroidism in patients with chronic kidney disease (CKD) by lowering the parathyroid hormone its use was, however, limited due to the development of hypercalcemia and hyperphosphatemia, which could lead to bone disease. 1, , 2, Paricalcitol, which is a selective vitamin D-receptor activator, was first proposed in 1998 for the treatment of secondary hyperparathyroidism due to advanced CKD 3, , 4, and patients on hemodialysis. 5, It can suppress the parathyroid hormone in secondary hyperparathyroidism without significant effects on calcium (Ca)-phosphorous (P) products in patients with CKD. 6, Several studies on the effects of paricalcitol on bone mass showed that this therapy could prevent renal insufficiency associated with bone mineral loss. 6, - 10, Nonetheless, only a few studies have evaluated the effects of paricalcitol on body composition and fat mass. 11, Since uremia and hemodialysis could affect body composition in patients suffering from CKD, 12, , 13, we evaluated the effects of paricalcitol on dual-energy X-ray absorptiometry (DXA)-derived body composition in a rat model of VDD. There is a paucity of information regarding the effects of paricalcitol on body composition among animals, or patients using paricalcitol. Commonly, paricalcitol is used in patients with CKD, who develop VDD and secondary hyperparathyroidism. 7, To evaluate the effects of paricalcitol on body composition, we used a rat model with VDD because, in patients with CKD, uremia, as a confounding factor, could affect the results of paricalcitol itself. Therefore, the present study aimed to investigate the effects of paricalcitol on body composition in VDD rats.Materials and MethodsThe study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences and its Vice-Chancellorship of Research (code, IR.SUMS.REC.1398.1114).ChemicalsParicalcitol (131918-61-1) was acquired from Sigma (Taufkirchen, Germany). A VDD diet was provided by Royan Institute (Isfahan, Iran). Enzyme-linked immunosorbent assay (ELISA) kits for examining 25-hydroxy vitamin D (25[OH]D) in rats (Catalog No., ZB-11556C-H9648) were purchased from ZellBio GmbH, Germany. Serum Ca, P, and alkaline phosphatase (ALP) kits were purchased from Biosystems Company, Spain. Thirty adult male Sprague-Dawley rats (age=10 weeks and weight=300&,plusmn 20 g) were included in this research, which was performed in the Shiraz Endocrinology and Metabolism Research Center, affiliated with Shiraz University of Medical Sciences, in 2020. The rats were obtained from the Animal Laboratory of the Shiraz Endocrinology and Metabolism Research Center.All the rats underwent one week&,rsquo s adaptation in the animal laboratory facilities before the study commencement. The animals were kept in standard cages (five in each cage), with 12/12 hours light/dark cycles at 23&,plusmn 2 &,deg C. 14, Simple randomization method was used to divide the rats into three groups of 10 rats each, (1) the control group, which was given a normal standard rodent chow diet and free access to tap water (2) the VDD group, which was given the standard VDD diet (TD. 87095 Brown C.C.), containing 20% lactose, 2% Ca, and 1.25% phosphate for three weeks and (3) the VDD+paricalcitol (VDD+P) group, which was given the VDD diet plus intraperitoneal injections of 32 ng of 9-nor-1,25-dihydroxy-vitamin D2 (paricalcitol Zemplar, Germany) on days 1, 3, 5, 8, 10, and 12 of the study, according to the Stavenuiter protocol. 15, At the end of the experiment, all the rats were anesthetized with ketamine 10% (100 mg/kg, Alfasan, the Netherlands) and xylazine 2% (10 m/kg, Alfasan, the Netherlands) solutions injected intraperitoneally and sacrificed by using thiopental (100 mg/kg).Biochemical StudiesSerum Ca, P, ALP, and 25(OH)D were checked for all the rats. On day 22 of the study, serum Ca (mg/dL), P (mg/dL), and ALP (Iu/L) were evaluated via the colorimetric assay with the AutoAnalyzer (Biosystems, S.A., Spain). Serum 25(OH)D (ng/mL) was measured via the ELISA method (ZellBio GmbH, Germany) with a 3.3% intra-assay coefficient of variation and a 5.1% inter-assay coefficient of variation. Body Composition MeasurementBody composition was assessed using the Hologic system DXA (Discovery W[S/N84107], USA))) with small animal software in Shiraz Endocrinology and Metabolism Research Center. First, the Rat Step Phantom (Hologic P/N 010-0758 Rev.004) scan was set up (figure 1 A,). In this method, when the system motion is complete, the Step Phantom is centered on the table along the long axis of the laser, with the laser crosshair placed at 3/4&,Prime (2 cm) of the right edge of the thinnest step (figure 1 B,). Thereafter, the Continue button is pressed to start the scan. After the Hologic system was set up, the rats were anesthetized with 50 mg/kg of body weight of ketamine hydrochloride and 5 mg/kg of body weight of xylazine hydrochloride injected intramuscularly in the gluteus region. Finally, the animals were placed in the prone position under the Hologic system and scanned for the whole body, lumbar spine, tibia, and femur.Figure 1. A, The Rat Step Phantom (Hologic P/N 010-0758 Rev.004) scan was set up. B, The Step Phantom is centered. Statistical AnalysisThe data were analyzed with SPSS Statistics, version 23, (SPSS, Chicago, IL., U.S.A.), and they were described as mean&,plusmn SD. The paired-sample t test was used to analyze values within the same group at baseline and the end of the study. The one-way analysis of variance (ANOVA) with the Tukey post hoc test was utilized to compare biochemical data between the groups. Statistical significance was defined as a P value of less than 0.05. ResultsAt the onset of the study, the three groups were not statistically significantly different in terms of body weight, as well as serum Ca, P, ALP, and 25(OH)D (P=0.786, P=0.397, P=0.643, P=0.112, and P=0.240, respectively). All the data are summarized in table 1,. Daily food intake was approximately 22&,plusmn 2.9 g, without any significant difference between the three groups (P=0.194). During the study, there were no noticeable changes in the rats&,rsquo behavior. Two rats, one in the VDD group and one in the VDD+P group, died. The mean body weight on the first day of the study was similar in all the groups (P=0.394), whereas body weight was lower in the VDD and VDD+P groups than in the control group at the end of the study (P&,lt 0.001) (figure 2 D,). Global lean mass in the VDD and VDD+P groups was lower than that in the controls (P&,lt 0.001) (figure 2 A,). Global fat mass was lower in the VDD and the VDD+P groups than in the control group (P=0.014 and P=0.003, respectively), with a more pronounced decline in the VDD+P group (figure 2 B,). Global fat percentage was reduced significantly in the VDD+P group (P=0.029) (figure 2 C,). Spine lean mass was not different between the three groups (figure 3 A,), while spine fat mass, spine fat percentage, and total spine mass were lower in the VDD+P group than in the other two groups (P=0.016, P=0.010, and P=0.001, respectively) (figure 3 B,, C,, and D,). Femur lean mass was decreased in the VDD and VDD+P groups (P=0.049 and P=0.033) (figure 4 A,). Still, there was no significant decrease in fat mass and fat percentage in the three groups (figure 4 B, and C,). Total femur mass was lower in the VDD+P group than in the control group (P=0.039) (figure 4 D,). There was no significant decrease in tibia lean mass and total tibia mass in all the groups (figure 5 A, and D,). Tibia fat mass and fat percentage were decreased in both VDD and VDD+P groups (P=0.024 and P=0.013, respectively) (figure 5 B, and C,). After the experiments, the animals were kept under the aforementioned conditions until they were sacrificed. Figure 6, illustrates three sample pictures of DXA reports in our three study groups. It appears that the rats in the VDD and VDD+P groups were thinner than the control rats.Studied ParametersTimeControlP value (first day vs. final day)Vitamin D-Deficient GroupP value (first day vs. final day)Vitamin D-Deficient Group Treated with ParicalcitolP value (first day vs. final day)P value of the Comparisons between the Three Groups Bodyweight (g)First day 300.10&,plusmn 6.23a,0.027305.40&,plusmn 1.88a,0.284301.80&,plusmn 6.91a,0.4460.786Day 22325.55&,plusmn 7.42a,300.00&,plusmn 5.36b,295.20&,plusmn 6.84b,0.00725(OH)D (nmol/mL)First day 89.25&,plusmn 3.95a,0.46079.7&,plusmn 4.25a,0.00082.37&,plusmn 3.95a,0.0000.240Day 2282.35&,plusmn 3.94a,19.03&,plusmn 1.59b,14.27&,plusmn 0.71c,&,lt 0.001Calcium (mg/dL)First day 9.76&,plusmn 0.13a,0.7629.26&,plusmn 0.38a,0.4499.01&,plusmn 0.56a,0.4080.397Day 229.80&,plusmn 0.18a,9.47&,plusmn 0.07a,9.57&,plusmn 0.21a,0.419Phosphorous (mg/dL)First day 6.10&,plusmn 0.18a,0.7795.88&,plusmn 0.29a,0.3816.34&,plusmn 0.51a,0.4990.643Day 226.37&,plusmn .065a, 6.23&,plusmn 0.19a, 6.02&,plusmn 0.20a, 0.813Alkaline phosphatase (IU/L)First day 482.80&,plusmn 31.3a,0.414388.80&,plusmn 24.68a,0.553410.55&,plusmn 40.66a,0.9040.112Day 22 457.11&,plusmn 44.6a,360.22&,plusmn 37.84a,393.30&,plusmn 21.95a,0.173a,b,cThe same letter shows no significant difference between the experimental groups (P&,gt 0.05), whereas different letters show significant differences. The one-way ANOVA with the Tukey post hoc test was used for the comparison between the groups, and the paired sample t test was applied to evaluate differences before and after the intervention in each group. VDD, Vitamin D-deficient group VDD+P, VDD+paricalcitol group |