Experimental Vitamin D Deficiency in Rats: Clinical Chemistry, Histopathological, and Immunological Evaluation

Abstract

Background: Vitamin D deficiency is a significant global health concern. Experimental models are essential to elucidate the biochemical, histopathological, and immunological consequences of this deficiency. This study established a vitamin D deficiency rat model to mimic insufficient vitamin D intake and examine the resulting health impacts, particularly on liver, kidney, and immune functions.

Materials and methods: Sprague-Dawley male rats were randomly assigned to two groups. The control group received a standard rodent diet, while the experimental group was fed a modified diet with reduced vitamin D for three months. Analyses included serum vitamin D levels, clinical chemistry, renal and liver histopathology, and blood immunophenotyping and cytokine analysis for both the control (n=7) and experimental (n=7) groups.

Results: Serum vitamin D 25-OH levels were threefold lower in the experimental group (p < 0.001), indicating the induction of vitamin D deficiency. No significant differences in weight gain were observed between the groups. All clinical chemistry parameters remained within reference ranges. However, the experimental group showed significant declines in triglycerides (TG, p=0.0441), alkaline phosphatase (ALP, p=0.0021), and alanine aminotransferase (ALT, p=0.0002). Histopathology revealed normal liver and kidney architecture in the control group, while the experimental group exhibited hepatic cord deterioration, severe vacuolization in the liver, and edema and dilatation in the renal cortex tubular epithelium. Immunophenotyping analysis of lymphocyte subsets and assessment of serum cytokines did not reveal any differences between the two groups.

Conclusion: A vitamin D deficiency model without complications such as obesity, parathyroid issues, or mortality was established in rats. This method could be applied in specific disease experimental models.

Keywords: clinical chemistry; cytokines; histopathology; immunophenotyping; vitamin d deficiency.

Figure 1. Body weights (g) and body weight changes of control and vitamin D-deficient (experiment) Sprague Dawley male rats over the experimental period.

Values are expressed as mean ± standard deviation (SD). Mann-Whitney U test was performed to evaluate the statistical difference.

Figure 2. Final serum vitamin D 25-OH levels of control and vitamin D-deficient (experiment) Sprague Dawley male rats (ng/mL).

Values are expressed as mean ± SD. Student’s t-test was performed to evaluate the statistical difference. The mean vitamin D 25-OH level of the experimental group was significantly different from that of the control group (***p<0.001).

Figure 3. Microscopic images of the liver for control (A-B) and experimental (C-D) groups. Histopathological evaluation scores of control and vitamin D-deficient (experiment) Sprague Dawley male rats (E).

The central vein located in the center of the classical liver lobule is seen in both groups. While regular hepatic cords around the central vein were observed in the control group, hepatic cord deterioration, severe vacuolization, congestion, and necrosis in hepatocytes were observed in the experimental group (hematoxylin and eosin staining, bar: 50 µm, A and C x125, B and D x400). Values are expressed as mean ± SD. ***Significantly different from control at P<0.001. The Mann-Whitney U test was performed to evaluate the statistical difference.

Figure 4. Microscopic images of the kidney for control (A-B) and experimental (C-D) groups. Histopathological evaluation scores of control and vitamin D-deficient (experiment) Sprague Dawley male rats (E).

In both groups, glomeruli, proximal, and distal tubules are visible in the renal cortex region. While the glomeruli and tubules exhibited normal morphology in the control group, dilatation, edema, shedding, and congestion were observed in the tubular epithelium in the experimental group (hematoxylin & eosin staining, bar: 50 µm, A and C x125, B and D x400). Values are expressed as mean ± SD. ***Significantly different from control at P<0.001. The Mann-Whitney U test was performed to evaluate the statistical difference.