Demonstrative Experiment on the Favorable Effects of Static Electric Field Treatment on Vitamin D3-Induced Hypercalcemia

Simple Summary Static electric field (SEF) treatment by high-voltage alternating current is a traditional complementary medicine in Japan. Although it is believed that the SEF-induced electric current serves to regulate cellular or humoral responses in patients, the mechanism for SEF treatment remains poorly understood. There have been very few experimental reports on the biological action with SEF treatment. The aim of this study was to elucidate the effects of SEF treatment on vitamin D3 (Vit D3)-induced abnormalities in mice. SEF treatment improved the abnormalities in the renal function tests and the imbalance of serum electrolytes. In addition, this treatment remarkably attenuated the Vit D3-induced tissue injuries (severe tissue calcification in the kidneys, hearts, and stomachs). It was likely that the SEF treatment had some favorable effects on the metabolism of calcium. In conclusion, this study provides important evidence that SEF treatment can reduce hypercalcemia and remove calcium deposits from the renal, cardiac, and gastric tissues. SEF treatment is useful in the regulation of disorders caused by an imbalance of serum electrolytes. This study experimentally demonstrates the favorable effects of SEF treatment on Vit D3-induced hypercalcemia. For small animals, the larger the body surface area per body weight becomes, the higher the therapeutic efficacy with SEF treatment. Abstract The purpose of this study was to elucidate the effects of static electric field (SEF) treatment on vitamin D3 (Vit D3)-induced hypercalcemia and renal calcification in mice. The mice were assigned to three groups: Vit D3-treated mice, mice treated with Vit D3 and SEF (Vit D3 + SEF), and untreated mice. After the administration of Vit D3, the Vit D3 + SEF-treated mice were exposed to SEF treatment by a high-voltage alternating current over five days. Serum biochemical examinations revealed that both the creatinine and blood urea nitrogen concentrations were significantly higher in the Vit D3-treated group. Significantly, decreased Cl concentrations, and increased Ca and inorganic phosphorus concentrations, were found in the Vit D3-treated group. In the Vit D3 + SEF-treated group, these parameters returned to the levels of the untreated group. In the Vit D3-treated group, histopathological examinations showed marked multifocal calcification in the lumens of the renal tubules and the renal parenchyma. The myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers. The stomach showed calcification of the cardiac mucosa. SEF treatment remarkably attenuated the Vit D3-induced hypervitaminotic injuries. In conclusion, this study provides important evidence that SEF treatment can reduce hypercalcemia and remove calcium deposits from the renal, cardiac, and gastric tissues. SEF treatment is useful in the regulation of disorders caused by an imbalance of serum electrolytes.


Introduction
Static electric field (SEF) treatment by high-voltage alternating current is a traditional complementary medicine used for headache, shoulder stiffness, chronic constipation, and insomnia. The widespread use of static electric field treatment has been accepted as a self-care physical intervention for pain reduction [1]. The safety of long-term continuous exposure has been confirmed by a study using mice [2]. The effect of the alternating current electrostatic high-voltage potential load was reported by a Japanese investigator in 1961 [3]. SEF exposure induced changes in the serum electrolytes (Ca, Mg, and phosphorus) of rabbits. It is believed that the SEF-induced electric current serves to regulate the cellular or humoral responses in patients [4]. There have been very few experimental reports on the biological action with SEF treatment (one study reports the inhibition of a rheumatoid arthritis model in mice) [5].
It is well known that hypervitaminosis D 3 causes an imbalance of the electrolyte metabolism characterized by hypercalcemia. The presence of microcalcification in the soft tissues is pathognomonic of the microscopic finding [6]. Although the exact mechanism of vitamin D toxicity remains unclear, recent studies report that excessive vitamin D supplementation caused critical clinical signs, such as myocardial infarction, altered mental status, or neuromyelitis spectrum disorder in elderly patients, as well as myositis, rhabdomyolysis, and severe hypercalcemia in a body builder [7,8]. In addition, the clinical presentation of an infant patient who was vulnerable to vitamin D toxicity included irritability, anorexia or poor feeding, diarrhea, the failure to gain weight, constipation, nausea, vomiting, dehydration, and physiological manifestations (hypercalcemia, hyperphosphatemia, and nephrocalcinosis) [9].
In the present study, we used this experimental model to investigate the regulation of the electrolyte imbalance after SEF treatment. The purpose of this study was to elucidate the effects of SEF treatment on vitamin D 3 (Vit D 3 )-induced hypercalcemia and renal calcification in mice. In addition, we biochemically and histopathologically investigated the sera and found that SEF treatment was useful in the field of complementary and integrative medicine.
The mice were housed in polysulfone cages (W 184 × D 332 × H 147 mm, Clea Japan Inc., Tokyo, Japan), with bedding made from pure-pulp (Care-feeaz, Hamri Co., LTD., Tsukuba, Ibaraki, Japan), in our specific-pathogen-free (SPF) barrier facilities. The animal room was maintained at constant temperature (23 ± 1 • C) and relative humidity (55 ± 10%). The room air was ventilated 10 to 15 times per hour automatically, and a 12 h/12 h light-dark cycle (lighting 06:00-18:00) was imposed. The animals received commercial radiation-sterilized diets (CE-2, Clea Japan Inc., Tokyo, Japan) and water ad libitum. These diets were previously sterilized by γ-irradiation at a dose of 30 kGy. The health statuses of the animals in our SPF facilities were checked four times a year for the pathogens listed by the Japanese Association of Laboratory Animal Facilities of National University Corporations, and the animals were free from all these pathogens.
All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Yamaguchi University and followed the Guidelines of Animal Care and Experiments of Yamaguchi University. The animal care and use program for the Advanced Research Center for Laboratory Animal Science in Yamaguchi University has been accredited by AAALAC International since 2018. The Institutional Animal Care and Use Committee of Yamaguchi University approved this specific study under Approval No. 368.
After completion of the administration of Vit D 3 , the mice were exposed to an SEF treatment of 60 Hz at 110 mT intensity by a high-voltage alternating current (1414 V to 9899 V) for 60 min/day. This treatment was performed for five consecutive days.
In this study, we used a high-voltage potential treatment device (Live-max 1/f for veterinary medicine, COCOROCA Corporation, Tokyo, Japan), which was approved by the Ministry of Health, Labor and Welfare (Approval Number 227AIBZX00017000). The cages containing the mice were placed on an electrical potential loading mat (304 × 420 mm, COCOROCA Corporation, Tokyo, Japan), and then the cages were exposed to static electric field treatment. All of the procedures were conducted on a high-voltage insulating mat (800 × 1200 mm, COCOROCA Corporation, Tokyo, Japan).

Serum Biochemistry
Under systemic anesthesia with isoflurane, the blood samples were collected from the caudal vena cava of the mice, using no anticoagulant. At 30 min after collection of the blood samples, sera were separated by centrifugation at 1500× g for 10 min for biochemical analysis.

Histopathology
Tissue specimens were obtained from the main organs (lungs, hearts, livers, and kidneys). The tissue specimens were fixed in 10% neutral buffered formalin, and 4 µm sections were stained with hematoxylin and eosin (HE), and by von Kossa stain.

Statistical Evaluation
Values were expressed as the mean ± standard deviation (SD), and statistical analysis was performed using the one-way repeated measures analysis of variance (ANOVA), and a multiple comparison test for parametric data (Tukey-Kramer method) and for nonparametric data (Steel's method). A comparison review of the aforementioned parameters was carried out among the three groups, and statistical significance was established at p < 0.05 or p < 0.01. Statistical analyses were performed with statistic software (Statcel-the Useful Add in Forms on Excel, 4th ed. OMS Publication Ltd., Tokorozawa, Saitama, Japan).

Serum Biochemical Findings
The results of the TP and ALB concentrations and the A/G ratios are shown in Figure 1. Although there were, statistically, no significant differences in the TP and ALB concentrations among the three groups. In the Vit D 3 -treated group, declined ALB concentrations Biology 2021, 10, 1116 4 of 10 brought a relative decrease in A/G ratios, leaving the TP levels within the unaffected range (p < 0.01).

Serum Biochemical Findings
The results of the TP and ALB concentrations and the A/G ratios are shown in Figure  1. Although there were, statistically, no significant differences in the TP and ALB concentrations among the three groups. In the Vit D3-treated group, declined ALB concentrations brought a relative decrease in A/G ratios, leaving the TP levels within the unaffected range (p < 0.01). The changes in the renal function tests are shown in Figure 2. The CRE concentrations in the Vit D3-treated group were significantly higher than those in the Vit D3 + SEF-treated group and the untreated group (p < 0.05). There were no differences in the CRE concentrations between the Vit D3 + SEF-treated group and the untreated group. The BUN concentrations in the Vit D3-treated group were also significantly higher than those in the Vit D3 + SEF-treated group and the untreated group (p < 0.01). The BUN concentrations did not differ between the Vit D3 + SEF-treated group and the untreated group. The changes in the renal function tests are shown in Figure 2. The CRE concentrations in the Vit D 3 -treated group were significantly higher than those in the Vit D 3 + SEF-treated group and the untreated group (p < 0.05). There were no differences in the CRE concentrations between the Vit D 3 + SEF-treated group and the untreated group. The BUN concentrations in the Vit D 3 -treated group were also significantly higher than those in the Vit D 3 + SEF-treated group and the untreated group (p < 0.01). The BUN concentrations did not differ between the Vit D 3 + SEF-treated group and the untreated group.
The changes in the electrolytes are shown in Figure 3. The Na, K, and Mg measurements exhibited no differences among the three groups. In contrast, significant decreases in the Cl levels in the Vit D 3 -treated group were statistically found, as compared with those in the Vit D 3 + SEF-treated group and the untreated group (p < 0.01). There were no differences in the Cl levels between the Vit D 3 + SEF-treated group and the untreated group. The Ca levels in the Vit D 3 -treated group indicated significantly higher increases than those in the Vit D 3 + SEF-treated group and the untreated group (p < 0.05 and p < 0.01, respectively). There were no differences in the Ca levels between the Vit D 3 + SEF-treated group and the untreated group. The IP levels in the Vit D 3 -treated group were significantly higher than those in the untreated group (p < 0.05). There were no differences in the IP levels between the Vit D 3 + SEF-treated group and the untreated group. The changes in the electrolytes are shown in Figure 3. The Na, K, and Mg measurements exhibited no differences among the three groups. In contrast, significant decreases in the Cl levels in the Vit D3-treated group were statistically found, as compared with those in the Vit D3 + SEF-treated group and the untreated group (p < 0.01). There were no differences in the Cl levels between the Vit D3 + SEF-treated group and the untreated group. The Ca levels in the Vit D3-treated group indicated significantly higher increases than those in the Vit D3 + SEF-treated group and the untreated group (p < 0.05 and p < 0.01, respectively). There were no differences in the Ca levels between the Vit D3 + SEF-treated group and the untreated group. The IP levels in the Vit D3-treated group were significantly higher than those in the untreated group (p < 0.05). There were no differences in the IP levels between the Vit D3 + SEF-treated group and the untreated group. There were statistically no significant differences in the hepatic function enzymes (AST, ALT, and ALP) among the three groups (Figure 4). Although the AST and ALT There were statistically no significant differences in the hepatic function enzymes (AST, ALT, and ALP) among the three groups ( Figure 4). Although the AST and ALT activities tended to increase in the mice treated with Vit D 3 , the Vit D3-treated group did not show significant differences as compared with the Vit D 3 + SEF-treated group and the untreated group.

Histopathological Findings
In the Vit D3-treated group, the gross findings revealed that the kidneys were bilaterally pale with irregular, granular, and white spotted surfaces. The renal specimens showed shrinkage of the vascular glomeruli, tightness of the glomerular space, and the degeneration of the epithelial lining of the renal tubules. Histopathologically, coagulation necrosis of the renal tubular epithelium was found, along with degenerative glomerulus. Marked multifocal calcification was observed in the lumens of the renal tubules and renal parenchyma ( Figure 5A, stained area ratio: 25-40% in 10 tissue sections). The tubular basement membranes contained calcified deposits, and the tubular epithelium was sloughed within the lumen. The calcification disappeared from the renal tissues in the Vit D3 + SEFtreated group ( Figure 5B).

Histopathological Findings
In the Vit D 3 -treated group, the gross findings revealed that the kidneys were bilaterally pale with irregular, granular, and white spotted surfaces. The renal specimens showed shrinkage of the vascular glomeruli, tightness of the glomerular space, and the degeneration of the epithelial lining of the renal tubules. Histopathologically, coagulation necrosis of the renal tubular epithelium was found, along with degenerative glomerulus. Marked multifocal calcification was observed in the lumens of the renal tubules and renal parenchyma ( Figure 5A, stained area ratio: 25-40% in 10 tissue sections). The tubular basement membranes contained calcified deposits, and the tubular epithelium was sloughed within the lumen. The calcification disappeared from the renal tissues in the Vit D 3 + SEF-treated group ( Figure 5B). Three mice showed some whitish spots on the cardiac surfaces. Myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers ( Figure 6, stained area ratio: 15-20% in 10 tissue sections). The foci had fibrosis with moderate lymphocytic infiltration and mild calcification of the blood vessels. The stomach showed calcification of the muscularis externa, muscularis mucosa, and mucosa, admixed with vessel calcification (Figure 7, stained area ratio: 10-15% in 10 tissue sections).   Three mice showed some whitish spots on the cardiac surfaces. Myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers ( Figure 6, stained area ratio: 15-20% in 10 tissue sections). The foci had fibrosis with moderate lymphocytic infiltration and mild calcification of the blood vessels. The stomach showed calcification of the muscularis externa, muscularis mucosa, and mucosa, admixed with vessel calcification (Figure 7, stained area ratio: 10-15% in 10 tissue sections). Three mice showed some whitish spots on the cardiac surfaces. Myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers ( Figure 6, stained area ratio: 15-20% in 10 tissue sections). The foci had fibrosis with moderate lymphocytic infiltration and mild calcification of the blood vessels. The stomach showed calcification of the muscularis externa, muscularis mucosa, and mucosa, admixed with vessel calcification (Figure 7, stained area ratio: 10-15% in 10 tissue sections).   Three mice showed some whitish spots on the cardiac surfaces. Myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers ( Figure 6, stained area ratio: 15-20% in 10 tissue sections). The foci had fibrosis with moderate lymphocytic infiltration and mild calcification of the blood vessels. The stomach showed calcification of the muscularis externa, muscularis mucosa, and mucosa, admixed with vessel calcification (Figure 7, stained area ratio: 10-15% in 10 tissue sections).   In the Vit D 3 + SEF-treated group, this treatment remarkably attenuated the Vit D 3 -induced hypervitaminotic injuries. Calcification, in particular, in the renal tubules, apparently decreased, as compared with that observed in the Vit D 3 -treated group. The glomeruli were unaffected, and the multifocal mineralized deposits resolved with SEF treatment in the the tubular lumen and basement membranes. No apparent changes were found in the hearts and livers, indicating the restoration of tissue damage by SEF treatment.
The untreated group showed no histopathological changes in any of the tissues, including the renal structure.

Discussion
SEF treatment, the placing of patients in the SEF by a high-voltage alternating current, has been utilized as one of the effective physical therapies. Few reports are available to evaluate the clinicopathological changes in the living body following SEF treatment [12][13][14][15]. Other investigators have described that extremely low-frequency electric fields had an impact on the lipid metabolism parameters (significant decreases in triglycerides and free fatty acids) in a hind-limb ischemic rat [4]. In addition, exposure to static magnetic fields had time-dependent effects on the glucose and lipid metabolisms in rats [16,17].
In the mice used in this study, the changes in the electrolytes, and the calcium deposition in the soft tissues due to Vit D 3 toxicity, were in good agreement with those of the other reported investigations [18,19]. Vit D 3 treatment caused hypercalcemia and hyperphosphatemia to a certain degree.
Previous studies have reported that extremely low-frequency electric fields had an impact on cellular Ca 2+ regulation in mouse splenocytes and human vascular endothelial cells [12,20]. Several studies have found that static magnetic fields influenced cellular calcium homeostasis [21,22]. The present study demonstrates that SEF treatment has the ability to participate in the regulation of Vit D 3 -induced hypercalcemia. Exposure to a high-voltage alternating current histologically inhibits the development of experimental collagen-induced arthritis in mice [5]. It was likely that SEF treatment had some favorable effects on the metabolism of calcium. The significant reduction in the Cl and IP concentrations was also accompanied by a decrease in the Ca concentrations following SEF treatment. Iatrogenic vitamin D toxicity in an infant was managed during hospital stay by the administration of fluids, calcitonin, and bisphosphonates. After therapy, the infant showed normal levels of Ca and PI, and there was no specific intervention to reverse the nephorocalcinosis, despite the good long-term renal function prognosis [9]. Our results suggest that SEF treatment should provide relief of tissue calcification, resulting in the normalization of electrolytes, such as Ca, PI, and Cl.
According to the experimental results of the spinal cords of rats, the Mg + concentrations were unchanged by acute exposure to static magnetic fields [23]. Our results of the serum Mg + concentrations were very different from those stated in the above-described report. This difference was probably dependent on the characteristics of the specimens (spinal fluid and sera) taken from the laboratory animals.
A recent review described that exposure to static magnetic fields altered the plasma levels of vitamins A, C, D, and E. Exposure to SEF probably had a direct effect on the metabolism of Vit D 3 , leading to the remarkable changes in the electrolytes [17].
The Vit D 3 -treated mice showed significant increases in the CRE and BUN values and the A/G ratios, suggesting decreases in the renal function. The CRE and BUN values and the A/G ratios in the Vit D 3 + SEF-treated mice were approximately equal to those obtained from the untreated normal mice. These findings reveal that SEF treatment restored the renal function to relatively normal limits.
Although there was a variation in measurements, depending on individual differences, Vit D 3 treatment caused an elevation in AST and ALT activities. SEF treatment provided the restoration of these elevated parameters to the state in the untreated mice, and the hepatic function consequently returned to normal.
Kidneys have naturally regenerative potentialities and can fully recover after acute injury under favorable conditions [24]. Our histopathological examinations demonstrated that SEF treatment brought remarkable improvements to the renal tubular epithelial cells and the glomerular membranes suffering from acute renal calcification. The calcium deposits were prominently removed from the renal tubules. These histopathological findings accurately reflected the changes in the serum chemical profiles, particularly in the electrolyte and renal functional examinations. Electric fields affect the selective transport of ions or molecules through membranes [25][26][27][28]. A previous experiment reported that the calcium influx was elevated 1.5-fold when the lymphocytes were exposed to Con-A plus 60 Hz magnetic fields [26]. In calcium transport and binding, the cell surface is implicated as a major site of interaction for extremely-low-frequency electromagnetic fields [29][30][31]. For small animals, the larger the body surface area per body weight becomes, the higher the therapeutic efficacy with SEF.
In this examination, although echocardiograms could not be recorded in the mice, the Vit D 3 -treated group seemed to be slow in their behavior, suggesting the changes in the cardiac legions and function.
In the Vit D 3 + SEF-treated mice, there were no histopathological changes in the other tissues, including the hearts and stomachs. These histopathological results were in close accord with the aforementioned serum chemical profiles, such as the enzymatic activities and the electrolyte levels. SEF treatment resulted in the elimination of Vit D 3 -induced invasiveness (calcification).

Conclusions
In conclusion, on the basis of the biochemical and histopathological investigation of sera, this study provides important evidence that SEF treatment can reduce hypercalcemia and remove calcium deposits from renal tissues, resulting in the restoration of Vit D 3induced renal injury. SEF treatment is useful in the regulation of disorders caused by an imbalance of serum electrolytes.