Abstract
This report describes cardiovascular and renal soft tissue mineralization and renal intratubular crystals in 13 out of 16 guinea pigs that were given very hard drinking water for 9 months. These animals, aged 14 to 20 months, were experimentally naïve. No clinical symptoms were observed, but 1 guinea pig was found dead in its cage. Necropsy did not reveal any gross findings; however, histologic examination revealed mineralization and crystal formations. Despite no known changes in the feed sourcing or formulation, the possibility that the incident was feed-related was considered. The most recent analysis of the feed obtained from the manufacturer during this period, which was conducted by an accredited laboratory authorized by the Ministry of Agriculture and Forestry, was appropriate. No similar lesions were reported at other centers using the same feed; however, drinking water analysis for total dissolved solids revealed extremely hard water, with elevated levels of calcium and calcium carbonate and low magnesium levels, due to a malfunctioning water treatment system. After installing a new system to balance calcium and magnesium, no new cases appeared over the next 2 years. It was determined that the mineralization and crystal formations were most likely caused by water hardness. This study demonstrates that mineralization typically attributed to feed in guinea pigs can also result from high calcium content in drinking water, highlighting the importance of water analysis in such cases.
Metastatic calcification is the abnormal deposition of calcium salts in normal tissues, often due to elevated blood calcium levels. It is commonly caused by conditions such as hyperparathyroidism, excessive vitamin D intake, chronic kidney disease, imbalances in calcium and phosphate levels, and certain types of malignancies. This condition typically affects soft tissues, such as the lungs, pleura, endocardium, blood vessels, kidneys, and stomach.6,8,11,15,16 This syndrome, known as metastatic mineralization in guinea pigs, is most common in males over 1 year of age.4,6,13,15,16,17,18 Experimental and spontaneous studies have linked these conditions to diets low in magnesium and imbalanced calcium-phosphate ratios.4,13,17,18 In addition, guinea pigs fed diets with excessive amounts of vitamin D can develop metastatic mineralization due to hypervitaminosis D. 6
Guinea pigs have rapid calcium metabolism, and the balance between calcium and phosphorus plays a crucial role in the development of metastatic mineralization, which often remains subclinical.4,13,15,16,17 These animals also have a higher rate of calcium absorption compared with other species, with their kidneys excreting a significant amount of excess calcium in urine.6,13,18 Crystalluria and nephrolithiasis are common health issues in various species, including guinea pigs, with the incidence increasing with age and females being more frequently affected. Among guinea pigs, calcium oxalate stones are the most common, although calcium carbonate (CaCO3), calcium phosphate, and struvite stones can also form.2,5,7,13 The consumption of CaCO3 in the diet enhances intestinal calcium absorption, increasing the risk of hypercalcemia and urolithiasis.1,13
A male, white Hartley guinea pig was found dead in its cage without any prior clinical symptoms, and a necropsy was performed to determine the cause of death. No gross abnormalities were observed in the visceral organs; however, histologic examination revealed mineralization in the heart, kidneys, and large and medium blood vessels. For educational purposes, necropsies were subsequently conducted on 15 clinically healthy guinea pigs including 10 males and 5 females, between 14 and 20 months of age, which had not been part of any experiments. Similar to the initial case, no gross findings were observed in any organs.
These necropsies were conducted following a long-standing protocol between Burdur Mehmet Akif Ersoy University’s Experimental Animal Production and Experimental Research Center and the Faculty of Veterinary Medicine. According to this protocol, animals that are dead, ill, tumor-bearing, or exhibiting clinical symptoms and deemed unsuitable for experimental studies are used for necropsy practice by veterinary students and are histologically examined. This protocol ensures continuous health monitoring of the animals and provides educational material for necropsy training. The use of these animals is ethically approved by the local animal experiments ethics committee, and they are raised in accordance with national and international regulations, with the center inspected annually. The guinea pigs in this study were subjected to necropsies under this protocol.
Following the gross postmortem examination, tissue samples from all organs, including the liver, lung, spleen, brain, intestine, kidneys, uterus or testis, heart, and vessels, were collected and fixed in 10% buffered formalin for 24 hours. The tissue samples were then routinely processed, embedded in paraffin, and 5 µm-thick serial sections were prepared. At least 4 sections from each block were examined. The sections were stained using hematoxylin and eosin and von Kossa’s techniques, and microscopic examinations were conducted with both light microscopy and polarized filters. Histologic examinations showed medial and adventitial mineralization of large and medium arteries with less extensive involvement of veins in the heart and kidneys, as well as crystals in the kidney tubules. Microscopic mineralization was detected in 13 of the 16 guinea pigs, including the one that had previously died. The 3 guinea pigs in which no mineralization was observed were the youngest in the group, and all 3 were 14 weeks old. The most severe mineralization was found in the guinea pig that was discovered dead in its cage.
Microscopic examination identified metastatic mineralization in 13 out of 16 guinea pigs. The analysis revealed extracellular amorphous or granular basophilic deposits in renal tubules, the myocardium, and the medial and adventitial layers of large (including the aorta and pulmonary artery) and medium-sized arteries, with less extensive involvement of veins of the heart and kidneys. Mineralization was primarily located in the interstitial tissue and, to a lesser extent, in the lumen of renal tubules. In the kidneys, mineralized areas were associated with interstitial fibrosis, inflammatory cell infiltration (mainly macrophages), tubulointerstitial damage, and tubular atrophy, while the glomeruli remained largely unaffected (Fig. 1a). Multifocal, chronic renal cortical infarcts, with lobular infarcts of mild to moderate severity, were also observed. Von Kossa staining revealed black calcium deposits in renal tubules (Fig. 1b). In addition, polarized light microscopy of kidney tissue stained with hematoxylin and eosin demonstrated bright, transparent, and colorless intratubular crystal deposits that were birefringent (Figs. 1c, d) along with tubular injury and mild interstitial inflammatory infiltrate.
Histologic features of metastatic calcification in guinea pigs. (a) Kidney, basophilic extracellular calcium deposits in renal tubules (arrows). Hematoxylin and eosin (HE). (b) Kidney, black calcium deposits near the corticomedullary junction, corresponding to the region shown in (a). von Kossa method. (c, d) Kidney, intratubular crystals associated with tubular epithelial attenuation and mild interstitial inflammatory cell infiltrates are identified under non-polarized light (c), and these crystals are birefringent under polarized light (d). HE. (e) Heart, myocardial calcification (arrow). HE. (f) Heart, the same region as in (e) shows black calcium deposits in the myocardium. von Kossa method. (g) Significant calcification in the tunica media (arrow) of aorta accompanied by small hemorrhagic areas in the vasa vasorum. HE. (h) In the same area as (g), black calcium deposits are identified in the media layer. von Kossa method.
Histologic analysis of cardiac tissues revealed multifocal myocardial mineralization in cardiomyocytes characterized by prominent extracellular basophilic granular deposits within the ventricular wall. Fibrosis associated with mineralization was evident in the myocardium, along with cardiomyocyte degeneration and necrosis (Fig. 1e). Von Kossa staining confirmed the presence of calcification in the myocardium (Fig. 1f).
Mineralization was also observed in the medial and adventitial layers of large and medium arteries, with less extensive involvement of veins, and was present in both the heart and kidneys. Other vascular changes included subendothelial interstitial edema and hemorrhage in the vasa vasorum of the aorta (Fig. 1g). Medial vascular calcification was confirmed with von Kossa methods (Fig. 1h). The distribution and severity of the histopathologic lesions are detailed in Table 1.
Distribution and severity of lesions in guinea pigs.
Histologic lesion assessment.
_, none; +, slight; ++, moderate; +++, severe.
After the histologic diagnosis of mineralization, it was hypothesized that the issue might be related to the feed and supplementary food. The laboratory animal centers at nearby universities (Suleyman Demirel and Akdeniz Universities) using the same feed were consulted, and none reported similar problems. According to Ministry of Agriculture and Forestry regulations, animal feed producers are required to have their feed analyzed every 6 months by an accredited laboratory authorized by the Ministry of Agriculture and Forestry for nutritional and chemical content, toxic substances, and bacterial load. These analysis results are available to purchasing companies or institutions upon request, and certain parameters are listed on the feed bag labels. For laboratory animal feed, these analysis reports are specifically requested and are used without further testing. The recent feed analysis reports from the relevant period were reviewed, and no abnormalities were found in the mineral composition or vitamin D levels. The mineral analysis results, conducted by an accredited laboratory, are presented in Table 2. The feed is procured every 6 months and stored in special rooms maintained at 18°C to 22°C. In addition, a semi-annual laboratory report on the nutritional composition of the feed is requested from the manufacturer (Korkuteli Yem, Antalya Türkiye). The animal feed, supplied by the same producer for years and specifically formulated to meet the nutritional needs of the guinea pigs, had never encountered any issues before this problem arose. According to laboratory analysis results, the feed adequately met the animals’ dietary requirements.
Composition of commercial feed according to bag label, laboratory analysis data, and reference values.
An investigation into supplementary food revealed that each guinea pig was given only 30 g of cucumber per week. Finally, an analysis of the water provided to the animals showed it to be extremely hard, with high levels of calcium and CaCO3 (Table 3). It was discovered that the guinea pigs had been consuming this water for 9 months due to an unnoticed malfunction in the main water treatment system, which had gone unaddressed during the university’s closure due to COVID-19.
Reference values for drinking water for human consumption and results of water analysis at the center before and after the water treatment system installation.
Abbreviation: TDS, total dissolved solids.
Water hardness levels or total dissolved solids are classified as follows: below 75 mg/L is considered soft, between 76 and 150 mg/L is moderately hard, between 151 and 300 mg/L is hard, and above 300 mg/L is very hard. 13 Water with hardness levels above 250 mg/L is deemed unsuitable for human and animal health.9,11,12
The Experimental Animal Production and Experimental Research Center is located on the lower floor of the Faculty of Veterinary Medicine. The city’s extremely hard tap water enters the facility’s purification system and is distributed throughout the building after treatment. However, the system requires monthly maintenance, which was not performed during the COVID-19 period. As a result, the filter became blocked, and untreated tap water entered the experimental animal center’s drinking water system. This issue went unnoticed for 9 months, as the faculty was closed during the pandemic. The guinea pigs consumed this hard water, containing high levels of calcium and CaCO3, which likely contributed to the problem.
After discovering that the drinking water was extremely hard, a new water purification device specifically designed for the animal drinking water supply was installed 1 week later. The water was reanalyzed immediately after installation, and the results are provided in Table 3. Despite no changes being made to the feed and supplements, no new cases of mineralization were observed in 47 guinea pigs examined over a 2-year period, reinforcing the belief that the previous cases were caused by the water.
Burdur is known for having some of the hardest water in Türkiye,11,14 which is why water purification devices are commonly used in homes and institutions, and the city’s tap water is not used for drinking without purification. All units at Burdur Mehmet Akif Ersoy University are equipped with water purification systems, including a general system in the Veterinary Faculty building. However, during the pandemic, this system’s malfunction went unnoticed, resulting in the guinea pigs being given very hard city tap water for 9 months.
Due to the suspension of experimental studies during the pandemic, the guinea pigs aged beyond the suitable age for experiments. After transitioning from remote to face-to-face education at the faculty, these aged guinea pigs, which had not been used in any experiments, were utilized for student training. Histologic examination of these animals revealed metastatic mineralization.
In guinea pigs, metastatic mineralization is rarely observed but generally affects males and is often linked to dietary factors. Potential causes include high calcium-to-phosphorus ratios, low magnesium levels, and excessive vitamin D intake. 17 Urolithiasis and nephrolithiasis in guinea pigs typically affect middle-aged and older females and are associated with the formation of calcium salt crystals. In our study, no gross abnormalities were found in the postmortem examinations of 16 guinea pigs. However, microscopic analysis revealed metastatic mineralization in the kidneys; heart; large arteries, including the aorta; and medium-sized arteries and veins of the heart and kidneys in 13 of the 16 guinea pigs. No calcification was detected in other examined organs and soft tissues. Histologic evaluations indicated that the lesions had become chronic, with long-lasting impacts on the animals’ health. These findings are consistent with observations reported in previous experimental and spontaneous studies.7,13 However, this study documented mineralization specifically in the kidneys, heart, and blood vessels.
Metastatic mineralization in guinea pigs is attributed to imbalances in dietary calcium, phosphorus, and magnesium. 3 Maintaining a proper balance between calcium and phosphorus is crucial, as disturbances can lead to metastatic mineralization. Elevated dietary phosphorus levels are known to contribute to soft tissue mineralization in guinea pigs.13,17 In addition, diets low in magnesium but high in phosphorus can exacerbate calcium accumulation and contribute to metastatic mineralization.15,16,18 Therefore, diets with excessive calcium and phosphorus require increased magnesium to mitigate the risk of mineralization.
Guinea pigs use cation exchange and phosphate anions to remove excess hydrogen ions rather than excreting protons through ammonium ion excretion in the kidney. Their rapid calcium turnover and the role of phosphate ions in metastatic mineralization underscore the need for magnesium supplementation to counteract hyperphosphatemia. 13 Another cause of metastatic mineralization is hypercalcemia induced by hypervitaminosis D, which enhances calcium absorption in the intestines. CaCO3 supplementation has been shown to increase calcium absorption, potentially leading to hypercalcemia. 13
In our study, metastatic mineralization was incidentally discovered during the histologic examination of guinea pigs used for educational purposes. Consequently, blood values were not assessed, and evaluating hypercalcemia and hyperphosphatemia was not feasible. The guinea pig diet met daily mineral requirements and had normal vitamin D levels. However, the drinking water analysis revealed high levels of calcium and CaCO3 and low magnesium levels. These findings are consistent with previous studies on metastatic mineralization.4,6,13,15,16,17,18 The absence of new cases following the installation of a water treatment device suggests that the issue was related to the drinking water source. While past studies have linked metastatic mineralization primarily to dietary factors, our study identifies hard drinking water as a significant contributing factor.
Other significant pathologic findings in guinea pigs are crystalluria and urolithiasis.2,5,7,13 Research has shown that the incidence of nephrolithiasis increases with age, with common minerals including CaCO3, calcium phosphate, struvite, and most frequently, calcium oxalate.2,7 CaCO3 is known to increase calcium absorption in the intestines, which raises the risk of crystalluria and nephrolithiasis formation.2,13
In this study, no uroliths were detected in the urinary bladder, ureters, or urethra during necropsy. However, examination of kidney sections under a polarized filter revealed intratubular crystal structures with birefringent properties. Histologic sections alone are insufficient to differentiate whether the crystals are composed of calcium carbonate or calcium oxalate, as both types exhibit similar microscopic features and birefringence.2,5 The composition of the crystals in this study could not be determined. Although bacterial infections are known to contribute to urolith formation,2,10 bacteriologic cultures could not be performed, and no evidence of infection was observed during the histologic examinations of the kidneys. It was concluded that the observed kidney changes were likely due to the high concentration of calcium and CaCO3 in the drinking water.
In this case, the company’s analysis results were accepted, and no feed samples were available for further analysis when the issue arose. However, the detection of a malfunction in the water treatment facility, the absence of issues in the feed analysis from that period, and the lack of problems reported by other nearby universities (Suleyman Demirel and Akdeniz Universities) using the same feed led us to conclude that the issue was related to the water.
In conclusion, our study demonstrated that high levels of calcium and CaCO3 in drinking water, as well as in the diet, led to the development of metastatic mineralization and crystal formations in kidney tubules in guinea pigs. This underscores the importance of both balanced nutrition and water quality in guinea pig husbandry. Our findings suggest that elevated calcium levels can adversely affect animal health, leading to mineralization and stone formation, and may predispose animals to kidney, heart, and vascular diseases.
Footnotes
Acknowledgements
The authors thank Burdur MAKU Scientific and Technological Application and Research Center for water analysis.
Author Contributions
MA performed the necropsy and OO performed histologic evaluations; the manuscript was written by MA and revised by OO.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
