Qom University of Medical Sciences Journal

Introduction
Urinary stones are among the common problems among people admitted to the emergency departments. Kidney or urinary stones usually affect adults over 20 years old of both sexes, but it is more common in men. Kidney stones consist of organic and crystalline components unified with proteins. It is the third most common disease of the urinary system after urinary tract infection and prostate pathology [1]. Its prevalence in different countries is increasing due to changes in diet and lifestyle, climate, amount of drinking water, the used drugs, genetics, age, and gender [2, 3].  stones Eighty percent of kidney stones are composed of calcium compounds, mainly calcium oxalate and calcium phosphate [1]. The formation of these crystals is due to the increase of calcium and uric acid or the decrease of its pH [3, 6] . The origin of calcium oxalate in the urine is either endogenous sources (cell destruction or formation in the liver from ascorbate and glyoxylate) or exogenous sources (due to the absorption of oxalate from the small intestine). The oxalate in plasma and the tissue cannot be metabolized; hence, it is excreted in the urine. In case of high consumption of ethylene glycol, oxalate is excreted in urine, which leads to precipitation of calcium oxalate crystals. The use of ethylene glycol in drinking water to form calcium oxalate stones is a confirmed method used in different studies, which mentioned by Lee et al. in 1991 as the inductor of stone formation in one of the kidneys [11-16].
Genetic continuity and similarities between closely related species have caused various diseases and cycles to be investigated in other organisms and generalized to humans. In the in-vivo environment, it is not possible to directly test on humans; therefore, laboratory animals are used to induce various diseases and investigate their etiology and treatment [17, 18]. Therefore, this study aims to find a method to create kidney stones similar to human oxalate and struvite stones at the right time and in both kidneys, bladder, and other organs of rats so that various therapeutic interventions can be done in the tested rats in the future studies by creating stones.
Methods
This experimental study was conducted in the animal house of the Urology Research Center of Sina Hospital in Tehran, Iran. Twenty 4-week-old young Wistar rats (10 males and 10 females, weighing 100-150 g) were purchased from the Pasteur Institute of Iran. Then, they were equally and randomly divided into two groups of control (5 males and five females received drinking water) and case (5 males and five females, received drinking water containing ammonium chloride 1% and ethylene glycol 0.75%). All five same-sex mice were kept in separate cages to avoid isolation, stress, or overpopulation. To create kidney and bladder stones in rats, ammonium chloride (1%) and ethylene glycol (0.75%) (Merck Co., Germany) were added to the drinking water. First, animals received 1% ammonium chloride in drinking water for three days. Then, 0.75% ethylene glycol (3 cc) was added to 300 cc drinking water and was given to the rats for 30 days.
The urine samples were collected to measure the amounts of oxalate (using Hitachi automatic analyzer), uric acid, pH, calcium, phosphorus, and creatinine. After molding, microscopic sections with a thickness of 5 microns were prepared from the paraffin blocks of kidney and stained (using hematoxylin-eosin stain method), and observations were made by a light microscope (Olympus).
Results
The volume of urine collected on the first day of the study was not significantly different between the groups. On the last day of the study, the volume of urine was very different; a significant decrease in the volume of urine was observed in the case group compared to the control group (P<0.05). The amount of drinking and eating also increased in the case group. Finally, an increase in body weight was observed in the case group. The number of oxalate crystals in Hounsfield units increased significantly on the 30th day after receiving ethylene glycol. The urine pH was 6.38 in the control group and 6.79 in the case group (P>0.05). Ethylene glycol significantly increased uric acid serum level, urea serum level, creatinine serum level, AST (aspartate aminotransferase) enzyme serum level, and ALT (alanine aminotransferase) enzyme serum level (P<0.05). Also, the urinary citrate level significantly decreased compared to the control group (P<0.05).
Based on the histological study on the kidney tissue, it was found that the kidney tissue cells in both cortex and medulla had a normal shape. Regular epithelial cells were observed in the central part of the tissue in the collecting ducts. In proximal and distal convoluted tubules, epithelial cells also had a normal shape. In the case group, the size of glomeruli was reduced compared to normal tissue, and bleeding was observed inside the Bowman capsule. The percentage of lymphocyte cells in the tissue increased compared to other groups, and the epithelial cell death in the Bowman capsule wall was observed in the glomeruli. In the central part of the tissue, the number of cells in the proximal and distal convoluted tubes was unclear, indicating the change in the cell structure. There were stones in the case group whose size in the left kidney of males and females was bigger than in their right kidney and had more inflammation than the other groups.
Based on the histological study on the bladder tissue, it was found that the number of stones with small sizes was more in the bladder tissue of male rats in the case group. The stone size in female rats was much bigger, but the number of observed stones was more. In addition, the degree of the bladder epithelial layer destruction was observed in some damaged areas in both sexes. On the other hand, in the bladder tissue of female rats, tissue bleeding in the superficial layers and the underlying connective tissue layers was observed in some parts of the transitional tissue. According to the concentration of oxalate, calcium, or phosphate in the urine, the type of these stones can be identified. The degree of bladder epithelial layer destruction in female rats in the case group was higher than in male rats because the thickness of the transitional epithelium was lower in female rats.
Conclusion
In this study, the formation of bilateral kidney stones and bladder stones was observed in male and female Wistar rats by administrating drinking water containing 1% ammonium chloride and 0.75% ethylene glycol for 30 consecutive days. This finding can be useful for using new treatments in rats.
Ethical Considerations
Compliance with ethical guidelines
This research was approved by the Ethics Research Committee of the Tehran University of Medical Sciences (Code: IR.TUMS.VCR.REC.1398.680).
Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.
Authors contributions
Conceptualization: Seyed Mohammad Kazem Aghamir Mohammadali; Statistical analysis and writing: Akram Mirzaei; Pathology: Diana Taheri; Implementation of the plan and laboratory animals: Leila Zareian Baghdadabad.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
Special thanks to the Urology Research Center at Sina Hospital, Tehran University of Medical Sciences.