Therapeutic and Preventive Effects of Aqueous Extract of Date Palm (Phoenix dactylifera L.) Pits on Ethylene Glycol-Induced Kidney Calculi in Rats Pouria Mohammadparast Tabas1, Hamed Aramjoo1, Ali Yousefinia1, Mahmoud Zardast2, Mohammad Reza Abedini3 & 4, Mohammad Malekaneh5* Purpose: Urinary tract stones are one of the most common diseases in the urinary tract. Lack of kidney stone treatment causes irreparable damages to the kidneys, which has many harmful effects. Date palm pits are recom- mended in traditional medicine as an effective drug in the treatment of kidney stones. The aim of this study was to investigate the effect of aqueous extract of date palm pits on kidney stones induced by ethylene glycol in male rats. Methods: In this study, 40 rats were classified into five groups (n = 8), including the healthy group receiving normal water, the negative control group, the therapeutic groups with doses of 150 mg/kg and 300 mg/kg, and the prevention group with a dose of 300 mg/kg. In order to induce kidney stones, ethylene glycolated water (1%) was used as drinking water in the studied groups. Blood and urine of rats were collected on days 14 and 28 of the study to assess urinary parameters of calcium, creatinine, uric acid and phosphorus, and serum parameters of blood urea nitrogen, creatinine, uric acid, calcium, and phosphorus. Also, the kidneys of rats were removed from the body on day 28 of the study and were given to a pathologist for examination. Results: Results of serum parameters show that the use of date palm pits extract in the treatment and prevention groups with a dose of 300 mg/kg significantly (P < .05) has reduced the levels of blood urea nitrogen, uric acid, calcium, creatinine, and phosphorus. Also, the results of urinary parameters show that the use of the extract caused a significant decrease (P < .05) in creatinine, uric acid, and calcium in the prevention group and a significant de- crease (P < .05) in creatinine and uric acid in the therapeutic group with a dose of 300 mg/kg. Pathological results show a decrease in the number and size of calcium oxalate crystals in renal tubules in the treatment and prevention groups in a dose-dependent manner. Conclusion: The results of this study showed that the use of aqueous extract of date palm pits has been effective in the treatment and prevention of kidney stones induced by ethylene glycol in rats. Keywords: calcium oxalate; date palm pits; ethylene glycol; kidney calculi; rat INTRODUCTION Kidney stone (KS) is the most common urinary tract disease due to urinary tract infections and pros- tate disorders. KSs are formed from organic and inor- ganic crystals in combination with protein(1). About 80 percent of kidney stones are calcium stones, which are composed of a combination of calcium oxalate (CaOx) and calcium phosphate(2). The prevalence of KS has been increasing globally over the last three decades. As this prevalence has increased, we have also seen an in- crease in the expenses associated with this disease. By 2030, the United States is expected to spend more than $ 5 billion a year to treat patients with KSs(3). KS formation is associated with a variety of factors including lifestyle, race, genetic background (herita- bility of ~45-60%), gender, and diet. Underlying dis- 1Student Research Committee, Birjand University of Medical Sciences, Birjand, Iran. 2Cardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran. 3Chronic Disease Program and Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada. 4Cellular and Molecular Medicine Research Center, Department of Pharmacology, Birjand University of Medical Sciences, Birjand, Iran. 5Department of clinical Biochemistry, Birjand University of Medical sciences, Birjand, Iran. *Correspondence: Associate Professor of Biochemistry, Department of Clinical Biochemistry, School of Medicine, Birjand University of Medical sciences, Birjand, Iran. Email: Drmalekaneh21@gmail.com. Received October 2020 & Accepted June 2021 eases such as diabetes, obesity and inactivity, gout, hy- perparathyroidism, hyperoxaluria, increased calcium, and changes in urine pH play an important role in the formation of KS(4-6). People suffering from KSs have severe colic pain, and on the other hand, obstruction caused by these stones reduces the urine output and, in some cases, hematuria, and if left untreated, can lead to kidney damage, kidney failure, and urinary tract infec- tions(7). There are currently several methods to remove KSs; supportive methods such as fluid intake and the use of analgesics to remove stones spontaneously and in the case of larger stones, methods such as chemical- ly dissolving stones, removing stones from the urethra, breaking stones with ultrasonic waves and, if necessary, open surgery could be used. In addition to many side ef- fects such as urinary tract infections, tissue damage and the possibility of stone re-formation can impose many Urology Journal/Vol 18 No. 6/ November-December 2021/ pp. 612-617. [DOI: 10.22037/uj.v18i.6530] ENDOUROLOGY AND STONE DISEASE costs on the patient(8,9). Nowadays, considering the side effects and severe detrimental effects of chemical drugs, the use of herbal and natural medicines has been considered by contemporary researchers, and in several studies, the effects of various herbs in the treatment of kidney stone disease have been studied(10,11). Monocotyledonous date palm tree belongs to the Are- caceae or Palmae family is from the Phoenix genus with the scientific name Phoenix dactylifera. Date palms are grown in desert areas, especially in the Middle East, and are native to Iraq and western and southern re- gions of Iran(12). Date palm pits extract enriches from polyphenols(13), which exerts many biological proper- ties, including antioxidant, anti-inflammatory, antiviral, and antibacterial activities(14) as well as anti-cancer ef- fects, and could be used for the treatment of diabetes. Moreover, it is useful to prevent neurological disorders and Alzheimer's disease, enhance sexual potency and improve anemia(15-17). Pits consist of 10 to 15 percent of the date palms weight and contain compounds that are chemically composed of saturated fatty acids such as palmitic acid and stearic acid, and unsaturated fatty acids such as oleic acid and linoleic acid, as well as elements such as zinc, cadmi- um, calcium, and potassium(18). It also contains antioxi- dants such as carotenoids, anthocyanins, glycosidic fla- vonoids from flavones, flavonols, and flavoxanthines classes, and due to the high percentage of antioxidant activity, they protect the body against oxidative damage (19,20). Owing to the fact that the medicinal plants have fewer side effects, and patients show higher emotional compliance, date palm pits is recommended as a treat- ment for kidney and bladder stones in traditional med- icine; however, and to our knowledge, no relevant sci- entific research has been reported so far in this regard. Therefore, the present study was conducted to investi- gate the therapeutic and preventive effects of date palm (Phoenix dactylifera) pits on kidney stones induced by ethylene glycol in rats. MATERIALS and METHODS Preparation of date palm pits extract To prepare date palm pits extract, some dates known in the market as Zahedi dates were purchased from the local market and after approval in the herbarium section of Birjand University of Medical Sciences were used. the date palm pits were first removed from the fruit and then thoroughly washed to remove any fruit residue and surface contaminants. After the pits dried in the shade, they were grinded using a grinder. To prepare the aqueous extract, 5 g of dried and grinded date palm pits powder was poured into 100 mL of distilled water, twice distilled at 95 °C, and after stirring, brewed for 15 minutes. Then, using filter paper (Blue Ribbon, Grade 589, Germany), the obtained solution was filtered in the specific containers of freeze-dryer (Dena Vacuum In- dustry, model FD-5005-BT, Iran) and placed in a -20° C freezer. After freezing, the solution was placed in a freeze-dryer and freeze-dried at a -50 °C and vacuum conditions. The dry powder obtained was stored in spe- cific polyethylene containers at 4 °C until use. The ex- tract solution was provided daily. Animals and study design In this study, 40 male Wistar rats (200 ± 25 g, 2 months) were kept in stable physical conditions at a tempera- ture of 25 ± 2 °C and a 12-hour light and dark cycle on research center of experimental medicine Birjand Data palm pit effects on kidney calculi in rats-Mohammadparast Tabas et al. Figure 1. Effect of date palm pits on histopathology of kidney. Histopathological sections of the kidney sample showed that there were no intratubular crystal deposition in the NC group and the tissue showed normal architecture of the renal epithelia and tubular structure (Fig. 1A). The ethylene glycol treated group showed the presence of intratubular deposition of the crystals and necrotic degeneration of the renal epithelia leading to damage to nephron and collecting system (Fig. 1B). Sections from 150mg/kg date palm pits (Fig. 1C) and 300mg/kg date palm pits (Fig. 1D) treated rats also demonstrated less retention of crystals in tubules and prevented necrotic damage. The sections from the kidney of rats treated with 150mg/kg date palm pits (Fig. 1C) exhibited the prevention of retention and tubular deposi- tion of crystals and lesser degree of necrotic damage to renal epithelia (Type of staining: Hematoxylin and Eosin; Magnification ×400). Vol 18 No 6 November-December 2021 613 Endourology and Stones diseases 614 University of medical sciences, Birjand, Iran. The study was approved by the Deputy of research and technology and ethics committee of Birjand University of Medical Sciences (Ethics code: IR.BUMS.REC.1398.156). The rats had free access to standard food (Javan- neh-Khorasan co, Iran) and drinking water, and were grouped 72 hours before the study began and placed in relevant cages to adapt to the new conditions. The du- ration of the study was considered 28 days, according to previous studies(21-24). The rats were divided into 5 groups (n = 8): Group 1: Received regular drinking water and receive 1 mL of normal saline orally per day (gavage) during the study (NC). Group 2: Received 1 mL of normal saline per day orally and 1% ethylene glycol (Merk, Germany) is added to their drinking water (EG). Group 3: Received the date palm pits extract 150 mg/kg per day from the 14th day until the end of the study and 1% ethylene glycol was added to their drinking water (T150). Group 4: Received the date palm pits extract 300 mg/kg per day from the 14th day until the end of the study and 1% ethylene glycol was added to their drinking water (T300). Group 5: Received 300 mg/kg of date palm pits extract per day orally and 1% ethylene glycol was added to their drinking water (P300). Collecting urine samples Each rat was kept in a separate metabolic cage for 24 hours and their urine was collected on days 14 and 28 of the study. Calcium, creatinine, uric acid, and phospho- rus in the rats’ urine were measured and analyzed with an auto-analyzer (Tokyo Bokei Prestige 24i, Japan). Collecting blood samples Blood samples from rats were taken on day 14 of the study through the retro-orbital sinus, and on day 28 of the study through their heart following anesthesia, and the serum was separated by blood centrifugation at 2500 rpm for 10 minutes at 4 °C, and was kept at -80 °C until biochemical analyzes were performed. Serum lev- els of blood urea nitrogen (BUN), creatinine, uric acid, calcium, and phosphorus were measured by commer- cially available kits and analyzed. Histopathological evaluation To study the kidney’s pathology, following anesthesia of the rats with ketamine and xylazine (65:10 mg/kg) on day 28, rats’ kidneys were removed, washed with normal saline (0.9%), and placed in 10% formalin solu- tion. After dehydration and embedding in paraffin, 5 microns-thick sections were prepared. From the pre- pared sections, 4 sections from each kidney (2 sections from each kidney lobe) were selected and stained by hematoxylin and eosin method and were given to the blinded pathologist to examine the possible damage of kidney tissue and formation of calcium oxalate crystals. Statistical analysis Finally, all data were entered into SPSS software (Ver- sion 19). After testing the normality of the data dis- tribution using a Kolmogorov-Smirnov test, one-way analysis of variance (ANOVA) was performed. Tukey HSD multiple comparisons were performed as a post- hoc test to see any significant differences between each group. Non-parametric variables were compared using the Kruskal-Wallis test followed by Mann-whitney test. The data were presented as Mean ± SD and a P-value of .05 or less was considered statistically significant. RESULTS Effects of date palm pits on plasma biochemical param- eters Groups BUN (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) NC 27 ± 10.23 0.55 ± 0.12 2.12 ± 0.55 8.77 ± 0.3 8.07 ± 0.34 EG 32.5 ± 3.41 0.8 ± 0.08* 3.57 ± 0.26* 10.4 ± 0.75** 8.37 ± 1.34 T150 38.5 ± 2.51 0.72 ± 0.05 2.72 ± 0.48 9.55 ± 0.49 8.12 ± 1.72 T300 40.75 ± 17.32 0.85 ± 0.1 2.72 ± 0.74 9.5 ± 0.34 6.92 ± 1.35 P300 37 ± 5.47 0.82 ± 0.22 3.8 ± 0.75 8.77 ± 0.29## 7.8 ± 0.73 Chi-Square = 7.29 F = 3.45 F = 5.45 F = 8.18 F = 0.86 P = 0.12 P = 0.03 P = 0.006 P = 0.001 P = 0.5 Table1. The pre-operative, baseline characteristics of the two groups Abbreviations: BUN, blood urea nitrogen. All data were expressed as mean ± SD (n = 8). # P < .05, ## P < 0.01, and ### P < .001 compared with NC group; * P < .05, ** P < .01, and *** P < .001 compared with EG group. Groups BUN (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) NC 26.85 ± 12.64 0.57 ± 0.32 2.28 ± 0.77 8.3 ± 1.48 8.14 ± 2.48 EG 66.5 ± 14.08*** 1.08 ± 0.41** 3.22 ± 0.79* 11.45 ± 2.36** 11.37 ± 1.97** T150 50.75 ± 8.87 0.87 ± 0.21 2.65 ± 0.36 10.63 ± 1.21 9.52 ± 2.1 T300 44.87 ± 18.61# 0.76 ± 0.35# 2.45 ± 0.57# 9.46 ± 1.69 9.48 ± 1.28# P300 43.87 ± 13.06## 0.7 ± 0.27# 2.05 ± 0.65## 8.9 ± 2.1# 8.1 ± 1.52## F = 7.42 Chi-Square = 10.84 Chi-Square = 12.85 F = 3.76 Chi-Square = 13.03 P < 0.0001 P = 0.01 P = 0.01 P = 0.01 P = 0.01 Table2. The effect of date palm pits extract on plasma parameters on day 28. Abbreviations: BUN, blood urea nitrogen. All data were expressed as mean ± SD (n = 8). # P < .05, ## P < 0.01, and ### P < .001 compared with NC group; * P < .05, ** P < .01, and *** P < .001 compared with EG group. Data palm pit effects on kidney calculi in rats-Mohammadparast Tabas et al. Examination of BUN and plasma phosphorus levels of rats on day 14 showed an increase in the groups that received ethylene glycol compared to the control group. BUN and plasma phosphorus levels on day 28 in the EG group showed a significant increase compared to the control group (P < .05). Consumption of date palm pits extract decreased the amount of BUN and plasma phosphorus in the therapeutic and prevention groups, which was significant in the therapeutic group with a dose of 300 mg/kg (T300) and prevention (P300) (P < .05). Examination of creatinine and uric acid levels in the plasma showed a significant increase in the EG group compared to the control group on days 14 and 28, and consumption of date palm pits extract caused a significant decrease in creatinine and uric acid levels of the therapeutic group with a dose of 300 mg/kg (T300) and prevention group with a dose of 300 mg/kg (P300) (P < .05). Plasma calcium levels on days 14 and 28 had a significant increase in the EG group compared to the control group, which consumption of the extract caused a significant decrease in plasma calcium levels in the prevention group with a dose of 300 mg/kg (P300) compared to the EG group (P < .05). Effects of date palm pits on urinary biochemical param- eters The urinary levels of creatinine showed a significant decrease and levels of uric acid and calcium showed a significant increase in the EG group compared with the control group on day 28 of the study (P < .05). The urinary level of creatinine show significantly increased and uric acid and calcium showed a significant de- crease in the prevention group with a dose of 300 mg/ kg (P300) in comparison to the EG group (P < .05). Also, creatinine showed a significant increase and uric acid showed a significant decrease at the dose of 300 mg/kg (T300 group) compared with the EG group (P < .05) (Table 2). Pathological results While Pathological results did not show any stones or tissue damage in the healthy group, a large number of calcium oxalate crystals were visible in the tubules from the rats in the negative control group. Date palm pits ex- tract decreased calcium oxalate crystals formation and also prevented stone formation in rats’ nephrons in a dose-dependent manner (Figure 1). DISCUSSION In this study, the therapeutic and preventive effects of aqueous extract of date palm (Phoenix dactylifera L.) pits on kidney stones induced by ethylene glycol in male rats were investigated. The results of the study demonstrated that oral treatment of date palm pits ex- tract effectively reduced kidney damage and stone for- mation in a dose-dependent manner compared with the control group. The results also showed that date palm pits extract had a preventive effect on kidney stone for- mation. Ethylene glycol is naturally converted to glycine in the body after being converted to glyoxylic acid by the en- zyme alanine-glyoxylate aminotransferase; when the amount of glyoxylic acid reaches too high, it is convert- ed by lactate dehydrogenase to oxalic acid, which in the presence of calcium is converted to insoluble calcium oxalate and deposited in body tissues, especially the kidneys(25). The study of chemical factors in urine could consider as a good indicator of stone formation rate and type of stone. Urinary saturation with stone constituents is the most important factor in the formation of kidney stones. Previous studies have shown that taking ethylene glycol for fourteen days causes the formation of kidney stones, mainly from calcium oxalate stones in rats, and this is due to the increased concentration of calcium and uric acid in the urine(26-28). Uric acid plays an important role in calcium solubility. Increasing uric acid prevents the formation of calcium-phosphate and calcium-citrate soluble stones and the production of calcium oxalate stones, thus increasing the production of urinary stones Groups Creatinine (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) NC 34.75 ± 19.8 3.85 ± 2.38 2.52 ± 1.02 23.7 ± 2.04 EG 34.25 ± 10.71 5.82 ± 5.73 5.12 ± 2.03 20.65 ± 0.63 T150 34.75 ± 19.68 5.7 ± 2.48 8 ± 1.59 23.42 ± 0.68 T300 36.75 ± 18.44 4.97 ± 3.1 7.6 ± 2.05 22.52 ± 1.77 P300 28.75 ± 6.29 3.95 ± 0.42 5.9 ± 7.21 19.25 ± 2.67 Chi-Square = 0.47 F = 0.32 F = 1.5 F = 4.73 P = 0.97 P = 0.86 P = 0.25 P = 0.01 Table 3. The pre-operative, baseline characteristics of the two groups for proximal All data were expressed as mean ± SD (n = 8). # P < .05, ## P < .01, and ### P < .001 compared with NC group; * P < .05, ** P < 0.01, and *** P < .001 compared with EG group. Groups Creatinine (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) NC 33.75 ± 12.94 4.47 ± 4.02 2.94 ± 1.53 22.3 ± 3.04 EG 18.5 ± 9.05** 12.28 ± 3.23** 8.2 ± 3.12** 25.25 ± 7.78 T150 22.85 ± 8.6 7.55 ± 3.6# 7.1 ± 1.56 25.7 ± 7.2 T300 27.96 ± 7.78# 5.96 ± 3.13### 6.7 ± 3.7 24.63 ± 6.7 P300 32.2 ± 12.1## 5.51 ± 3.39## 3.02 ± 1.44## 24.15 ± 7.25 Chi-Square = 11.37 Chi-Square = 16.05 F = 7.43 Chi-Square = 1.78 P = 0.02 P = 0.003 P < 0.0001 P = 0.77 Table4. The effect of date palm pits extract on urinary parameters on day 28. Data was presented as n (%) and mean ± SD; *Ureteral injury as in European association guideline including I: Mucosal abrasion; II: Ureteral perforation; III: Intussus- ception / avulsion Data palm pit effects on kidney calculi in rats-Mohammadparast Tabas et al. Vol 18 No 6 November-December 2021 615 Endourology and Stones diseases 616 (29,30). Consumption of date palm pits extract in the treat- ment of kidney stones significantly reduced uric acid and also prevented the increase in urinary uric acid and calcium in a dose-dependent manner thus reduced the risk of formation of kidney stones. Glomerular filtration rate (GFR) in urolithic rats de- creases due to the blockage of urinary tubules by stones formation inside them, and this reduction causes the ac- cumulation of excretory substances such as uric acid, calcium, phosphate, creatinine, and urea in the blood and increases the serum level of these substances and also reduces the excretion substances such as creatinine into the urine and reduces its urinary amounts(27,31,32). Previous studies have shown that the use of ethylene glycol increases lipid peroxidation and reduces the anti- oxidant potential of renal tissue, which is an important factor in damaging the tubules and decrease of renal filtration(33). Treatment with date palm pits extract sig- nificantly reduces serum levels of urea, calcium, phos- phate, creatinine, and uric acid and also dramatically prevents the increase in serum levels of urea, calcium, phosphate, creatinine, and uric acid and also treatment with date palm pits extract significantly increases uri- nary creatinine level and also dramatically prevents the reduces urinary creatinine. Increased GFR in therapeu- tic groups, as well as prevention group, is attributed to antioxidant activity and anti-lipid properties (peroxida- tion reactions) of date palm pits extract(14,34). Study of Baghbani et al.(34) on the antioxidant and an- timicrobial properties of date palm pits extract and its effects on physicochemical, microbial and sensory properties of cupcake have shown that date palm pits can be considered as an important source of natural antioxidants for medicinal and commercial purposes. Catechins and rutin are found in abundance among the 7 active compounds in date palm pits, including gallic acid, catechin, chlorogenic acid, rutin, vanillin, p-cou- maric acid, and sinapic acid. Catechin is the most im- portant polyphenolic compound extracted from date palm pits and flavonoids with significant antioxidant activity. The antioxidant activity of catechins is mostly related to the ortho dihydroxyl groups in the beta ring of the catechin structure, which has anti-free radical activity(35). It seems that the antioxidant properties of catechins and consequently dates have been one of the main factors in showing the protective properties of date seeds against damage to kidney tissue due to expo- sure to ethylene glycol. Previous studies on date palm pits have proven its an- ti-inflammatory properties, so it could be stated that the aqueous extract of date palm pits may have reduced kidney inflammation. The microscopic study of kidneys of urolithic rats shows the presence of irregular shapeless crystals inside the tubules, which causes tubule inflammation. Date palm pits extract reduced the number and size of calci- um oxalate crystals in the tubules. The results show that the use of date palm pits extract reduces and prevents the formation of urinary stones. It seems that date palm pits extract is effective in preventing recurrent kidney stones. The precise mechanism of action of date palm pits extract may be due to its antioxidant properties or decrease of the main stone-former constituents in the urine. However, according to our studies knowledge, no study has been reported the effect of date palm pits on kidney stones and the resulting damages; therefore, it is not possible to comment on the exact effects of date palm pits on kidney stones and their possible mecha- nisms. CONCLUSIONS The results of this study show that aqueous extract of date palm pits has been effective in the treatment and prevention of kidney stones induced by ethylene gly- col in rats. Its precise mechanism on kidney stones is not clear and requires further investigation. However, its effect may be attributed to the antioxidant and an- ti-inflammatory properties and the decrease of the main stone-former constituents in the urine. CONFLICT OF INTEREST The authors declared that they have no conflict of in- terest. ACKNOWLEDGMENTS This article is the outcome of a research project ap- proved by the Research Council of Birjand University of Medical Sciences (IR.BUMS.REC.1398.153). We hereby would like to thank the Deputy of Research and Technology of Birjand University of Medical Scienc- es for financing the project (Grant No. 5044), and the Clinical Research Development Unit (CRDU) of Val- iasr Hospital, Birjand University of Medical Sciences, Birjand, Iran, for their support, cooperation and assis- tance throughout the period of study. REFERENCES 1. Winoker JS, Bamberger JN, Chandhoke RA, Atallah W, Gupta M. What Factors Drive Staghorn vs Nonstaghorn Pattern Growth in Patients with Metabolic Stones? J Endourol. 2019;33:954-9. 2. Evan AP. Physiopathology and etiology of stone formation in the kidney and the urinary tract. Pediatr Nephrol. 2010;25:831-41. 3. Howles SA, Wiberg A, Goldsworthy M, Bayliss AL, Gluck AK, Ng M, et al. Genetic variants of calcium and vitamin D metabolism in kidney stone disease. Nat. Commun. 2019;10:1-0. 4. Coe FL, Worcester EM, Evan AP. Idiopathic hypercalciuria and formation of calcium renal stones. Nat. Rev. Nephrol. 2016;12:519. 5. 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