Nephrotoxicity of hydroalcoholic extract of Teucrium polium in Wistar rats

Background and Objectives: Teucrium polium L. (Calpoureh) is a wild-growing flowering plant, found abundantly in South-West of Asia, Europe and North Africa. Traditionally, Teucrium polium L. (Calpoureh) has been used for different pathological conditions. In traditional Iranian medicine, the tea of Teucrium polium L. is used for treating many diseases such as type 2 diabetes. It is believed that this plant has beneficial therapeutic properties. However, further studies are necessary to identify its toxic effects. The aim of this study was to evaluate the nephrototoxicity of hydroalcoholic extract of T. polium in male Wistar

In addition, lowering blood lipids,  induction of vascular relaxation), 25 positive inotropic and chronotropic 26 and decreasing of blood pressure 27 has been reported by this plant. The use of herbal remedies containing T. polium extracts without any scientific guidance has resulted in several cases of hepatotoxicity. [27][28][29] Other Teucrium species including other plants widely used in folk medicine have been demonstrated to be responsible for liver injuries. 28,29 In France, all preparations containing germander as an herbal medicine were prohibited. 28,29 On the other hand, the number of the reports concerned with the toxicity potential of T. polium are not adequate. Therefore, the present study was conducted to investigate the nephrotoxicity of the hydroalcoholic extract of TP on Wistar rats.

METHODOLOGY
Plant Material: Aerial parts of TP were collected from mountains around Chaharmahal and Bakhtiyari Province, Iran, in July 2010 and the plant was authenticated at the Medical Plants Research Center of Shahrekord University of Medical Sciences. A voucher specimen was deposited there (voucher 298). Extraction Preparation: The T. polium leaves were dried in a ventilated room at 45 0 C for 48 h and powdered. 300 grams of aerial parts of TP were macerated with ethanol (70%) at 30°C for 24 hours and was shaken intermittently. The extraction procedure was repeated two times and then was concentrated in a rotary evaporator under low pressure to give one third of the primary volume. The solution was then dried by oven at 40°C. The dried extract was reconstructed with distilled water to prepare suitable concentrations. Animal Studies: In this experimental study, 100 adult male Wistar rats (150-200 g) were used. The animals were divided randomly into 10 groups of 10, including, 2 control and 8 case groups. Animals of five groups received normal saline, 50, 100, 150 or 200 mg/kg extract, respectively, for 28 days, and then were killed (Phase 1). The animals of other five groups received the same drug regimen; however, they were kept for 28 days, without drug injection, and then were killed (Phase 2). Animals in control groups received the same volume of distilled water (1ml/kg). After collecting the blood samples, they were left for half an hour, to be coagulated. Then, blood serum was separated by centrifugation for 10 minutes at 4000 rpm. Histopathology Study: After the rats were anesthetized with ether, systematic method of dissection was done. Sterile incision was made in the specific location. The kidneys were removed and examined. Then, a longitudinal incision was made on kidneys. One half of the kidney was placed in 10% buffered formalin solution for 24 hours for staining with Hematoxylin and Eosin (H&E) (31)(32)(33). The staining routine method with H&E was done and histopathology slides were prepared. Using optical microscopy, the microscopic evaluations were done based on the following criteria. The lesion were classified scoring 1 to 4, based on tubular dilatation, degeneration of cells, the percentage of vacuolization tubular cells, hyaline cast and debris tubule cells (Table-I) [30][31][32][33][34][35] Statistical Analysis: Data were expressed as mean ± SEM. To compare the percentage of hyaline cast, debris, vacuolization, flattening of tubular cells, degeneration of tubular cells and dilatation of tubular lumen of kidney tissue between and within the groups and between each phase of study, Mann-Whitney or Kruskal-Wallis were applied. Values of P<0.05 were considered statistically significant.

RESULTS
The percentage of vacuolization, flattening, degeneration and destruction in tubular cells and in kidney tissue is demonstrated in Fig. 1   results indicate significant differences between two phases and between each group as demonstrated in the Fig.1. Comparison between groups showed that, vacuolization was increased with increasing TP dose.
In phase one of the study, no significant difference of mentioned morphologic lesions of the tubules was found between cases and control groups. However, after a period of 28 days (Phase 2), morphologic lesions regarding the effect of drug on tubular cells were appeared. The percentage of debris and dilatation in kidney tissue in group one of phase one was 0.4±0.266 and 0.5 ±0.5 respectively. In phase II, the percentage of dilatation of tubular lumen in groups 3 and 5 were 1.5±1.067 and 3.5 ±2.134 respectively. These parameters (debris of tubular cells and dilatation of tubular lumen) as well as hyaline cast in both phases of each group were zero.
There were not significant differences of flattening between the groups in phase one and control group; however after a period of 28 days of drug cessation, flattening was appeared. Also comparison between groups showed that flattening was increased by increasing the drug dosage. Degeneration occurred even with low doses of TP. Also according to the ascending curve, degeneration was also increased with increasing the dose of TP. Comparing the groups in phase one and two, the injuries were increased after 28days in phase 2 (P <0.05). Fig.3.
showed that degeneration occurred even with low doses of TP. Figure also showed that degeneration of tubular cells was dose dependent.
There were no significant differences of tubular cell destruction between the cases groups of phase 1 and control group; however increased destruction of tubular cells could be seen by increasing the doses of the herb in phase 2. The serological results are shown in Table-II. The results showed that only in group 5, blood urea nitrogen (BUN) was increased. Higher doses of TP could increase BUN. Furthermore, based on Table-II, taking low doses of TP has not had significant effect on creatinine but 200 mg/kg dose had significant effect. The results showed that only in group 5 blood urea nitrogen was increased, while applying low doses of the herb, was not accompanied by changes in renal Nephrotoxicity of Teucrium polium in rats    function test. This study also showed that using 200mg/kg of this herb raised the serum creatinine.

DISCUSSION
The results showed that injection of TP with different doses had toxic effects on renal tubule cells; however, the toxicity was significant after cessation of drug administration for 28 days. It can be concluded that TP injections did not show its effects immediately following drug injection, but after a period of 28 days, it has shown its effectiveness. The results also showed that destruction percentage was dose dependent. The plant teucrium polium is used worldwide in traditional and herbal medicine. [36][37] However, it has been found to cause hepatotoxicity in humans. [37][38][39][40][41] Although the mechanism of Teucrium polium hepatotoxicity is unclear, teucrin A and several neoclerodanediterpenoids, present in the aerial parts of the plant, have been reported as the probable hepatotoxic precursors of this herb. 41 In some reports, the liver injury has been associated with the presence of autoantibodies in the serum. 41 Studies concerning renal side effects of Teucrium polium are quite scarce. The study conducted by Khleiefat et al., showed that chronic use of the T. polium extract for six weeks increased blood urea, which is consistent with our results. 42 Also Khleiefat et al showed that the kidney was markedly damaged, consequently an increase in cytoplasmic vacuolation of the kidney cells after chronic treatment with 50 mg/kg of the plant was observed under the conditions of prolonged herb administration. 42 In the study conducted by Khleiefat et al an increase in blood urea was observed, after six weeks treatment with this herb. In conclusion, the results of the present study demonstrates phytotoxic effect of the medicinal plant T. polium on the tubular cells of the kidney. To the best of our knowledge, this is the first report on the nephrotoxicity of this herb. [43][44][45][46][47][48] Indeed, some of the medicinal plant can be a common source of kidney damage. [48][49][50][51] Drugs found to cause renal toxicity exert their toxic effects by one or more common mechanism. [46][47][48][49][50][51] This toxicity tends to be more common among certain patients and in specific clinical situations. Hence, successful prevention requires knowledge of pathogenic mechanisms of renal injury, related risk factors and preemptive measures, coupled with vigilance and early intervention. [46][47][48][49][50][51][52][53][54][55][56][57]

CONCLUSION
In this study we concluded that T. polium extract has renal tubular toxicity. The results suggest that the consumption of TP for different conditions be carried out carefully because the renal injury in rats is significant and in humans can also have similar effects. Given that increasing the dose of TP, increased injury, we suggest that taking high doses of T. polium should be strictly avoided.

Conflict of Interest:
The author declared no competing interests.