Abstract
PURPOSE: Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as “gokhru” which is often used in ayurveda to treat various urinary diseases including urolithiasis. MATERIALS AND METHODS: The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells. RESULTS: Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.
phytotherapy; urolithiasis; calcium oxalate; NRK 52E, Tribulus terrestris
BASIC AND TRANSLATIONAL UROLOGY
Diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of Tribulus terrestris
A. AggarwalI; S. TandonI; S. K. SinglaII; C. TandonI
IDepartment of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, India
IIDepartment of Biochemistry, Panjab University, Chandigarh, India
Correspondence Correspondence to Dr. C. Tandon Biotechnology and Bioinformatics Jaypee University of Information Technology Waknaghat, 173215, Solan, India E-mail: tandonchanderdeep@yahoo.com
ABSTRACT
Purpose: Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as “gokhru” which is often used in ayurveda to treat various urinary diseases including urolithiasis.
Materials and Methods: The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells.
Results: Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner.
Conclusion: The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.
Key words: phytotherapy; urolithiasis; calcium oxalate; NRK 52E, Tribulus terrestris
INTRODUCTION
Nephrolithiasis is common, affecting up to 10% of the population at some point during their lifetime
Various authors have suggested the role of crystal induced cell injury in the development of kidney stones by providing the sites for crystal attachment and retention within the kidneys
Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin
Various mechanisms have been proposed to explain crystal retention
The surgical methods available to treat kidney stones like extracorporeal shock wave lithotripsy have serious side effects. Therefore, it is worthwhile to look for an alternative for the management of urolithiasis. Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established through systematic and pharmacological studies, except for some composite herbal drugs and plants
Fruits of Tribulus terrestris (Zygophyllaceae) locally named as “gokhru” in India are commonly used in folklore to treat urolithiasis. So far, its diuretic properties have been documented in literature and it is actively used in various drug formulations of kidney stone treatments.
The present study aimed at investigating the efficacy of Tribulus terrestris on calcium oxalate crystal nucleation and growth in vitro as well as further examining the potency of Tribulus terrestris on oxalate induced injury in NRK 52E (rat renal tubular epithelial) cells.
MATERIALS AND METHODS
Preparation of the Tribulus terrestris Extract
The dried and matured fruits of Tribulus terrestris were obtained from “Natural Remedies Pvt. Ltd.” at Bangalore in India. A collection of voucher specimens is available at the company.
The air-dried fine powdered plant fruits were boiled in distilled water. The extract was then filtered using Whatman No. 1 filter paper and the filtrate was evaporated in vacuum and dried using a rotary evaporator at 60º C
For cell culture studies a stock solution of the dried aqueous Tribulus terrestris extract was dissolved in dimethyl sulfoxide (DMSO) [final concentration of the DMSO in the highest concentration of plant extract tested did not exceed 0.4% (v/v) and did not affect the cell proliferation]. Further dilutions of the stock were done using serum free DMEM (Dulbecco’s Modified Eagle’s Media) and filtered by 0.3 mm syringe filter
Nucleation Assay
The method used was similar to that described by Hennequin et al. with some minor modifications
Growth Assay
Inhibitory activity against CaOx crystal growth was measured using the seeded, solution-depletion assay described previously by Nakagawa and colleagues
Cell Culture
Normal rat epithelial derived renal tubular epithelial (NRK 52E) cells were obtained from National Centre of Cell Sciences (NCCS, Pune). The cells were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) with 2.0 mM L-glutamine adjusted to contain 3.7 g/L sodium bicarbonate, 4.5 g/L glucose. Media was supplemented with 1% Penicillin (100 units/mL)-Streptomycin (10,000 µg/mL) and 10% fetal bovine serum. Cells were cultured in 25 cm2 tissue-culture treated flasks at 37o C and 5% CO2 in humidified chambers.
Oxalate-induced Cell Injury
NRK 52E cells were incubated in DMEM containing 1 mM sodium oxalate in the presence of different concentrations of the aqueous extract of the test sample (10 µg/mL, 25 µg/mL and 50 µg/mL) for 72 hours 14,
Cytotoxicity - Trypan Blue Assay
The cytotoxicity of the aqueous extract of T. terrestris was assessed by cell viability using trypan blue exclusion method. For the determination of cell viability, cells were plated at the density of 4 × 104 cells/well and cultured for 72 h. The medium was replaced with serum-free medium and the cells were treated with various concentrations of the plant extracts (10 µg/mL, 25 µg/mL and 50 µg/mL) for a further 72 h. The percentage viability for the cells was calculated as (live cells/total cells) x 100.
LDH Leakage Assay
LDH leakage assay was performed by the method described by Wagner et al.
Statistical Analysis
Data were expressed as mean values of three independent experiments (each in triplicate) and analyzed by the analysis of variance (p < 0.05) to estimate the differences between values of extracts tested.
RESULTS
Inhibition of Nucleation of CaOx Crystals by Tribulus terrestris Extract
Figure-1 displays the effect of the different concentration of the aqueous extract of Tribulus terrestris on the nucleation of calcium oxalate crystals. As regards control (with no plant sample), the percentage inhibition was constant at 71.4 ± 0.001 with increase in the concentration of Tribulus terrestris extract of 25 µg/mL, 50 µg/mL and 100 µg/mL. As the concentration of Tribulus terrestris extract was increased to 200 µg/mL, the percentage inhibition increased to 100 ± 0.001 but was reduced to 85.7 ± 0.002 for 400 µg/mL. The percentage inhibition was restored to 100 ± 0.001 with 1000 µg/mL of the extract.
Inhibition of CaOx Crystal Growth by Tribulus terrestris Extract
Figure-2 demonstrates the percentage inhibition shown by Tribulus terrestris on the calcium oxalate crystal growth. Tribulus terrestris extract showed inhibition in a concentration dependent manner. The percentage inhibition with 25 µg/mL of plant sample was 17.6 ± 0.004. With 50 µg/mL, 100 µg/mL and 200 µg/mL, the inhibition was almost constant in the range of 65-70% but inhibition increased significantly with 400 µg/mL and 1000 µg/mL of Tribulus terrestris extract to 126.4 ± 0.001 and 169.2 ± 0.001 respectively.
Diminution of Oxalate-induced Renal Tubular Epithelial Cell Injury by Tribulus terrestris Extract
Figure-3 depicts the protective effect of the aqueous extract of Tribulus terrestris towards the renal tubular epithelial cells. The oxalate induced a significant injury to the cells which could be ascertained by a decrease in viability from 100% in the controls (untreated cells) to 73.9%. However, the injury due to oxalate was significantly reduced in those cells treated with the Tribulus terrestris extracts. As the concentration of the extract increased from 10 µg/mL to 50 µg/mL, the percentage viability improved showing that the plant has an inhibitory activity towards the oxalate which caused injury to the renal cells in a concentration dependent manner. The plant extract alone (50 µg/mL, containing 0.4% DMSO) had no effect on the cell injury in the absence of oxalate indicating that even at the highest concentration of DMSO used there was no cytotoxicity to the cells. The percentage viability with 10 µg/mL, 25 µg/mL and 50 µg/mL was 81.6 ± 6.9, 84.9 ± 1.9 and 89.1 ± 6.9 respectively.
Lactate dehydrogenase is a stable cytosolic enzyme that is released when the cell is lysed or there is any injury on the cell membrane. A significant increase in LDH release was seen when the NRK 52E cells were exposed to oxalate alone. When NRK 52E cells were treated with the plant extract at varying concentrations(10, 25 and 50 µg/mL) along with oxalate (1 mM) for 72 h, a reduction in oxalate-induced cell injury was observed as assessed by a decreased LDH release (Figure-4), Again it was seen that the plant extract alone had no significant effect on the measures of cell injury in the absence of oxalate. The percentage LDH release for 10 µg/mL, 25 µg/mL and 50 µg/mL was observed to be 126.5 ± 4.2, 112.6 ± 5.2 and 109.8 ± 1.0 respectively after treatment with oxalate and the plant extract with respect to control.
COMMENTS
There is growing evidence that CaOx nephrolithiasis is associated with renal injury. Hyperoxaluria is a major risk factor for calcium oxalate nephrolithiasis, and calcium oxalate urinary stones are the most common type of urinary stone. High level of oxalate produced a variety of changes in the renal epithelial cells, such as an increase in free radical production and a decrease in antioxidant status, followed by cell injury and cell death. These changes are significant predisposing factors for the facilitation of crystal adherence and retention 5,14.
Due to significant side effects and failure to prevent recurrence by the present day treatment procedures for urolithiasis, alternative treatment modalities using herbal products have assumed importance. A dramatic advancement in using phytotherapy for urolithiasis treatments has been observed in recent years and many investigators have proposed to further scientific study on its efficacy. Many medicinal plants have been employed for centuries to treat urinary stones though the rationale behind their use is not well established.
In the present study, the anticalcifying properties of Tribulus terrestris commonly called “gokhru” were explored in vitro. The inhibitory potency of the plant was tested on the nucleation and growth of the most commonly occurring kidney stones, calcium oxalate monohydrate. A concentration dependent trend of inhibition was observed using Tribulus terrestris extract with maximum inhibition of 100% and 170% for CaOx nucleation and the growth assay respectively with 1000 µg/mL of the extract.
In our study with NRK 52E, Tribulus terrestris proved to have a protective effect towards the renal epithelial cells again in a concentration dependent manner. When NRK-52E cells were injured by exposure to oxalate for 72 h, the plant extract prevented the injury in a dose-dependent manner. The mechanism of inhibition /reduction in the injury needs to be studied further. Studies have shown that inhibition of the inflammatory response induced by injury due to crystal formation helps in restoring normalcy.
Beghalia et al.
Our studies are in agreement with the studies previously reported as regards the anti-urolithiatic potency of Tribulus terrestris on the growth COM crystals using double diffusion gel growth technique
Recently several plants including Herniaria hirsuta
CONCLUSION
In conclusion, the aqueous extract of Tribulus terrestris has been shown to possess an ability to inhibit CaOx crystallization in vitro. In addition this extract has also shown cytoprotective properties towards the NRK 52E cells by lowering LDH leakage and increasing the cell viability. Our study suggests the possibility of using Tribulus terrestris as a therapeutic agent to treat urolithiasis and further characterization of its active compound(s) could lead to a new candidate drug for patients with urolithiasis.
ACKNOWLEDGEMENT
The Department of Biotechnology, Government of India, provided funds for this research work.
CONFLICT OF INTEREST
None declared.
Accepted after revision: October 29, 2009
EDITORIAL COMMENT
Kidney stone disease is a major health problem in modern societies. As technology evolved, surgical options have gained more acceptance as they provide less invasive approaches, more efficacious results and lesser collateral effects. However, the costs involved are significant and an increasing effort should be continuously made in order to optimize prevention. The article presented by Aggarwal et al. clarifies the efficacy of the herbal Tribulus terrestris on the inhibition of calcium oxalate calculi formation. Herbal medicine has been long used to treat different health conditions including stone disease. However, only more recently efforts began to be made to determine the mechanisms involved and their objective efficacy. In the present evidence-based medicine era this is of utter importance. Herbal medicines may be an alternative to the currently existing medicines providing the additional advantage of minimal or inexistent collateral effects. Other herbal medicines should undergo evaluations in vitro to amplify the urologist’s clinical armamentarium to combat kidney stones.
Dr. Ricardo Miyaoka
Department of Urologic Surgery
University of Minnesota
Minneapolis, MN, USA
E-mail:miyao002@umn.edu
EDITORIAL COMMENT
In this paper, the authors addressed the potential use of Tribulus terrestris as the therapeutic agent to treat urolithiasis. Urolithiasis is characterized by high recurrence rate and among the treatments used are extracorporeal shock wave lithotripsy and drug treatment, although there is no satisfactory drug to use in clinical therapy. Thus the prevention of this disease or its recurrence would be of great interest. Phytotherapy is a common method used in folk medicine as an alternative for primary health care in many countries and particularly the potential effect of many plants to treat urolithiasis has been reported over the past years. The precipitation of calcium oxalate (CaOx) inside the renal tubules and the interaction between CaOx crystals and tubular epithelium plays an important role in the genesis and evolution of urolithiasis, since renal tubular cells selectively bind and uptake CaOx crystals, a phenomenon followed by a series of intracellular events that culminate in a cell damage and death. It was shown that aqueous extract of Tribulus terrestris was able to inhibit CaOx crystallization in vitro and showed cytoprotective properties increasing the cell viability.
The extract of plants with antilithiatic properties (Tribulus terrestris, Phyllanthus niruri, Herniaria hirsute, etc.) has been shown effective to prevent calculi development in the experimental models in vivo and in vitro, showing significant effects on many stages of stone formation including crystallization, aggregation, cellular adherence and adsorption of macromolecules into the calculi, however, its effects in lithiatic patients are much less clear. Many reasons can be raised for this difference such as the treatment onset, number of patients, time of treatment, adhesion to the treatment, etc. Moreover, it was previously shown (1) that rats with already formed vesical calculi, the administration of Phyllanthus niruri had no effect on the calculi size or elimination rate but it induced a shift in the calculi shape toward a smoother surface and probably more fragile form, which could contribute to elimination and/or dissolution of calculi. Overall the available data point to a useful therapeutic application of these plants, including Tribulus terrestris in lithiatic patients, mainly as prophylactic agent in those persons who are at high risk to develop stones since they can potentially interfere with the pathogenesis of urolithiasis and may represent an attractive alternative for the prevention of lithiasis of the urinary tract.
REFERENCES
Dr. Mirian A. Boim
Associate Researcher, Renal Division
Federal University of São Paulo
São Paulo, SP, Brazil
E-mail: mirian@nefro.epm.br
- 1 Kumar V, Farell G, Deganello S, Lieske JC: Annexin II is present on renal epithelial cells and binds calcium oxalate monohydrate crystals. J Am Soc Nephrol. 2003; 14: 289-97.
- 2 Prasad KVSRG, Sujatha D, Bharathi K: Herbal Drugs in Urolithiasis - A Review. Phycog Rev. 2007; 1: 175-9.
- 3 Verkoelen CF, Romijn JC, de Bruijn WC, Boevé ER, Cao LC, Schröder FH: Association of calcium oxalate monohydrate crystals with MDCK cells. Kidney Int. 1995; 48: 129-38.
- 4 Verkoelen CF, Verhulst A: Proposed mechanisms in renal tubular crystal retention. Kidney Int. 2007; 72: 13-8.
- 5 Khan SR: Calcium oxalate crystal interaction with renal tubular epithelium, mechanism of crystal adhesion and its impact on stone development. Urol Res. 1995; 23: 71-9.
- 6 Maroni PD, Koul S, Chandhoke PS, Meacham RB, Koul HK: Oxalate toxicity in cultured mouse inner medullary collecting duct cells. J Urol. 2005; 174: 757-60.
- 7 Jonassen JA, Kohjimoto Y, Scheid CR, Schmidt M: Oxalate toxicity in renal cells. Urol Res. 2005; 33: 329-39.
- 8 Kok DJ, Khan SR: Calcium oxalate nephrolithiasis, a free or fixed particle disease. Kidney Int. 1994; 46: 847-54.
- 9 Jethi RK, Duggal B, Sahota RS, Gupta M, Sofat IB: Effect of the aqueous extract of an Ayurvedic compound preparation on mineralization & demineralization reactions. Indian J Med Res. 1983; 78: 422-5.
- 10 Barros ME, Schor N, Boim MA: Effects of an aqueous extract from Phyllantus niruri on calcium oxalate crystallization in vitro. Urol Res. 2003; 30: 374-9.
- 11 Kieley S, Dwivedi R, Monga M: Ayurvedic medicine and renal calculi. J Endourol. 2008; 22: 1613-6.
- 12 Miyaoka R, Monga M: Use of traditional Chinese medicine in the management of urinary stone disease. Int Braz J Urol. 2009; 35: 396-405.
- 13 Kandil O, Radwan NM, Hassan AB, Amer AM, el-Banna HA, Amer WM: Extracts and fractions of Thymus capitatus exhibit antimicrobial activities. J Ethnopharmacol. 1994; 44: 19-24.
- 14 Moriyama MT, Miyazawa K, Noda K, Oka M, Tanaka M, Suzuki K: Reduction in oxalate-induced renal tubular epithelial cell injury by an extract from Quercus salicina Blume/Quercus stenophylla Makino. Urol Res. 2007; 35: 295-300.
- 15 Hennequin C, Lalanne V, Daudon M, Lacour B, Drueke T: A new approach to studying inhibitors of calcium oxalate crystal growth. Urol Res. 1993; 21: 101-8.
- 16 Nakagawa Y, Abram V, Parks JH, Lau HS, Kawooya JK, Coe FL: Urine glycoprotein crystal growth inhibitors. Evidence for a molecular abnormality in calcium oxalate nephrolithiasis. J Clin Invest. 1985; 76: 1455-62.
- 17 Jeong BC, Kwak C, Cho KS, Kim BS, Hong SK, Kim JI, et al.: Apoptosis induced by oxalate in human renal tubular epithelial HK-2 cells. Urol Res. 2005; 33: 87-92.
- 18 Wagner A, Marc A, Engasser JM, Einsele A: The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors. Biotechnol Bioeng. 1992; 39: 320-6.
- 19 Beghalia M, Ghalem S, Allali H, Belouatek A, Marouf A: Inhibition of calcium oxalate monohydrate crystal growth using Algerian medicinal plants. J Med Plants Res. 2008; 2: 66-70.
- 20 Wesson JA, Worcester EM, Wiessner JH, Mandel NS, Kleinman JG: Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules. Kidney Int. 1998; 53: 952-7.
- 21 Joshi VS, Parekh BB, Joshi MJ, Vaidya AB: Herbal extracts of Tribulus terrestris and Bergenia ligulata inhibit growth of calcium oxalate monohydrate crystals in vitro. J Crystal Growth. 2005; 275: e1403-8.
- 22 Anand R, Patnaik GK, Srivastava S, Kulshreshtha DK, Dhawan BN: Evaluation of antiurolithiatic activity of Tribulus terrestris. Int J Pharmacog. 1994; 32: 217-24.
- 23 Anand R, Patnaik GK, Kulshreshtha DK, Dhawan BN: Activity of certain fractions of Tribulus terrestris fruits against experimentally induced urolithiasis in rats. Indian J Exp Biol. 1994; 32: 548-52.
- 24 Atmani F, Farell G, Lieske JC: Extract from Herniaria hirsuta coats calcium oxalate monohydrate crystals and blocks their adhesion to renal epithelial cells. J Urol. 2004; 172: 1510-4.
- 25 Campos AH, Schor N: Phyllanthus niruri inhibits calcium oxalate endocytosis by renal tubular cells: its role in urolithiasis. Nephron. 1999; 81: 393-7.
- 26 Bashir S, Gilani AH: Antiurolithic effect of Bergenia ligulata rhizome: an explanation of the underlying mechanisms. J Ethnopharmacol. 2009; 122: 106-16.
- 27 Bijarnia RK, Kaur T, Singla SK, Tandon C: A novel calcium oxalate crystal growth inhibitory protein from the seeds of Dolichos biflorus (L.). Protein J. 2009; 28: 161-8.
- 28 Kaur T, Bijarnia RK, Singla SK, Tandon C: Purification and characterization of an anticalcifying protein from the seeds of Trachyspermum ammi (L.). Protein Pept Lett. 2009; 16: 173-81.
- 1. Barros ME, Lima R, Mercuri LP, Matos JR, Schor N, Boim MA: Effect of extract of Phyllanthus niruri on crystal deposition in experimental urolithiasis. Urol Res. 2006; 34: 351-7.
Publication Dates
-
Publication in this collection
21 Oct 2010 -
Date of issue
Aug 2010
History
-
Received
29 Oct 2009 -
Accepted
29 Oct 2009