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Department of Biological & Biomedical Sciences Medical College, Pakistan

January 2011

Antiurolithic effect of berberine is mediatedthrough multiple pathwaysSamra BashirAga Khan University

Anwar H. GilaniAga Khan University

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Recommended CitationBashir, S., Gilani, A. (2011). Antiurolithic effect of berberine is mediated through multiple pathways. European Journal ofPharmacology, 651(2012-01-03), 168-175.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/37

Pulmonary, Gastrointestinal and Urogenital Pharmacology

Antiurolithic effect of berberine is mediated through multiple pathways

Samra Bashir a,b, Anwar H. Gilani a,⁎a Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistanb Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan

a b s t r a c ta r t i c l e i n f o

Article history:Received 10 May 2010Received in revised form 7 October 2010Accepted 29 October 2010Available online 27 November 2010

Keywords:BerberineNephrolithiasisAntioxidantDiureticAnimal model

Berberine is an isoquinoline alkaloid, occurring in nature as the main constituent of several plants withmedicinal use in kidney stone disease. This work was undertaken to evaluate its antiurolithic potential andexplore the possible underlying mechanism(s). Berberine was tested in vitro for the antioxidant effect and invivo for diuretic and antiurolithic effects on an animal model of calcium oxalate urolithiasis. Berberineexhibited concentration-dependent (50–150 μg/ml) antioxidant effect against ferrous-ascorbate inducedlipid peroxidation in rat kidney homogenate with potency slightly higher than the reference antioxidant,butylated hydroxytoluene. In Wistar rats, berberine (5–20 mg/kg) increased urine output accompanied byincreased pH and Na+ and K+ excretion and decreased Ca2+ excretion, similar to hydrochlorothiazide. In ananimal model of calcium oxalate urolithiasis developed in maleWistar rats by adding 0.75% ethylene glycol indrinking water, berberine (10 mg/kg) prevented as well as eliminated calcium oxalate crystal deposition inrenal tubules and protected against deleterious effects of lithogenic treatment including weight loss, impairedrenal function and oxidative stress, manifested as increased malondialdehyde and protein carbonyl contents,depleted GSH and decreased antioxidant enzyme activities of the kidneys. In naïve rats, berberine (10 mg/kg)increased urine volume and pH and decreased Ca2+ excretion. Results of this study suggest the presence ofantiurolithic effects in berberine against calcium oxalate stones mediated through a combination ofantioxidant, diuretic, urinary alkalinizing and hypocalciuric effects. These data invite future studies onberberine to establish its efficacy for clinical use.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Urinary tract stones are worldwide, sparing no geographical,cultural or racial groups (Moe, 2006). Calcific stones composed ofcalcium oxalate (CaOx) alone or mixture of CaOx and calciumphosphate are hitherto the most common, accounting for more than80% of uroliths. Mechanisms involved in the formation of suchconcretions are not fully understood but it is generally agreed thaturinary lithiasis is multifaceted involving crystal nucleation, aggrega-tion and growth of insoluble particles. Urine is always supersaturatedwith common stone forming minerals, however, crystallizationinhibiting capacity of urine does not allow urolithiasis to happen inmost of the individuals, whereas, this natural inhibition is in deficit instone formers (Robertson et al., 1969; Tiselius, 2005). Studies havealso shown that tubular cell injury facilitates CaOx crystal formationand deposition in the renal tubules. Experimental studies havedemonstrated that both oxalate and CaOx crystals directly inducerenal epithelial cell injury through lipid peroxidation and involveoxygen free radical generation (Khan, 1995; Santosh Kumar andSelvam, 2003; Tsujihata et al., 2006).

Urolithiasis is largely recurrent with a relapse rate of 50% in 5–10 years, thereby exists with substantial economic consequences anda great public health importance (Moe, 2006). Endoscopic stoneremoval and extracorporeal shock wave lithotripsy have revolution-ized the treatment of urolithiasis but do not prevent the likelihood ofnew stone formation (Goldfarb and Coe, 2005). Various therapiesincluding thiazide diuretics and alkali-citrate are being used inattempt to prevent recurrence of hypercalciuria- and hyperoxaluria-induced calculi but scientific evidence for their efficacy is lessconvincing (Hess, 2003).

Berberine is an isoquinoline alkaloid, widely distributed in nature.It exists as main constituent of several plants including Hydrastiscanadensis (goldenseal), Coptis chinensis (coptis or goldenthread),Berberis aquifolium (Oregon grape), Berberis aristata (tree turmeric)and Berberis vulgaris (barberry). Plants rich in berberine have widemedicinal applications in virtually all traditional systems and haveseveral therapeutic uses in common including bladder, kidney andgall stones and as diuretic (Duke et al., 2002). We have previouslyreported antiurolithic potential in Berberis vulgaris which possessesberberine as the main constituent (Bashir et al., 2010).

Berberine has wide clinical applications. The predominant usesinclude bacterial diarrhea, intestinal parasite and ocular trachomainfections. Berberine is safely used up to the dose as high as200 mg/kg orally 2 to 4 times daily in most clinical situations and

European Journal of Pharmacology 651 (2011) 168–175

⁎ Corresponding author. Tel.: +92 21 4864571; fax: +92 21 493 4294, 494 2095.E-mail address: anwar.gilani@aku.edu (A.H. Gilani).

0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2010.10.076

Contents lists available at ScienceDirect

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has not been shown as cytotoxic or mutagenic (Birdsall and Kelly,1997).

Considering the well reputed use of the parent plant species inurolithiasis, current investigation was undertaken to evaluate theantiurolithic effect of berberine. Berberine was studied on an animalmodel of CaOx urolithiasis for both preventive and curative effectsand for antioxidant and diuretic activities as possible mechanisms ofthe antiurolithic effect.

2. Materials and methods

2.1. Drugs and standards

All the chemicals used were of analytical grade available.Ammonium chloride, berberine chloride, ethylene glycol, thymol,reduced glutathione, 5-5′-dithiobis, 2-nitrobenzoic acid (DTNB),thiobarbituric acid (TBA), 1,1-diphenyl-2-picrylhydrazyl (DPPH),Folin-Ciocalteu's phenol reagent, hydrochlorothiazide, potassiumcitrate tribasic hydrate, reduced glutathione, H2O2, trichloroaceticacid (TCA), butylated hydroxytoluene (BHT), 1,1,3,3,-tetraethoxypropane, sodium oxalate (Na2C2O4), calcium chloride (CaCl2) andguanidine hydrochloride were obtained from Sigma ChemicalCompany, St. Louis, MO, USA. Reagents used for histological prepara-tions were eosin spirit soluble, hematoxylin, xylene (BDH ChemicalLimited, Poole, England), paraffin wax (Merck, Darmstadt, Germany),silver nitrate (AgNO3), nuclear fast red and poly-L-lysine (SigmaChemical Company, St. Louis, MO, USA). Kits used in this study for thedetermination of Ca2+, Mg2+, blood urea nitrogen (BUN), creatinine,superoxide dismutase (SOD) and glutathione peroxidase (GPx) werepurchased from Randox Laboratories Ltd., Ardmore, Diamond Road,Crumlin, Co., Antrim, UK. Oxalate estimation was done by the kit fromTrinity Biotech Plc, IDA Business Park, Bray, Co., Wicklow, Ireland, andfor citrate estimation, the kit was purchased from R-Biopharm AG, D-64293 Darmstadt, Germany.

2.2. Animals

Experiments were performed in compliancewith the rulings of theInstitute of Laboratory Animal Resources, Commission on LifeSciences, National Research Council (1996) and approved by theEthical Committee for Research on Animals (ECRA) of the Aga KhanUniversity, Karachi, Pakistan.

Wistar rats (180–220 g) of either sex used for this study weresourced locally and housed at the animal house of the Aga KhanUniversity, kept in plastic cages (47×34×18 cm3) with saw dust(renewed after every 48 h), under a controlled temperature of 23–25 °C and 12 h light–dark cycle. Animals had access to food and waterad-libitum throughout the study except 24 h before and during 6 h ofdiuretic study and while collecting 24 h urine samples, food waswithdrawn.

2.3. Determination of antioxidant activity

Antioxidant potential of berberine was estimated in vitro by itsinhibitory effect against lipid peroxidation induced in rat kidneyhomogenate with ferrous-ascorbate system, as described previously(Kang et al., 2003).

2.4. Determination of diuretic activity

The diuretic activity of the test material was studied onWistar ratsof either sex (180–220 g) as described previously (Consolini et al.,1999). Animals were divided with matched body weight and sex intogroups of 6 animals each. Negative and positive control groups weregiven, by gavage, saline (20 ml/kg) and standard diuretic drug:hydrochlorothiazide, 10 mg/kg of body weight, respectively. The rests

of the groups were given different doses of the test material dissolvedin saline. Subsequently, the animals were placed individually inmetabolic and diuretic cages (Techniplast, 21020 Buguggiate-Va-Italy). The urine was collected in graduated cylinders for 6 h at 1 hintervals. Total urine excreted out was collected and the volume wasdetermined. The pH of the pooled urine from each animal wasdetermined by using pH meter, Na+ and K+ concentrations on flamephotometer (Flame Photometer 410, Corning, UK) and Ca2+ concen-tration by using commercially available kit.

2.5. Study on animal model of urolithiasis

Male Wistar rats weighing 180–220 g were divided with matchedbody weights into seven groups of six animals each, which were thenrandomly selected to receive various treatments. In study onpreventive effect of berberine, groups I, II and III, while for curativeeffect, groups IV, V and VI served as vehicle control, stone formingcontrol and treated group, respectively. Vehicle controls receivedintraperitoneal injections of vehicle (2.5 ml/kg) once in 24 h and nostone inducing treatment while both stone forming control andtreated groups received stone inducing treatment, for up to 3 weeks,which comprised of 0.75% (w/v) ethylene glycol with 1% (w/v)ammonium chloride for 5 days, following this the water supply wasswitched to 0.75% ethylene glycol alone in water (Atmani et al., 2003).For the determination of stone preventing effect, vehicle (2.5 ml/kg)and berberine (10 mg/kg) were started simultaneously with litho-genic treatment to groups II and III, respectively, while for the curativeeffect, respective treatments to groups V and VI were started at theend of 3 weeks of stone inducing treatment and continued for a periodof 2 weeks. Groups VII received berberine alone for 3 weeks andserved as berberine control. The dose of berberine selected for CaOxantinephrolithic effect in vivo was the one which has causedmaximum diuresis with increase in the urine pH. The weight andactivity were regularly monitored to assess their overall health so thatany animal looking lethargic or excessively losing weight could beexcluded from the study.

At the end of the respective treatment periods, the animals wereindividually housed in metabolic and diuretic cages. After collecting3 h morning urine for the crystalluria study, 24 h urine was collected.Following volume and pH determination, part of each 24 h urinesample was acidified to pH 2 with 5 M HCl. Both acidified and nonacidified urine samples were then centrifuged at 1500×g for 10 minto remove debris and supernatants were stored at −20 ºC untilanalyzed. Blood was collected through cardiac puncture from animalsunder ether anaesthesia for serum separation in order to assess serumcreatinine and BUN.

Animals were sacrificed and both the kidneys were excised, rinsedin ice cold physiological saline and weighed. The right kidney wasfixed in 10% neutral buffered formaline, processed and stained withHaematoxylin and Eosin (H & E) or by Pizzolato's method; whichselectively stains CaOx (Pizzolato, 1971) for microscopic examination.To count the number of crystalline deposits, a sagittal section of eachrenal specimen was divided into 8 equal sized regions by four virtuallines (Fig. 2) according to the method of Tsai et al. (2008). A field of100× was then randomly selected from each region and CaOxdeposits were counted. The total number of CaOx deposits in eachspecimen was reported as average of the eight readings. The leftkidney was worked into 10% homogenate in phosphate bufferedsaline (50 mM, pH 7.4), centrifuged at 1500×g and the supernatantswere used to assess in the antioxidant enzymes activities, reducedglutathione levels and markers of peroxidative injury to lipid andprotein, malondialdehyde (MDA) and protein carbonyl content (PCC),respectively.

In acidified urine samples, oxalate, calcium (Ca2+) andmagnesium(Mg2+) contents were determined by using commercially availablekits, while inorganic phosphate excretion was determined by the

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method of Daly and Ertingshausen (1972) In non-acidified urinesamples, citrate, creatinine and uric acid (UA), while in serum,creatinine and BUN were estimated with the help of kit-basedmethods. Creatinine clearance (CC) was calculated using the formula

CC ml=minð Þ = mg creatinine= dl urineð Þ × ml urine= 24 hð Þmg creatinine = dl serumð Þ × 1440

:

In the kidney homogenates, MDA content was estimated bythiobarbituric acid reactive method (Wong et al., 1987) and theamount of MDA was determined from the standard curve of 1,1,3,3-tetraethoxy propane.

The PCC was estimated by the protein derivatization withdinitrophenyl hydrazine (DNPH) into chromophoric dinitrophenylhydrazones using by the method of Levine et al. (1990) and thecarbonyl content was calculated using the DNPH molar extinctioncoefficient of 22,000 M−1 cm−1.

Reduced glutathione (GSH) was estimated as total non-proteinthiol (SH) group by the method described by Moron et al. (1979). Forthe purpose of quantitation, a calibration curve was prepared usingGSH as a standard.

Superoxide dismutase (SOD) and glutathione peroxidase (GPx)were determined by using commercially available methods.

Catalase activity was determined by monitoring the decomposi-tion of H2O2 at 240 nm with a spectrometer (Aebi, 1983). Catalaseactivity was measured by using 240 (molar extinction coefficient)=0.0394 mmol−1 min−1 for H2O2 and expressed as U/mg proteinwhere one U of catalase activity decomposes 1 μmol of H2O2 per minunder standard conditions at 25 °C.

Protein contents of urine and kidney homogenates were deter-mined by Lowry et al. (1951).

2.6. Statistical analysis

The data expressed are mean±standard error of mean (S.E.M.)and the median effective concentration (EC50 value) with 95%confidence intervals (CI). All statistical comparisons between thegroups are made by means of OneWay Analysis of Variance (ANOVA)with post hoc Dunnett's test or by Student's t-test. The P-value lessthan 0.05 is regarded as significant. The concentration-responsecurves were analyzed by non-linear regression using GraphPad Prism(GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Antioxidant effect

Berberine at concentrations of 50 and 150 μg/ml inhibited in vitrolipid peroxidation induced in rat kidney homogenate similar to thecontrol drug BHT, as shown in Table 1.

Fig. 1. Represented images of CaOx crystals viewed in 3 h morning urine under lightmicroscope (400×), from vehicle control (A), stone forming (B) and berberine (10 mg/kg) treated animals (C) in the preventive study.

Fig. 2. Lines drawn on saggital section of kidney to divide it in 8 equal parts for crystalcounting.

Table 1Inhibitory effect of berberine and butylated hydroxytoluene (BHT) on in vitro lipidperoxidation induced in rat kidney homogenate.

Concentration % Inhibition

Berberine BHT

50 mg/ml 53.5±2.12a 36±3.6150 mg/ml 97.5±3.54a 74.6±4.7

a Pb0.01 vs. BHT.

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3.2. Diuretic effect

Results of the study for diuretic effect of berberine are given inTable 2. At the doses of 5 (Pb0.05), 10 (Pb0.01) and 20 mg/kg(Pb0.01), berberine increased the urine output similar to hydrochlo-rothiazide, indicating diuretic effect. In addition to its effect on urinevolume, berberine also increased urine excretion of Na+ and K+ butdecreased Ca2+, like that exhibited by hydrochlorothiazide. Berberine(5 and 10 mg/kg) also caused an increased in the urine pH as didhydrochlorothiazide. With further dose increment (20 mg/kg), ber-berine, despite of exhibiting diuretic effect, did not increase urine pHabove that of the control animals.

3.3. Effect on nephrolithic rats

Various parameters recorded from groups of animals at the end ofthe treatments to determine the preventive and curative effects ofberberine are given in Tables 3 and 4, respectively. Among groups inthe preventive study, after 3 weeks on stone inducing regimen, therewere significantly abundant (Pb0.01) and visibly bigger CaOx crystalspredominately of calcium oxalate dihydrate as compared to thevehicle control in 3 h morning urine of the stone forming control(Fig. 1A and B), whereas a co-treatment with berberine (10 mg/kg)significantly decreased urinary crystal count (Pb0.01 vs. stoneforming) as well as visibly reduced crystal size (Fig. 1C). Volume of24 h urine and water intake was higher in stone forming groupcompared to those of the vehicle control animals (Pb0.01). Urine pHwas also reduced though not to a significant extent. A simultaneoustreatment with berberine significantly prevented the increase in urinevolume and water intake (Pb0.05 vs. stone forming), although boththe parameters remained higher than those of the vehicle control(Pb0.05). Berberine also caused a significant increase in urine pHcompared to the stone group (Pb0.05). In parallel with crystalluria,oxalate excretion was also significantly enhanced (Pb0.01) in stoneforming animals whereas Ca2+ excretion was decreased (Pb0.05).Urine contents of citrate, UA and Mg2+ did not alter to any significantextent. In berberine treated group, ethylene glycol treatmentincreased oxalate excretion but not to a significant extent comparedto those of the vehicle control but urinary Ca2+ excretion wassignificantly reduced (Pb0.05).

Lithogenic treatment caused impairment of renal functions of theuntreated rats as evident from the markers of glomerular and tubulardamages: raised BUN and serum creatinine and reduced creatinineclearance (Pb0.01), which were prevented in the animals simulta-neously treated with berberine.

A significant loss in body weights (Pb0.01) by the stone inducingtreatment was observed in untreated group during the study period,while in berberine treated group there was a net gain in bodyweight over 3 weeks of treatment similar to that of the controlanimals.

Kidneys excised from stone formers were enlarged and heavier(Pb0.01) in stone forming animals, whereas, berberine treated groupkidneys were not significantly different from those of the vehiclecontrol. When observed under polarized light microscope, manybirefringent crystalline deposits in the histological preparations wereseen in tubules of all regions of kidneys: cortex, medulla and papilla,of all the animals in the stone forming group but were found in only 2out of 6 rats treated with berberine which were also less abundant(Pb0.05) and visibly smaller (Fig. 3). The crystals were shown as CaOxwhen stained black with Pizzolato's method. The renal tubules werealso markedly dilated in the entire kidneys of the rats in stone group.

Stone inducing treatment enhanced MDA and PCC contents ofkidneys (Pb0.01), and decreased reduced glutathione levels andactivity of the antioxidant enzymes of untreated group (Pb0.05).Berberine treatment protected against the changes associated withoxidative stress.

In berberine control group there was a significant increase in urinevolume and pH and decrease in Ca2+ excretion (Pb0.05) compared tothe vehicle control group, whereas other urinary, serum and renalparameters were not different.

In the curative study, withdrawal of stone inducing treatmentinitiated spontaneous recovery of the animals in untreated control assuggested by a net gain in the body weights during post inductionperiod and slight, though not significant, decrease in urine volume,water intake and increase in urine pH. Urine crystal count was alsoreduced to a level similar to the control animals and similarly urinarycontent of oxalate was decreased (Pb0.05) compared to that of thestone forming animals in the preventive study after 3 weeks oflithogenic treatment. Discontinuation of lithogenic treatment alsoreversed decreased urinary Ca2+ content, there was rather a slight,although insignificant, increase in urinary Ca2+ excretion. There wasan improvement in kidney functions as reflected by the levels ofserum creatinine and creatinine clearance, which became comparableto those of the vehicle control animals. Kidney weight was alsosignificantly less (Pb0.01) than the untreated group of the preventivestudy. CaOx crystal deposits could be seen in kidneys of all rats inuntreated group even after two weeks of lithogenic treatment freeperiod, which were however less extensive (P b 0.05) than theuntreated group in the preventive study and similarly renal tubuleswere less dilated (Fig. 4). A spontaneous reduction in oxidative stressover two weeks of post induction period was also evident fromdecreased MDA and PCC (Pb0.05) and increased levels of reducedglutathione and antioxidant enzyme activities measured in kidneyhomogenates of the untreated animals. Post induction treatment withberberine enhanced the spontaneous recovery of urolithic animals.After two weeks of berberine treatment, kidney weight, BUN andoxalate excretion were similar to those of the vehicle control animalsbut similar to that of treated animals in the preventive study, meanurine volume remained significantly higher than that of the vehiclecontrol, pH was significantly higher than that of the untreated groupand calcium contents were lower than both the vehicle control andthe untreated group (pb0.05).

CaOx deposits were found in kidneys of 4 out of 6 animals in thetreated group which were however significantly less than theuntreated control of the curative study (Pb0.05).

4. Discussion

Berberine is the main constituent of several plant speciestraditionally reputed as therapy for urinary stone disease. To exploreits potential antiurolithic effect, berberine was tested for theantioxidant and diuretic effects, being properties likely to contributein its medicinal effect, and on animal model of urolithiasis, to confirmstone preventive and curative activities in vivo.

Animal and cellular studies have shown that exposure to highlevels of oxalate and CaOx crystals produce cellular injury mediated

Table 2Diuretic effect of berberine and the reference diuretic hydrochlorothiazide (HCT).

/100 g bodyweight/6 h

Vehiclecontrol

Berberine HCT

(5 mg/kg) (10 mg/kg) (20 mg/kg) (10 mg/kg)

Urine volume(ml)

1.07±0.31 1.47±0.4a 1.79±0.56b 1.91±0.39b 2.38±0.89b

Na+(mmol) 0.09±0.02 0.12±0.01 0.15±0.02a 0.22±0.04a 0.34±0.02b

K+(mmol) 0.07±0.01 0.08±0.01 0.103±0.01 0.15±0.02a 0.19±0.03b

Ca++ (μmol) 9.32±1.6 6.84±0.68 4.7±1.10a 4.55±0.5a 2.29±0.52a

pH 5.9±0.3 6.35±0.4a 6.4±0.39b 5.7±0.41 6.74±0.7b

Values given are mean±S.E.M. (n=6).a Pb0.05 vs. vehicle control.b Pb0.01 vs. vehicle control.

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by membrane lipid peroxidation through intracellular oxygen freeradical generation. It has been demonstrated that epithelial cell injuryfacilitates the events of CaOx crystal nucleation, aggregation bylowering concentration at which crystal forms and promotes crystalretention in renal tubules crucial for subsequent stone development(Khan and Hackett, 1991; Khan 1995; Moro et al., 2005). Recentlyobtained human data are also suggestive of the development ofoxidative stress in kidney stone patients (Huang et al., 2003).Antioxidant potential of berberine was estimated by lipid peroxida-tion inhibitory activity. Berberine inhibited ferrous-ascorbate induced

lipid peroxidation of rat kidney in vitro, similar to BHT, a standardantioxidant. Several experimental studies have shown that antiox-idants such as vitamin E, catechin and selenium can protect againstoxidative injury by oxalate and crystal deposition while some urinarymacromolecules such as glycosaminoglycans, bikunin, uropontin,uromodulin etc. are also shown to possess protective effect againstoxalate injury (Thamilselvan and Menon, 2005; Itoh et al., 2005;Santosh Kumar and Selvam, 2003; Tsujihata et al., 2006).

When tested for diuretic effect, berberine increased urine excretionof the rats. Increase in the urine volume was also accompanied by an

Table 4Various parameters recorded after 14 days of post stone induction treatment for the curative effect of berberine.

Vehicle control Untreated control Berberine treated (10 mg/kg)

Gain in b.wtA (%) 13.9±2.7 8.5±3.3 14.7±2.2CUB (count/mm3) 25±7.6 33±9.8 38±5.9Kidney wts (g) 0.65±0.04 0.94±0.04a 0.73±0.02b

CDC/field (10x) 0 32±5.9a 14.5±4.3c

Water intake (ml) 8.56±0.75 16.9±2.3a 12.9±1.53Urine/24 h Volume (ml) 9.01±1.53 15.9±1.32d 13.8±1.47d

pH 6.53±0.07 6.28±0.15 6.78±0.12c

Ca++ (mg) 3.7±0.73 5.2±0.53 3.9±0.63Mg++ (mg) 2.97±0.27 3.15±0.45 2.93±0.18OxD (mg) 0.34±0.03 0.69±0.08a 0.43±0.04b

Citrate (mg) 28±1.90 26±2.3 29±1.6UAE (mg) 0.66±0.16 0.76±0.13 0.58±0.07

Kidney function tests BUNF 23.6±0.87 39.13±3.84b 23.01±2.38b

SCG mg/dl 0.90±0.06 1.03±0.08 0.83±0.04CCH ml/min 0.89±0.02 0.78±0.10 0.86±0.05

MDAI (nmol/mg protein) 0.76±0.05 6.5±1.8a 3.16±0.59PCCJ (nmol/mg protein) 4.35±0.52 7.08±0.87d 5.34±0.44GSHK (nmol/mg protein) 19±2.7 11.7±1.4d 17.0±0.9SODL (U/mg protein) 9.42±0.84 6.37±1.44 8.19±0.58GPXM (U/mg protein) 1.19±0.08 0.79±0.14 1.09±0.15Catalase (U/mg protein) 31.5±2.7 25.2±3.1 32.7±1.8

Values given are mean±S.E.M. (n=6).ABody weight; BCrystalluria; CCrystal deposits; DOxalate; EUric acid; FBlood urea nitrogen; GSerum creatinine; HCreatinine clearance; IMalondialdehyde; JProtein carbonyl content;KReduced glutathione; LSuperoxide dismutase; and Mglutathione peroxidase.

a Pb0.01 vs. vehicle control.b Pb0.01 vs. stone forming group.c Pb0.05 vs. stone forming group.d Pb0.05 vs. vehicle control.

Table 3Various parameters recorded after 21 days of treatment for the preventive effect of berberine.

Vehicle control Stone forming control Berberine treated (10 mg/kg) Berberine control (10 mg/kg)

Change in b.wtA (%) 16.7±3.89 6.10±2.95a 11.5±3.09b 20.7±2.89CUB (count/mm3) 38±8.5 172±26a 85±13b 41±7.6Kidney wts (g) 0.68±0.02 1.41±0.05a 0.80±0.10b 0.65±0.03CDC/field (10×) 0 53.6±7.5a 6.17±2.57c 0Water intake (ml) 7.1±0.76 21.5±3.1a 12.3±2.65c,d 12.6±3.5a

Urine/24 h Volume (ml) 6.67±0.5 22.4±3.12a 13.7±2.25c,d 13.1±2.5d

pH 6.35±0.13 5.67±0.07 6.59±0.16c 6.67±0.11d

Ca++ (mg) 3.33±0.39 1.89±0.16d 2.05±0.21d 2.19±0.25d

Mg++ (mg) 3.7±0.65 2.75±0.49 3.21±0.23 4.11±0.62OxD (mg) 0.36±0.11 1.41±0.25a 0.54±0.16b 0.48±0.05Citrate (mg) 35±4.7 27±3.5 30±2.9 33±4.2UAE (mg) 0.58±0.16 0.67±0.19 0.61±0.23 0.59±0.18

Kidney function tests BUNF 25.7±1.82 63.7±12.87d 24.8±3.1c 27.8±2.49SCG mg/dl 0.91±0.05 1.43±0.15a 0.95±0.05b 0.93±0.04CCH ml/min 0.86±0.04 0.53±0.03a 0.81±0.06b 0.83±0.07

MDAI (nmol/mg protein) 0.56±0.01 13.3±2.8a 1.63±0.09b 0.45±0.07PCCJ (nmol/mg protein) 3.87±0.59 11.20±1.08a 6.39±1.36b 3.54±0.71GSHK (nmol/mg protein) 21±1.9 11.6±2.7d 17.2±1.2 20±1.8SODL (U/mg protein) 7.17±0.65 3.91±1.30d 6.12±0.49 7.49±0.67GPXM (U/mg protein) 1.23±0.06 0.64±0.14a 0.93±0.15b 1.26±0.10Catalase (U/mg protein) 35.7±1.9 22.2±3.7d 29.7±4.6c 29.4±1.6

Values given are mean±S.E.M. (n=6).ABody weight; BCrystalluria; CCrystal deposits; DOxalate; EUric acid; FBlood urea nitrogen; GSerum creatinine; HCreatinine clearance; IMalondialdehyde; JProtein carbonyl content;KReduced glutathione; LSuperoxide dismutase; and Mglutathione peroxidase.

a Pb0.01 vs. vehicle control.b Pb0.01 vs. stone forming group.c Pb0.05 vs. stone forming group.d Pb0.05 vs. vehicle control.

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increase in the Na+ and K+ excretion similar to the standard diuretic,hydrochlorothiazide, suggesting that berberine induced diuresis iscaused by its saluretic effect. Treatment with berberine and similarlyhydrochlorothiazide also increased urine pH and decreased urine Ca2+

content. Urinary supersaturation with stone forming minerals is aprimary requisition for crystal precipitation and a major risk factor forthe stone development (Hess and Kok, 1996). Thiazide diuretics due totheir hypocalciuric and diuretic effects reduce urinary supersaturationof calcium salts and are commonly used to treat calcium stone disease(Goldfarb and Coe, 2005).

Renal calcium oxalate deposition induced by ethylene glycol andammonium chloride in rats is frequently used to mimic the urinarystone formation in humans (Thamilselvan et al., 1997; Atmani et al.,2003; Tsai et al., 2008). Therefore, we evaluated both the preventionand curative effects of berberine on urolithiasis in this model.

In preventive study, the analysis of crystalluria showed thatuntreated animals were excreting abundant and larger crystals thanthe treated animals. Crystalluria could occur similarly in both healthyand stone forming individuals where the latter tend to excrete largerand aggregated particles than the formers (Robertson et al., 1969).CaOx crystal agglomerates tend to retain in kidney by trapping in

renal tubules and develop into renal stones (Atmani et al., 2003). Thepresence of significantly less oxalate content in urine of berberinetreated rats is an obvious reason for less abundant crystals but thepresence of crystallization inhibitory constituents in berberine and itspH increasing effect cannot be ignored as solubility of CaOx isincreased in alkaline medium (Tiselius, 2005). Berberine significantlyprevented the increase in urine volume associated with lithogenictreatment (Fan et al., 1999), though urine output remained still higherthan that of control animals similar to the berberine control group,suggestive of intrinsic diuretic effect of berberine as accounted for theraised urine volume. Consistent with some previous reports, stoneinduction by hyperoxaluric agents caused an increase in oxalate anddecrease in Ca2+ excretion in untreated group (Fan et al., 1999; Parket al., 2008). When administered simultaneously, berberine pre-vented hyperoxaluric effect of the stone inducing treatment but didnot affect hypocalciuria which can also be attributed to its intrinsiceffect on Ca2+ excretion, as evident from the diuretic study.

There was hypertrophy and extensive calcium oxalate crystaldeposition in kidneys of untreated rats accompanied by oxidativedamage as reflected from increased levels of markers of peroxidativeinjury: MDA and PCC, and decreased activities of antioxidant enzymes

Fig. 3. Representative microscopic images (100×) of kidney sections from the animals in study for the preventive effect of berberine. (A), (B) and (C) are H & E stained kidneysections from vehicle control, stone forming and treated groups, respectively and (D), (E) and (F) are the respective sections stainedwith Pizzolato's method.White and black arrowspoint to crystal deposits and dilated tubules, respectively.

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and reduced glutathione levels as well as deteriorated renal functions.The renal tubules were markedly dilated in the entire kidney ofall these rats, and this might have been caused by distal obstructionof renal tubular flow by large crystals. Hyperoxaluria is a majorrisk factor for calcium oxalate nephrolithiasis. Several in vivo andin vitro studies have demonstrated that exposure to high level ofoxalate results in greater production of superoxide and hydroxylfree radicals, leading to antioxidant imbalance and has beenmanifested as antioxidant depletion, peroxidation of lipid and protein(Thamilselvan et al., 2000; Hackett et al., 1994; Kumar et al., 1991;Kwak et al., 2002; Selvam, 2002; Thamilselvan et al., 1997), changes inmembrane integrity and cell death (Thamilselvan and Menon 2005;Itoh et al., 2005; Jeong et al., 2006; Santosh Kumar and Selvam, 2003).These changes facilitate CaOx crystal adherence and retention in renaltubules (Wiessner et al., 2001; Khan 1995; Rashed et al., 2004;Thamilselvan et al., 2000). The inhibitory effect of berberine oncalcium oxalate crystal retention in renal tubules thus could have alsobeen caused by its antioxidant activity.

In berberine control group, berberine treatment increased urinevolume and pH and decreased Ca2+ excretion, thus confirming thediuretic, urine alkalinizing and hypocalciuric effects of berberineobserved in the diuretic and animal model studies.

In curative study, withdrawal of stone inducing treatmentevoked a spontaneous recovery of nephrolithic animals in theuntreated groups. There was a gain in body weight, significantdecrease in urinary oxalate, renal crystal deposition, oxidative stressand kidney weights, and improvement in renal functions comparedto the untreated rats in the preventive study after 21 days on stoneinducing regimen, although the renal CaOx deposits, urine volumeand oxalate, BUN, and oxidative stress were significantly higher thanthose of the control animals. Despite of high urinary oxalate content,the increased urinary volume of the untreated animals appears asaccounted for the presence of negligible crystalluria as that of thecontrol animals. Post induction treatment with berberine extractenhanced spontaneous recovery of the rats. In berberine treatedgroup, after two weeks of ethylene glycol discontinuation, kidneyfunctions, kidney weights, urinary oxalate and MDA content andoxidative enzyme levels of kidneys became similar to those of therespective control animals. Kidney deposits of CaOx crystals werealso significantly lower than the respective untreated group butwere present in greater number of the animals than the treatedgroup of the preventive study receiving similar dose of berberine,suggesting that the prophylactic effect of berberine is stronger thanits curative effect.

Fig. 4. Representative microscopic images (100×) of kidney sections from the animals in study for the curative effect of berberine. (A), (B) and (C) are H & E stained kidney sectionsfrom vehicle control, stone forming and treated groups, respectively and (D), (E) and (F) are the respective sections stained with Pizzolato's method.

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5. Conclusion

Results of this study suggest that plant alkaloid berberine istherapeutically effective for both prevention as well as treatment ofcalcium oxalate urolithiasis, exhibiting these effects through acombination of antioxidant, diuretic, hypocalciuric and urine alkala-nizing activities. These findings also suggest berberine as activeprincipal of the plants used in urolithiasis, however, the coexistence ofother biologically active constituents cannot be excluded. As berber-ine is in wide clinical use for some other pathological conditions andexcept a few contraindications it is generally considered safe, thesedata invite clinical trials on berberine to establish its efficacy forhuman use.

Acknowledgements

This studywasfinanced in part by theHigher Education Commissionof Pakistan and the University Research Council of the Aga KhanUniversity, Karachi, Pakistan.

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