Download Antiurolithic effect of berberine is mediated through multiple pathways

eCommons@AKU
Department of Biological & Biomedical Sciences
Medical College, Pakistan
January 2011
Antiurolithic effect of berberine is mediated
through multiple pathways
Samra Bashir
Aga Khan University
Anwar H. Gilani
Aga Khan University
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Recommended Citation
Bashir, S., Gilani, A. (2011). Antiurolithic effect of berberine is mediated through multiple pathways. European Journal of
Pharmacology, 651(2012-01-03), 168-175.
Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/37
European Journal of Pharmacology 651 (2011) 168–175
Contents lists available at ScienceDirect
European Journal of Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Antiurolithic effect of berberine is mediated through multiple pathways
Samra Bashir a,b, Anwar H. Gilani a,⁎
a
b
Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan
Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan
a r t i c l e
i n f o
Article history:
Received 10 May 2010
Received in revised form 7 October 2010
Accepted 29 October 2010
Available online 27 November 2010
Keywords:
Berberine
Nephrolithiasis
Antioxidant
Diuretic
Animal model
a b s t r a c t
Berberine is an isoquinoline alkaloid, occurring in nature as the main constituent of several plants with
medicinal use in kidney stone disease. This work was undertaken to evaluate its antiurolithic potential and
explore the possible underlying mechanism(s). Berberine was tested in vitro for the antioxidant effect and in
vivo for diuretic and antiurolithic effects on an animal model of calcium oxalate urolithiasis. Berberine
exhibited concentration-dependent (50–150 μg/ml) antioxidant effect against ferrous-ascorbate induced
lipid 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 by
increased pH and Na+ and K+ excretion and decreased Ca2+ excretion, similar to hydrochlorothiazide. In an
animal model of calcium oxalate urolithiasis developed in male Wistar rats by adding 0.75% ethylene glycol in
drinking water, berberine (10 mg/kg) prevented as well as eliminated calcium oxalate crystal deposition in
renal tubules and protected against deleterious effects of lithogenic treatment including weight loss, impaired
renal 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 of
antiurolithic effects in berberine against calcium oxalate stones mediated through a combination of
antioxidant, diuretic, urinary alkalinizing and hypocalciuric effects. These data invite future studies on
berberine 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 of
calcium oxalate (CaOx) alone or mixture of CaOx and calcium
phosphate are hitherto the most common, accounting for more than
80% of uroliths. Mechanisms involved in the formation of such
concretions are not fully understood but it is generally agreed that
urinary lithiasis is multifaceted involving crystal nucleation, aggregation and growth of insoluble particles. Urine is always supersaturated
with common stone forming minerals, however, crystallization
inhibiting capacity of urine does not allow urolithiasis to happen in
most of the individuals, whereas, this natural inhibition is in deficit in
stone formers (Robertson et al., 1969; Tiselius, 2005). Studies have
also shown that tubular cell injury facilitates CaOx crystal formation
and deposition in the renal tubules. Experimental studies have
demonstrated that both oxalate and CaOx crystals directly induce
renal epithelial cell injury through lipid peroxidation and involve
oxygen free radical generation (Khan, 1995; Santosh Kumar and
Selvam, 2003; Tsujihata et al., 2006).
⁎ Corresponding author. Tel.: + 92 21 4864571; fax: + 92 21 493 4294, 494 2095.
E-mail address: [email protected] (A.H. Gilani).
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.10.076
Urolithiasis is largely recurrent with a relapse rate of 50% in 5–
10 years, thereby exists with substantial economic consequences and
a great public health importance (Moe, 2006). Endoscopic stone
removal and extracorporeal shock wave lithotripsy have revolutionized the treatment of urolithiasis but do not prevent the likelihood of
new stone formation (Goldfarb and Coe, 2005). Various therapies
including thiazide diuretics and alkali-citrate are being used in
attempt to prevent recurrence of hypercalciuria- and hyperoxaluriainduced calculi but scientific evidence for their efficacy is less
convincing (Hess, 2003).
Berberine is an isoquinoline alkaloid, widely distributed in nature.
It exists as main constituent of several plants including Hydrastis
canadensis (goldenseal), Coptis chinensis (coptis or goldenthread),
Berberis aquifolium (Oregon grape), Berberis aristata (tree turmeric)
and Berberis vulgaris (barberry). Plants rich in berberine have wide
medicinal applications in virtually all traditional systems and have
several therapeutic uses in common including bladder, kidney and
gall stones and as diuretic (Duke et al., 2002). We have previously
reported antiurolithic potential in Berberis vulgaris which possesses
berberine as the main constituent (Bashir et al., 2010).
Berberine has wide clinical applications. The predominant uses
include bacterial diarrhea, intestinal parasite and ocular trachoma
infections. Berberine is safely used up to the dose as high as
200 mg/kg orally 2 to 4 times daily in most clinical situations and
S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
has not been shown as cytotoxic or mutagenic (Birdsall and Kelly,
1997).
Considering the well reputed use of the parent plant species in
urolithiasis, current investigation was undertaken to evaluate the
antiurolithic effect of berberine. Berberine was studied on an animal
model of CaOx urolithiasis for both preventive and curative effects
and for antioxidant and diuretic activities as possible mechanisms of
the antiurolithic effect.
169
of the groups were given different doses of the test material dissolved
in saline. Subsequently, the animals were placed individually in
metabolic and diuretic cages (Techniplast, 21020 Buguggiate-VaItaly). The urine was collected in graduated cylinders for 6 h at 1 h
intervals. Total urine excreted out was collected and the volume was
determined. The pH of the pooled urine from each animal was
determined by using pH meter, Na+ and K+ concentrations on flame
photometer (Flame Photometer 410, Corning, UK) and Ca2+ concentration by using commercially available kit.
2. Materials and methods
2.5. Study on animal model of urolithiasis
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, potassium
citrate tribasic hydrate, reduced glutathione, H2O2, trichloroacetic
acid (TCA), butylated hydroxytoluene (BHT), 1,1,3,3,-tetraethoxy
propane, sodium oxalate (Na2C2O4), calcium chloride (CaCl2) and
guanidine hydrochloride were obtained from Sigma Chemical
Company, St. Louis, MO, USA. Reagents used for histological preparations were eosin spirit soluble, hematoxylin, xylene (BDH Chemical
Limited, Poole, England), paraffin wax (Merck, Darmstadt, Germany),
silver nitrate (AgNO3), nuclear fast red and poly-L-lysine (Sigma
Chemical Company, St. Louis, MO, USA). Kits used in this study for the
determination of Ca2+, Mg2+, blood urea nitrogen (BUN), creatinine,
superoxide dismutase (SOD) and glutathione peroxidase (GPx) were
purchased from Randox Laboratories Ltd., Ardmore, Diamond Road,
Crumlin, Co., Antrim, UK. Oxalate estimation was done by the kit from
Trinity Biotech Plc, IDA Business Park, Bray, Co., Wicklow, Ireland, and
for citrate estimation, the kit was purchased from R-Biopharm AG, D64293 Darmstadt, Germany.
2.2. Animals
Experiments were performed in compliance with the rulings of the
Institute of Laboratory Animal Resources, Commission on Life
Sciences, National Research Council (1996) and approved by the
Ethical Committee for Research on Animals (ECRA) of the Aga Khan
University, Karachi, Pakistan.
Wistar rats (180–220 g) of either sex used for this study were
sourced locally and housed at the animal house of the Aga Khan
University, 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 water
ad-libitum throughout the study except 24 h before and during 6 h of
diuretic study and while collecting 24 h urine samples, food was
withdrawn.
2.3. Determination of antioxidant activity
Antioxidant potential of berberine was estimated in vitro by its
inhibitory effect against lipid peroxidation induced in rat kidney
homogenate 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 on Wistar rats
of either sex (180–220 g) as described previously (Consolini et al.,
1999). Animals were divided with matched body weight and sex into
groups of 6 animals each. Negative and positive control groups were
given, by gavage, saline (20 ml/kg) and standard diuretic drug:
hydrochlorothiazide, 10 mg/kg of body weight, respectively. The rests
Male Wistar rats weighing 180–220 g were divided with matched
body weights into seven groups of six animals each, which were then
randomly selected to receive various treatments. In study on
preventive effect of berberine, groups I, II and III, while for curative
effect, groups IV, V and VI served as vehicle control, stone forming
control and treated group, respectively. Vehicle controls received
intraperitoneal injections of vehicle (2.5 ml/kg) once in 24 h and no
stone inducing treatment while both stone forming control and
treated 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 was
switched 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 lithogenic treatment to groups II and III, respectively, while for the curative
effect, respective treatments to groups V and VI were started at the
end of 3 weeks of stone inducing treatment and continued for a period
of 2 weeks. Groups VII received berberine alone for 3 weeks and
served as berberine control. The dose of berberine selected for CaOx
antinephrolithic effect in vivo was the one which has caused
maximum diuresis with increase in the urine pH. The weight and
activity were regularly monitored to assess their overall health so that
any animal looking lethargic or excessively losing weight could be
excluded from the study.
At the end of the respective treatment periods, the animals were
individually housed in metabolic and diuretic cages. After collecting
3 h morning urine for the crystalluria study, 24 h urine was collected.
Following volume and pH determination, part of each 24 h urine
sample was acidified to pH 2 with 5 M HCl. Both acidified and non
acidified urine samples were then centrifuged at 1500 ×g for 10 min
to remove debris and supernatants were stored at −20 ºC until
analyzed. Blood was collected through cardiac puncture from animals
under ether anaesthesia for serum separation in order to assess serum
creatinine and BUN.
Animals were sacrificed and both the kidneys were excised, rinsed
in ice cold physiological saline and weighed. The right kidney was
fixed in 10% neutral buffered formaline, processed and stained with
Haematoxylin and Eosin (H & E) or by Pizzolato's method; which
selectively stains CaOx (Pizzolato, 1971) for microscopic examination.
To count the number of crystalline deposits, a sagittal section of each
renal specimen was divided into 8 equal sized regions by four virtual
lines (Fig. 2) according to the method of Tsai et al. (2008). A field of
100× was then randomly selected from each region and CaOx
deposits were counted. The total number of CaOx deposits in each
specimen was reported as average of the eight readings. The left
kidney was worked into 10% homogenate in phosphate buffered
saline (50 mM, pH 7.4), centrifuged at 1500 ×g and the supernatants
were used to assess in the antioxidant enzymes activities, reduced
glutathione levels and markers of peroxidative injury to lipid and
protein, malondialdehyde (MDA) and protein carbonyl content (PCC),
respectively.
In acidified urine samples, oxalate, calcium (Ca2+) and magnesium
(Mg2+) contents were determined by using commercially available
kits, while inorganic phosphate excretion was determined by the
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S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
Fig. 2. Lines drawn on saggital section of kidney to divide it in 8 equal parts for crystal
counting.
The PCC was estimated by the protein derivatization with
dinitrophenyl hydrazine (DNPH) into chromophoric dinitrophenyl
hydrazones using by the method of Levine et al. (1990) and the
carbonyl content was calculated using the DNPH molar extinction
coefficient of 22,000 M− 1 cm− 1.
Reduced glutathione (GSH) was estimated as total non-protein
thiol (SH) group by the method described by Moron et al. (1979). For
the purpose of quantitation, a calibration curve was prepared using
GSH as a standard.
Superoxide dismutase (SOD) and glutathione peroxidase (GPx)
were determined by using commercially available methods.
Catalase activity was determined by monitoring the decomposition of H2O2 at 240 nm with a spectrometer (Aebi, 1983). Catalase
activity was measured by using 240 (molar extinction coefficient) =
0.0394 mmol− 1 min− 1 for H2O2 and expressed as U/mg protein
where one U of catalase activity decomposes 1 μmol of H2O2 per min
under standard conditions at 25 °C.
Protein contents of urine and kidney homogenates were determined 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 the
groups are made by means of One Way Analysis of Variance (ANOVA)
with post hoc Dunnett's test or by Student's t-test. The P-value less
than 0.05 is regarded as significant. The concentration-response
curves were analyzed by non-linear regression using GraphPad Prism
(GraphPad Software, San Diego, CA, USA).
3. Results
3.1. Antioxidant effect
Fig. 1. Represented images of CaOx crystals viewed in 3 h morning urine under light
microscope (400×), from vehicle control (A), stone forming (B) and berberine (10 mg/
kg) treated animals (C) in the preventive study.
method of Daly and Ertingshausen (1972) In non-acidified urine
samples, citrate, creatinine and uric acid (UA), while in serum,
creatinine and BUN were estimated with the help of kit-based
methods. Creatinine clearance (CC) was calculated using the formula
ðmg creatinine = dl urineÞ × ðml urine = 24 hÞ
CC ðml=minÞ =
:
ðmg creatinine = dl serumÞ × 1440
In the kidney homogenates, MDA content was estimated by
thiobarbituric acid reactive method (Wong et al., 1987) and the
amount of MDA was determined from the standard curve of 1,1,3,3tetraethoxy propane.
Berberine at concentrations of 50 and 150 μg/ml inhibited in vitro
lipid peroxidation induced in rat kidney homogenate similar to the
control drug BHT, as shown in Table 1.
Table 1
Inhibitory effect of berberine and butylated hydroxytoluene (BHT) on in vitro lipid
peroxidation induced in rat kidney homogenate.
Concentration
50 mg/ml
150 mg/ml
a
P b 0.01 vs. BHT.
% Inhibition
Berberine
BHT
53.5 ± 2.12a
97.5 ± 3.54a
36 ± 3.6
74.6 ± 4.7
S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
3.2. Diuretic effect
Results of the study for diuretic effect of berberine are given in
Table 2. At the doses of 5 (P b 0.05), 10 (P b 0.01) and 20 mg/kg
(P b 0.01), berberine increased the urine output similar to hydrochlorothiazide, indicating diuretic effect. In addition to its effect on urine
volume, berberine also increased urine excretion of Na+ and K+ but
decreased Ca2+, like that exhibited by hydrochlorothiazide. Berberine
(5 and 10 mg/kg) also caused an increased in the urine pH as did
hydrochlorothiazide. With further dose increment (20 mg/kg), berberine, despite of exhibiting diuretic effect, did not increase urine pH
above that of the control animals.
3.3. Effect on nephrolithic rats
Various parameters recorded from groups of animals at the end of
the treatments to determine the preventive and curative effects of
berberine are given in Tables 3 and 4, respectively. Among groups in
the preventive study, after 3 weeks on stone inducing regimen, there
were significantly abundant (P b 0.01) and visibly bigger CaOx crystals
predominately of calcium oxalate dihydrate as compared to the
vehicle 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 (P b 0.01 vs. stone
forming) as well as visibly reduced crystal size (Fig. 1C). Volume of
24 h urine and water intake was higher in stone forming group
compared to those of the vehicle control animals (P b 0.01). Urine pH
was also reduced though not to a significant extent. A simultaneous
treatment with berberine significantly prevented the increase in urine
volume and water intake (P b 0.05 vs. stone forming), although both
the parameters remained higher than those of the vehicle control
(P b 0.05). Berberine also caused a significant increase in urine pH
compared to the stone group (P b 0.05). In parallel with crystalluria,
oxalate excretion was also significantly enhanced (P b 0.01) in stone
forming animals whereas Ca2+ excretion was decreased (P b 0.05).
Urine contents of citrate, UA and Mg2+ did not alter to any significant
extent. In berberine treated group, ethylene glycol treatment
increased oxalate excretion but not to a significant extent compared
to those of the vehicle control but urinary Ca2+ excretion was
significantly reduced (P b 0.05).
Lithogenic treatment caused impairment of renal functions of the
untreated rats as evident from the markers of glomerular and tubular
damages: raised BUN and serum creatinine and reduced creatinine
clearance (P b 0.01), which were prevented in the animals simultaneously treated with berberine.
A significant loss in body weights (P b 0.01) by the stone inducing
treatment was observed in untreated group during the study period,
while in berberine treated group there was a net gain in body
weight over 3 weeks of treatment similar to that of the control
animals.
Table 2
Diuretic effect of berberine and the reference diuretic hydrochlorothiazide (HCT).
/100 g body
weight/6 h
Vehicle
control
Urine volume
(ml)
Na+ (mmol)
K+ (mmol)
Ca++ (μmol)
pH
1.07 ± 0.31 1.47 ± 0.4a
0.09 ± 0.02
0.07 ± 0.01
9.32 ± 1.6
5.9 ± 0.3
Berberine
(5 mg/kg)
HCT
(10 mg/kg)
(20 mg/kg)
Values given are mean ± S.E.M. (n = 6).
a
P b 0.05 vs. vehicle control.
b
P b 0.01 vs. vehicle control.
Kidneys excised from stone formers were enlarged and heavier
(P b 0.01) in stone forming animals, whereas, berberine treated group
kidneys were not significantly different from those of the vehicle
control. When observed under polarized light microscope, many
birefringent crystalline deposits in the histological preparations were
seen 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 2
out of 6 rats treated with berberine which were also less abundant
(P b 0.05) and visibly smaller (Fig. 3). The crystals were shown as CaOx
when stained black with Pizzolato's method. The renal tubules were
also markedly dilated in the entire kidneys of the rats in stone group.
Stone inducing treatment enhanced MDA and PCC contents of
kidneys (P b 0.01), and decreased reduced glutathione levels and
activity of the antioxidant enzymes of untreated group (P b 0.05).
Berberine treatment protected against the changes associated with
oxidative stress.
In berberine control group there was a significant increase in urine
volume and pH and decrease in Ca2+ excretion (P b 0.05) compared to
the vehicle control group, whereas other urinary, serum and renal
parameters were not different.
In the curative study, withdrawal of stone inducing treatment
initiated spontaneous recovery of the animals in untreated control as
suggested by a net gain in the body weights during post induction
period and slight, though not significant, decrease in urine volume,
water intake and increase in urine pH. Urine crystal count was also
reduced to a level similar to the control animals and similarly urinary
content of oxalate was decreased (P b 0.05) compared to that of the
stone forming animals in the preventive study after 3 weeks of
lithogenic treatment. Discontinuation of lithogenic treatment also
reversed decreased urinary Ca2+ content, there was rather a slight,
although insignificant, increase in urinary Ca2+ excretion. There was
an improvement in kidney functions as reflected by the levels of
serum creatinine and creatinine clearance, which became comparable
to those of the vehicle control animals. Kidney weight was also
significantly less (P b 0.01) than the untreated group of the preventive
study. CaOx crystal deposits could be seen in kidneys of all rats in
untreated group even after two weeks of lithogenic treatment free
period, which were however less extensive (P b 0.05) than the
untreated group in the preventive study and similarly renal tubules
were less dilated (Fig. 4). A spontaneous reduction in oxidative stress
over two weeks of post induction period was also evident from
decreased MDA and PCC (P b 0.05) and increased levels of reduced
glutathione and antioxidant enzyme activities measured in kidney
homogenates of the untreated animals. Post induction treatment with
berberine enhanced the spontaneous recovery of urolithic animals.
After two weeks of berberine treatment, kidney weight, BUN and
oxalate excretion were similar to those of the vehicle control animals
but similar to that of treated animals in the preventive study, mean
urine volume remained significantly higher than that of the vehicle
control, pH was significantly higher than that of the untreated group
and calcium contents were lower than both the vehicle control and
the untreated group (p b 0.05).
CaOx deposits were found in kidneys of 4 out of 6 animals in the
treated group which were however significantly less than the
untreated control of the curative study (P b 0.05).
4. Discussion
(10 mg/kg)
1.79 ± 0.56b 1.91 ± 0.39b 2.38 ± 0.89b
0.12 ± 0.01
0.15 ± 0.02a 0.22 ± 0.04a
0.08 ± 0.01 0.103 ± 0.01 0.15 ± 0.02a
6.84 ± 0.68
4.7 ± 1.10a 4.55 ± 0.5a
6.35 ± 0.4a
6.4 ± 0.39b
5.7 ± 0.41
171
0.34 ± 0.02b
0.19 ± 0.03b
2.29 ± 0.52a
6.74 ± 0.7b
Berberine is the main constituent of several plant species
traditionally reputed as therapy for urinary stone disease. To explore
its potential antiurolithic effect, berberine was tested for the
antioxidant and diuretic effects, being properties likely to contribute
in its medicinal effect, and on animal model of urolithiasis, to confirm
stone preventive and curative activities in vivo.
Animal and cellular studies have shown that exposure to high
levels of oxalate and CaOx crystals produce cellular injury mediated
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S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
Table 3
Various parameters recorded after 21 days of treatment for the preventive effect of berberine.
Change in b.wtA (%)
CUB (count/mm3)
Kidney wts (g)
CDC/field (10×)
Water intake (ml)
Urine/24 h
Kidney function tests
MDAI (nmol/mg protein)
PCCJ (nmol/mg protein)
GSHK (nmol/mg protein)
SODL (U/mg protein)
GPXM (U/mg protein)
Catalase (U/mg protein)
Volume (ml)
pH
Ca++ (mg)
Mg++ (mg)
OxD (mg)
Citrate (mg)
UAE (mg)
BUNF
SCG mg/dl
CCH ml/min
Vehicle control
Stone forming control
Berberine treated (10 mg/kg)
Berberine control (10 mg/kg)
16.7 ± 3.89
38 ± 8.5
0.68 ± 0.02
0
7.1 ± 0.76
6.67 ± 0.5
6.35 ± 0.13
3.33 ± 0.39
3.7 ± 0.65
0.36 ± 0.11
35 ± 4.7
0.58 ± 0.16
25.7 ± 1.82
0.91 ± 0.05
0.86 ± 0.04
0.56 ± 0.01
3.87 ± 0.59
21 ± 1.9
7.17 ± 0.65
1.23 ± 0.06
35.7 ± 1.9
6.10 ± 2.95a
172 ± 26a
1.41 ± 0.05a
53.6 ± 7.5a
21.5 ± 3.1a
22.4 ± 3.12a
5.67 ± 0.07
1.89 ± 0.16d
2.75 ± 0.49
1.41 ± 0.25a
27 ± 3.5
0.67 ± 0.19
63.7 ± 12.87d
1.43 ± 0.15a
0.53 ± 0.03a
13.3 ± 2.8a
11.20 ± 1.08a
11.6 ± 2.7d
3.91 ± 1.30d
0.64 ± 0.14a
22.2 ± 3.7d
11.5 ± 3.09b
85 ± 13b
0.80 ± 0.10b
6.17 ± 2.57c
12.3 ± 2.65c,d
13.7 ± 2.25c,d
6.59 ± 0.16c
2.05 ± 0.21d
3.21 ± 0.23
0.54 ± 0.16b
30 ± 2.9
0.61 ± 0.23
24.8 ± 3.1c
0.95 ± 0.05b
0.81 ± 0.06b
1.63 ± 0.09b
6.39 ± 1.36b
17.2 ± 1.2
6.12 ± 0.49
0.93 ± 0.15b
29.7 ± 4.6c
20.7 ± 2.89
41 ± 7.6
0.65 ± 0.03
0
12.6 ± 3.5a
13.1 ± 2.5d
6.67 ± 0.11d
2.19 ± 0.25d
4.11 ± 0.62
0.48 ± 0.05
33 ± 4.2
0.59 ± 0.18
27.8 ± 2.49
0.93 ± 0.04
0.83 ± 0.07
0.45 ± 0.07
3.54 ± 0.71
20 ± 1.8
7.49 ± 0.67
1.26 ± 0.10
29.4 ± 1.6
Values given are mean ± S.E.M. (n = 6).
A
Body weight; BCrystalluria; CCrystal deposits; DOxalate; EUric acid; FBlood urea nitrogen; GSerum creatinine; HCreatinine clearance; IMalondialdehyde; JProtein carbonyl content;
K
Reduced glutathione; LSuperoxide dismutase; and Mglutathione peroxidase.
a
P b 0.01 vs. vehicle control.
b
P b 0.01 vs. stone forming group.
c
P b 0.05 vs. stone forming group.
d
P b 0.05 vs. vehicle control.
by membrane lipid peroxidation through intracellular oxygen free
radical generation. It has been demonstrated that epithelial cell injury
facilitates the events of CaOx crystal nucleation, aggregation by
lowering concentration at which crystal forms and promotes crystal
retention in renal tubules crucial for subsequent stone development
(Khan and Hackett, 1991; Khan 1995; Moro et al., 2005). Recently
obtained human data are also suggestive of the development of
oxidative stress in kidney stone patients (Huang et al., 2003).
Antioxidant potential of berberine was estimated by lipid peroxidation inhibitory activity. Berberine inhibited ferrous-ascorbate induced
lipid peroxidation of rat kidney in vitro, similar to BHT, a standard
antioxidant. Several experimental studies have shown that antioxidants such as vitamin E, catechin and selenium can protect against
oxidative injury by oxalate and crystal deposition while some urinary
macromolecules such as glycosaminoglycans, bikunin, uropontin,
uromodulin etc. are also shown to possess protective effect against
oxalate 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 excretion
of the rats. Increase in the urine volume was also accompanied by an
Table 4
Various parameters recorded after 14 days of post stone induction treatment for the curative effect of berberine.
Gain in b.wtA (%)
CUB (count/mm3)
Kidney wts (g)
CDC/field (10x)
Water intake (ml)
Urine/24 h
Kidney function tests
MDAI (nmol/mg protein)
PCCJ (nmol/mg protein)
GSHK (nmol/mg protein)
SODL (U/mg protein)
GPXM (U/mg protein)
Catalase (U/mg protein)
Volume (ml)
pH
Ca++ (mg)
Mg++ (mg)
OxD (mg)
Citrate (mg)
UAE (mg)
BUNF
SCG mg/dl
CCH ml/min
Vehicle control
Untreated control
Berberine treated (10 mg/kg)
13.9 ± 2.7
25 ± 7.6
0.65 ± 0.04
0
8.56 ± 0.75
9.01 ± 1.53
6.53 ± 0.07
3.7 ± 0.73
2.97 ± 0.27
0.34 ± 0.03
28 ± 1.90
0.66 ± 0.16
23.6 ± 0.87
0.90 ± 0.06
0.89 ± 0.02
0.76 ± 0.05
4.35 ± 0.52
19 ± 2.7
9.42 ± 0.84
1.19 ± 0.08
31.5 ± 2.7
8.5 ± 3.3
33 ± 9.8
0.94 ± 0.04a
32 ± 5.9a
16.9 ± 2.3a
15.9 ± 1.32d
6.28 ± 0.15
5.2 ± 0.53
3.15 ± 0.45
0.69 ± 0.08a
26 ± 2.3
0.76 ± 0.13
39.13 ± 3.84b
1.03 ± 0.08
0.78 ± 0.10
6.5 ± 1.8a
7.08 ± 0.87d
11.7 ± 1.4d
6.37 ± 1.44
0.79 ± 0.14
25.2 ± 3.1
14.7 ± 2.2
38 ± 5.9
0.73 ± 0.02b
14.5 ± 4.3c
12.9 ± 1.53
13.8 ± 1.47d
6.78 ± 0.12c
3.9 ± 0.63
2.93 ± 0.18
0.43 ± 0.04b
29 ± 1.6
0.58 ± 0.07
23.01 ± 2.38b
0.83 ± 0.04
0.86 ± 0.05
3.16 ± 0.59
5.34 ± 0.44
17.0 ± 0.9
8.19 ± 0.58
1.09 ± 0.15
32.7 ± 1.8
Values given are mean ± S.E.M. (n = 6).
A
Body weight; BCrystalluria; CCrystal deposits; DOxalate; EUric acid; FBlood urea nitrogen; GSerum creatinine; HCreatinine clearance; IMalondialdehyde; JProtein carbonyl content;
K
Reduced glutathione; LSuperoxide dismutase; and Mglutathione peroxidase.
a
P b 0.01 vs. vehicle control.
b
P b 0.01 vs. stone forming group.
c
P b 0.05 vs. stone forming group.
d
P b 0.05 vs. vehicle control.
S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
173
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 kidney
sections from vehicle control, stone forming and treated groups, respectively and (D), (E) and (F) are the respective sections stained with Pizzolato's method. White and black arrows
point to crystal deposits and dilated tubules, respectively.
increase in the Na+ and K+ excretion similar to the standard diuretic,
hydrochlorothiazide, suggesting that berberine induced diuresis is
caused by its saluretic effect. Treatment with berberine and similarly
hydrochlorothiazide also increased urine pH and decreased urine Ca2+
content. Urinary supersaturation with stone forming minerals is a
primary requisition for crystal precipitation and a major risk factor for
the stone development (Hess and Kok, 1996). Thiazide diuretics due to
their hypocalciuric and diuretic effects reduce urinary supersaturation
of calcium salts and are commonly used to treat calcium stone disease
(Goldfarb and Coe, 2005).
Renal calcium oxalate deposition induced by ethylene glycol and
ammonium chloride in rats is frequently used to mimic the urinary
stone formation in humans (Thamilselvan et al., 1997; Atmani et al.,
2003; Tsai et al., 2008). Therefore, we evaluated both the prevention
and curative effects of berberine on urolithiasis in this model.
In preventive study, the analysis of crystalluria showed that
untreated animals were excreting abundant and larger crystals than
the treated animals. Crystalluria could occur similarly in both healthy
and stone forming individuals where the latter tend to excrete larger
and 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). The
presence of significantly less oxalate content in urine of berberine
treated rats is an obvious reason for less abundant crystals but the
presence of crystallization inhibitory constituents in berberine and its
pH increasing effect cannot be ignored as solubility of CaOx is
increased in alkaline medium (Tiselius, 2005). Berberine significantly
prevented the increase in urine volume associated with lithogenic
treatment (Fan et al., 1999), though urine output remained still higher
than that of control animals similar to the berberine control group,
suggestive of intrinsic diuretic effect of berberine as accounted for the
raised urine volume. Consistent with some previous reports, stone
induction by hyperoxaluric agents caused an increase in oxalate and
decrease in Ca2+ excretion in untreated group (Fan et al., 1999; Park
et al., 2008). When administered simultaneously, berberine prevented hyperoxaluric effect of the stone inducing treatment but did
not affect hypocalciuria which can also be attributed to its intrinsic
effect on Ca2+ excretion, as evident from the diuretic study.
There was hypertrophy and extensive calcium oxalate crystal
deposition in kidneys of untreated rats accompanied by oxidative
damage as reflected from increased levels of markers of peroxidative
injury: MDA and PCC, and decreased activities of antioxidant enzymes
174
S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
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 sections
from vehicle control, stone forming and treated groups, respectively and (D), (E) and (F) are the respective sections stained with Pizzolato's method.
and reduced glutathione levels as well as deteriorated renal functions.
The renal tubules were markedly dilated in the entire kidney of
all these rats, and this might have been caused by distal obstruction
of renal tubular flow by large crystals. Hyperoxaluria is a major
risk factor for calcium oxalate nephrolithiasis. Several in vivo and
in vitro studies have demonstrated that exposure to high level of
oxalate results in greater production of superoxide and hydroxyl
free radicals, leading to antioxidant imbalance and has been
manifested 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 in
membrane 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 renal
tubules (Wiessner et al., 2001; Khan 1995; Rashed et al., 2004;
Thamilselvan et al., 2000). The inhibitory effect of berberine on
calcium oxalate crystal retention in renal tubules thus could have also
been caused by its antioxidant activity.
In berberine control group, berberine treatment increased urine
volume and pH and decreased Ca2+ excretion, thus confirming the
diuretic, urine alkalinizing and hypocalciuric effects of berberine
observed in the diuretic and animal model studies.
In curative study, withdrawal of stone inducing treatment
evoked a spontaneous recovery of nephrolithic animals in the
untreated groups. There was a gain in body weight, significant
decrease in urinary oxalate, renal crystal deposition, oxidative stress
and kidney weights, and improvement in renal functions compared
to the untreated rats in the preventive study after 21 days on stone
inducing regimen, although the renal CaOx deposits, urine volume
and oxalate, BUN, and oxidative stress were significantly higher than
those of the control animals. Despite of high urinary oxalate content,
the increased urinary volume of the untreated animals appears as
accounted for the presence of negligible crystalluria as that of the
control animals. Post induction treatment with berberine extract
enhanced spontaneous recovery of the rats. In berberine treated
group, after two weeks of ethylene glycol discontinuation, kidney
functions, kidney weights, urinary oxalate and MDA content and
oxidative enzyme levels of kidneys became similar to those of the
respective control animals. Kidney deposits of CaOx crystals were
also significantly lower than the respective untreated group but
were present in greater number of the animals than the treated
group of the preventive study receiving similar dose of berberine,
suggesting that the prophylactic effect of berberine is stronger than
its curative effect.
S. Bashir, A.H. Gilani / European Journal of Pharmacology 651 (2011) 168–175
5. Conclusion
Results of this study suggest that plant alkaloid berberine is
therapeutically effective for both prevention as well as treatment of
calcium oxalate urolithiasis, exhibiting these effects through a
combination of antioxidant, diuretic, hypocalciuric and urine alkalanizing activities. These findings also suggest berberine as active
principal of the plants used in urolithiasis, however, the coexistence of
other biologically active constituents cannot be excluded. As berberine is in wide clinical use for some other pathological conditions and
except a few contraindications it is generally considered safe, these
data invite clinical trials on berberine to establish its efficacy for
human use.
Acknowledgements
This study was financed in part by the Higher Education Commission
of Pakistan and the University Research Council of the Aga Khan
University, Karachi, Pakistan.
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