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Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

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Alteration in Oxidative/nitrosative imbalance, histochemical


expression of osteopontin and antiurolithiatic efficacy of Xanthium
strumarium (L.) in ethylene glycol induced urolithiasis
Padma Nibash Panigrahia,b,* , Sahadeb Deya , Monalisa Sahooc,
Shyam Sundar Choudharya , Sumit Mahajana
a
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly Uttar Pradesh-243122, India
b
Division of Medicine, Faculty of Veterinary and Animal Science, Banaras Hindu University, Varanasi, Uttar Pradesh-221005, India
c
Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India

A R T I C L E I N F O A B S T R A C T

Article history:
Received 20 September 2016 Xanthium strumarium has traditionally been used in the treatment of urolitiasis especially by the rural
Received in revised form 24 October 2016 people in India, but its antiurolithiatic efficacy was not explored scientifically till now. Therefore, the
Accepted 8 November 2016 present study was designed to validate the ethnic practice scientifically, and explore the possible
antiurolithiatic effect to rationalize its medicinal use. Urolitiasis was induced in hyperoxaluric rat model
Keywords: by giving 0.75% ethylene glycol (EG) for 28 days along with 1% ammonium chloride (AC) for first 14 days.
Xanthium strumarium Antiurolithiatic effect of aqueous-ethanol extract of Xanthium strumarium bur (xanthium) was evaluated
Urolithiasis based on urine and serum biochemistry, oxidative/nitrosative stress indices, histopathology, kidney
Rat
calcium and calcium oxalate content and immunohistochemical expression of matrix glycoprotein,
Immunohistochemistry
osteopontin (OPN). Administration of EG and AC resulted in hyperoxaluria, crystalluria, hypocalciuria,
Osteopontin
Oxidative/Nitrosative stress polyurea, raised serum urea, creatinine, erythrocytic lipid peroxidise and nitric oxide, kidney calcium
content as well as crystal deposition in kidney section in lithiatic group rats. However, xanthium
treatment significantly restored the impairment in above kidney function test as that of standard
treatment, cystone. The up-regulation of OPN was also significantly decreased after xanthium treatment.
The present findings demonstrate the curative efficacy of xanthium in ethylene glycol induced
urolithiasis, possibly mediated through inhibition of various pathways involved in renal calcium oxalate
formation, antioxidant property and down regulation of matrix glycoprotein, OPN. Therefore, future
studies may be established to evaluate its efficacy and safety for clinical use.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction epidemiological studies have shown that majority (70%) of stones


commonly contain calcium oxalate (CaC2O4) [1,2]. In spite of
Urolithiasis is one of the important constraints in human and substantial progress in the study of physiology of urolithiasis, its
animal health globally since last two decades, irrespective of exact mechanism is still not clearly understood. The recent
geographical, racial and cultural boundaries [1,2]. It is also proposed mechanism of stone formation involves urinary super-
considered as the third most common problem of the urinary saturation, crystal nucleation, precipitation, growth, aggregation of
tract with an estimated lifetime risk of around 1–5% in Asia, 8–15% crystals and their retention in renal tubular epithelial cells [1,5].
in America and Europe, and 20% in the Middle East countries [3,4]. These processes are modulated by a variety of urinary macro-
The incidence of urolithiasis had been increases in India now a day molecules which become incorporated in the growing crystals and
and two “stone belts” had also been scientifically identified in India eventually constitute organic component or matrix. Osteopontin
[3]. Oxalate, struvite, urate, brushite, cystine were the most (OPN) is a negatively charged aspartic acid rich important matrix
commonly reported urolith in man and animal. However, glycoprotein usually associated with calcium oxalate stone
formation [6–8]. It is mainly present in the loop of Henle and
distal nephrons in normal kidneys in animal and humans, but its
* Corresponding author at: Division of Medicine, Faculty of Veterinary and expression is significantly up regulated after renal damage.
Animal Science, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India. Although, OPN is a well known component of stone matrix, but
E-mail address: pnpvetmed@gmail.com (P.N. Panigrahi).

http://dx.doi.org/10.1016/j.biopha.2016.11.029
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1525

whether it acts as a promoter or inhibitor of stone formation is 2.3. Phytochemical screening


controversial till date [6,9,10].
Modern diagnostic and therapeutic aids like extracorporeal The preliminary phytochemical screening of xanthium was
shock wave lithotripsy, ureteroscopy and percutaneous nephro- carried out as per the standard procedures described by Harborne
lithotomy had revolutionized the urological practices but cannot [27]. For qualitative screening, Mayer’s test and Dragendroff test
altered the recurrence of stone formation and also have adverse were done to determine the presence of alkaloids; Benedict test,
side effects. Pharmaceutical agents like thiazide and citrate Barfoed test and Fehling test for carbohydrate; Foam test for
diuretics, alkali therapy, allopurinol etc have also limited efficacy saponin; Biuret test and Ninhydrin test for protein; Alkaline
in addition to their less tolerability [11]. Indeed, urolithiasis is a reagent test for flavonoids; Sodium hydroxide test for coumarin;
multifactorial disease and involves in alteration of several Noller’s test for triterpinoids; and Gelatine test was done for
biochemical pathways. Therefore, the present situation demands detection of tannins.
a newer approach of therapy. Several recent studies have
highlighted the effectiveness of medicinal plants and natural 2.4. Study on animal model of urolithiasis
compounds for treatment and management of urolithiasis [2–
4,12–15]. Moreover, herbal remedies are known to contain Antiurolithiatic activity of the test plant material was deter-
multiple constituents, acting through multiple pathways such as mined by hyperoxaluric rat model of calcium oxalate urolithiasis as
antioxidant, analgesic, diuretic, pH neutralising etc. Xanthium described previously [4,28]. Thirty male rats were divided with
strumarium has traditionally been used as an herbal medicine in matched body weights into five groups of six animals each, which
India, China, Europe, Malaysia and America [16]. The whole plant were then randomly selected to receive various treatments. Rats of
especially the root, bur and leaves had been reported to possess Group I served as healthy control without receiving any treatment.
antiulcerogenic [17], anti-cell proliferative [18,19], anti-inflamma- Group II rats act as lithiatic control received stone-inducing
tory and analgesic [20], antidiabetic and hypoglycaemic [16], treatment comprised of 0.75% (w/v) ethylene glycol (EG) along
antiarthritic [21], diuretic and renoprotective [3,16], antimicrobial with 1% (w/v) ammonium chloride in drinking water for 14 days for
[22], antitrypanosomal [23], antihelmintic [24] and anti-plasmo- induction of crystal followed by EG alone for next 14 days. Group III
dial [25] properties. The bur extract had traditionally been used in rats received stone inducing treatment for 14 days followed by
the treatment and management of urolithiasis and renal ailments 500 mg/kg hydo-ethanolic (1:1) extract of xanthium (EC50) orally
in some parts of India [3] but its antiurolithiatic efficacy was not for next 14 days orally through gastric gavages (The EC50 dose was
explored scientifically till now. Hence the study was designed to calculated from earlier study, detailed not presented in this
validate the ethnic practice scientifically in ethylene glycol induced manuscript, Panigrahi et al., 2016, PhD thesis submitted to Deemed
lithiatic rat model, and to explore the possible mechanism of University- Indian Veterinary Research Institute). Group IV rats
antiurolithiatic effect of xanthium to rationalize its medicinal use. received stone inducing treatment for 14 days followed by
standard drug-cystone at 100 mg/kg for next 14 days orally
2. Materials and methods through gastric gavages. Group V served as vehicle control and
received stone inducing treatment for 14 days followed by drinking
2.1. Animals water for next 14 days without any treatment to check auto healing
process.
The animal care and handling was performed in accordance
with the internationally accepted standard guidelines for use of 2.5. Sampling
animals and the protocol was approved by institutional animal
ethical committee, Indian Veterinary Research Institute, India Twenty four hour urine samples were collected at day 0, day 14
(vide letter no- F.26-1/2015-16/JDR). Thirty male albino Wistar rats and day 28, housing rats individually in the metabolic cages.
(weighing 150–200 g) were procured from the laboratory animal Following volume and pH determination, part of each urine sample
research division and housed in clean polypropylene cages under was acidified to pH 2 with 5 M hydrochloric acid. Both acidified and
controlled temperature 25  2  C, humidity 45–55% and 12 h light- non-acidified urine sample were centrifuged at 1500g for 10 min to

dark cycle throughout the experimental period. Animals were remove debris and supernatants and were stored at 20 C until
given standard diet and had free accesses to food and water ad analysed. Blood was collected through cardiac puncture from
libitum throughout the study. However, food was withdrawn while individual animals under ether anaesthesia at day 0, day 14 and day
collecting 24 h urine samples inside metabolic cage. 28 for harvesting serum required for kidney function test. Animals
were sacrificed after end of study and both the kidneys were
2.2. Plant material and extraction excised for histopathology, estimation of tissue calcium content
and Immunohistochemistry.
The plant Xanthium strumarium was collected from horticulture
section of the institute and was identified and authenticated from 2.6. Biochemical analysis of urine and serum
Botanical Survey of India, Central National Herbarium, Howrah,
India. The bur of X. strumarium was separated from whole plant, In acidified urine sample calcium, phosphorus, sodium and
washed with distilled water, dried and uniformly powdered using potassium were determined using commercial kits. Oxalate
an electric grinder. Hydro-ethanol extract of the powdered X. content of acidified urine was estimated using quantitative ELISA
strumarium bur (xanthium) was extracted in room temperature kit (Quibiotech, China). Microscopic examination of urine was

(37 C) for 24 h with occasional shaking using solvent system performed in non acidified first 3 h morning samples. Urine urea
(aqueous: ethanol 1:1) and filtered using filter paper (Whatman nitrogen (UUN) and urine creatinine was estimated in non acidified
no. 40). The solvent was evaporated using rotary evaporator under urine sample. Blood urea nitrogen (BUN), creatinine, total protein
pressure. The extract was dried in vacuo and stored refrigerated and albumin were estimated in serum using commercial kits.
until use [26]. The recovery percentage was 18% (w/w).
1526 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

2.7. Estimation of oxidative/nitrosative stress indices 2.9. Immunohistochemistry for OPN

Lipid peroxidases (LPO) level in 10% RBC haemolysate was The left kidneys were fixed in 10% neutral buffered formalin,
estimated spectrophotometrically following the method of Placer sectioned at 5 mm thicknesses, deparaffinised by immersion in
[29]. Lipid peroxidation was calculated using 1.56  105 as xylene and dehydrated in a graded series of ethanol. Then, the
extinction coefficient to express the value in nanomole of kidney sections were boiled in 10 mM sodium citrate buffer (pH
Malonaldehyde (MDA) per millilitre of haemolysate [30]. Super- 6.0) for 10 min for antigen retrieval and cooled at room
oxide dismutase (SOD) was measured in the supernatant of 10% temperature. Endogenous peroxidase activity was inhibited by
RBC haemolysate following the method of Marklund [31] with incubation with 0.3% H2O2 for 10 min to prevent non-specific
certain modifications suggested by Menami and Yoshikawa [32]. bindings sections were blocked by incubation in 3% normal goat
Each unit of SOD activity is defined as the quantity of enzyme that serum for 60 min. The sections were stained with primary antibody
inhibits auto oxidation of pyrogallol by 50% under suitable for OPN (mouse monoclonal to osteopontin, 1: 50 dilutions, Santa

experimental conditions. Catalase (CAT) activity in 10% RBC Cruz Biotechnologies, USA) and incubated for overnight at 4 C.
haemolysate was estimated spectrophotometrically at wave length After three successive washing with PBS for 10 min, the sections
of 240 nm after appropriate dilution following the method of were incubated with goat-anti mouse immunoglobulin-G horse-
Cohen [33] and the values were expressed in units per milligram of radish peroxidase-conjugated secondary antibody (1: 200 dilu-
haemoglobin. Glutathione (GSH) was estimated in packed RBC tions, Santa Cruz Biotechnologies, USA) for 1 h. Thereafter, sections
following DTNB (di-thiobis2-nitro benzoic acid) method [34]. were washed 3 times with PBS and stained with 3.30 -diamino-
Nitric oxide (NO) level of blood plasma was measured by nitrate benzidine (Abcam Biotechnologies, India) and counterstained with
reduction on copper cadmium alloy (Cu–Cd alloy) followed by Mayer’s hematoxylin solution (Sigma Aldrich biochemical, USA).
colour development with Griess reagent (0.1% naphthalene Finally, the sections were mounted with CC/mount (Sigma Aldrich
diamine dihydrochloride in 3 N hydrochloric acid and 1% biochemical, USA) and photographed under light microscope.
sulphanilamide 1:1) as described by Sastry [35].
2.10. Statistical analysis
2.8. Histopathology and calcium oxalate crystal deposition study
The data were expressed as mean  standard error of mean
The kidneys were rinsed in ice cold physiological saline and (SEM) and median effective concentration (EC50 value) with 95%
weighed. The left kidney was fixed in 10% neutral buffered confidence interval (CI). All statistical comparisons between the
formalin, sectioned at 5 mm thickness and stained with Hematox- groups were made by t-test (comparison between two groups) or
ylin and Eosin (H and E) for histopathological examination and also ANOVA with post hoc Dunnett’s test using SPSS. P value <0.05 is
stained with Pizzolato’s method for in situ demonstration of considered as significant.
calcium oxalate crystal deposition [36]. The right kidney was taken
for estimation of tissue calcium content by atomic absorption 3. Results
spectrophotometer.
Calcium oxalate crystal deposition within the kidneys was 3.1. Phytochemical screening
determined using the semiquantitative scoring methods described
previously [14]. Briefly, ten randomly selected microscopic fields The preliminary phytochemical screening of xanthium revealed
were examined under 10X magnification and crystal deposits were positive for alkaloids, flavonoids, triterpenoids, tannins, saponins
graded according to the scale: 0 = <1 crystal, 1 = 1-10, 2 = 11–30, and proteins, and negative for carbohydrate and coumarin.
3 = 31–50, 4 =  51–75 and 5 = >75 crystals [4].

Table 1
Effect of Xanthium strumarium and standard drug on 24 h urinary biochemical parameters.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle


Volume (ml) 14 6.533  1.091 11.333  1.453** 11.333  0.882** 10.700  0.814* 10.900  1.320*
28 6.500  0.764 14.867  0.926** 8.633  0.895## 7.533  0.712## 11.600  0.757**
pH 14 7.100  0.115 6.567  0.088 6.433  0.338 6.367  0.384 6.700  0.355
28 6.600  0.305 5.467  0.437* 6.300  0.264# 6.600  0.312# 5.817  0.361*
Oxalate (mg) 14 0.727  0.044 1.700  0.156** 2.123  0.072** 2.050  0.201** 1.883  0.034**
28 0 0.697  0.045 2.703  0.086** 0.903  0.075## 0.740  0.064## 2.007  0.118**##
Calcium (mg) 14 6.935  0.452 3.837  0.238** 4.753  0.320** 4.287  0.424** 4.006  0.471**
28 7.239  0.704 3.374  0.492** 5.750  0.308## 6.084  0.206## 3.670  0.329**
Phosphorus (mg) 14 4.963  0.422 6.102  0.629 6.042  0.251 5.262  0.746 5.683  0.361
28 5.014  0.191 7.411  0.329 5.633  0.477 5.077  0.310 6.501  0.290
UUN (mg) 14 8.526  0.346 12.483  0.800* 14.219  1.671** 14.567  0.652** 12.721  1.193*
28 7.335  0.736 20.677  1.753** 11.894  0.904*## 7.803  0.460## 18.262  2.678**
Creatinine (mg) 14 0.344  0.052 0.681  0.088** 0.774  0.026** 0.814  0.089** 0.799  0.034**
28 0.362  0.014 1.389  0.026** 0.697  0.055**## 0.544  0.037*## 1.268  0.064**
Potassium (mg) 14 10.2190.147 9.483  0.219 10.192  0.108 10.399  0.395 9.840  0.254
28 13.057  0.116 12.267  0.333 12.733  0.316 12.556  0.378 11.482  0.376
Sodium (mg) 14 81.10  9.71 95.40  7.32 98.77  13.15 89.59  5.71 88.93  5.96
28 74.78  12.50 101.91  9.09 89.65  8.45 89.04  1.80 84.38  0.90

UC = untreated healthy control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle =
vehicle control group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and # #p < 0.01 vs. Lithogenic group (group B). UUN = urinary urea nitrogen.
P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1527

Table 2
Effect of Xanthium strumarium and standard drug on serum biochemical parameters.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle


BUN (mg/dl) 14 16.194  0.531 45.456  1.564** 41.615  3.247** 48.893  2.141** 41.298  1.957**
28 21.795  1.873 53.655  3.459** 28.856  3.314*## 23.776  1.596## 36.581  1.815**##
Creatinine (mg/dl) 14 0.324  0.032 0.466  0.023* 0.614  0.014** 0.562  0.050** 0.541  0.046**
28 0.282  0.047 1.445  0.075** 0.407  0.018*## 0.354  0.052## 0.801  0.043**##
Total Protein (g/dl) 14 4.239  0.212 5.949  0.244** 5.747  0.125** 5.379  0.114** 5.131  0.195**
28 5.220  0.210 6.742  0.177** 5.636  0.105## 5.626  0.168## 6.334  0.237**
Albumin (g/dl) 14 2.735  0.056 3.061  0.136 3.123  0.089 3.048  0.218 3.123  0.125
28 3.152  0.169 3.518  0.167 3.053  0.150 3.127  0.071 3.361  0.095

UC = untreated control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle = vehicle control
group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and ##p < 0.01 vs. Lithogenic group (group B).

3.2. Urine and serum biochemistry stone inducing treatment and further decreased (p < 0.01) on day
28 in lithiatic control rats as compared to untreated rats. But, they
All the day 0 parameters of urine and serum biochemistry were increased significantly after treatment with xanthium and
recorded just before the start of experiment were statistically the values were statistically similar to standard treatment and
similar among all the groups; hence the values were not given in untreated control (Table 3).
the table. Tables 1 and 2 summarise the change in urine and serum
biochemistry among different groups at different interval of study, 3.4. Histopathology and calcium oxalate crystal deposition study
respectively. The volume of urine, urinary oxalate concentration,
UUN and urine creatinine concentration increased significantly The microscopy of histological section of kidney revealed
(p < 0.01) on day 14 in all four grouped rats received stone inducing presence of calcium oxalate crystals mostly in renal tubules in
treatment as compared to healthy untreated control rats (Table 1). cortex region and less commonly in medulla both in Pizzolato’s
The values were again increased on day 28 in lithiatic control (Fig. 1) and H&E (Fig. 3) method of staining in lithiatic rats. The
(group B) and vehicle control (group E) rats but treatment with healthy control rats did not show any calcium oxalate crystals in
xanthium significantly reduced the above parameters as that of kidney section whereas, a high score of crystal deposits were
standard treatment (group D). On contrary, the pH of urine and Ca recorded in lithiatic and vehicle group by Pizzolato staining (Fig. 2).
concentration decreased significantly (p < 0.01) after stone induc- However, xanthium treatment significantly (p < 0.01) reduce the
ing treatment, but the above parameters were increased signifi- calcium oxalate crystal deposits as compared to lithiatic group and
cantly after treatment with xanthium and the values were the crystal deposits score is statistically similar with standard
statistically similar to standard treatment. The inorganic phos- treatment (Fig. 2). Lithiatic treatment affected proximal nephrons,
phate, sodium and potassium concentration differ non-signifi- distal nephrons, collecting ducts and CaC2O4 crystals induced
cantly before and after treatment in all groups and the values were injury to the renal epithelium resulting in leakage of cellular
statistically similar (Table 1). EG treatment cause significant content into the lumen which was observed from the histology
impairment of renal functions as evident from significantly raised examination of the kidney tissue section stained with H & E stain
BUN, creatinine and TP concentration in all four grouped rats (Fig. 3). There was severe mononuclear cell infiltration, necrosis,
except healthy control on day 14; and lithiatic group on day 28, tubular degeneration, dilatation of tubules casts, and fibrin
which were prevented after treatment with xanthium and cystone deposits in kidney of lithiatic control (Fig. 3B) and vehicle control
(Table 2). On contrary, only drinking water (group E) could not rats (Fig. 3E). The renal parenchyma was almost intact, tubules
resolve the problem as observed from kidney function test. appeared normal and significant reduction in cellular infiltration
and haemorrhages were observed in rats treated with xanthium
3.3. Alteration of oxidative/nitrosative indices (Fig. 3C) and standard treatment (Fig. 3D). While there was
complete absence of crystals observed in the healthy control rats
The mean LPO and NO activity increased significantly (p < 0.01) (Fig. 3A).
in all the rats received stone inducing treatment after induction of The tissue Ca concentration was significantly increased in
urolithiasis (Table 3). However, xanthium treatment significantly lithiatic rats and vehicle control (p < 0.01) as compared to healthy
reduced the above oxidative/nitrosative stress and the mean values control (Fig. 4). However, treatment with xanthium significantly
were statistically similar to rats treated with standard drug, reduces the tissue Ca content and the value is statistically similar to
cystone. On contrary, the antioxidant enzymes GSH, SOD and CAT healthy control as that of standard treatment.
decreased significantly (p < 0.01) on day 14 in all the rats received

Table 3
Effect of Xanthium strumarium and standard drug on oxidative/nitrosative indices.

Parameters Day UC Lithiatic Xanthium Cystone Vehicle


LPO (nmol MDA/mg Hb) 14 2.368  0.275 3.887  0.193** 3.661  0.207** 4.095  0.171** 4.063  0.189**
28 3.155  0.175 5.783  0.209** 3.688  0.276## 3.181  0.242## 4.362  0.216**##
GSH (mmol/mg Hb) 14 0.304  0.034 0.162  0.032** 0.191  0.028 ** 0.206  0.048* 0.182  0.011**
28 0.310  0.027 0.139  0.021** 0.213  0.020 * 0.214  0.022 * 0.150  0.010 **
CAT (U/mg Hb) 14 1.252  0.034 0.773  0.042** 0.839  0.037** 0.722  0.026** 0.834  0.036**
28 1.205  0.118 0.534  0.041** 1.022  0.114## 1.137  0.068## 0.654  0.044**
SOD (U/mg Hb) 14 0.864  0.032 0.553  0.035** 0.587  0.027** 0.684  0.030** 0.524  0.023**
28 0.970  0.073 0.374  0.021** 0.720  0.031*## 0.838  0.048## 0.425  0.026**
NO (mmol/ml) 14 3.288  0.039 6.155  0.197** 6.384  0.320** 6.438  0.225** 6.448  0.190**
28 2.876  0.038 10.285  0.519** 3.791  0.148## 3.047  0.216## 7.320  0.404**#

UC = untreated control, Lithiatic = lithogenic control group, Xanthium = 500 mg/kg Xanthium strumarium, Cystone = 100 mg/kg standard drug cystone, Vehicle = vehicle control
group. *p < 0.05 and **p < 0.01 vs Healthy control (group A), #p < 0.05 and ##p < 0.01 vs. Lithogenic group (group B).
1528 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

Fig. 1. Histology of representative kidney section stained with Pizzolato’s stain (X100).
Black colour particles showing calcium oxalate crystal. A = Untreated healthy control, B = Lithiatic control, C = 500 mg/kg Xanthium strumarium, D = 100 mg/kg Cystone,
E = Vehicle control

3.5. Immunohistochemistry and expression of OPN 4. Discussion

Immunohistochemical staining for detection of OPN revealed India has been known to be rich repository of medicinal plants
expression of OPN in all groups of rats (Fig. 5). However, it was since ancient civilization. Xanthium strumarium is a commonly
barely detectable in the healthy control group only mild expression available weed in India and has traditionally been used as a
in the distal limb of Henle in the outer medulla (Fig. 5A). On medicinal herb in most parts of India, China, Europe and America
contrary, its expression was intensely increased throughout the [16]. The whole plant especially the root, bur and leaves had been
renal tubules, proximal and distal convoluted tubules, collecting reported to possess antiulcerogenic [17], anti-cell proliferative
ducts in lithiatic group (group B) (Fig. 5B). Treatment with [18,19], anti-inflammatory and analgesic [20], antidiabetic and
xanthium significantly reduced the expression of OPN (Fig. 5C) hypoglycaemic [16], antiarthritic [21], diuretic and renoprotective
which was comparable to the standard treatment (Fig. 5D). The [3,16], antimicrobial [22], antitrypanosomal [23], antihelmintic
quantitative analysis of immunohistochemical expression of OPN [24] and anti-plasmodial [25] properties. The bur extract contains
in kidney tissue also revealed significant (p < 0.01) reduction in rich source of bioactive compounds [37] and has traditionally been
expression of OPN after treatment with xanthium in rats (Fig. 6). used in the treatment and management of urolithiasis and renal
ailments in some parts of India [3] but its antiurolithiatic efficacy
6 was not explored scientifically till now. Hence, the study was
designed to evaluate the antiurolithiatic and reno-protective
CaOx crystal deposition Score

efficacy of xanthium in ethylene glycol induced rat model.


5
Calcium oxalate stone formation is a multi-factorial process
4.67 involving various etiological factors. Hyperoxaluric rat model is the
4
## 4.17 most potent experimental model for preclinical evaluation of
## antiurolithiatic efficacy of medicinal herbs because the physiolog-
3
ical process mimics the etiology of kidney stone formation in
2.53 human and animal [38]. The hepatic enzymes metabolize EG to
2 2.27 oxalic acid by glyoxalate mechanism, which combine with calcium
ion in the renal tubular epithelium to form calcium oxalate crystals
1
[39]. The lithogenesis can be induced either by ethylene glycol
0 alone [38], or in combination with ammonium chloride [4,14,15].
0 Administration of EG and AC resulted in increased calcium oxalate
A B C D E
crystalluria, hypocalciuria and polyuria in the present study in
Fig. 2. Calcium oxalate crystal deposition score of kidney section stained with lithiatic group as compared to healthy control, which might be due
Pizzolato’s stain. to impaired renal function [4]. The impairment of renal function
Severity grade were assigned as 0 = <1 crystal, 1 = 1-10, 2 = 11-30, 3 = 31-50, was also clearly indicated by elevation of serum creatinine, BUN, TP
4 =  51–75 and 5 = >75 crystals; data are expressed as mean SEM. #p < 0.05,
in lithiatic group as compared to untreated healthy control, which
##p < 0.01 vs Lithiatic control (group B). A = Untreated healthy control, B = Lithiatic
group, C = 500 mg/kg Xanthium strumarium, D = 100 mg/kg cystone, E = Vehicle has been restored by xanthium treatment and the values were
control. statistically similar to standard cystone treatment. Similar finding
P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1529

Fig. 3. Histology of representative kidney section stained with H and E stain (X 100).
A. Kidney section of untreated healthy control rat showing normal architecture; B. Kidney section of Lithiatic control rat showing crystals, haemorrhages, tubular
degeneration and dilatation; C. Kidney section of rat treated with Xanthium @ 500 mg/kg showing almost normal architecture with mild tubular dilatations; D. Kidney section
of rat treated with Cystone @ 100 mg/kg showing almost normal architecture with mild tubular dilatations and mono nuclear infiltration; E. Kidney section of vehicle control
rat showing crystal, tubular dilatation and infiltration of cells.

were also reported by taking various herbs such as Origanum the major cause of hypercalciuria. In the present study, hyper-
vulgare [4], Astilbin containing rhizome of Smilax china [12], calciuria was not observed in the EG group as previously described
Holarrhena antidysenterica [14], Gokshuradi polyherbal ayurvedic in some of the study [42,43]. A possible explanation for this result
formulation [28], Punica granatum [40] and Adiantum capillus is that the formation of CaC2O4 crystal consumes free calcium (Ca)
veneris [41]. However, withdrawal of lithogenic treatment after and normalizes Ca levels in the urine. However, oxalate is a more
14 days in vehicle control group (group E) caused mild recovery in important risk factor in the process of urinary calculi formation
kidney function test was observed but the values were significantly than hypercalciuria [28,44,45]. Because, hyperoxaluria is associat-
different from healthy control as well as xanthium and cystone ed with production of free radical resulting in oxidative damage of
treatment group. Khan [4] also reported similar findings while renal epithelium thereby providing a nidus for crystal attachment
evaluate the curative effect of Origanum vulgare (Linn.) against and ultimately causes crystal aggregation and retention in renal
urolithiatic rats. tubules [46–48]. Therefore, decrease in oxalate concentration in
Hyperoxaluria and hypercalciuria are considered as the major urine may explain its decrease in oxidative stress and renal crystal
risk factor in pathogenesis of kidney stone formation. Continuing deposition which was observed after xanthium treatment.
hypercalciuria promotes the nucleation and subsequent precipita- Moreover, the antioxidant property of xanthium was also recently
tion of calcium oxalate crystals from the urine. Some studies have reported by some researchers due to presence of various bioactive
suggested that disorders of renal tubular calcium reabsorption are compounds [16,37].
It has been reported that reactive oxygen species (ROS) and
reactive nitrogen species (RNS) play an important role in renal
Renal ssue Calcium stone formation as a signalling molecule, as well as agent of injury
and inflammation [49,50]. ROS are produced through the involve-
2
** ment of both mitochondria [51,52] and NADPH oxidase, but NADPH
1.8 **
oxidase is a major source of ROS in the kidney [45,50].
1.6 Experimental studies suggest that renal cellular exposure to high
1.4
## oxalate, calcium oxalate or Calcium phosphate crystals results in
1.2 ## increased gene expression and production of molecules involved in
mg/g

1 tissue remodelling, inflammation and biomineralization [50].


0.8 Hyperoxaluria and CaC2O4 crystal deposition induce rennin up-
0.6 regulation and generation of angiotensin II which activate the non
0.4 phagocytic NADPH oxidase [53] leading to the production of ROS/
0.2 RNS. ROS induced damage to the cells leads to cell death and the
0 formation of membrane bound vesicles which support crystal
A B C D E nucleation [50]. As a result crystallization inhibitors became
Fig. 4. Renal tissue calcium content. defective or damaged by exposure to excess free radicals and thus
A = Untreated healthy control, B = Lithiatic group, C = 500 mg/kg Xanthium strumar- not able to provide adequate protection resulting in crystal growth
ium, D = 100 mg/kg cystone, E = Vehicle control. *p < 0.05 and **p < 0.01 vs healthy and aggregation. Cell death also leads to the formation of new cells
control group (group A), #p < 0.05, ##p < 0.01 vs Lithiatic control (group B)
1530 P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532

Fig. 5. Immunohistichemical expression of OPN in kidney section (X100).


A. Mild expression of OPN in outer medulla in healthy untreated control rat; B. Increased expression of OPN throughout renal tubules of cortex and medulla in Lithiatic control
rat; C. Mild expression of OPN in rat treated with xanthium @ 500 mg/kg; D. Mild expression of OPN in rat treated with Cystone @ 100 mg/kg; E. Increased expression of OPN in
vehicle control rat. DAB counterstained with Mayer’s Hematoxylene

to repopulate the epithelium. The surfaces of the new cells as well [45], and Jagannath [42] also reported the increased activity of LPO
as the exposed basement membrane are conducive to crystal in both the renal tissue and urine in rats with hyperoxaluria and
attachment and retention [54]. Crystals retained in the terminal CaC2O4 nephrolithiasis. Similar renoprotective and antioxidant
collecting ducts produce Randall’ plugs which will act as stone effect was also reported in other medicinal herbs namely rhizome
nidus when exposed to the pelvic urine. of Smilax china containing Astilbin [12], Artemisia arborescens [55]
In the present study, there was significantly increased activity and green tea extract [56] against oxidative related renal injury.
of LPO and NO; and significantly decreased activity of SOD, CAT and Additionally, Selvam [57] and Sumitra [58] reported that treatment
GSH in lithiatic rats as compared to healthy control which might be with antioxidant like vitamin E improved the tissue levels of
due to production of ROS and RNS. However, treatment with antioxidant enzymes like SOD, CAT and GSH, reduced injury and
xanthium significantly reduced the activity of LPO and NO, and totally eliminated CaC2O4 crystal deposition in the kidneys.
increased the mean activity of SOD, CAT and GSH, and the values The microscopic examination of renal histology showed intra-
were statistically similar to standard treatment demonstrating the tubular and interstitial CaC2O4 crystal deposits, mononuclear cell
optimal antioxidant effect of both the plant extract. Shirfule [28], Li infiltration, dilated renal tubule in untreated rats, consistent with
finding of other researchers [7,14]. The presence of polycrystalline,
rosette like arranged CaC2O4 crystals are evidence of adhesion and
80 retention of particles within the renal tubules. In contrast,
** treatment with xanthium significantly reduced the CaC2O4 crystal
70 deposit evident by Pizzolato’s method of staining and renal
% of OPN positive cells

** histology was also nearly same as healthy control.


60
Osteopontin is an important component of stone matrix and
50 thought to be involved in many pathogenic and physiologic
*## *## processes, including cell migration, adhesion, inflammation, renal
40
injury and cancer [6,9,10]. OPN is mainly expressed in specific sites
30 like the loop of Henle and distal nephron in normal kidneys in
animal and humans. However, OPN expression may be significant-
20
ly up-regulated in all tubular segments after renal injury especially
10 in nephrolithiasis [6,15,45]. OPN is always associated with renal
stone formation, but whether it acts as a promoter or inhibitor of
0
stone formation is controversial. It was thought that, OPN may act
A B C D E
as a modulator of CaC2O4 crystallization, involved in nucleation,
Fig. 6. Quantitative analysis of immunohistochemical expression OPN in kidney growth and aggregation of crystal in the kidney [6,8]. In the present
tissue of rats. study, there was up-regulation of OPN in urolithiatic rats without
The number of stained (OPN positive) cells/100cells was counted across 10 fields/
any treatment. However, xanthium significantly decreased the
section. A = Untreated healthy control, B = Lithiatic group, C = 500 mg/kg Xanthium
strumarium, D = 100 mg/kg cystone, E = Vehicle control. Data represented mean  OPN expression and associated renal injury induced by EG
SD. * p < 0.05, ** p < 0.01 vs healthy control (group A); #p < 0.05, ##p < 0.01 vs suggesting a potent anti-inflammatory and anti-urolithiatic effect.
Lithiatic control (group B).
P.N. Panigrahi et al. / Biomedicine & Pharmacotherapy 84 (2016) 1524–1532 1531

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