Effect of aqueous extract from Herniaria hirsute L. on experimentally nephrolithatic rats

Abstract

Despite considerable progress in medical therapy, there is no satisfactory drug to treat kidney stones. Therefore, this current study is aimed to look for an alternative treatment by using Herniaria hirsuta on nephrolithiasic rats as a preventive agent against the development of kidney stones. The experiment was conducted in normal and calcium oxalate (CaOx) nephrolithiasic rats during 3 weeks. Several parameters were followed weekly including water intake, urinary volume and pH, some urinary chemistries, and crystalluria. At the end, kidneys were analyzed by light microscope. The results showed that water intake and urinary volume increased in nephrolithiasic rats, but their urinary pH decreased especially in the third week of treatment. Urinary oxalate increased significantly during the second week for untreated rats and remained constant in rats treated with Herniaria decoction. However, urinary calcium decreased significantly in week 2 in untreated rats and remained constant in treated rats. Qualitative analysis of crystalluria showed that untreated rats excreted large CaOx monohydrate and few dihydrate crystals while treated animals excreted mostly small CaOx dihydrate crystals. The examination of kidney sections revealed that CaOx deposition was limited in treated rats when compared to untreated ones. These results obtained in vivo confirmed the beneficial effect of Herniaria hirsuta and may justify its use as a preventive agent against the formation of calcium oxalate kidney stones.

Full text

Journal of Ethnopharmacology 95 (2004) 87–93
Effect of aqueous extract from Herniaria hirsuta
L. on experimentally nephrolithiasic rats
Fouad Atmania,∗, Yamina Slimania, Mostapha Mimounib, Mohammed Aziza,
Brahim Hachtb, Abderrahim Ziyyata
aLaboratory of Cellular Physiology and Pharmacology, Department of Biology, Faculty of Sciences,
University Mohammed 1, Oujda 60000, Morocco
bLaboratoire de Sp´eciation et de Surveillance de la Pollution en M´editerran´ee, Universit´e Mohammed 1,
Facult´e des Sciences, D´epartement de Chimie, Oujda, Morocco
Received 15 September 2003; received in revised form 9 June 2004; accepted 29 June 2004
Available online 23 August 2004
Abstract
Despite considerable progress in medical therapy, there is no satisfactory drug to treat kidney stones. Therefore, this current study is aimed
to look for an alternative treatment by using Herniaria hirsuta on nephrolithiasic rats as a preventive agent against the development of kidney
stones.
The experiment was conducted in normal and calcium oxalate (CaOx) nephrolithiasic rats during 3 weeks. Several parameters were followed
weekly including water intake, urinary volume and pH, some urinary chemistries, and crystalluria. At the end, kidneys were analyzed by light
microscope.
The results showed that water intake and urinary volume increased in nephrolithiasic rats, but their urinary pH decreased especially in the
third week of treatment. Urinary oxalate increased significantly during the second week for untreated rats and remained constant in rats treated
with Herniaria decoction. However, urinary calcium decreased significantly in week 2 in untreated rats and remained constant in treated
rats. Qualitative analysis of crystalluria showed that untreated rats excreted large CaOx monohydrate and few dihydrate crystals while treated
animals excreted mostly small CaOx dihydrate crystals. The examination of kidney sections revealed that CaOx deposition was limited in
treated rats when compared to untreated ones.
These results obtained in vivo confirmed the beneficial effect of Herniaria hirsuta and may justify its use as a preventive agent against the
formation of calcium oxalate kidney stones.
© 2004 Published by Elsevier Ireland Ltd.
Keywords: Nephrolithiasis; Calcium oxalate crystallization; Kidney stones; Preventive treatment; Herniaria hirsuta L.
1. Introduction
Kidney stone formation or urolithiasis is a com-
plex process that results from a succession of several
physico-chemical events including supersaturation, nucle-
Abbreviations: EG, ethylene glycol; CaOx, calcium oxalate; COM, cal-
cium oxalate monohydrate; COD, calcium oxalate dihydrate; ESWL, extra-
corporeal shock wave lithotripsy
∗Corresponding author. Tel.: +212 56 50 06 01; fax: +212 56 50 06 03.
E-mail address: atmani@sciences.univ-oujda.ac.ma (F. Atmani).
ation, growth, aggregation, and retention within renal tubules
(Khan, 1997). Epidemiological data collected during several
decades showed that the majority of stones, up to 80%, are
composed mainly of calcium oxalate (CaOx) (Daudon et al.,
1993). It is important to point out that urolithiasis is charac-
terized by high recurrence rate requiring therefore a preven-
tive treatment. Among the treatments used are extracorporeal
shock wave lithotripsy (ESWL) and drug treatment. Even
improved and beside the high cost that imposes, compelling
data now suggest that exposure to shock waves in therapeutic
doses may cause acute renal injury, decrease in renal func-
0378-8741/$ – see front matter © 2004 Published by Elsevier Ireland Ltd.
doi:10.1016/j.jep.2004.06.028

88 F. Atmani et al. / Journal of Ethnopharmacology 95 (2004) 87–93
tion, and an increase in stone recurrence (Begun et al., 1991;
Kishimoto et al., 1986). In addition, persistent residual stone
fragments and possibility of infection after ESWL represent a
serious problem in the treatment of stones. Also, even though
drug treatment has shown some feasibility in many random-
ized trials, it is not accomplished without side effects, which
are some times very serious (Ruml et al., 1997; Yendt et al.,
1970). Therefore, it is worthwhile to look for an alternative
to these means by using medicinal plants or phytotherapy. In-
deed, herbal medicine is as ancient as the history of mankind.
Actually, herbal medicine has gained popularity in Europe
and is becoming increasingly in United States as well. In this
regard, many plants have been used to treat kidney stones and
showed to be effective among them Phyllanthus niruri,Zea
mays,Agropyron repens, and Herniaria hirsuta (Alexander
and Nestor, 1999; Freitas et al., 2002; Grases et al., 1993,
1995; Yasui et al., 1999; Atmani and Khan, 2000). This later
plant, is a Mediterranean traditional medicinal plant widely
used in Morocco to treat lithiasis patients. Our previous study
carried out in vitro showed that extract from Herniaria hir-
suta promoted nucleation of CaOx crystals, increasing their
number but decreasing their size (Atmani and Khan, 2000).
It also promoted the formation of CaOx dihydrate crystals
despite CaOx monohydrate crystals. Finally, the extract in-
hibited significantly CaOx crystal aggregation. In the present
work, we evaluated the possible therapeutic potential of Her-
niariahirsutaasapreventiveagentinexperimentallyinduced
CaOx nephrolithiasis in rats.
2. Material and methods
2.1. Preparation of herb decoction
Herniaria hirsuta L. (Caryophyllaceae) was collected in
Spring near to Oujda city (East of Morocco). A voucher spec-
imen (AS43) was deposited at the Agronomic and Veterinary
Institute of Hassan II at Rabat City, Morocco. The extract was
prepared according to a similar procedure used often by pa-
tients with some minor modifications. Fresh herb was dried
at 45◦C overnight, boiled in distilled water, and then the fi-
nal solution was lyophilized. The powder was reconstituted
to prepare a solution of 50mg/ml in distilled water.
2.2. Animals and treatments
Male rats of the wistar strain weighing about 280g were
housed in metabolic cages 3 days prior to the start of the ex-
periment for acclimatization. The experiment was conducted
in accordance to internationally accepted standard guidelines
for use of animals. They were fed regular chow and had free
access to tap water ad libitum. They were then divided into
three groups comprising six animals each. Group I was used
as control, group II which was given ethylene glycol (EG)
0.75% only was used as untreated nephrolithiasic rats group,
and group III, used as treated nephrolithiasic rats group, was
given EG 0.75% and 1ml of decoction of Herniaria hirsuta.
EG was added in their drinking water to induce a chronic
low grade hyperoxalluria and generate CaOx deposition
into kidneys. Twenty-four hour urine samples from each
rat were collected weekly in presence of sodium azide as
antibacterial agent during 3 weeks to analyze some urinary
components, to measure urinary volume and pH, and to
analyze qualitatively crystalluria as well. All animals were
kept under a controlled 12 h light dark cycle and temperature
of 22 ±2◦C. At the end of the experimental study, all
animals were sacrificed after ether anesthesia. Kidneys
harvested from animals were analyzed histologically after
they were fixed in formalin and embedded in paraffin. The
7␮m thick sections were stained by eosin solution and
examined by plain and polarized light microscopy.
2.3. Analytical procedures
Urinaryoxalatewasestimatedaccordingtothemethodde-
scribed by Hodgkinson et al. (Hodgkinson, 1970) with some
modifications. Briefly, 1ml of urine was acidified beforehand
by concentrated HNO3to solubilize crystals and then ad-
justed to pH 7 by NaOH in the presence of color indicator, the
bromothymol blue. About 2 ml of saturated CaSO4and 14 ml
of pure ethanol were added to precipitate oxalate overnight.
The samples were centrifuged at 450 ×gfor 10 min and then
filtered on filter paper. The precipitate obtained was solubi-
lized in 10 ml of water acidified by 2 ml of concentrated sul-
furicacid. The samples were titrated by a solutionof KMnO4.
Calcium and magnesium analysis was performed by using
a Varian A20 double beam spectrophotometer equipped with
a Varian hollow cathode and a Deuterium background cor-
rector. All standards were made sufficiently acidic to avoid
metal hydrolysis and to match content in the sample using
nitric acid 65% suprapure. All dilutions were made in 50ml
polypropylene tubes.
2.4. Data analysis
Student’s t-test was used for statistical comparison of data
between groups. Pvalues <0.05 were considered significant.
Results are presented as means ±standard errors (S.E.).
3. Results
The values reported in Table 1 showed that water intake
and urinary volume were similar in all groups at the begin-
ning of the experiment. During the experiment, the values
remained mostly constant for control group but increased for
both groups receiving EG only and EG plus Herniaria hir-
suta decoction. In terms of water intake, there was significant
difference between untreated nephrolithiasic rats (group II)
and controls (group I) in week 2. Regarding urinary volume,
there was a significant difference for both groups II and III
in week 2 and 3 compared to control group (group I).
Urinary pH remained stable for control group through-
out the experiment. However, it was decreased for both

F. Atmani et al. / Journal of Ethnopharmacology 95 (2004) 87–93 89
Table 1
Urinary parameters in normal and ethylene glycol-fed rats, on and off herbal treatment
Groups Water intake (ml) Urinary volume (ml) Urinary pH
Control Untreated Treated Control Untreated Treated Control Untreated Treated
Week 0 34.0 ±8.3 33.5 ±6.3 28.0 ±10.3 14. 8 ±3.7 14.4 ±3.3 16.2 ±5.1 7.97 ±0.27 7.65 ±0.49 7.57 ±0.23
Week 1 28.5 ±4.4 32.7 ±8.2 23.5 ±6.8 13.0 ±2.8 16.3 ±6.4 13.8 ±2.6 7.74 ±0.40 7.51 ±0.38 8.11 ±0.67
Week 2 27.0 ±3.5 35.5 ±7.2a38.5 ±15.0 10.8 ±1.3 19.7 ±6.4a24.8 ±10.7b8.14 ±0.40 7.54 ±0.55 7.54 ±0.86
Week 3 35.7 ±3.2 39.7 ±9.2 49.8 ±16.5 10.3 ±1.0 18.0 ±5.1a31.3 ±15.6b7.93 ±0.53 7.04 ±0.24a6.82 ±0.83b
aUntreated vs. control.
bTreated vs. control.
groups receiving EG and reached significant values on
week 3.
The values for urinary analysis reported in Table 2 showed
that oxalate was significantly higher in untreated nephrolithi-
asic rats compared to control group especially in week 2.
However, it remained constant in rats treated with Herniaria
extract.Urinarycalciumleveldecreasedsignificantlyinweek
2 in untreated rats and remained to a comparable level to con-
trol in treated rats. For magnesium, we noticed some fluc-
tuations in the values during the experiment that make the
interpretation difficult.
Qualitative crystalluria analysis demonstrated the pres-
ence of a large amounts of struvite particles in control rats
(Fig. 1a). However, for all untreated nephrolithiasic rats
(group II), calcium oxalate monohydrate (COM) and calcium
oxalate dihydrate (COD) crystals were detected throughout
the experiment (Fig. 1b). For all nephrolithiasic rats treated
with herb extract, crystalluria was composed mostly of COD
particles and only with few COM particles were present in
their urine samples (Fig. 1c). Interestingly, the size of both
type of crystals was smaller when compared to untreated
nephrolithiasic rats.
The examination of paraffin kidney sections at the time
of sacrifice revealed that four control animals had only few
crystals scattered in some parts of the kidney (Fig. 2a) and
none for two animals. However, the untreated nephrolithiasic
animals receiving EG only (group II) had crystals in all three
major areas of the kidney. Interestingly, in many tubules, es-
pecially in the medulla, crystals were agglomerated in large
aggregatesor“mini-stones”(Fig. 2b). In contrast, the kidneys
of group III receiving Herniaria extract in addition to EG had
Table 2
Urinary chemistries in normal and ethylene glycol-fed rats, on and off herbal treatment
Groups Oxalate (mg/24 h) Calcium (mg/24 h) Magnesium (mg/24 h)
Control Untreated Treated Control Untreated Treated Control Untreated Treated
Week 0 0.30 ±0.08 0.23 ±0.03 0.25 ±0.05 3.87 ±0.88 3.22 ±0.54 2.80 ±1.36 12.5±3.8 12.7 ±0.8 9.2±1.5 c
Week 1 0.30 ±0.06 0.29 ±0.10 0.26 ±0.05 2.39 ±0.69 1.11 ±0.36 2.11 ±0.58 13.9±2.6 15.6 ±6.8 16.7±8.0
Week 2 0.18 ±0.03 0.28 ±0.09a0.25 ±0.06b1.15 ±0.60 1.01 ±0.66a1.16 ±1.40c12.3±2.6 23.5 ±7.5 7.4±3.3bc
Week 3 0.16 ±0.02 0.21 ±0.09 0.24 ±0.12 1.19 ±0.46 1.39 ±0.49 1.73 ±0.70 9.5±1.0 11.8 ±4.8 13.8±4.3b
aUntreated vs. control.
bTreated vs. control.
cUntreated vs. treated.
limited CaOx deposition in four animals and mostly no de-
position in two animals compared to control rats (Fig. 2c).
4. Discussion
Urinary stone disease is mainly the result of supersatura-
tion of urine with certain urinary salts such as CaOx, the most
common constituent of kidney stones (Daudon et al., 1993).
Many in vivo models have been developed to understand the
mechanisms involved in the formation of urinary stones and
to ascertain the effects of various therapeutic agents on de-
velopment and progression of the disease (De Bruijn et al.,
1993; Khan, 1985). Rat is the most frequent used animal to
induce CaOx deposition into kidneys and mimic the etiol-
ogy of the formation of stones in humans. Accordingly, we
evaluated the effectiveness of a medicinal plant, Herniaria
hirsuta, on rats rendered nephrolithiasic by administration of
EG. In fact, the plant is widely used in Morocco in the treat-
mentof kidney stone formation as an antilithiasic and diuretic
agent.
The interesting finding of our study is the diuretic effect
of Herniaria hirsuta extract on rats. Even the values were
not statistically significant, we noticed that urinary volume
increased in rats receiving EG and the plant extract compared
to those receiving EG only. In a separate and preliminary
experiment, we noticed a significant diuretic effect at week 3
in rats receiving Herniaria hirsuta only. All parameters were
comparable to control rats. Our finding is in accordance with
the results obtained by Grases et al. (1993) who showed a
diuretic effect exerted by the plant extract on rats.

90 F. Atmani et al. / Journal of Ethnopharmacology 95 (2004) 87–93
Fig. 1. Microscopic examination of urinary crystals excreted by (a) the control rats consisted predominately of large amount of struvite particles (b) rats
receiving EG 0.75% only in which large oval COM (m) and small pyramidal COD (d) crystals can be seen, and (c) rats receiving EG 0.75% and herb extract,
few oval COM (m) and large amount of small pyramidal COD (d) crystals can be seen.

F. Atmani et al. / Journal of Ethnopharmacology 95 (2004) 87–93 91
Fig. 2. Paraffin section viewed under polarized light of (a) a control rat kidney, (b) a kidney from a rat that received EG 0.75% only where numerous crystals in
the cortex and medulla can be seen, and (c) a kidney from a rat that received EG 0.75% and herb extract. Note reduced crystal deposition compared to control
group (a).

92 F. Atmani et al. / Journal of Ethnopharmacology 95 (2004) 87–93
The analysis of urinary chemistries seemed to be not con-
vincing and it is difficult to draw a clear and definite con-
clusion. Nevertheless, if we make a general look over the
data we can underline two important points. First, oxalate
level remained constant in the urine of nephrolithiasic rats
treated with the plant extract while it increased in untreated
rats group. Second, urinary calcium of treated rats remained
at a level comparable to control rats. Considering the stud-
ies quoted in the literature (Freitas et al., 2002; Grases et al.,
1993, 1995) it is important to point out that medicinal plants
used in the treatment of urolithiasis seemed to have little ef-
fect on urinary chemistries. Further investigations including
large number of rats are highly recommended to reach a con-
clusive result.
EG administration resulted in the development of per-
sistent crystalluria in all rats. CaOx crystals were present
as monohydrates (COM) and dihydrates (COD) crystals.
COM crystals were generally in the form of biconcave ovals.
COD crystals were generally dipyramidals in shape. Inter-
estingly, COM with large size were more abundant in the
urine of untreated nephrolithiasic rats. However, crystalluria
of nephrolithiasic rats treated with Herniaria extract showed
few COM but abundant COD particles with small size. Such
effect has been observed in vitro when crystallization was
induced in the urine in presence of herb extract (Atmani and
Khan, 2000). We suggest that the plant may contain sub-
stances that inhibit COM crystallization and promote the for-
mation of COD particles. Since the formation of such crystals
depends on Ca/Ox we suggest also that the plant has the abil-
ity to modify this ratio by binding at least one element either
directly or indirectly. Based on all modifications observed
in crystalluria after the treatment of nephrolithiasic rats by
Herniaria hirsuta extract, two major benefit effects of this
plant can be attributed. First, the extract induced more crys-
tals in urine, thereby it reduced supersaturation which is an
initial and prerequisite step for lithogenesis. Second, the size
of crystals was reduced and thus can simply be swept by
the urine flow out off the kidneys before they become large
enough to be entrapped physically in the urinary tract.
Microscopic examination with polarized light of kidney
sections derived from nephrolithiasic rats showed that crys-
tal deposits were intensely birefringent, polycrystalline, and
arranged in rosette characteristic of CaOx crystals. The pres-
ence of such deposits is an evidence of adhesion and retention
of particles within renal tubules. However, four nephrolithi-
asic rats treated with Herniaria hirsuta extract had limited
CaOx deposition and in two animals none comparable to
control rats. In this regard, it is actually admitted that as-
sociation of crystals with renal tubular cells is considered to
be a potential factor in the process of renal stones forma-
tion. Indeed, considering the rate of crystal growth even at
its maximum speed and tubular fluid flow, calculations sug-
gest that a single crystal would not become large enough to
be retained and occlude the lumen during its normal transit
through the nephron (Finlayson and Reid, 1978). Neverthe-
less, based on new calculations taking into consideration the
size increasing effect of agglomeration process, crystalline
particles can become large enough to seal the nephron (Kok
and Khan, 1994). Furthermore, it is established that crystals,
especiallyCOM crystals, can be retained by attachmentto the
surface of renal epithelial cells and be internalized (Lieske
et al., 1997). At this level we can consider that Herniaria
hirsuta may have two additional beneficial effects in the pre-
vention of kidney stones formation. First, we have demon-
strated that the plant efficiently inhibited CaOx aggregation
(Atmani and Khan, 2000). Therefore, even nucleation and
growth of crystals cannot be prevented, their agglomeration
into large particles is prohibited. Consequently, crystals re-
main dispersed in the urine and can be flashed easily out off
the kidney. Accordingly, it is agreed that crystalluria is an in-
dicator of urinary supersaturation that occurs similarly both
in healthy and stone formers (Werness et al., 1981). How-
ever, these later tend to excrete large and aggregated crystals
(Robertson et al., 1969). Second, we have mentioned earlier
that the plant promoted the formation of small COD particles.
Such crystals adhere less to renal epithelial cells than COM
counterpart (Wesson et al., 1998). To this, we can add that the
plant extract may interfere directly in the inhibition of crystal
adhesion to the epithelium by blocking the attachment sites
located either into the cell surfaces or into the surface of the
crystals them selves. This later hypothesis was confirmed in
our recent work showing that aqueous extract obtained from
Herniaria hirsuta significantly inhibited COM crystal attach-
ment to renal epithelium cells in a concentration-dependent
manner (Atmani et al., 2004). We suggested that the extract
containssubstances that coat COM crystals, thereby blocking
their adhesion to the cell surface.
Overall, our data presented in this current paper indicate
that administration of Herniaria hirsuta extract to experi-
mentally CaOx-induced nephrolithiasic rats reduced the de-
position of crystals into kidneys confirming its antilithiasic
effect. Further studies are necessary to clarify the mechanism
underlying this effect, which is still not clear so far.
Acknowledgments
This research was supported in part by the Interna-
tional Foundation for Sciences, Stockholm, Sweden, through
Grant F/3071-1. We thank the members of Laboratoire de
P´
etrologie, Min´
eralogie et G´
eologie Economique, University
Mohammed 1, Faculty of Sciences, Department of Geology,
Oujda, Morocco, for the use of their microscope facility.
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