Urolithiasis (2013) 41:205–215
DOI 10.1007/s00240-013-0556-9
ORIGINAL PAPER
Molecular mechanisms involved in the protective effect
of the chloroform extract of Selaginella lepidophylla
(Hook. et Grev.) Spring in a lithiasic rat model
Estévez-Carmona Marı́a Mirian • Narvaéz-Morales Juanita •
Barbier Olivier Christophe • Meléndez-Camargo Marı́a Estela
Received: 7 January 2013 / Accepted: 13 March 2013 / Published online: 30 March 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Urolithiasis is a multifaceted process, pro-
gressing from urine supersaturation to the formation of
mature renal calculi. Calcium oxalate, the main component
of kidney stones, has toxicological effects on renal epi-
thelial cells. Some medicinal plants have shown pharma-
cological effects against renal lithiasis, such as Selaginella
lepidophylla (Hook. et Grev) Spring, a plant empirically
used in Mexico for its diuretic and antilithiasic activity.
The plant was identified and ground, and a chloroform
extract (CE) was obtained. Urolithiasis was induced in
Wistar female rats by administration of ethylene glycol and
ammonium chloride for 21 days. Urolithiasis rats were
treated with the CE (50 mg/kg) for 21 days. Osmolality,
creatinine, sodium and potassium concentrations were
measured in blood and urine. Glomerular filtration rate
(GFR), and electrolytic and water balances were calcu-
lated. Urinary oxalic acid concentration was measured.
Apoptosis, lipoperoxidation, ROS and p-amino hippuric
acid were determined in cortical tissue. Urolithiasis rats
showed a decrease of urinary flow, GFR, electrolytic bal-
ance, renal tubular secretion and ATP concentration and
increase of urinary oxalic acid, lipoperoxidation, oxidative
stress and apoptosis in cortical tissue. After treatment with
E.-C. M. Mirian  M.-C. M. Estela (&)
Laboratorio de Farmacologı́a y Toxicologı́a Renal y Hepática,
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico
Nacional, Av. Wilfrido Massieu, Esq. Manuel Luis Stampa S/N,
U.P. Adolfo López Mateos, 07738, México, D.F., México
e-mail: mcamargo@ipn.mx; emelendezc@hotmail.com
N.-M. Juanita  B. O. Christophe
Departamento de Toxicologı́a, Centro de Investigación
y de Estudios Avanzados del Instituto Politécnico Nacional,
Av. Instituto Politécnico Nacional 2508 Col. San Pedro
Zacatenco, 07360 México, D.F., México
the CE, urinary flow rate, GFR and renal tubular secretion
levels were recovered; on the other hand, serum creatinine
and urinary oxalic acid decreased on day 21. CE of
Selaginella lepidophylla prevented the damage caused by
lithiasic process by improving the active secretion in the
proximal tubules, counteracting the ROS and lipoperoxi-
dation effects by oxalate and decreased the OAT3
expression on kidney.
Keywords Urolithiasis  Proximal tubule  Apoptosis 
Lipoperoxidation  Oxalic acid  OAT3  MRP2
Introduction
Renal lithiasis is a common disease worldwide, with a high
prevalence of 5.2 % in the global population [1]. The
lifetime risk of stone formation is 13 % in men and 7 % in
women with a recurrence rate of 30–40 % at 5 years [2].
Approximately 5 % of people are expected to form a uri-
nary tract stone within their lifetime [3]. Urolithiasis, a
multifaceted process, involves a number of events includ-
ing urine supersaturation, formation of microcrystals,
growth and maturation of the kidney stone. The retention
of microcrystals by the urothelium is believed to be a
necessary and critical event in the growth of renal calculi
[4]. Observations of stone disease in rat models and
humans suggest that crystal retention might involve the
urothelial cell membrane [5, 6]. In addition, crystal-mem-
brane interactions have been implicated in several disease
states including lung inflammation in silicosis. This bind-
ing process seems to be mediated by specific interactions
between molecular structures on the surface of stone
crystals and molecular arrays on the cell membrane surface
[7].
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Calcium oxalate (CaOx), is the most common compo-
nent of renal calculi, representing up to 80 % of analyzed
stones [8]. In the kidney, the mature CaOx kidney stones
can be located along the urinary tract. It is known that
calculi can migrate through the collecting duct to the
papillary area or sometimes even to the bladder [9].
However, it is in the cortical region, specifically proximal
tubules where the oxalate is excreted from body by active
tubular secretion pathway and is in the tubular epithelial
cells where oxalate is stored and the oxidative and
inflammatory damages start [10]. Crystals of CaOx exist
under physiological conditions as two crystalline poly-
morphs: monoclinic CaOx monohydrate (COM) and
tetragonal CaOx dihydrate (COD), however, the exposure
of cells to COM was reported to cause cellular injury seven
times more than COD [11]. COM causes nephrotoxicity in
epithelial cells through free radical-mediated reactions that
eventually lead to lipid peroxidation causing renal injury
[12], and membrane and organelle damage [13] followed
by cell necrosis and apoptosis [14]. Cellular damage by
CaOx mainly takes place in the tubular portion of the
nephron, such damage is a retroactive process, since it
allows for the formation and growth of crystals, which
migrate throughout the kidney from collector tubules to
renal papilla furnishing an encrustation platform or a nidus
for future development of a mature kidney stone [15].
However, in the genesis of CaOx crystals in the proximal
tubules, there are several cellular pathways that have been
little studied and may form the basis of new treatments
against CaOx urolithiasis.
Available treatments, such as the extracorporeal shock
wave lithotripsy (ESWL) and drug therapy, revolutionized
urological practice and have become the standard proce-
dure for eliminating kidney stones. However, shock waves
have traumatic effects, persistence of residual stone frag-
ments and favor renal infections. Moreover, ESWL may
cause acute renal injury, renal function impairment [16],
hemorrhage and hypertension [17]. Pharmacologic therapy,
on the other hand, is not specific for preventing or treating
renal lithiasis and, hence, the recurrence of kidney stones is
high, 70–81 % in males and 47–60 % in females [18].
Therefore, new alternatives for the treatment of kidney
stones are being tested using medicinal plants or phyto-
therapy [19]. A number of plant drugs with antilithiasic
effect, both preventive and therapeutic, have been used in
many parts of the world [20, 21]. The main mechanisms
reported of studied plants are inhibition of CaOx endocy-
tosis in vitro [22], normalization of kidney deposits,
recovery of renal impairments in urolithiasis [23], reduc-
tion of CaOx crystal growth, antioxidant activity in renal
cells [24] and increment of diuresis in rats [25].
Selaginella lepidophylla (Hook. et Grev.) Spring
(Selaginellaceae) is a plant native to Mexico where it is
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Urolithiasis (2013) 41:205–215
commonly known as ‘‘doradilla’’ (goldenish), ‘‘siempre
viva’’ (evergreen), ‘‘planta de la resurección’’ (resurrection
plant), and ‘‘flor de piedra’’ (stone flower), and is used in
traditional medicine as a diuretic and as remedy for treating
kidney stones [26]. Organic extracts of Selaginella lepid-
ophylla, specifically alkaline chloroform extract (CE), have
shown diuretic and antilithiasic properties in rats [27].
However, the action mechanisms involved in this thera-
peutic activity is still unknown. In this study, we investi-
gate the pharmacologic effect of Selaginella lepidophylla
CE in an experimental model of lithiasis for studying the
cellular mechanisms involved in the filtration and active
tubular secretion pathways.
Methods
Plant material
Selaginella lepidophylla plants were collected in the State
of Tlaxcala, Mexico, and taxonomically authenticated by a
specialist at Escuela Nacional de Ciencias Biológicas
(ENCB), Instituto Politécnico Nacional. A voucher speci-
men was deposited in the ENCB herbarium under entry
number 47098. The dried plant material was ground in a
mill (FITZÒ MILL model D Comminutor, Industrial Drive
Elmhorst, Ilinois 60126).
Preparation of ethanol and chloroform extracts
of Selaginella lepidophylla
All the plant material (6 kg) was percolated with ethanol
(J.T. BakerÒ 96 grade) for 7 days at room temperature. The
solvent was then evaporated under vacuum and the solid
residue (180 g) was the ethanol extract (EE). Acid–base
reactions were made with the EE, using 5 % hydrochloric
acid and 10 % ammonium hydroxide. The alkaline phase
was extracted three times with chloroform (J.T. BakerÒ
reagent grade) and concentrated under reduced pressure
[28]; the organic residue (30 g) obtained was the chloro-
form extract (CE). Phytochemical screening for alkaloids
was made to CE. Dragendorff, Mayer, Hagar and Marquin
reactions were tested according to standard methods
described by Aiyelaagbe and Osamudiamen [29].
Animals
Adult female Wistar rats were used. They were housed and
maintained in the animal house at room temperature
(22–24 °C) and 50–55 % relative humidity, with a 12/12-h
day/night cycle. Rats were fed with standard rodent diet
and water ad libitum. Care and handling of animals fol-
lowed internationally accepted procedures according to the
Urolithiasis (2013) 41:205–215
Institute for Laboratory Animal Research’s Guide for Care
and Use of Laboratory Animals.
The experiments were carried out in un-anesthetized
adult female Wistar rats. These were randomly allocated to
two groups: the control group (I, n = 8), which received
regular rat food and drinking water ad libitum, and the
lithiasis group (II, n = 16) which had a standard diet with
ethylene glycol (0.75 %) and ammonium chloride (0.5 %)
added to the drinking water for 21 days to induce COM
lithiasis [21, 30]. Once the CaOx kidney stones were
induced, the urolithiasis rats were then randomly allocated
to two groups: lithiasis without treatment (n = 6) and rats
treated with chloroform extract (CE, 50 mg/kg body
weight) for 21 days (III, n = 10) [31]. The body weight
and water intake of animals were recorded during both
urolithiasis induction and treatment periods.
At the beginning and the end of treatment, renal lithiasis
and the antilithiasic effect of CE of Selaginella lepido-
phylla were demonstrated by functional renal tests. To start
the experiment, the bladders of animals were emptied by
gentle compression on the abdomen and then the rats were
kept in metabolic cages for 6 h without food and fluids.
After this period of time, urine samples were collected, also
by abdominal compression to insure the complete empty-
ing of the bladder. Anesthetized rats were sacrificed by
decapitation. Blood was collected, the serum separated by
centrifugation at 10,000g for 10 min and the kidneys
removed for further molecular studies.
Effect of CE on the glomerular filtration rate of lithiasic
rats
Serum and urinary creatinine were determined using the
Jaffe alkaline picrate method as modified by Melendez
et al. [32]. Glomerular filtration rate (GFR) was estimated
from clearance of endogenous creatinine using the con-
ventional equation:
GFR ¼ Ccrea ¼ ðUcrea  Jv =Pcrea Þ
where GFR or Ccrea is the creatinine clearance rate
(ml/min); Ucrea and Pcrea are the creatinine concentrations
(mg/dl) in urine and serum, respectively; and Jv is the
urinary flow rate (ml/min). To avoid the error due to
tubular secretion of creatinine, only female animals (in
which no secretion occurs) were used [33].
Effect of CE on the electrolyte balance of lithiasic rats
Osmolality of serum and urine were measured in a vapor
pressure osmometer (Wescor, Logan Utah) by triplicate.
Urinary flow rate was measured and the osmolar (Cosm) and
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free water (CH2 O ) clearances were calculated using con-
ventional equations:
Cosm ¼ Uosm  Jv =Posm
CH2 O ¼ Jv  Cosm
where Cosm is the osmolal clearance (ml/min); Uosm and
Posm are the osmolality values in urine and serum,
respectively (mosm/l); Jv is the urinary flow rate (ml/min)
and CH2 O is the water clearance (ml/min).
Sodium and potassium concentrations were measured
both in urine and serum samples using a Flame photometer
Coleman 51 Perkin-Elmer, Norwalk, Conn. Urine clear-
ances of sodium and potassium were calculated using the
conventional equation:
FEx ¼ ðCx =Ccrea Þ  100
Cx ¼ ðUx  Jv =Px Þ
where FEx is the fractional excretion (%) of substance x;
Cx and Ccrea are the clearance (ml/min) of substance x and
creatinine, respectively; Ux and Px, are the concentrations
(lEq/l) of substance x in urine and serum, respectively; and
Jv is the urinary flow rate (ml/min).
Determination of urinary oxalate in lithiasis and CE-
treated rats
The concentration of oxalic acid in urine was measured
using the Hodgkinson [34] method of oxalic acid titration
with potassium permanganate.
Effects of the chloroform extract (CE) on glucose
and protein urinary excretion in lithiasic rats
Glucose concentration in urine samples was measured
using a standard kit (GL2623, RANDOX, Laboratories
Ltd., UK). Urinary protein was quantified using the Brad-
ford method [35]. Glucosuria and proteinuria were deter-
mined from the urinary concentration of glucose and
protein, respectively, and the urinary flow rate [31].
Effect of lithiasis and CE on the uptake
of p-aminohippuric acid
p-Aminohippuric acid (PAH) was used as an indicator of
the renal secretory pathway of organic anions. Slices were
obtained from the kidney’s cortex of rats from each
experimental group and then incubated in Ringer solution
containing PAH (1 mM), for 1 h. The slices were then
removed from the incubation media, the wet weight of the
tissue pieces was recorded, the PAH concentration was
measured using the method of Bratton and Marshall and
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the results were finally expressed as the tissue/medium 1 lg/ml of quinine sulfate in 0.05 M H2SO4. The results
ratio [36]. were expressed as arbitrary units of relative fluorescence
per gram of protein [31].
Determination of cortical ATP content in lithiasic
and CE rats Effect of lithiasis and CE on oxidative stress

ATP content was quantified using a colorimetric method
based on the phosphorylation of glycerol to generate a
product that is easily quantified (OD = 570 nm) with a
common commercial kit (K 354-100, Bio Vision Inc.,
USA). The results were expressed as nmol per gram of
protein [37].
Determination of apoptosis in lithiasic and CE-treated
rats by quantitation of Caspase 3
Renal cortical tissue was mechanically homogenized with
lyses solution, and the activity of Caspase 3 was measured
using a Caspase-3/CPP32 Colorimetric Assay Kit (K106-
100, BioVision Inc., USA). The protein content of each
sample was determined using the bicinchoninic acid (BCA)
method. The results were expressed as Arbitrary Units per
gram of protein.
Determination of lipoperoxidation in renal cortex
of lithiasic and CE rats
Lipid peroxidation was evaluated by the formation of lipid-
soluble fluorescence. 500 ll aliquots of cortical homoge-
nate were added to 5 ml of chloroform–methanol (2:1, v/v)
solution. After stirring for 15 s, the mixture was cooled in
ice for 30 min to allow phase separation. The chloroform
phase was measured in a Perkin-Elmer LS55 Lumines-
cence Spectrophotometer at 370 nm (excitation) and
430 nm (emission) wavelengths. The spectrophotometer
sensitivity was adjusted to 140 fluorescence units with
Reactive oxygen species (ROS) in cortical tissue homog-
enates were determined using a commercial kit based on
the dichlorofluorescein method, in which 5-(and-6)-car-
boxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxy-
H2DCFDA) formed acts as a reliable fluorogenic marker
for ROS in cortical tissue [38]. The results were expressed
as arbitrary units of fluorescence per gram of protein.
Effect of lithiasis and CE on OAT3 expression
in cortical tissue
Cortical slice homogenates were boiled for 3 min in the
presence of 5 % b-mercaptoethanol, 2 % sodium dodecyl
sulfate (SDS), 10 % glycerol and 0.01 % bromophenol
blue. Samples were applied to a 12 % acrylamide gel,
separated by electrophoresis and then electroblotted onto
nitrocellulose membranes. To ascertain an equal protein
load, an antibody against human b-actin was used. The
nitrocellulose membranes were incubated for 1 h with 5 %
non-fat dry milk in phosphate-buffered saline containing
0.2 % Tween 20 (PBST). After rinsing with PBST, the
membranes were incubated at 4 °C overnight with a
commercial goat polyclonal antibody against rat OAT3 (sc-
107833) at a 1:10,000 dilution, or with mouse monoclonal
antibody against multidrug-resistant protein 2 (MRP2) (sc-
59609) at 1:10,000 dilution, or with commercial mouse
monoclonal antibody against human b-actin (sc-130300)
(at 1:1,000 dilution); all antibodies were purchased from
Santa Cruz Biotechnology Inc., CA. The membranes were
incubated for 1 h with a peroxidase-coupled goat antibody

Table 1 Effect of urolithiasis and CE treatment on body weight and water intake in rats
Groups Body weight (g) Water intake (ml/day)
Day 21 (induction) Day 21 (treatment) Day 21 (induction) Day 21 (treatment)

Control 230.674 ± 1.894 249.882 ± 2.025 17.600 ± 1.398 22.421 ± 1.650

(8) (8) (8) (8)
Lithiasis 215. 626 ± 2.963a 235.125 ± 2.317a,c 12.250 ± 1.712a 17.500 ± 1.787a
(10) (10) (10) (10)
CE 215.757 ± 2.625a 245.756 ± 2.945b 12.336 ± 1.661a 19.375 ± 1.652
(6) (6) (6) (6)
Data are mean ± standard error
Data in parentheses indicate number of animals per group
a
P \ 0.001 compared to the control group
b
P \ 0.001 compared to lithiasis group
c
P \ 0.001 compared to the CE group

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Urolithiasis (2013) 41:205–215 209

IgG (Santa Cruz, CA). After further washing with PBST,

blots were exposed to autoradiography plates for 5 min and
revealed, fixed and dried. A densitometric quantification of
the Western blot signal intensity of membranes was per-
formed using the ImageJ software [39].

Statistical analysis

Data were analyzed with one-way ANOVAs followed by

Student Newman–Keuls multiple range tests. A difference
was considered statistically significant when P \ 0.05.

Results

Chloroform extract (CE)

Chloroform extract was obtained from the ethanol extract

of Selaginella lepidophylla, with a 4 % yield. The alkaloids
tests were positive for all the reactions carried out.

Effect of CE on renal parameters in urolithiasis rats

It was observed a decrement in the body weight and in the

water intake in urolithiasis rats during the induction of
kidney stone disease in comparison to control animals;
however, there was a recovery of these variables in CE-
treated rats with respect to lithiasis group (Table 1).
As shown in Fig. 1a, the urinary flow rate of lithiasic
rats decreased in comparison to control rats (3.982 ± 0.611
vs. 7.294 ± 0.682 ll/min, respectively; P \ 0.001), whereas
CE-treated animals showed a recovery compared with lithiasic Fig. 1 Urinary flow rate (a), serum creatinine concentration (b) and
rats (7.252 ± 0.643 ll/min; P \ 0.001). creatinine clearance (c) during lithiasis and after CE treatment. Data
are mean ± SE, aP \ 0.001 compared to the control, bP \ 0.001
At the same time, serum creatinine increased in uro-
compared to the lithiasis group, nControl = 8, nLithiasis = 6 and
lithiasis (1.444 ± 0.021 vs. 2.410 ± 0.121 mg/dl; P \ 0.001) nCE = 10
but this increase was improved by the CE treatment (1.491 ±
0.033 mg/dl; P \ 0.001) (Fig. 1b).
These changes affected the GFR. Thus, urolithiasis increased highly in this group compared to the control and
induced a drop in GFR (1.122 ± 0.07 vs. 0.454 ± 0.08 ml/ lithiasis groups (Tables 2, 3).
min; P \ 0.001) and, as expected, the CE treatment pro- Osmolal (Cosm) and free water (CH2 O ) clearances, which
tected GFR from this reduction (0.850 ± 0.064 ml/min; diminished during lithiasis, recovered after CE adminis-
P \ 0.001 compared with lithiasis) but not completely tration (Table 3).
(P \ 0.001 with control) (Fig. 1c).
Effects of CE on the urinary excretion of glucose
Effect of CE on renal handling of electrolytes and protein
in urolithiasis rats
Glycosuria increased in the lithiasic rats (P \ 0.001) and
Whereas lithiasic rats showed a decrease in the clearance of proteinuria augmented in both the lithiasic rats and the CE-
potassium (CK) and sodium (CNa), and an increase of treated groups. Interestingly, only proteinuria in lithiasic
fractional excretions, the CE group showed a recovery with group was significant compared with control (P \ 0.001)
respect to the lithiasis group, especially in CK, which but not with CE group (Table 2).

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Table 2 Effect of urolithiasis and CE treatment on electrolytic balance, glycosuria and proteinuria
Groups Glycosuria (lg/min) Proteinuria (lg/min) CK (ml/min) CNa (ll/min)

Control 0.038 ± 0.002 1.418 ± 0.908 0.614 ± 0.078 7.991 ± 1.000

(8) (8) (8) (8)
Lithiasis 0.061 ± 0.005a 1.948 ± 0.573a 0.554 ± 0.090a 5.247 ± 1.011a
(6) (6) (6) (6)
CE 0.029 ± 0.003b 1.859 ± 0.157 0.943 ± 0.101a,b 8.708 ± 0.766b
(10) (10) (10) (10)
Data are mean ± standard error
Data in parentheses indicate number of animals per group
CK, potassium clearance; CNa, sodium clearance
a
P \ 0.001 compared to the control group
b
P \ 0.001 compared to lithiasis group

Table 3 Effect of urolithiasis and CE treatment on osmolal and free water clearances and fractional excretion of potassium and sodium
Groups Cosm (ml/min) CH2 O (ml/min) FEK (%) FENa (%)

Control 0.026 ± 0.003 -0.019 ± 0.002 9.644 ± 2.007 0.124 ± 0.023

(8) (8) (8) (8)
Lithiasis 0.015 ± 0.002a -0.011 ± 0.002a 21.428 ± 2.059a 0.198 ± 0.020a
(6) (6) (6) (6)
CE 0.028 ± 0.003b -0.021 ± 0.002b 10.471 ± 0.746b 0.098 ± 0.008b
(10) (10) (10) (10)
Data are mean ± SE
Cosm, osmolal clearance; CH2 O , free water clearance; FEK, fractional excretion of potassium; FENa, fractional excretion of sodium
a
P \ 0.001 compared to the control
b
P \ 0.001 compared to the lithiasis group

Urinary oxalate in urolithiasis and CE-treated groups
As expected, the urinary concentration of oxalic acid
(Fig. 2a) increased in rats with renal lithiasis (5.091 ±
0.330 mg/ml) compared to the control group (1.414 ±
0.132; P \ 0.001). However, after the 21 days of treatment
with CE, a reduction was observed (3.202 ± 0.304 mg/ml;
P \ 0.001 compared to the lithiasis group). However,
this was not a full recovery as it was still significantly
(P\ 0.001) higher than in the control group.
Effect of urolithiasis and CE on the uptake
of p-aminohippuric acid
Both experimental groups showed a significant decrease
in PAH uptake: the lithiasic group had a PAH tissue/
medium ratio (1.994 ± 0.082) significantly (P \ 0.001)
lower than that in the control group (3.033 ± 0.114), but
the decrease in the CE-treated group (2.522 ± 0.060) was
significantly (P \ 0.001) lower than in the lithiasic group
(Fig. 2b).
Changes of ATP content in urolithiasis and CE-treated
rats
Compared to the control group, lithiasic rats showed a
significantly (P \ 0.001) lower content of ATP in cortical
tissue (5.251 ± 0.574 vs. 3.542 ± 0.193 nmol/g protein).
However, after the CE treatment, no recovery in the
ATP content of cortical cells was observed (3.982 ±
0.250 nmol/g protein; P \ 0.001) (Fig. 3a).
Increase of apoptosis in urolithiasis and CE-treated rats
Apoptosis was estimated in terms of the Caspase 3 content
in cortical tissue (Fig. 3b). Caspase 3 increased signifi-
cantly (P \ 0.001) in lithiasic rats (0.342 ± 0.023 Arbi-
trary Units/g protein) compared to control animals
(0.274 ± 0.012). The CE treatment induced a significant
reduction (0.311 ± 0.013 Arbitrary Units/g protein;
P \ 0.001 compared to the lithiasic group), although this
was not a full recovery as it was still significantly
(P \ 0.001) higher than the control group.

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Fig. 2 Urinary concentration of oxalic acid (a), tissue/medium ratio
of PAH uptake (b). Data are mean ± SE, aP \ 0.001 compared to
the control group, bP \ 0.001 compared to the lithiasis group,
nControl = 8, nLithiasis = 6 and nCE = 10
Effect of urolithiasis and CE treatment on oxidative
stress
Rats with calculi showed a significant increase in oxidative
stress parameters. Lipid peroxidation in renal cells
(Fig. 4a) increased in lithiasic rats (0.035 ± 0.001 vs.
0.056 ± 0.022 Fluorescence Arbitrary Units/g protein;
P \ 0.001), whereas rats treated with CE showed a clear
but not significant decrease (0.046 ± 0.002 Fluorescence
Arbitrary Units/g protein).
Another parameter that can also be used to estimate
oxidative stress is the cellular content of ROS. As expec-
ted, the ROS content in cortical tissue of lithiasic rats
(17,174 ± 1183) increased significantly compared to that
in the control group (12,687 ± 780; Fluorescence Arbitrary
Units/g protein; P \ 0.001). Interestingly, the CE treatment
significantly reduced the ROS content (13,268 ± 675 Fluo-
rescence Arbitrary Units/g protein; P \ 0.001 compared to the
lithiasis group) (Fig. 4b).
Determination of OAT3 and MRP2 expression
in cortical tissue
Figure 5a shows the semi-quantification by Western blot of
the expression of OAT3. It is well known that OAT3 is one
of the basolateral transporters of oxalate in proximal tubule
cells.
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Fig. 3 ATP (a) and Caspase 3 (b) contents in renal cortical tissue.
Data are mean ± SE, aP \ 0.001 compared to the control,
b
P \ 0.001 compared to the lithiasis group, nControl = 8, nLithiasis = 6
and nCE = 10
Interestingly, OAT3 expression increased significantly
during lithiasis (1.000 ± 0.083 vs. 1.782 ± 0.281 relative
OD corrected with actin; P \ 0.05). OAT3 expression in
the CE-treated group was significantly lower than in the
lithiasic group (1.571 ± 0.344 relative OD corrected with
actin; P \ 0.05).
Another transporter involved in the secretion of oxalate
is the MRP2, located in the apical membrane of proximal
epithelial cells. Figure 5b shows that the expression of
MRP2 underwent the same change pattern as OAT3: a
significant increase during lithiasis (1.000 ± 0.031 vs.
1.363 ± 0.082 relative OD corrected with actin; P \ 0.05)
and then a significant reduction caused by the CE treatment
(1.201 ± 0.113 relative OD corrected with actin; P \ 0.05
compared to the lithiasis group).
Discussion
Urolithiasis, the formation of stones in the urinary tract, is a
common disease that has affected humans since antiquity.
Despite the progress made in understanding the molecular
mechanisms governing the formation of renal calculi, and
in the general management of this disease, it still has a high
prevalence and recurrence in stone formers as neither a
specific therapy for renal lithiasis nor a clear understanding
of the molecular mechanisms involved in its development
do exist [40].
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Fig. 4 Oxidative stress: lipid peroxidation (a) and ROS content (b) in
cortical tissue. Data are mean ± SE, aP\ 0.001 compared to the con-
trol group, bP \ 0.001 compared to the lithiasis group, nControl = 8,
nLithiasis = 6 and nCE = 10
Selaginella lepidophylla has been widely used in Mex-
ico as a remedy for renal lithiasis and other kidney diseases
[41]. In particular, chloroform extracts of this plant have
shown pharmacological activity in kidney. In previous
studies, in our laboratory a bioassay of Selaginella lepid-
ophylla was made to identify the organic extract respon-
sible of the antiurolithic effect of the plant, the major
activity against CaOx kidney stones was attributed to
alkaline chloroform extract of plant (CE) in a dose of
50 mg/kg body weight [27].
The urolithiasis process involves several changes in both
renal balance and epithelial tissue, due to crystal interac-
tion with renal cells [42]. CaOx calculi, the most common
type of renal calculi, are related to low urinary volume and
a deficient renal function, which cause changes in the
body’s electrolytic and hydric balance; it has been reported
that the crystals produced during CaOx urolithiasis are of
COM and COD, however, the formed monohydrated
crystals have the major harmful properties to renal epi-
thelial cells, with respect to the dihydrate form. The result
of injury cell by CaOx crystals is the promotion of the
formation and the growth of new crystals during the kidney
stone disease [11].
Calcium oxalate calculi induced in an experimental
model by administering ethylene glycol were able to alter
the general health state in animals. The final metabolite
formed since EG is the oxalic acid, which acts mainly in
123
Urolithiasis (2013) 41:205–215
Fig. 5 Expression of oxalate transporters in cortical tissue: OAT3
(a) and MRP2 (b). Micrographs of Western blot show a representative
image of one of three independent experiments. Histograms represent
relative OD corrected by actin. Data are mean ± SE, aP\ 0.001
compared to the control group, bP \ 0.001 compared to the lithiasis
group
the kidney and causes acute poisoning symptoms [43]. Due
to the disease state during kidney stones induction, uro-
lithiasis rats stopped eating and drinking, so the body
weight and water intake decreased. On the other hand, it is
probably that the bitter taste of drinking water by the
ammonium chloride content, necessary to maintain an acid
urinary pH during all the induction period [44], makes the
water intake also decrease in urolithiasis rats. Additionally
in CE group, there was a recovery of body weight and
volume of water intake similar to the control rats.
Another variable altered during urolithiasis was the
glomerular function causing a reduction in GFR and the
subsequent accumulation of endogenous creatinine in
serum in lithiasic rats. This demonstrates that the glomer-
ular function is related to ethylene glycol metabolites with
high affinity for kidney cells in the presence of CaOx [45].
Urolithiasis (2013) 41:205–215
The recovery of GFR was probably due to the scarcity of
oxalate on kidney after treating the animals with CE, since
as can be observed in this work, the lower excretion of
oxalic acid in urine in the CE group may be related with a
minor quantity of oxalate in kidney. Then if there are no
hyperoxaluria, the CaOx is not more in contact to renal
cells, so the alterations in cells produced during urolithiasis
could be reversed.
On the other hand, it is known that the urinary excretion
of protein and glucose is related to renal tubular damage
[8, 46]. Despite the high concentration of these molecules
in urine during nephrolithiasis, due to a poor reabsorption
of endogenous molecules in the proximal tubules by the
CaOx content in tubular cells and its nephrotoxicity, in our
experiments, urinary excretion of glucose and proteins
increased significantly in rats with renal calculi in spite of
their low urinary volume during kidney stone disease.
Renal lithiasis alters tubular secretion as oxalate anions
are actively secreted by the kidney proximal tubules and
they cause renal epithelial cell injury, favoring crystal
retention in the urothelium [45]. An excess of oxalate can
alter the secretory pattern of the PAH marker in renal
tubules in the same way as other organic anionic com-
pounds [22]. In our study, lithiasic rats showed a reduction
in the PAH tissue/medium ratio as a consequence of the
increase in oxalate anions in cortical tissue, as this anion
surpasses the secretory capacity of the proximal tubular
cell transporters and then gets accumulated [45]. On the
other hand, in this study, we have shown that the CE
treatment might noticeably improve the secretory function
as substances in the chloroform extract of Selaginella
lepidophylla avoid the build-up of oxalate in kidney.
The chloroform extract shows a potential pharmaco-
logical effect against urolithiasis, as the renal alterations
induced by oxalate lithiasis can be reverted by treatment
with a CE of Selaginella lepidophylla. This protective
effect can be explained by the diuretic activity induced by
this plant: the increase in urine volume [41] can prevent
oxalate from being stored for a long time in the kidney.
Lipoperoxidation and ROS are natural cell processes;
however, when their concentration is increased, functional
alterations occur. There is a relationship between oxidative
stress molecules and apoptosis, inflammation, kidney stone
formation or several chronic diseases [47]. In this study, the
increase in lipoperoxidation and ROS was a consequence of
CaOx in the tubular cells; these events have been related to
damages to cell membranes and mitochondria (the energy
source for active processes and proapoptotic initiators) that
play an important role in the formation of renal calculi.
Several studies have reported that aqueous extracts, organic
extracts and isolated molecules from medicinal plants
act by counteracting oxidative stress produced by CaOx
crystals in vitro or in vivo studies [24, 48] or through the
213
inhibition of the formation and the aggregation of new
crystals by several pathways [49], thereby reducing the
inflammatory response associated with CaOx calculi [50]. It
is likely that the CE has antioxidant properties that could
account for the reduction of ROS in cortical tissue, or
perhaps the scarcity of oxalate in the kidney suppresses
ROS production in renal cells.
Apoptotic events commonly occur in calcium oxalate
renal lithiasis due to the toxicological effect of this com-
pound on the kidney’s epithelial cells [51]. In this work, the
presence of apoptosis was confirmed by the quantification
of the proapoptotic enzyme Caspase 3 in cells, an indicator
of programmed cell death. This evidenced the relationship
between renal damage and cell death with the build-up of a
matrix of calcium oxalate renal calculi in the urothelium
[7]. Therefore, it was necessary to examine the effect of CE
on apoptosis by oxalic calculi. We found that CE caused a
significant reduction in apoptotic tissue death, which might
be explained by a possible antioxidant activity of the plant
material related to the prevention of cell death [52] and to
the decrement of calcium oxalate in renal tissue [53]. In
this study, we confirmed the relationship between oxalate
renal calculi and apoptosis and the fact that CE reduces cell
death related to oxalate urolithiasis.
Different processes take place in cells damaged by toxic
compounds. In nephrolithiasis, renal cells accumulate
oxalate, causing an imbalance in the cell osmosis and in the
active transport of compounds through kidney cells [54].
Renal tubular secretion is diminished in oxalate urolithia-
sis, and the decrease in PAH uptake is associated with a
lower energy expenditure of ATP [55]. However, the
expression of OAT3 increased in lithiasic rats probably as a
kidney’s defensive mechanism that aimed to eliminate the
excess oxalate from the basolateral membrane toward the
apical side of tubular cells. Organic anion transporters
require energy for the active transport of xenobiotics from
the basolateral membrane toward the cell apical side [56].
When the concentration of oxalate anions inside the cell is
high, a conservative action to save vital compounds to
prevent larger injury takes place: the expression and
functionality of OAT3 increase and oxalate might either
enter the cell competing with PAH in the proximal tubules
or impair the anion elimination mechanisms, probably
mediated by the oxalate’s toxicity-dependent inhibition of
some enzymes specific for OAT3 [39]. On the other
hand, MRP2, located on the apical membranes, mediates
the ATP-dependent transport PAH and supports the
OAT3 activity against oxalate damage as the oxalate
excess in tubular cells can go out by active process by
luminal transporters [57]. In this study, we showed that
oxalate damage as the oxalate calculi induce significant
modifications in critical steps of the active processes
involved in cellular excretion by organic transporters and
123
214
that the CE treatment contribute to the recovery of the
secretory function.
Conclusions
The chloroform extract of Selaginella lepidophylla (Hook.
et Grev.) Spring prevented calcium oxalate renal calculi in
rats. It reduced oxalic acid in urine and serum creatinine,
and also decreased ROS, lipoperoxidation, apoptosis and
OAT3 expression in cortical tissue. On the other hand, the
CE increased urinary flow and GFR after a 21-day treat-
ment in calcium oxalate urolithiasis rats.
Acknowledgments This research was partially supported by the
Secretarı́a de Investigación y Posgrado (IPN) and the Consejo Nac-
ional de Ciencia y Tecnologı́a (Conacyt) under grant number 152416.
M.M.E.C is a PhD fellow from Conacyt (Grant number 211857).
Conflict of interest None.
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