Ameliorative effects of the ethanolic extract of Allium saralicum R . M . Fritsch on CCl 4-induced nephrotoxicity in mice : A stereological examination

The present study was carried out to investigate the nephroprotective effect of the ethanolic extract of Allium saralicum R.M. Fritsch (ASRMF) in mice. Thirty-five male mice were divided into five groups (n=7). Group 1 (positive control) received 1 mL/kg olive oil intraperitoneally (i.p.) and 0.5 mL distilled water orally; Group 2 (negative control) received CCl4 (50% in olive oil, 1 mg/kg; i.p.); Groups 3, 4 and 5 received CCl4 and 200, 800 and 1600 μg/kg of ASRMF extract, respectively. The renal volume and cortex in Groups 1 and 2 were increased by 55% and 62% (p≤0.001) following CCl4 administration, respectively, and were improved after ASRMF administration. The volume of proximal convoluted tubules (PCTs), glomeruli, vessels and interstitial tissue increased 80%, 150%, 83% and 64% (p≤0.05), respectively, in CCl4treated mice, and decreased significantly with 800 and 1600 μg/kg of ASRMF. The length of PCTs and vessels increased 51% and 45% and decreased (p≤0.05) with 200, 800 and 1600 μg/kg of ASRMF, respectively. CCl4-treated mice lost 22.5% of glomeruli; the loss was inhibited significantly (p≤0.05) by ASRMF. Urea and creatinine concentrations were increased (p≤0.05) in CCl4-induced nephrotoxicity as compared to the controls, whereas different doses of ASRMF restored the levels of these biomarkers compared to the negative controls. In conclusion, ASRMF has a potent nephroprotective property and can improve renal structural and serum biomarkers in CCl4-induced nephrotoxicity in mice.


INTRODUCTION
Carbon tetrachloride (CCl 4 ), a clear, colorless, volatile, heavy and nonflammable liquid is a potent environmental hepatotoxin and nephrotoxin that causes steatosis, necrosis and cirrhosis [1].CCl 4 intoxication causes free radical generation in many tissues, such as the liver, kidneys, heart, lung, testes, brain and blood [2][3][4][5].The initial step in the tissue injury induced by CCl 4 is the cytochrome P450-mediated transfer of a single electron to the C-Cl bond, which gives rise to a radical anion as a transient intermediate that eliminates chlorine to form a carbon-centered radical, the trichloromethyl radical •CCl 3 , and chloride [6,7].The •CCl 3 in the presence of oxygen is subsequently converted into an even more reactive trichloromethyl peroxyl radical (•OOCCl 3 ) that initiates the process of lipid peroxidation [8] and production of membrane-damaging products such as malondialdehyde (MDA).It is believed that the free radical-induced lipid peroxidation is one of the major causes of cell membrane damage that leads to a number of pathological states causing acute and chronic renal injuries [9][10][11].Furthermore, various documented case studies have established that CCl 4 produces renal disease with an altered antioxidant status in humans [12,10].
Findings from the screening of different medicinal plants describe their antioxidant properties and indicate that they could protect organs against CCl 4induced oxidative stress by altering the levels of antioxidant enzymes [13,14].
Allium saralicum (Valak Soori in Persian) is an endemic plant of Iran that grows widely in the western parts of the country.The genus Allium is placed in the family Amaryllidaceae and subfamily Allioideae.The cooking and consumption of parts of the plant is due to the large variety of flavors and textures of the species.Various Allium species have been cultivated from the earliest times and about a dozen species are economically important as crops or garden vegetables, and some species are important as ornamental plants [15,16].
In the present study, we investigated the ameliorative effect of the ethanolic extract of Allium saralicum by studying the microscopic structural changes in mice kidney after CCl 4 -induced nephrotoxicity using modern design-based stereological methods.The stereological variables are volume of the renal cortex, medulla, glomeruli, connective tissues, proximal and distal convoluted tubules, vessels, collecting ducts, Henle's loop and lengths of the five last-mentioned tubular structures.Renal functions were also investigated by examining serum biomarkers, urea and creatinine.

Plant collection
Mature Allium saralicum plants were collected from around Kermanshah city during April 2015.The plant was identified for the first time and a voucher specimen (no.2738RUH) was deposited at the herbarium of the Research Center of the Faculty of Agriculture, Razi University, Kermanshah, Iran.

Plant extraction
All parts of the plant (leaves, stem, flowers, seeds and roots) were shade-dried for one week.The dried aerial parts of the plants were ground and about 150 g of the obtained powder was extracted with 450 mL of 100% ethanol for 2 h at 40°C with continuous shaking.The extract was left for 24 h at room temperature and then filtered through Whatman filter paper #2.The obtained extract was concentrated under reduced pressure using a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) at 80°C until a semi-solid sticky mass was obtained.Since all ethanol evaporates completely during the extraction process, the extract does not induce any ethanol-related adverse effects following administration.The components of this extract were analyzed by gas chromatography/mass spectrometry (GC/MS) in the Research Center of Razi University.The complete details of this GC/MS are presented in Table 1.

Animals and treatments
Thirty-five healthy male BALB/c mice weighing between 35 and 40 g were obtained from the laboratory animal center of Kermanshah University of Medical Sciences.The animals were kept under constant humidity and temperature.All animals were allowed access to food and water ad libitum during the experiment.The animals were treated according to the standard directive, as recommended in the Guide for Care and Use of Laboratory Animals [17] approved by the research authorities of Kermanshah University of Medical Sciences.The mice were divided randomly into five groups (n=7) as follows: Group I (control) received 1 mL/kg olive oil i.p. and 0.5 mL distilled water by gavage; Group II received 1 mg/kg CCl 4 mixed with olive oil at a ratio of 5:5 i.p., and 0.5 mL distilled water by gavage; Group III received CCl 4 mixed with olive oil at a ratio of 5:5 i.p. and Allium saralicum R.M. Fritsch (ASRMF) (200 µg/kg bw) by gavage; Group IV received CCl 4 mixed with olive oil at a ratio of 5:5 i.p. and ASRMF (800 µg/kg bw) by gavage; Group V received CCl 4 mixed with olive oil at a ratio of 5:5 i.p. and ASRMF (1600 µg/kg bw) by gavage.All administrations were performed twice a week (on Saturday and Wednesday) for 45 days.After completion of the experiment, the animals were weighed and killed by chloroform inhalation.Blood samples were obtained from the animals' heart tubes that were centrifuged to obtain the serum.The left kidney of each animal was removed and cleaned of perirenal fat and connective tissue.The kidney was then weighed and the primary volume was measured using the immersion method [18].The kidney was fixed in neutral buffered formaldehyde for 5 days.To prevent reference trap, the reference volume or the final volume of the kidney must be estimated [19,20].
The reference volume was estimated by calculating shrinkage after tissue processing and staining without any need for serial sections.Estimation of shrinkage and tubule length requires isotropic uniform random sections [19,21].These sections were obtained by the orientator method.Briefly, each kidney was placed on a circle on which each half was divided into 10 equal distances (φ-clock), a random number between 0 and 10 was selected and the kidney was sectioned into two halves with a blade in this direction.The cut surface of one half of the kidney was then placed along the 0-0 direction of the second circle with 10 unequal sinusweighted divisions (θ-clock) and the second cut was done by selecting a random number.The cut surface of the other half of the kidney was placed vertically on the θ-clock.The second cut was done by selecting a random number.The entire kidney was sectioned with a blade, placed in the direction of the second cuts with an interval of 1 mm.The slabs (7-10 slabs) were then collected.A circle was punched from a kidney slab by a trocar.The diameters of the circular piece of the kidney were measured by a micrometer and the area of the circle was estimated using the usual formula for calculating the area of a circle.The slabs and circular pieces were embedded in paraffin and 5 µm-thick sections were prepared and stained by the Periodic Acid Schiff (PAS) method.After staining, the area of the circular piece was measured again and the volume shrinkage was calculated as: where AA and AB are the areas of the circular piece after and before tissue processing, respectively.Af-ter estimating the shrinkage, the final volume of the kidney (the reference space) was corrected using the following formula: V final = V primary × (1-volume shrinkage)

Stereological study: volume estimation
Each sampled section was analyzed using a video-microscopy system equipped with a microscope (Olympus CX2, Japan) linked to a video camera (Dinocapture ver.5; dino-lit.com;30.5 mm), a P4 PC computer and a monitor to determine the parameters.Between 10 and 14 microscopic fields were examined in each kidney.The point probe (composed of 20 points) was superimposed on the images of the tissue sections viewed on the monitor and volume densities (V v ) of the renal cortex, medulla, glomeruli, proximal convoluted tubule (PCT), distal convoluted tubule (DCT), collecting ducts (CD), loop of Henle (LH), vessels and connective tissue were obtained using a point-counting method (Fig. 1) and the following formula [19]: where P structure and P preference are the number of test points falling on the structure's profile and on the reference space, respectively.One section and 10-14 microscopic fields were examined in each kidney.The absolute volume of the parameters was estimated by multiplying the fractional volume by the final volume of the kidney to prevent the reference trap [21,22].

Length estimation
The length densities of the renal tubules and vessels were estimated by randomly superimposing an unbiased counting frame (740×740 µm) on the monitor live images.The tubule profiles completely inside the counting frame or partly inside the counting frame but only touching the top and right lines were counted.The tubule profiles touching the bottom and left lines and its extensions were ignored (Fig. 2).The length density (L v ) of the each tubule was calculated as: where ΣQ denotes the total number of the tubule profiles counted per mouse kidney; a(frame) equals the area associated with a frame, 547600 µm 2 and Σ frame is the total number of frames counted.The total length of each tubule in mouse kidney (L) was calculated by multiplying the length density (L v ) by the final volume of the kidney [19,21].

Number estimation
The total number of glomeruli per kidney was estimated by the physical dissector method [23].From each kidney, a section pair 20 µm apart (the 1 st and 5 th sections) was obtained.Two separate projecting systems with similar equipment were used.Two dissector probes (740×740 µm) with exclusion lines (the left and lower borders) and inclusion lines (the right and upper borders) were superimposed on the images of the 1 st section, which served as the reference section (Fig. 3a), and the 7 th section as the look-up section (Fig. 3b) at 135× magnification.A glomerulus was counted if it was presented in a reference section but not in the lookup section and if it did not touch the exclusion lines.At least 100 glomeruli per kidney were counted.The numerical density of the glomeruli was estimated using: where ΣQ -denotes the number of counted glomeruli, a (frame) is the area of the dissector frame, ΣP is the sum of studied field and h is the dissector height.The total glomerular number was estimated by multiplying the numerical density (N v ) by the reference volume (renal cortex).

Biochemical analysis
The serum biomarkers, urea and creatinine, were measured by a spectrophotometric procedure and determined as markers of kidney function using kits (Ziest Chem Diagnostics) according to the manufacturers' instructions.All biochemical measures were done in duplicate.

Statistical analysis
The data are expressed as the means±standard deviation.A statistical comparison between group means was performed by one-way ANOVA followed by Tukey's post-hoc test.P≤0.05 was considered as significant.

Effects of CCl 4 and ASRMF on the weight and volumes of the kidney and of its components
The data regarding kidney weight, mean absolute volume of kidney and its components in control and treated groups are presented in Tables 2-4.The kidney weight and volume were increased by 44% and 55% (p≤0.001),respectively, in CCl 4 -treated mice in comparison with the control group.The volume of the cortex increased 62% (p≤0.001) in this group, but the medulla only increased 22%, which was not significant (p>0.05) in comparison with the control group.Treatment of CCl 4 -treated mice with 800 and 1600 µg/kg of ASRMF significantly (p≤0.05)improved kidney weight and kidney volume.Further, the volume of the cortex was decreased significantly (p≤0.05) in both ASRMF-treated groups as compared to the CCl 4treated group, whereas, the medulla volume did not exhibit a significant decrease (p>0.05).
The volumes of PCT, GLOM, vessels and interstitial tissue were increased 80%, 150%, 83% and 64% (p≤0.05),respectively, in the CCl 4 -treated mice as compared to the controls (Tables 3 and 4).The volumes of the collecting duct (CD) and loop of Henle (LH) did not show significant differences (p>0.05).Treatment of mice with 800 and 1600 µg/kg of AS-RMF significantly (p≤0.05)decreased the volume of PCTs, vessels and interstitial tissue as compared to the CCl 4 -treated group, while the glomerular volume was decreased significantly (p≤0.05) after treatment with the highest dose of ASRMF in comparison to the CCl 4 -treated mice.

Effects of CCl 4 and ASRMF on renal tubule and vessel lengths
The length of the PCT and vessels increased 51% and 45%, respectively (p≤0.05), following CCl 4 administration.ASRMF (800 and 1600 µg/kg) shifted these values toward the control values (Table 5).The increases in length of DCT, CD and LH were not significant (p>0.05) in the CCl 4 -treated mice as compared to the control.

Effects of CCl 4 and ASRMF on glomerular number
The obtained results showed that the number of glomeruli per kidney in the CCl 4 -treated group were significantly (22.5%) lower than in control animals.Coadministration of either 800 or 1600 µg/kg ASRMF and CCl 4 significantly inhibited the decrease in the number of glomeruli (p≤0.05) in comparison with the CCl 4 -treated group.The glomerular number did not significantly differ between 800 and 1600 µg/kg ASRMF (Table 4).

Effects of CCl 4 and ASRMF on the serum biochemical profile
The serum concentrations of creatinine and urea serve as indicators of kidney injury and dysfunction.The effect of CCl 4 administration on changes in these biochemical markers are presented in Table 6.The concentrations of urea and creatinine were increased in the serum in CCl 4 -induced nephrotoxicity as compared to the control group.However, the increase in urea was not significant (p>0.05).Coadministration of both CCl 4 and different doses of ASRMF significantly reversed the levels of these markers, pointing to recovery and normalization.

DISCUSSION
The present in vivo study demonstrated the nephroprotective potential of the ethanol extract of Allium saralicum R.M. Fritsch on CCl 4 -induced nephrotoxicity by stereological methods.Numerous experimental studies showed that CCl 4 causes tissue damage in many organs, albeit mainly in the liver, and changes in several blood biochemical parameters [1,[24][25][26].It has been reported that CCl 4 systemically administered to rats is distributed at higher concentrations in the kidney than in the liver [27,28].Since the kidney has an affinity for CCl 4 and contains cytochrome P450 predominantly in the cortex [1,29,30], CCl 4 is likely to contribute to nephrotoxicity.
In this study, after exposure of experimental animals to 1 mg/kg bw of CCl 4 , we observed a significant increase in serum biomarkers, creatinine and urea, which is in agreement with previous reports [26,31,32].The increase in serum urea and creatinine was inhibited significantly in groups that received simultaneously 800 and 1600 µg/kg body weight of ASRMF and CCl 4 in comparison with CCl 4 -treated mice.Similar investigations have also documented that different plant extracts significantly recovered biochemical marker fluctuations induced by CCl 4 intoxication [5,31].Such effects might be related to the antioxidant properties of these plant extracts.Based on the results of GC/MS analysis, linolenic acid constitutes the main part of the ethanolic extract of AS-RMF.Recent research has shown that oral intake of α-linolenic acid is beneficial in experimental colitis, myocardial infarction, arterial thrombus formation and osteoporosis.There is also evidence of the protective role of plant Ω3 against nephrotoxicity induced by gentamicin [32].Based on previous investigations, α-linolenic acid has antioxidant and anti-inflammatory activities.This fatty acid has a strong inhibitory effect on the production of NO and iNOS and TNF-α gene expression by blocking NF-κB and MAPK activation.The ability of α-linolenic acid to regulate the expression of NF-κB, TNF-α and inflammatory interleukins may account for its protective effects [33].Furthermore, other compounds of Allium saralicum, such as phytol, neophytadiene and vitamin E, are potent antioxidant and anti-inflammatory agents [34].
As our results show, CCl 4 -treated animals exhibited a significant increase in kidney weight and volume.The main effect was seen on the cortex.It is well known that the renal cortex is composed mainly of the PCT and DCT.However, only the PCT was affected by CCl 4 as its volume and length were significantly increased.Accordingly, it was revealed that reversible alterations occur in the renal proximal tubular epithelium following CCl 4 administration, which is manifested by loss of basilar interdigitations, cellular swelling and swollen microvilli [36].On the other hand, administration of CCl 4 resulted in an insignificant increase in the volumes of vessels and connective tissue.Oxidative stress induced by CCl 4 can promote the formation of a variety of vasoactive mediators that can affect renal function directly by initiating renal vasoconstriction [5].These events may explain the increase in kidney weight and volume observed in this study.In addition to the PCT, vessels and interstitial tissue, CCl 4 also causes hypertrophy of glomeruli, which could be due to vasoconstriction and congestion of capillary tufts in the renal corpuscles.
Our study, with its design based on unbiased stereological methods, showed that the administration of CCl 4 to animals treated with ASRMF significantly inhibited glomerular hypertrophy and loss of glomerular number in comparison with untreated animals.There are reports that plant extracts of Oxalis corniculata [37], Citharexylum spinosum [38] and Sonchus asper [39] can inhibit glomerular hypertrophy in CCl 4 -induced nephrotoxicity.
Although the glomerular number was reduced significantly in CCl 4 -treated animals, there was no significant difference between the control group and mice that received 800 and 1600 µg/kg of ASRMF.In fact, glomerular number loss is irreversible.Because the nephrons develop during embryonic development and as there is no nephrogenesis after birth, the maintenance of glomerular number in CCl 4 -induced nephrotoxicity is an important potent nephroprotective effect of ASRMF.
The results of the present study were obtained using stereological techniques.Several methods pro-vide potentially less variable and more sensitive and quantifiable measures of tissue changes that could be associated with biomarker appearance [40].It should be noted that biomarkers found in biological fluids are typical indicators of functional change or cellular injury.High fidelity scanning can convert glass slides to digital images, allowing software-based, image analysis [41,42] and quantification of the injury or association with a stainable tissue protein.Software applications for stereology evaluation of digital images enhance the ability to detect and quantify changes in tissue morphology [21,43].These methods offer potentially more sensitive methods for evaluating early and/or very mild changes in tissue morphology that might be relevant for early biomarker release and unperceivable to a trained pathologist working with light microscopy.
Finally, it can be concluded that the high dose of ASRMF extract has a nephroprotective effect against renal structural changes induced by CCl 4 in mice.This beneficial effect could be attributed to the antioxidant activities of compounds contained in the extract, such as linolenic acid, phytol, neophytadiene and vitamin E.

Fig. 1 .
Fig. 1.Microscopic section of the kidney showing the glomerulus, proximal and distal convoluted tubules.To estimate volume density, the total number of points hitting each component (the point is the right upper corner of the cross, the arrow) was divided by the total number of points hitting the reference space.Scale bar 50 µm PAS, 400×.

Fig. 3 .
Fig. 3.For estimating the numerical density of glomeruli according to the physical dissector method, the images of two sections 20 µm apart (1 st and 5 th sections) were used as (a) a reference and (b) the look-up section, respectively.An unbiased counting frame is superimposed on the sample sections.Glomerular profiles are counted if they are contained completely or partly in the frame and are not hit by exclusion (red lines), and if they disappeared in the look-up section (here 1).Scale bars 100 μm, PAS, 100×.

Fig. 2 .
Fig. 2. The length density of the renal tubules is estimated by an unbiased counting frame on the images.The tubule profiles completely inside the counting frame or partly inside the counting frame but only touching the top and right lines are counted (here 5 proximal convoluted tubules).The tubule profiles touching the bottom and left lines and its extensions are ignored.Scale bar 50 µm, PAS, 400×.

Table 1 .
The components of Allium saralicum R.M. Fritsch analyzed by GC/MS.

Table 2 .
Kidney weight (mg), absolute volume of the kidney (mm 3 ), and absolute volumes (mm 3 ) of the cortex and medulla of the control and experimental groups treated with Allium saralicum R.M. Fritsch.Results are given as means±standard deviation.
* p ≤ 0.05 vs the control group ** p≤0.05 vs the CCl 4 -treated group

Table 3 .
Absolute volume (mm 3) of the proximal and distal convoluted tubules (PCT, DCT), collecting ducts (CD), loop of Henle (LH), vessels (VES) and interstitial tissues (IT) in the control and experimental groups treated with Allium saralicum R.M. Fritsch.Results are given as means±standard deviation.

Table 4 .
Absolute volume (mm 3 ) and number of the glomeruli (GLOM) in the control and experimental groups treated with Allium saralicum R.M. Fritsch.Results are given as means±standard deviation.
* p≤0.05 vs the control group ** p≤0.05 vs the CCl 4 -treated group

Table 5 .
Absolute length (m) of the proximal and distal convoluted tubules (PCT, DCT), collecting ducts (CD), loop of Henle (LH) and vessels (VES) in the control and experimental groups treated with Allium saralicum R.M. Fritsch.Results are given as means±standard deviation.

Table 6 .
Serum profile parameters in the control and experimental groups treated with Allium saralicum R.M. Fritsch.Results are given as means±standard deviation.
* p≤0.05 vs the control group ** p≤0.05 vs the CCl 4 -treated group