Protective Effect of Quercetin on Histomorphometric Changes in Kidney of Retinoid Acid-Treated Rat Fetuses/Efecto Protector de la Quercetina sobre los Cambios Histomorfometricos en el Rinon de Fetos de Rata Tratados con Acido Retinoico.
Vitamin A (retinol) and its analogs (retinoids) are important regulators of cell proliferation, differentiation, immune function, and apoptosis. Retinoic acid (RA), which exists in both cis and trans isomeric forms, is the most biologically active metabolite of vitamin A and is also essential for normal development (Abu-Abed et al., 2002). All-trans-retinoic acid (atRA), is a signaling molecule indispensable for the formation of many organs, including eyes, heart, and kidneys) Duester, 2008) . AtRA is an important physiological regulator of cellular differentiation, proliferation, apoptosis, reproduction and embryonic development in many species (Maden, 2006). Retinoic action is mediated by specific nuclear retinoic acid receptors and retinoid receptors belonging to the steroid/thyroid super-family of transcription factors. Inadequate levels of retinoids (excess or deficiency) may result in a set of defects denoted retinoic acid embryopathy which may provoke defects in the development of the neural crista (Kam et al., 2012). The kidneys are target organs for vitamin A action. Retinoic acid (RA), a vitamin A metabolite, is involved in embryonic kidney patterning through the control of receptor tyrosine kinase expression, which modulates ureteric bud branching morphogenesis. Vitamin A status of the mother profoundly affects kidney organogenesis of the newborn (Bhat & Manolescu, 2006).
AtRA affects by serving as an activating ligand of nuclear atRA receptors (RAR a, b, and g) and peroxisome proliferator-activated receptors (PPAR b/d), which form heterodimers with retinoid X receptors (Mark et al., 2006). In kidney atRA receptors are cell specific in expression; these are therefore, practically responsible for activation of both receptors by their respective agonists (Wagner, 2001).
The concentration of atRA during embryonic development is tightly controlled in a spatial and temporal manner, and in adult tissues, it is maintained within a very narrow range that is specific for each given tissue. If the control mechanisms fail and the concentration of atRA exceed or fall below the optimal range, tissues and cells undergo pathophysiological changes that in most severe cases can lead to disease (Mark et al.). Development of the kidney is reported to be the result of interactions between the metanephric and ureteric bud mesenchyme in the presence of retinoids (Vilar et al, 1996).
Embryopathy due to RA is being intensely investigated in view of the teratogenic potential of retinols and of the crucial role played by their receptors in embryo development. AtRA increases the production of reactive oxygen species and oxidative stress (Notario et al., 2003).
Quercetin (3, 30, 40, 5, 7-pentahydroxyflavone) is a flavonoid commonly found in frequently consumed foods, including apples, berries, onion, tea, nuts, seed and vegetables that represent an integral part of the human diet. Quercetin is one of the most abundant representing the 60-75 % of the average polyphenol ingestion (Goldberg et al., 1995). Quercetin has been reported to have biological, pharmacological, and medicinal activities that are believed to arise from its antioxidant properties (Perez-Vizcaino et al., 2009). Quercetin could prevent oxidant injury and cell death by several mechanisms, such as scavengening oxygen radicals, protecting against lipid peroxidation and chelating metal ions. Quercetin directly scavenges the superoxide anion and inhibits several superoxide-generating enzymes such as xanthine oxidase (XO) or the neutrophil membrane NADPH oxidase complex (Maciel et al., 2013).
As above was mentioned atRA increases oxidative stress and quercetin acts as antioxidant; in present study, the preventive effect of quercetin on histomorphometrical changes in kidney of fetuses of rat treated by atRA was evaluated.
MATERIAL AND METHOD
Animals. Male and female healthy rats of Wistar strain, 3-4 month old, weighing 200-220 g were purchased (Joundishapour laboratory animal center, Ahvaz, Iran) and housed individually (males) or at 10 per polycarbonate cage (female) for a 2-week acclimation period. Rats were fed ad libitum by standard laboratory pellet (Pars khurakdam, Tehran, Iran.) and tap water. A 12 h light:12 h dark was mentioned. Room temperature was at 23[+ or -]2 [degrees]C with a relative humidity of 45-55 %. This experimental study was done in animal model in department of basic sciences of faculty of veterinary medicine of Shahid Chamram University (Ahvaz -Iran). The animal care was provided under the supervision of a qualified veterinarian.
Females were mated overnight with males. Pregnancy was ascertained the next morning by presence of a vaginal plug, and this time was designated as gestational day (GD) 0.
Drug administration. Pregnant rats (n=40) were randomly divided into seven groups and treated as follow:
Group 1: Control group: normal saline was administrated to pregnant rats for inducing similar condition (injection and handling) to other groups.
Group 2: atRA (25 mg/kg) was intraperitoneally administrated at 8-10th days of gestation.
Group 3: Dimethylsulfoxide as atRA solvent was intraperitoneally administrated to pregnant rats for inducing similar condition (effect of solvent) to other groups.
Group 4: quercetin (75 mg/kg) was intraperitoneally administrated at 8-10th days of gestation.
Group 5: quercetin (200 mg/kg) was intraperitoneally administrated at 8-10th day of gestation.
Group 6: atRA (25 mg/kg) plus quercetin (75 mg/kg) was intraperitoneally administrated at 8-10th day of gestation.
Group 7: atRA (25 mg/kg) plus quercetin (200 mg/kg) was intraperitoneally administrated at 8-10th day of gestation.
Sampling and staining. The animals were sacrificed by euthanized and cervical dislocation at 20th day of gestation and a midline longitudinal incision was given from xiphi-sternum to pubic symphysis to expose the uterus; implantation sites in the uterine horns, number of intact fetuses and resorbed embryonic masses were counted. Fetuses were dissected using dissecting stereomicroscope and their kidneys were removed and fixed in 10 % formalin for 72 h for histological preparation. The specimens were dehydrated through a graded series of alcohol, cleared in xylene and infiltrated with molten paraffin before preparing the paraffin blocks; 5 pm thick sections were obtained, using rotatory microtome. The sections were stained with standard hematoxylin and eosin method before examining them under the light microscope (Fig. 1). Measurement of diameter periglomerular thickness and diameter of renal corpuscles was made after calibrating eyepiece graticule with stage micrometer at various magnifications using x10 and x40 objectives.
Statistical. Statistical significance between groups was determined using SPSS program and compared by one-way analysis of variance (ANOVA) and Post hoc LSD. The minimum level of significance was p < 0.05.
Percentages of absorbed fetuses were 66.66, 47.05 and 45.71 in groups 2, 6 and 7, respectively, so quercetin decreased the resorption rate. No maternal deaths were observed throughout the course of this study.
Kidneys were bilaterally present and normal in shape in all fetuses of the total groups. On histological examination, the sagittal section of kidneys from the control group showed well developed cortical renal corpuscles. In control, DMSO and quercetin alone groups, kidney showed no pathological appearances (Fig. 2).
On histological evaluation of kidney from treated group of fetuses, it was observed that there was severe degeneration of glomeruli. There was increased periglomerular space in sections of treated groups of kidney (Table I).
The mean of number of renal corpuscles of animals' fetuses that received atRA in 8-10th days was significantly decreased in comparison with normal saline group. The mean of diameter of renal corpuscles of animals' fetuses that received atRA significantly increased in comparison with other groups except with atRA plus quercetin (75 mg/kg). The mean of diameter of periglomerular space of animals' fetuses that received atRA significantly increased in comparison with other groups (Fig. 3) (Table I).
In the present study for first time, the effect of quercetin on histomorphometrically changes in kidney of rat fetuses treated by atRA was evaluated. We demonstrated astRA (at dose 25 mg/kg, IP) decreased number of renal corpuscles and increased periglomerular space and diameter of renal corpuscles.
Elmazar et al. (1996) found hypoplasia of kidney, hydronephrosis and hydroureter after administration of RA (37.5 mg/kg body weight). In the present study we focused on the effects of RA on histological structure of fetal kidney. The RA was given on 8-10th of gestation, considered to be critical for developing kidney, since nephrogenic cords are reported to appear by 8th day and pronephric tubules and duct are observed to be suspended in the coelom by 10th day (Naseer & Tahir, 2012).
Examination of the sagittal section of kidneys from control group showed normal looking well developed organ with outer cortex and inner medulla, whereas those from experimental group, showed increase in periglomerular space and diameter of renal corpuscles induced by RA. Similar findings were reported earlier in which development of the collecting duct system was greatly impaired in RAR ab2-mutant mice embryo; fewer branches of ureteric bud were present, and their ends were positioned abnormally at a distance from the renal capsule (Mendelsohn et al., 1999).
The results presented here show that quercetin administration during the gestational period has a partial protective effect on atRA-induced teratogenesis (decreasing periglomerural space, increasing number of renal corpuscles). It is well established that atRA is an important physiological regulator of embryonic development; it regulates many processes in organogenesis such as development of important organs and systems including the heart, the cardiovascular system, the hindbrain, and the foregut, among others (Ali-Khan & Hales, 2006). However, both its deficiency and excess can result in abnormal embryonic development. When atRA is administered in large doses during this critical period GD 8-10, it causes embryonic malformations in a dose-dependent manner. Inappropriate gene expression has been proposed as a mechanistic basis for atRA teratogenicity. Morphological changes visible after atRA treatment of embryos could be explained by alterations in the spatial and temporal patterns of expression of genes controlling differentiation, proliferation, apoptosis, and morphogenesis in embryonic organization and in initial axial patterning (Mulder et al., 2000).
Interestingly, inactivation of both RARa and RARb in mice fetuses resulted in renal malformations (Mendelsohn et al., 1994). Further evidence showed that RARa and RARb were coexpressed with Ret, a receptor tyrosine kinase involved in renal development, in renal stromal mesenchyme, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth, and down-regulation of Ret in the ureteric bud. Moreover, studies in mice indicate that the RA signaling in ureteric bud cells mainly depends on atRA generated through Raldh2 in stromal cells (Malpel et al., 2000).
The kidney is an organ highly vulnerable to damage caused by reactive oxygen species (ROS), likely due to the abundance of polyunsaturated fatty acids in the composition of renal lipids. ROS are involved in the pathogenic mechanism of conditions such as glomerulosclerosis and tubulointerstitial fibrosis.
AtRA plays important role in the control of cell differentiation and morphogenesis during prenatal development. However atRA, used in the treatment of dermatological disorders, has been implicated in the production of congenital anomalies in infants born to mothers taking the drugs during the first trimester. The critical period for atRA exposure appears to be 2-5 weeks postconception for humans (Holson et al, 1997).
Also, we observed protective effect of quercetin on atRA teratogenicity. This effect reported by some researchers. For example; Prater et al. (2008) reported that low-dose quercetin (66 mg/kg supplemented in rodent chow throughout gestation; approximately 70 % of human dose), high-dose quercetin (333 mg/kg supplemented in rodent chow throughout gestation; approximately 3.5x daily human dose), impairs placental oxidative stress and fetal skeletal malformation induced by methylnitrosourea.
In one study, quercetin treatment prevents renal tubular damage and increased oxidative stress induced by chronic cadmium administration, most probably throughout its antioxidant properties (Morales et al., 2006).
Devi & Shyamala (1999) reported quercetin (20 mg/ kg, i.p., once a week x5) has significant cytoprotective effect in cisplatin-induced renal tubular damage in vivo in rats. In one study, quercetin could protect the rat kidney against lead-induced injury by improving renal function, attenuating histopathologic changes, reducing ROS production, renewing the activities of antioxidant enzymes, decreasing DNA oxidative damage and apoptosis (Liu et al., 2010).
Gupta et al. (2010) reported that quercetin (10, 30 and 100 mg/kg) for 5 consecutive days) ameliorates the diethylnitrosamine induced hepatotoxicity in rats and can be a candidate for a good chemoprotectant.
Also, quercetin reduced abnormal development of mouse embryos produced by hydroxyurea. Liang et al. (2009) demonstrated that quercetin (66 mg/kg supplemented diet) significantly improves high fatty saturated induced fetal skeletal maldevelopment, perhaps in part due to antioxidant effects of quercetin in placenta. This speculation is supported by previous reports that demonstrate quercetin prevention of oxidant injury and cell death by ROS scavenging and protection against lipid peroxidation
In another study, quercetin with dose 50 mg/kg orally was most effective in preventing arsenic poisoning by reducing oxidative stress (Dwivedi & Flora, 2011).
In conclusion, the present study showed the effects of quercetin for the first time on histomorphometrically changes induced atRA in kidney of rat fetuses. The present results indicate that exposure 25 mg/kg of atRA in 8-10th days of gestation of rat decreases number of renal corpuscles and increase diameter of renal corpuscles and periglomerural space in kidney of fetuses. The protective effect of quercetin in atRA -induced histomorphometrical changes in kidney of rat fetuses may, at least in part, be due to its antioxidant activity, which we believe deserves further investigation.
Hereby, research deputy of Shahid Chamran University of Ahvaz will be highly appreciated for financial funding of this study.
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Department of Anatomy and Embryology
Faculty of Veterinary Medicine
Shahid Chamran University of Ahvaz
Mahmood Khaksary-Mahabady (1), Reza Ranjbar (1), Hossein Najafzadeh-Varzi (2), Babak Mohammadian (3), & Nahid Gohari-Behbahani (4)
(1) Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
(2) Department of Pharmacology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
(3) Department of Pathology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
(4) Veterinary Anatomical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Caption: Fig. 1. Histological structure of sagittal section of fetal kidney of control showing cortex (C), medulla (M), nephrogenic (N), deep cortex (D), Renal corpuscles (G) and cross sections of cortical ducts (T), (H & E 10X).
Caption: Fig. 2. Histological structure of sagittal section of fetal kidney (H & E 10X). (A) Control, (B) DMSO , (C) quercetin (75 mg/kg), (D) quercetin (200 mg/kg). Orange arrow: periglomerular space, blue arrow: distal tubule, green arrow: proximal tubule, bilateral arrow: diameter of corpuscles
Caption: Fig. 3. Histological structure of sagittal section of fetal kidney (H &E 10X). (A) Control, (B) atRA , (C) atRA plus quercetin (75 mg/kg), (D) atRA plus quercetin (200 mg/kg). Orange arrow: periglomerular space, blue arrow: distal tubule, green arrow: proximal tubule, bilateral arrow: diameter of corpuscles.
Table I: Morphometric analysis of renal glomeruli in control and experimental animals. Groupl (control); Group 2 received atRA; Group 3 received DMSO; Group 4 received quercetin (75 mg/kg), Group 5 received quercetin (200 mg/kg); Group 6 received atRA plu Groups Mean number of corpuscles Mean Diameter of corpuscles Groupl 13.666 [+ or -] 0.186 a c 65.516 [+ or -] 0.975 a Group2 10.384 [+ or -] 0.407 b 74.843 [+ or -] 1.352 b Group3 12.928 [+ or -] 0.126 a c d 68.884 [+ or -] 1.232 a Group4 12.384 [+ or -] 0.212 c d 67.014 [+ or -] 1.131 a Group5 12.933 [+ or -] 0.118 a c d 68.428 [+ or -] 0.759 a Group6 10.833 [+ or -] 0.321 b 74.402 [+ or -] 1.035 b Group7 11.076 [+ or -] 0.264 b 69.452 [+ or -] 1.439 a Groups Mean diameter of periglomerural Groupl 4.624 [+ or -] 0.145 a Group2 15.926 [+ or -] 0.519 b Group3 5.886 [+ or -] 0.209 c Group4 4.303 [+ or -] 0.141 a Group5 4.539 [+ or -] 0.134 a Group6 11.165 [+ or -] 0.764 d Group7 6.672 [+ or -] 0.354 c * -Different letters indicate significant differences within the column (P<0.0001)