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A new model for fetal programming: maternal Ramadan type fasting programs nephrogenesisMohany, M.1, Ashton N.2, Harrath, H.1, Nyengaard, J.3, Alomar, S.1, Alwasel, S.11 Zoology Department, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.2 Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK3 Stereology and Electron Microscopy Laboratory, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. 1Zoology Department, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.Running Title: Maternal fasting and kidney developmentCORRESPONDENCE TO:Saleh AlwaselE-mail: salwasel@ksu.edu.saAbstractThe effect of maternal Ramadan fasting type (RTF) on pregnancy outcome, kidney development, and nephron number in male rat offspring was investigated in this study. Pregnant rats were given food and water ad libitum during pregnancy (control) or restricted for 16 h per day (RTF). Kidney structure was examined during fetal life, at birth, and in early and late adulthood. Maternal body weight, food intake and plasma glucose levels were significantly lower (p ? 0.001) in the RTF group. Litter and pup weights were significantly lower (p ? 0.05) in the RTF group at birth, with no difference in litter size. The RTF group had a longer gestation, delayed nephrogenesis with less well differentiated glomeruli, more connective tissue, fewer medullary rays, a wider nephrogenic zone, and increased apoptosis at birth. Maternal fasting reduced nephron number (by 31%) with unchanged kidney and total glomerular volume. Mean glomerular volume was significantly higher in RTF offspring. Qualitative assessment of renal structure revealed interstitial fibrosis, with enlarged lobulated glomeruli in the renal cortex and tubular dilation in the medulla of RTF kidneys. Taken together, gestational fasting delays nephrogenesis and reduces nephron number in the kidneys of the offspring, in part due to increased apoptosis. Key words: Fasting, pregnancy, nephrogenesis, apoptosisIntroductionRamadan is a month of day-time fasting that occurs annually in Islamic culture. The timing of Ramadan varies as it follows the lunar calendar; therefore it can fall in any season. Geographical and seasonal differences lead to wide variation in fasting hours ranging from 5 to 20 h per day. Although pregnant women are allowed to defer fasting until birth and weaning have occurred, most women in Saudi Arabia prefer to fast with their family and to share the spiritual practice.1 Inadequate nutrition during pregnancy is associated with fetal programming of adult diseases such as hypertension 2, diabetes mellitus 3 and cancer.4 The fetal programming hypothesis proposes that these diseases originate from adaptations that occur during early development, which may be vascular, metabolic and/or endocrine in nature. They permanently change the physiology of the offspring in adult life.5 A number of studies have provided evidence that maternal malnutrition contributes to abnormal structure and function of the kidney, which is linked to the development of hypertension.6,7 When nutrients are restricted in utero, their distribution to the kidney is reduced to ensure appropriate development of the brain and heart: this is denoted as a trade-off. As a result of this trade-off between the brain and kidney during organogenesis, the number of nephrons is diminished, thus increasing the risk of chronic kidney disease.8 Maternal food restriction reduces nephron number,9,10 decreases the glomerular filtration rate,11 and alters renal sodium transporters in the resulting offspring.12 These changes in the kidney are linked to the development of hypertension in humans 13 and in experimental animal models. 14 In humans, nephrogenesis begins during week 9 of gestation and is completed by 32-36 weeks.15 In rats, nephrogenesis begins at gestational day 13 and continues until approximately postnatal day 10.16 It has been postulated that maternal nutrient deficiency may lead to retardation of nephrogenesis and delayed maturation of the glomeruli.17 Investigators have utilized different levels of maternal malnutrition, including global food restriction,18 low iron,19 low protein diets 20 and other dietary regimes. Although most studies have shown that different types of maternal malnutrition induce permanent negative effects on the kidney, the mechanisms underlying this remain controversial. Despite the fact that over 1 billion Muslims fast during Ramadan every year, relatively few studies have investigated the effects of fasting on physiology. Some studies have shown that intermittent fasting and periodic fasting have positive impact on various organ systems such as the brain21 and heart 22. In contrast, fasting was found to reduce blood glucose, body weight and body mass index in adults.23 Fasting during pregnancy results in poor weight gain,24 shorter gestational length,25 smaller placenta,26 and occasionally, smaller baby size.1 No studies have investigated the effects of maternal fasting on kidney development of the offspring. Therefore, we hypothesized that fasting during pregnancy may reduce the availability of essential nutrients for the developing fetus and, subsequently, the fetal kidney might be vulnerable to permanent structural changes. The aim of this study was to introduce a new model to evaluate structural changes in the kidneys of rat offspring following maternal Ramadan type fasting.MethodsAnimals and housing conditionsThirty-two young adult virgin female Wistar rats weighing 230–250 g were obtained from the Central Animal House of Pharmacy College at King Saud University, and were maintained and monitored in a specific pathogen-free environment. All animal treatments were conducted according to the standards set forth in the guidelines for the care and use of experimental Animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The Research Ethics Committee in the Zoology Department, College of Science, King Saud University approved the protocol used in this study. All animals were allowed to acclimatize in polycarbonate cages inside a well-ventilated room for 1 week prior to experimentation. The animals were maintained under standard laboratory conditions (temperature of 23°C, relative humidity of 60–70%, and 12-h light/dark cycle). Standard rat chow (containing 20 % protein, 6 % ash, 4 % crude fats, 3.5 % crude fibers, 1 % calcium, 0.6 % Phosphorus, 0.5 % salt, 70 IU /Kg Vitamin E, 20 IU / Kg Vitamin A, 2.2 IU / Kg vitamin D and 2850 Kcal/Kg) was purchased from the Grain Silos & Flour Mills Organization (GSFMO), Riyadh, Saudi Arabia. The animals were provided with food and water ad libitum.MatingAnimals were mated in wire-mesh floored mating cages. Mating was confirmed by the appearance of a white vaginal plug on the cage floor. This was counted as day 0 of gestation. After that, all pregnant females were transferred to husbandry cages.Feeding regimen and experimental designPregnant females were randomly assigned into two groups. The control group (C, n = 16) received food and water ad libitum from day 0 of conception until birth. The second group was exposed to fasting for 16 h to mimic Ramadan fasting (RTF, n = 16), beginning on day 0 when conception was confirmed. In this model, food and water were withdrawn from 4:00 pm until 8:00 am, and over the next 8 h, rats were provided with food and water ad libitum. Maternal food intake and body weight were recorded daily until the end of pregnancy. After birth, both groups were provided with food and water ad libitum. Pup number, sex and weight were recorded on the day of birth. Litters were culled to four males and four females, wherever possible, in order to standardize milk availability. Mothers and offspring were monitored on a daily basis until the end of weaning. Sample collectionEight pregnant rats at gestational days 18 (GD 18) and 20 (GD 20) were anaesthetized with Inactin (100 mg/kg i.p.). Fetuses were dissected and weighed before fetal kidneys were harvested. Blood samples from pregnant rats at GD 18 and GD 20 were withdrawn from the heart and placed into heparinized tubes. In other subgroups (n = 10), offspring were euthanized on postnatal day 1, 28 and 112, and kidneys were immediately immersed and fixed in 10% neutral buffer formalin (NBF) (pH 7.4) for subsequent use in histopathological examinations. Blood biochemistry Plasma was separated by centrifugation at 800 g for 10 min, and the resulting plasma was stored at -80°C for subsequent use in blood biochemistry analyses. Plasma total protein was estimated according to the Biuret method, in which protein in alkaline solution form with copper(II) ions a colored complex , highly stable, which is spectrophotometrically measurable by commercially available diagnostic kits, according to the manufacturer’s instructions (Quimica Clinica Aplicada S.A., Spain). Estimation of plasma glucose based on the oxidation of glucose to gluconic acid is catalyzed by glucose oxidase producing hydrogen peroxide. The hydrogen peroxide reacts with 4-aminoantipyrine and p-hydroxybenzoic acid in the presence of peroxidase to give a quinone derivative, whose coloration was measurable by commercially available diagnostic kits, according to the manufacturer’s instructions (Quimica Clinica Aplicada S.A., Spain) which is proportional to the glucose concentration in the sample..Renal histopathologyAfter kidney fixation, all samples were placed in tissue cassettes. The cassettes were then placed in an automated histology tissue processor (Tissue-Tek VIP 5, Sakura, USA) and processed overnight for wax infiltration. The tissue cassettes were removed from the processor and embedded in paraffin wax using Tissue-Tek TEC 5 (Sakura, USA). Sections (5–7 ?m) were obtained using an automated microtome (Leica RM 2125 RM, Leica Microsystems, Nussloch, Germany), floated on warm (30°C) water, and placed onto glass slides. Sections were stained with haematoxylin and eosin (H&E) using an automated staining machine (Multistainer Leica ST5020, Leica, Germany) and cover slipped (Leica CV5030, Leica, Germany). A light microscope (Olympus BX 50, Japan) with a digital high-resolution camera (Olympus DP 70, Japan) was used for imaging.Cortex , medulla and nephrogenic zone assessment For the measurement of the cortex, medulla and the nephrogenic zone in day 1 kidneys, 4 ? sections from the central region of the kidney were H&E stained and examined by optical microscopy. For each section, 3 microphotographs were obtained at a magnification of 40 x. Three measurements for each parameters were recorded and measured using image analysis software (Imagej v. 1.46 r for Windows, NIH, USA). An average was determined for each kidney. The nephrogenic zone was defined as the area in the outer renal cortex exhibiting developing nephrons in the form of comma and S-shaped bodies. The renal cortex was defined as the area from the cortico-medullary junction to the superficial edge of the outer section.Glomerulosclerosis index and interstitial fibrosis Kidney specimens were fixed in 10% neutral buffer formalin (NBF) (pH 7.4), the tissue was processed to paraffin, sectioned at 4 ?m, and stained with periodic acid Schiff (PAS) for demonstration of glomerulosclerosis and interstitial fibrosis. Histological changes were assessed semi-quantitatively. Periodic acid - Schiff (PAS) stained sections were examined using a Nikon Eclipse E600 light microscope. One hundred glomeruli per section were randomly selected and the degree of glomerular damage assessed using a semiquantitative scoring method: grade 0, normal glomeruli; grade 1, sclerotic area up to 25% (minimal sclerosis); grade 2, sclerotic area 25 to 50% (moderate sclerosis); grade 3, sclerotic area 50 to 75% (moderate-severe sclerosis); grade 4, sclerotic area 75 to 100% (severe sclerosis). The glomerulosclerotic index (GSI) was calculated using the following formula: GSI = (1 x n1) + (2 x n2)+ (3 x n3) + (4 x n4)/ n0 + n1 + n2 + n3 + n4 where nx is the number of glomeruli in each grade of glomerulosclerosis (Saito et al., 1987). Additionally, interstitial fibrosis was assessed at 400x magnification on PAS-stained sections using 10 randomly selected fields for each animal and scored by the following criteria: 1, area of damage <25%; 2, 25–50%; 3, 50–75%; and 4, 75-100% as previously described (Leelahavanichkul et al. 2010).TUNEL assayKidneys (n = 5) were harvested following dissection from newborn rats, fixed in NBF for 18–24 h and preserved in ~70% alcohol. Paraffin sections were prepared at a thickness of 3 μm, mounted on slides, and then deparaffinized, rehydrated and washed in PBS. Paraffin sections were permeabilized in 0.1% Triton X-100, with 0.1% sodium citrate, and treated with pepsin (0.3% in HCl, pH 2) for 5 min at 37°C. Slides were placed in plastic jars containing 0.1 citrate buffer, pH 6, for microwave irradiation at 750 W for 45 s, and then rinsed twice with 1× PBS. TUNEL staining was performed using the In Situ Cell Death Detection Kit, TMR red 12156792910 (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions. Samples were rinsed twice with 1× PBS, stained with Hoechst, washed in TE buffer for 10 min and mounted in 50% glycerol/TE. Sections were observed with a Nikon TE 2000 fluorescent microscope and images were acquired using a Nikon DS-cooled camera head DS-5Mc connected to a Nikon DS camera control unit DS-L1. A quantification of apoptosis by the TUNEL assay was performed using a modification of the technique described by Malik et al. In brief, an observer blinded to the study group counted the total number of apoptotic nuclei per high power field (HPF) in six non-overlapping regions per section (x400). Morphometrical and stereological measurementsKidney samples were excised from 28-day-old rats, decapsulated and fixed in ~10% NBF (pH 7.4) for 24 h and then embedded in paraffin. Kidneys were extensively sectioned at thickness of 20 μm. During sectioning, every 30th and 31st section (a section pair) were collected and mounted on slides, with the first collected pair being chosen randomly between one and 30. All sections were placed in a 60°C oven for 48 h, deparaffinized in xylol, dehydrated in a series of ethanol and stained with H&E. All stereological analyses were carried out using a microscope (Olympus, BX-51) equipped with a digital camera (Olympus DP, 70) and a pro Scan III motorized stage (Prior Scientific, Rockland, MA) connected to a computer (Dell Optiplex GX110; Dell, Round Rock, TX) with two monitors. The system was controlled by the newCAST stereological software package (version 2.1.5.8, Visiopharm, Horsholm, Denmark) and the main objectives used were 1.25× and 10× dry lenses. Nephron number Glomerular number was counted in every 20 μm kidney section pair using a modified version of the physical fractionator technique as described by Schreuder et al.27 Approximately eight pairs of consecutive kidney sections per rat were used. Glomerular number was estimated using the following formula:N = 1SSF x 1ASF x Σ Q-2where, N is the total number of glomeruli, SSF is the section sampling fraction (=1/30), and ASF is the area sampling fraction, which is calculated as the counting frame area (~375000 ?m2) divided by the step lengths in the x- and y-direction (1580 ?m). ΣQ- refers to the sum of glomeruli counted per kidney. The total volume of the kidney was calculated from the weight using a kidney tissue density of 1.05 g/cm2 .28 Two different point-counting test grids were used to estimate the volume density of glomeruli in the kidney as follows:Vv(glom/kid) = ΣPglom . p(kidney)ΣPkidney. pglom ,where ΣP(glom) is the total number of points of the counting grid that hit glomeruli (renal corpuscles), p(kidney) = 4 is the number of points in the counting grid used to count kidney tissue, ΣP(kidney) is the total number of points of the counting grid that hit kidney tissue, and p(glom) = 81 is the number of points in the counting grid used to count glomeruli. Total glomerular volume was calculated by multiplying the total kidney volume with the glomerular volume density. Mean glomerular volume was then obtained by dividing the total glomerular volume with the total number of glomeruli per kidney. Total and mean glomerular volume are affected to an unknown degree by tissue shrinkage following paraffin embedding. Statistical analysisData were first tested for normality and homogeneity of variance prior to further statistical analyses. Data were normally distributed and are expressed as means ± standard errors (SEM). Maternal body weight and food intake were compared using a 2-way repeated measures analysis of variance (ANOVA); all other comparisons were made using 2-tailed Student’s unpaired t-tests (SPSS software, version 17). Differences were considered statistically significant at p < 0.05. ResultsModel characterizationBody weight was significantly (p < 0.001) lower in RTF dams compared to C animals (Fig. 1 A). Although animals in the RTF group had plenty of food during the day time, they ate significantly (p < 0.001) less food compared to the C group, a phenomenon that was observed on all gestation days (Fig. 1B). This represent a reduction of 180 Kcal during the whole gestation (C, 1305 ± vs RTF, 1125 ± Kcal, (p < 0.001). The calculation showed that relative food intake was significantly lower ( p ? 0.05) in RTF dams (Fig.1C). The total body weight gain throughout pregnancy was 114 g in the C group versus 71 g in the RTF group, resulting in a significant reduction (p < 0.001) in total weight gain in response to fasting. Analysis of blood samples obtained on GD 20 showed that the plasma glucose level was significantly lower in rats exposed to RTF than the C group. Plasma protein level was also decreased in RTF pregnant rats; however, this reduction did not reach statistical significance (Table, 1). Maternal fasting did not affect litter size of RTF (Table 1). Additionally, no differences were noted in maternal nursing behavior between the RTF and C mothers, and no pups were eaten by their mothers in either group.Litter weight was significantly reduced by ~8% in the RTF group. The average RTF pup weight was significantly lower than that of the control pups (Table 1). Gestation length was slightly but significantly increased in the fasting group. Kidney developmentGenerally, kidneys derived from the RTF group (Fig. 2) were less developed in comparison with age-matched controls at GD 18, GD 20 and birth, examined at 40× magnification. The kidney from RTF animals at GD 18 appeared to have a greater proportion of mesenchymal-like connective tissue compared with the controls. At GD 20, the cortex and medulla were well-differentiated in the control kidneys but were less-differentiated in RTF kidneys, and the latter seemed to have fewer medullary rays. RTF kidneys at birth exhibited a wider nephrogenic zone and had a greater proportion of connective tissue and relatively few medullary rays in comparison to the controls. At high magnification (400×), control GD 18 kidneys exhibited extensive glomerulogenesis at various stages, with the normal development of peripherally-induced, developing nephron sites and ureteric buds. The control nephrogenic developmental stages were identifiable as comma- and S-shaped, pre-capillary and immature glomeruli (Fig. 3 A). Few mature glomeruli were observed in the RTF group since most glomeruli were less well differentiated in the form of S- and comma-shaped bodies. There were also more interstitial mesenchymal cells in the interstitial area (Fig. 3 B). Kidneys from the GD 20 control group (Fig. 3 C) had immature and mature glomeruli under the capsule, whereas the metanephroi of the RTF group showed predominantly immature S-shaped bodies (Fig. 3 D). Many well-developed glomeruli were observed in the cortico-medullary region of the kidney at day 1 in the control group (Fig. 3 E), and the renal cortex showed two distinct cortical zones; the nephrogenic zone containing immature forms of renal development and the juxtamedullary zone containing mature renal corpuscles and convoluted tubules. The medullary rays were present in both zones. At day 1, the RTF kidneys showed retarded nephron maturation and many immature glomeruli in the nephrogenic zone (Fig. 3 F). The semiquantitative analysis indicated that the cortex/medulla ratio was not different between RTF and control groups, however, the nephrogenic-zone/cortex ratio was significantly higher in RTF kidney compared to control (C, 32.1 ± 2.2 vs. RTF, 44.5 ± 21.7; p < 0.001). Kidney apoptosisApoptosis was assessed by TUNEL assay, in which positive staining indicates DNA fragmentation indicative of apoptosis. The incidence of TUNEL-positive staining was higher in the RTF group compared with the controls (Fig. 4). The results showed that RTF significantly increased apoptotic nuclei by 77 % compared to control (C, 10.8 ± 0.85 vs RTF, 19.2 ±0.86; p < 0.001) (Fig. 4). Stereology Histological examination of the renal cortex from 28-day-old rats revealed no qualitative differences in either the glomeruli or distal and proximal convoluted tubules between the RTF and control groups (Fig. 5 A). However, stereology revealed glomerular enlargement and fewer nephrons in kidneys from the RTF group (Fig. 5 B). There was a significant reduction in nephron number (~31% fewer) in kidneys from the RTF group compared with those from the control group (C, 2680 ± 1680 vs. RTF, 18500 ± 1000; p < 0.001; Fig. 5 C). Conversely, the mean glomerular volume was significantly greater in kidneys from the RTF group compared with those from the C group (C, 0.50 ± 0.088 × 106 ?m3 vs. RTF, 0.75 ± 0.1 × 106 ?m3; p = 0.197; Fig. 5 F). Thus, the total volume of all glomeruli in RTF kidneys was not different from that of the control animals as shown in Fig. 5 E. Although nephron number and mean glomerular volume were altered in the RTF group, the overall size, structure and volume of the kidneys were not different between the RTF and C groups (C, 0.432 ± 0.0299 cm3 vs. RTF, 0.463 ± 0.0228 cm3; p = 0.454; Fig. 4 D).Histopathology Sections of renal cortex from 112-day-old rats revealed normal structure of the proximal convoluted tubules, distal convoluted tubules, Bowman's capsule and glomeruli in the control group (Fig. 6 A). RTF kidneys showed more tubular atrophy and interstitial fibrosis with enlarged lobulated glomeruli in the renal cortex (Fig. 6 B). Renal medulla from the control group (Fig. 6 C) showed normal microscopic architecture with collecting ducts, thin limb loops of Henle and thick descending limbs, whereas the medulla from RTF offspring exhibited changes, including tubular dilatation and infiltration of proteinaceous casts (Fig. 6 D). Glomerulsclerosis and interstitial fibrosis were assessed semi-quantitatively in PAS stained sections. The data of glomerulosclerosis damage score exhibited mild glomerulosclerosis in RTF renal cortex compared with control renal cortex (Fig.7 A-C). While, the degree of interstitial fibrosis was significantly increased (p ? 0.05) in RTF renal medulla compared with control renal medulla (Fig.8 A-C). DiscussionDespite the well-documented deleterious effects of maternal malnutrition in the development of disease,29,30 limited data are available on the long-term effects of fasting exposure during gestation, especially on renal programming. Food intake and body weight gain were significantly lower in the fasting group compared with the control. This level of reduction in food intake is similar to the food restriction employed by others;31,32 but differs in terms of the timing of food availability. This finding is also consistent with the well-established observation of fasting or food restriction in pregnant rats 33 and humans 34 when subjected to weight loss as a result of decreased energy intake. The present study shows that exposure of pregnant rats to a 16-h fast significantly increased gestation length. Reduced maternal food intake in sheep also increased gestational length.36 In contrast, Almond and Mazumder 35found that maternal fasting in humans resulted in a significant reduction of gestational length. Total protein was not affected by maternal fasting; however, blood glucose was significantly reduced in the maternal fasting group. A reduction in blood glucose can initiate a signaling cascade that leads to permanent physiological adaptations, which increase the risk of developing chronic disease.37 The results of our study showed that birth weight was significantly lower in RTF offspring, which may be due to a reduction in maternal food intake and overall weight gain. These findings are consistent with findings obtained from clinical studies in humans, in which in utero exposure to fasting during Ramadan has been associated with lower birth weight among Michigan Arab mothers38 and Saudi Arabian mothers.1 In the present study, exposure to fasting during pregnancy had no effect on litter size. Similar observations were made by Woodall et al. 39 who reported that 30% nutritional restriction did not affect litter size. Both low birth weight and early catch-up growth are considered developmental markers for later adult disease.40 An interesting finding in the current study is that Ramadan type fasting delayed nephrogenesis. Kidney sections of RTF displayed less well differentiated glomeruli, more connective tissue, fewer medullary rays and significant increase in area of nephrogenic zone. In addition, sections showed a significant increase in apoptosis in RTF rat kidneys at birth when compared with controls. These findings are in accordance with a study conducted by Tafti et al., (2011) which showed that maternal undernutrition upregulated most of genes involved in both the extrinsic and intrinsic pathways of apoptosis as well as the downstream caspase cascade in offspring nephrogenesis. Previous studies have shown that protein restriction in pregnancy was associated with decline in the final number of glomeruli with aberrant nephrogenesis induced by increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis (Welham et al., 2002).Another major finding of this study was that offspring exposed to maternal RTF exhibited a ~31% reduction in nephron number compared with the control, suggesting that maternal RTF may have slowed or halted nephrogenesis. Evidence from animal studies suggests that maternal protein or global nutrient restriction, uterine artery ligation, hyperglycaemia, and exposure to various agents, such as glucocorticoids or alcohol, lead to the production of offspring with fewer nephrons.41 Studies have suggested different mechanism(s) leading to reduced nephron number; for example, the depletion of stem cells by increased apoptosis,42,43inhibition of ureteric branching44 and early cessation of nephrogenesis.45 The second finding of this study was that maternal RTF had no effect on kidney volume or total glomerular volume. Consistent with the present findings, offspring exposed to 50% food restriction in utero had a similar kidney volume to the controls.46 Accordingly, mean glomerular volume of RTF offspring was significantly greater than that of the control animals, probably due to a decrease in nephron number. Additionally, some studies investigating nutritional programming have revealed an inverse relationship between nephron number and blood pressure 6,47. The results of the current study show that maternal RTF during pregnancy resulted in retarded nephrogenesis. Similar observations have been made in previous studies 45 using different models of nutritional programming. Delayed nephrogenesis may occur due to increased apoptosis, as observed in the present study. Pathological structural observations in adult kidneys may be the result of low nephron number. Similar observations have shown that 50% protein restriction during pregnancy induces renal injury with enhanced tubular dilatation, tubular atrophy and interstitial fibrosis.48 A limitation of our study is the duration over which fasting was imposed upon the pregnant rats. Pregnant women observing Ramadan fast for 1 month out of their 9 months of pregnancy, whereas in our rat model the animals were subject to daily fasting from conception to term. We chose this approach in order to illicit the maximum response, as in principle Ramadan could fall at any stage of human pregnancy, By fasting our rats throughout gestation we were able to impose the greatest challenge on the fetus; however we recognize that the magnitude of the impact on the developing kidneys may be less if the rats were fasted for shorter periods of time.In conclusion, we have developed a rat model of maternal Ramadan fasting, in which the kidneys of offspring exhibit retarded nephrogenesis and low nephron number, partly due to increased apoptosis.AcknowledgmentsThe authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group program. The Center for Stochastic Geometry and Advanced Bioimaging is supported by the Villum Foundation.Conflicts of InterestNone.Ethical StandardsThe authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guides on the care and use of laboratory animals.ReferencesAlwasel SH, Abotalib,Z, Aljarallah JS, Osmond C, Alkharaz SM, Alhazza IM, Barker, D. J. P. Sex differences in birth size and intergenerational effects of intrauterine exposure to Ramadan in Saudi Arabia. Am J Hum Biol. 2011;23, 651-654.?Van Abeelen AF, Veenendaal MV, Painter RC, De Rooij SR, Thangaratinam S, Van Der Post JA, Bossuyt PM, Elias SG, Uiterwaal CS, Grobbee DE, Saade GR, Mol BW,Khan KS, Roseboom TJ. The fetal origins of hypertension: a systematic review and meta-analysis of the evidence from animal experiments of maternal undernutrition. J Hypertens. 2012; 30, 2255-67.Sánchez-Muniz FJ, Gesteiro E, Espárrago Rodilla M, Rodríguez Bernal B, Bastida S. 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Schreuder MF, Nyengaard JR, Fodor M, van Wijk JA, Delemarre-van de Waal HA. Glomerular number and function are influenced by spontaneous and induced low birth weight in rats.?J Am Soc Nephrol. 2005;?16, 2913-2919.?Nyengaard JR. Stereologic methods and their application in kidney research.?J Am Soc of Nephrol. 1999; 10, 1100-1123.?Paulino-Silva KM, Costa-Silva JH. Hypertension in rat offspring subjected to perinatal protein malnutrition is not related to the baroreflex dysfunction. Clin Exp Pharmacol Physiol. 2016;43,1046-1053. Alzamendi A, Zubiría G, Moreno G, Portales A, Spinedi E, Giovambattista A. High Risk of Metabolic and Adipose Tissue Dysfunctions in Adult Male Progeny, Due to Prenatal and Adulthood Malnutrition Induced by Fructose Rich Diet. Nutrients. 2016; 8,178. Léonhardt M, Lesage J, Dufourny L, Dickès-Coopman A, Montel VER, Dupouy JP. Perinatal maternal food restriction induces alterations in hypothalamo-pituitary-adrenal axis activity and in plasma corticosterone-binding globulin capacity of weaning rat pups.?Neuroendocrinol. 2002;?75, 45-54.?Akitake Y, Katsuragi S, Hosokawa M, Mishima K, Ikeda T, Miyazato M, Hosoda H. Moderate maternal food restriction in mice impairs physical growth, behavior, and neurodevelopment of offspring.?Nutr Res. 2015;?35, 76-87.?Fleeman TL, Cappon GD, Chapin RE, Hurtt ME. The effects of feed restriction during organogenesis on embryo-fetal development in the rat.?Birth Defects Res B Dev Rep Toxicol. 2005;?74, 442-449.?Awwad J, Usta IM, Succar J, Musallam KM, Ghazeeri G, Nassar AH. The effect of maternal fasting during Ramadan on preterm delivery: a prospective cohort study.?BJOG. 2012; 119, 1379-1386.?Almond D, Mazumder B.?Health capital and the prenatal environment: the effect of maternal fasting during pregnancy?(No. w14428). Natl Bur Eco Res. 2008;14428.Cleal JK, Poore KR, Newman JP, Noakes DE, Hanson MA, Green LR. The effect of maternal undernutrition in early gestation on gestation length and fetal and postnatal growth in sheep.?Pediatr Res. 2007; 62, 422-427.?Sharp D. The Fetal Matrix: Evolution, Development and Disease.?J R Soc Med. 2005; 98,130-131.Almond D, Mazumder B. Health capital and the prenatal environment: the effect of Ramadan observance during pregnancy.?Ame Econ J: Appl Econ. 2011; 3,56-85.? Woodall SM, Johnston BM, Breier BH, Gluckman PD. Chronic maternal undernutrition in the rat leads to delayed postnatal growth and elevated blood pressure of offspring.?Pediatr Res. 1996; 40(3), 438-443.?Osmond C, Barker DJ, Winter PD, Fall CH, Simmonds SJ. Early growth and death from cardiovascular disease in women.?Bmj. 1993; 307, 1519-1524.?Singh RR, Moritz KM, Bertram JF, Cullen-McEwen LA. Effects of dexamethasone exposure on rat metanephric development: in vitro and in vivo studies.?Am J Physiolo Renal Physiol. 2007;?293(2), F548-F554.?Welham SJ, Wade A, Woolf AS. Protein restriction in pregnancy is associated with increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis.?Kidney internat.?2002; 61, 1231-1242.?Pham TD, MacLennan NK, Chiu CT, Laksana GS, Hsu JL, Lane RH. Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney. Am J Physiol Regul Integr Comp Physiol. 2003 285, R962-70. Hokke SN, Armitage JA, Puelles VG, Short KM, Jones L, Smyth, IM, et al. Altered ureteric branching morphogenesis and nephron endowment in offspring of diabetic and insulin-treated pregnancy.?PloS one. 2013;?8, e58243.?Awazu M, Hida M. Maternal nutrient restriction inhibits ureteric bud branching but does not affect the duration of nephrogenesis in rats.?Pediatr Res. 2015;?77, 633-639.?Vaccari B, Mesquita FF, Gontijo JA, Boer PA. Fetal kidney programming by severe food restriction: Effects on structure, hormonal receptor expression and urinary sodium excretion in rats.?J Renin-Angiotensin-Aldosterone Syst.?2015; 16(1), 33-46.?Benz K, Amann K. Maternal nutrition, low nephron number and arterial hypertension in later life.?Biochim Biophys Acta. 2010;1802, 1309-1317.?Woods LL, Weeks DA, Rasch R. Programming of adult blood pressure by maternal protein restriction: role of nephrogenesis.?Kidney int. 2004;?65, 1339-1348.?LegendsFig. 1: Body weight, food intake and relative food intake were recorded daily during gestation. Maternal body weight, food intake and relative food intake were significantly lower in the RTF (p ? 0.001, p ? 0.001and p ? 0.05 respectively)group compared to C animals on all gestation days. Statistical comparisons were performed by repeated measures ANOVA. Data are presented as means ± SEM.Fig. 2: Representative micrographs of kidney cross sections from the control and RTF groups at GD 18, GD 20, and day 1 stained with haematoxylin and eosin (H&E). RTF kidneys are immature, as illustrated by the higher level of connective tissue at GD 18, fewer medullary rays at GD 20, and wider nephrogenic zone at birth. H&E (40×).Fig. 3: Light micrographs of kidney tissue from the control (A, C, E) and RTF (B, D, F) groups at GD 18, GD 20, and day 1. Developing nephrons at various stages are seen in the renal cortex. At GD 18, the arrow indicates a cluster of mesenchymal cells around the ureteric duct (U). RV denotes renal vesicle, S-shaped body; C, comma shaped body with mitotic figure (arrow); G, glomerulus at the capillary loop stage, the parietal epithelium is cuboidal, and the renal tubule (RT). Note that kidneys from the RTF group have more mesenchymal-like connective tissue (CT) with fewer well differentiated glomeruli in the form of S- and comma-shaped bodies. GD 20 metanephros sections from control animals depict stages of glomerular maturity with more glomeruli (G). Although the metanephros of the RTF group at GD 20 shows considerable growth and differentiation, the most predominant nephron stages are the S-shaped bodies (s). Section from the renal cortex of a control kidney at day 1 reveals the presence of two cortical zones; the nephrogenic zone (NZ) containing immature forms of renal developmental stages (arrows), and the juxtamedullary zone (J) containing mature glomeruli (G), and Bowman's space (head arrow). The medullary rays (MR) are located in both zones. The RTF group shows delayed glomerular maturation (G) and fewer (immature) glomeruli in the nephrogenic zone (H&E).Fig. 4: Kidney on day 1 from C (A–C) and RTF (D–F) animals stained with Hoechst (nuclear staining) and TMR red (DNA fragmentation). TUNEL assay showing an increased rate of apoptosis (arrows) in nephrogenic zone of RTF kidney compared with a control kidney at birth. Scale bar = 100 ?m.Fig. 5: Representative micrographs of kidney tissue from the control (A) and RTF (B) groups at day 28, depicting a low nephron number with glomerular enlargement (stars) in the RTF group (H&E, 200×). The nephron number in rat kidneys was significantly (p ? 0.001) reduced in the RTF rats compared to the control rats (C). There was no significant difference in kidney volume between the two groups (D). There was no significant difference in total glomerular volume between RTF and C offspring (E). Mean glomerular volume was significantly higher in the RTF group compared with the C group (F), (n = 8 per group). Fig. 6: Light micrographs of renal cortex (A, B) from the control and RTF groups at day 112. Section from the renal cortex of the control group reveals the normal appearance of the proximal convoluted tubules (PT), distal convoluted tubules (DT), Bowman's capsule and glomerulus (G). The RTF renal cortex shows mild glomerulosclerosis with segmental sclerosis (head arrows), thickening of Bowman's capsule and hyaline changes. Light micrographs of renal medulla (C, D) from the control and RTF groups at day 112. The renal medulla from control rats shows normal collecting ducts (CD), thin limb loop of Henle, and thick descending limb. Medulla of RTF offspring shows tubular dilatation and infiltration of proteinaceous casts (head arrow) (H&E).Figures and TablesFigure (1):-53340-43180A00A-3429025400B00B184785-95250C00CTable 1: Characteristics of the Ramadan fasting model. Data are presented as the mean ± SEM; n = 16 per group, p ? 0.05*; p ? 0.001***, NS = not significant. Maternal glucose level, litter weight, and pup weight were significantly lower in the RTF group. Maternal fasting resulted in a longer gestation period in the RTF group. There were no significant differences in maternal plasma protein and litter size between groups.ParametersGroupsSignificanceCRTFPlasma glucose (mg/dl)54.4 ± 2.0528.7 ± 2.35***Plasma protein (g /dl)8. 71 ± 0.636.86 ± 0.71NSLitter size 11.8 ± 0.3311.8 ± 0.43NSLitter weight71.7 ± 2.367.6 ± 2.4*Pup weight6.22 ± 0.15 5.69 ± 0.06*Gestation length21.2 ± 0.1722 ± 0.09*Figure (2)Figure (3)Figure (4)Figure (5)16510128270C00C-1333585090D00D1143093980E00E4127592710F00FFigure (6) ................
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