Experimental Nephrology and Genetics: Original Paper Nephron 2015;129:283–292 DOI: 10.1159/000377634
Received: October 31, 2014 Accepted after revision: February 1, 2015 Published online: April 18, 2015
Fetal Kidney Programming by Maternal Smoking Exposure: Effects on Kidney Structure, Blood Pressure and Urinary Sodium Excretion in Adult Offspring Daniel B. Block Flávia F. Mesquita Ize P. de Lima Patricia A. Boer José A.R. Gontijo Department of Internal Medicine School of Medicine, State University of Campinas, Campinas, Brazil
Abstract Introduction: Fetal programming by different insults results in low birth weight and reduction in nephron number increasing the risk for adult development of cardiovascular and renal diseases. Maternal smoking is an important modifiable adverse fetal exposure worldwide and leads to a decrease in the offspring’s birth weight. Thus far, the specific adverse fetal smoking exposures and mechanisms underlying these associations on renal development and functional disorder are unclear. Methods: The present study investigates, in adult male rats, the effect of smoking exposure (Sk) in uteri on blood pressure (BP) by an indirect tail-cuff method using an electrosphygmomanometer, and its association with nephron structure by stereological estimation, immunohistochemical and histological techniques, in parallel with kidney function creatinine and lithium clearance. Results: The current study showed in a 16-week old Sk offspring enhanced arterial blood pressure associated with, reduced urinary sodium excretion and higher TGF-β1 glomerular expression. Sk glomeruli also presented an upregulated collagen and fibronectin deposition intrinsically related to fibrotic process as compared to age-matched control group. Conclusion: Here, we demonstrate that fetal-programmed
© 2015 S. Karger AG, Basel 1660–8151/15/1294–0283$39.50/0 E-Mail [email protected] www.karger.com/nef
Sk offspring present pronounced glomerular TGF-β1 and fibrotic marker expression that may, subsequently, promote a glomerular epithelial-mesenchymal transition activated process in an Sk offspring. Although the precise mechanism responsible for the subsequently renal morphological and functional response in Sk offspring is incompletely known, the current data suggest that changes in renal function are conducive to excess sodium tubule reabsorption that is associated with enhanced TGF-β1, fibronectin and collagen deposition, intrinsically related to fibrotic process, might potentiate the programming of adult hypertension. © 2015 S. Karger AG, Basel
Introduction
The developmental plasticity hypothesis suggests that various adverse intrauterine exposures lead to persistent fetal developmental adaptations. These adaptations may be beneficial in the short term, but may have adverse consequences in postnatal life [1]. Fetal programming by different insults results in low birth weight and reduction in nephron number [2–7], and increases the risk for adult development of cardiovascular and renal diseases [4–6]. Low birth weight (LBW) is also associated with impaired renal growth, raised blood pressure and impaired renal function [8–11]. A reduction in nephron number and, therefore, in the whole-kidney glomerular filtration area would result in reduced sodium excretory capacity, enDr. José A.R. Gontijo Departamento de Clínica Médica Faculdade de Ciências Médicas, Universidade Estadual de Campinas Campinas, SP 13083–592 (Brazil) E-Mail gontijo @ fcm.unicamp.br
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Key Words Fetal programming · Maternal smoking exposure · Arterial hypertension · Glomerular fibrosis · TGF-β1 · Epithelial-to-mesenchymal transition
Material and Methods Animals The experiments were conducted on age-matched rats of sibling-mated Wistar HanUnib rats (250–300 g) allowed free access to water and normal rat chow. The experimental protocol and study design was approved by Institutional Animal Ethics Com-
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mittee (protocol 2419–1) and the general guidelines established by the Brazilian College of Animal Experimentation (COBEA) were followed throughout the investigation. Our local colonies originated from a breeding stock supplied by CEMIB/Unicamp (CEMIB/Unicamp, Campinas, SP, Brazil). Immediately after weaning at 3 weeks of age, animals were maintained under controlled temperature (25 ° C) and lighting conditions (7: 00 a.m.– 7:00 p.m.), with free access to tap water and standard rodent laboratory chow (Nuvital, Curitiba, PR, Brazil) and followed up to 16 weeks of age. The dams were divided into two groups as follows: (1) control group (Co) and (2) smoker group (Sk). The method of smoking exposure was adapted from studies by Mello et al. [23] and Gomes et al. [24]. Briefly, rats were placed in a Perspex chamber containing one inlet and one outlet. The inlet was connected, via a two-way valve, to a multicigarette smoking apparatus, containing five cigarettes, and to the external atmosphere. The outlet was attached to a negative-pressure pump. Under these conditions, a volume of air was sucked through the chamber, a portion of which was drawn through the cigarettes, thereby carrying smoke into the chamber. Combustion of five cigarettes occurred in approximately 30 min, by which time the chamber had been cleared of smoke by the passage of clean air. The animals of the Sk group, five animals at each time, were exposed to the inhalation of tobacco by 30 min at day, throughout the first week (adjustment period). After the adjustment period, animals were then mated and the day that the sperm were seen in the vaginal smear was designated as day 0 (zero) of pregnancy. After establishing pregnancy, the rats were subjected to basic smoke dose of two such exposures over 30min each per day (1 h total exposure), throughout the entire threeweek gestational period. It is estimated that the amount of smoke inhaled from cigarettes derived during the period of 4 weeks correspond to inhalation of 14 mg tar, 11 mg nicotine and 15 mg carbon monoxide [23, 24]. Between each exposure, rats were removed from the smoking chamber and offered fresh air, tap water and standard rodent chow, ad libitum. The smoking dams body mass was obtained at 9th, 14th and 16th days of gestation. Delivered Co or Sk male pups were weighed weekly.
Blood Pressure Measurement The systemic arterial pressure was measured in conscious offspring in 5th 10th, and 16th weeks of age by an indirect tail-cuff method using an electrosphygmomanometer (IITC Life Science – BpMonWin Monitor Version 1.33) combined with a pneumatic pulse transducer/amplifier. This indirect approach allowed repeated measurements with a close correlation (correlation coefficient = 0.975) compared to direct intra-arterial recording. The mean of three consecutive readings represented the blood pressure. Stereological Estimation of Kidney and Glomerular Volume and Glomerular Number At postnatal day 12 (post complete kidney maturation), offspring were anesthetized with a mixture of ketamine (75 mg·kg–1 body weight, i.p.) and xylasine (10 mg·kg–1 body weight, i.p.) and the left kidney was removed, weighed and volume was estimated by the Cavalieri’s principle. Then, they were longitudinally divided into two halves and a 4-mm slice, from each kidney, was weighted. The slices were placed in 10% formalin for stereological estimation of total nephron number. After paraffin inclusion, the tissue blocks were exhaustively sectioned at 5 μm and then stained with haematoxilin-eosin. We used the ‘fractionators’ method to estimate the
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hanced susceptibility to hypertension and reduced renal reserve; therefore, the limiting compensation for renal injury may, at least in part, explain the higher prevalence of hypertension and renal disease observed in populations with a high prevalence of low birth weight [4–6, 10]. Maternal smoking is a very important modifiable adverse fetal exposure worldwide and leads to a decrease of offspring’s birth weight [12]. Studies suggested that maternal smoking exposure might also be associated with higher blood pressure in the offspring, independent of birth weight [1, 13], which might be explained by adverse effects on fetal kidney development [1, 14]. Also, the classical vasoactive tobacco action has been extended due to its properties as cytokine that actively participates in the renal physiopathology. Thus far, the specific adverse fetal smoking exposures and mechanisms underlying these associations on renal development and functional disorder are not known. Irreversible renal fibrosis is a common consequence after most renal injuries [15, 16]. Incidentally, the transforming growth factor β (TGFβ) is a key regulator of extracellular matrix (ECM) protein deposition in renal tissue [17]. Podocyte increased expression of TGF-β mRNA and glomeruli ECM protein deposition have been associated to focal segmental glomerulosclerosis [18], membranous nephropathy and diabetic nephropathy [19]. Renal TGF-β1 expression is implicated in the pathogenesis of fibrosis in both glomerular and interstitial compartments [20]. It is clear that well-described phenomenon of type II epithelial-to-mesenchymal transition (EMT) plays a pivotal role in organ fibrosis progression [20, 21]. It has been demonstrated that TGF-β1 may stimulate the accumulation of matrix and inflammation [21, 22]. In the current study, we hypothesized that maternal smoking exposure may affect the body and kidney growth, the renal morphological structure and function and, the arterial blood pressure levels in adulthood rats. Thus, the present study was performed in 12-days and 16-week-old maternal smoking male offspring compared to appropriated gender and age-matched animals evaluating the kidney expression of fibrotic and EMT markers in parallel with kidney function.
Renal Function Evaluation The renal function tests were performed and estimated by creatinine and lithium clearance on the last day at 5, and 13 weeks of age in unanaesthetized, unrestrained Co (n = 15) and Sk (n = 11) male offspring. Briefly, fourteen hours before the renal test, 60 μmol CLi 100 g–1 body weight was given by gavage. After an overnight fast, each animal received a load of tap water by gavage (5% of body weight), followed by a second load of the same volume, 1 h later, and spontaneously voided urine was collected over a 120 min period into a graduated centrifuge tube. At the end of the experiment, blood samples were drawn through cardiac puncture in anesthetized rats, and urine and plasma samples were collected for analysis [26–28]. Measurement of Proteinuria In the 13th week of age, male rats from all groups (Co, n = 5 and Sk, n = 6) received a load of tap water by gavage (5% of the body weight) and twenty minutes later were housed individually in metabolic cages; spontaneously voided urine was collected over a 2 h period and immediately stored at –20 ° C until processing. The proteinuria was detected using the BIOPROT U/LCR (Bioclin).
Data Presentation, Statistical and Biochemical Analysis Data obtained from this study are expressed as the mean ± SD or median and quartile deviation when appropriated. Plasma and urinary sodium, potassium and lithium concentrations were measured by flame photometry (B262; Micronal, São Paulo, Brazil). Creatinine was determined spectrophotometrically (362; Micronal, São Paulo, Brazil) by the alkaline picrate method. Creatinine clearance was used to estimate the glomerular filtration rate (GFR) and lithium clearance (CLi) was used to assess proximal tubule output [26–28]. Fractional sodium excretion (FENa) was calculated as CNa/ CCr × 100, where CNa is sodium clearance and CCr is creatinine clearance. The fractional proximal (FEPNa) and post-proximal (FEPPNa) sodium excretion was calculated as CLi/CCr × 100 and CNa/CLi × 100, respectively. Data obtained over time were analyzed using appropriate Kruskal-Wallis one-way analysis of variance. Post-hoc comparisons between selected means were made by Bonferroni’s contrast test when initial analysis indicated statistical differences between experimental groups. Comparisons involving only two samples of independent observations within or between groups were made using the Student t-test or Mann-Whitney U test when appropriated from GraphPad Prism 5.01 (GraphPad Software, Inc., USA). The level of significance was set at p ≤ 0.05.
Results
Immuno and Histochemical Procedures Sixteen week-old adult male offspring from different mothers of the Co (n = 5) and Sk (n = 5) groups were anesthetized with a mixture of ketamine (75 mg·kg–1 body weight, i.p.) and xylasine (10 mg·kg–1 body weight, i.p.), and the level of anesthesia was controlled by monitoring the corneal reflex. The animals were then perfused with saline containing heparin (5%) for 15 min under constant pressure, followed by perfusion with 0.1 M phosphate buffer (pH 7.4) containing 4% (w/v) paraformaldehyde and 0.1 M sucrose for 25 min. After perfusion, kidneys were removed and placed in the same fixative for 2 h, followed by PBS containing 0.1% glycine for 30 min and PBS containing 15% (w/v) sucrose overnight at 4 ° C. In the following day, the tissues were placed in an OCT compound cryoprotector (Tissue-tech), cut on a Leica cryostat and mounted on silane-coated slides. For immunohistochemistry, the sections were incubated sequentially with (1) phosphate-buffered saline (PBS) containing 2% milk for 45 min to minimize non-specific reactions, (2) mouse anti-fibronectin antiserum (Santa Cruz) or rabbit anti-TGF-β1 antiserum (Santa Cruz) and (3) anti-mouse or rabbit Alexa 488 labeled antibody (molecular Probes) for 2 h at room temperature. After incubation, the sections were rinsed in 0.1 M PBS and cover-slipped with Vectashield anti-fading medium containing DAPI (Vector). The sections were examined with a fluorescence microscope (Olympus BX51). No immunoreactivity was seen in control experiments in which primary antibodies was omitted. After picrosirius staining, we evaluated the density of collagen. Ten cortical and ten medullar fields of histological sections for each experimental (Co, n = 4) or control (Sk, n = 4) animals were analyzed and the average of collagen density readings were determined. Images were captured with photomicroscope and analyzed by the Leica Qwin 3.1 for windows.
The maternal smoking exposure during pregnancy caused a significant decreased dam’s body weight over the whole gestation (fig. 1a). However, the smoking exposure did not affect the number of offspring per litter and the proportion between male and female offspring (p = 0.3245). Sk male offspring also presented significant reduction in birth body mass (7.20 ± 0.051 g in Co, n = 27 vs. 6.36 ± 0.24 g in Sk, n = 26, p < 0.0007) (fig. 1b). The body weight was unchanged in smoke offspring group (n = 11) from the 4th to 9th week of age (fig. 1c) when compared to control (n = 11) age-matched rats. However, beyond the 9th week of age, Sk offspring shows a significant decrease in body mass (fig. 1c). Beyond 16 weeks of age, the Sk offspring (n = 11) showed an increased systolic blood pressure from (Sk: 130.5 ± 2.43 mm Hg vs. 130.7 ± 2.43 mm Hg in Co at 5th week of age; NS), to (Sk: 146.1 ± 3.5 mm Hg vs. 134.2 ± 3.01 mm Hg in Co, at 16th week of age; p = 0.0001), when compared to control (n = 15) group (fig. 1d). Stereological estimations, at postnatal 12 days, immediately after complete renal maturation (n = 5 for each group), we verified that Sk offspring although present a reduction of the kidney mass (Sk: 0.1095 ± 0.006 g/100 g body weight vs. 0.1264 ± 0.021 g/100 g body weight in Co, p = 0.0278; fig. 1e), the glomeruli number per kidney (Sk: 9,908 ± 3,178.96 units vs. 10,143 ± 3,533.54 units in Co, p = 0.3452; fig. 1f), and glomeruli volume (15,761.21 ± 2,202.24 μl vs. 16,488.34 ± 4,792.07 μl in Co, p = 0.500; fig. 1g) were unchanged when compared with Co group.
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number of glomeruli and estimated the total number of renal corpuscles per kidney considering the analyzed fraction of the kidney corrected to the entire organ as described at Mandarim-de-Lacerda [25].
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0.0020. The enhanced FENa+ in Sk rats was accompanied by unchanged proximal sodium excretion and increased fractional potassium excretion (FEK+) (Sk: 0.36 ± 0.036% vs. 0.26 ± 0.022% in Co, p = 0.0169) compared with the Co age-paired control group. However, this increased FENa+ occurred in parallel to significant augmentation in FEPPNa+ (Sk: 2.85 ± 0.047% vs. 1.13 ± 0.15% in Co, p = 0.0001; fig. 2a). On the other hand, in offspring at 13-week of age, the renal tubule sodium handling parameters, estimated by CLi and CCr, did not significantly differ among the groups Co and maternal smoking exposure offspring (fig. 2b).
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Renal Function Data The data for renal function in 5- and 13-week-old male offspring of both (Co, n = 15 and Sk, n = 11) groups are summarized in figure 2. In 5-week-old rats (fig. 2a), in both studied groups, the urinary flow rates (data not show) and the glomerular filtration rates, estimated by CCr, did not significantly differ during the renal tubule sodium handling studies. In an offspring at 5 weeks of age, the fractional urinary sodium excretion (FENa+) was significantly higher in Sk rats when compared with Co age-matched group, as follows: 1.11 ± 0.19% vs. 0.45 ± 0.07% in Co, p =
Proteinuria Thirteen-week-old Sk group observed a significant increased urinary protein excretion (7.19 ± 1.16 μg/dl, n = 6) when compared to age-matched control group (4.84 ± 0.78 μg/dl) (p = 0.0087, n = 5) (fig. 2f). Immuno and Histochemical Procedures The study shows an intensive rise of the TGF-β1 immunoreactivity in 16-week-old Sk (n = 5) in both glomeruli and parietal epithelium (fig. 3a–d) compared to that found in the control (n = 5) group. Additionally, fibronectin immunoreactivity was also highly expressed in Sk (n = 5) offspring glomeruli and peritubular spaces when compared to age-matched Co (n = 5) animals (fig. 3e–h). The density of collagen fibers deposition in the renal cortical zone of 16-week-old Sk (n = 4) offspring, semi-quantitative evaluated by picrosirius staining technique, showed a
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fractional sodium excretion (FENa+, b), proximal (FEPNa+, c) and post-proximal (FEPPNa+, d), fractional sodium excretion and fractional potassium excretion (FEK+, e) in 5-week-old and 13-week-old control (n = 15) offspring compared to Sk (n = 11) age-matched rats.
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The main purpose of the current study was to expose embryos and fetuses to smoking in conditions similar to those inherent in active smoker’s mothers. The developmental plasticity hypothesis supports that adverse intrauterine exposures lead to persistent fetal adaptations and organ disorders in adulthood. Therefore, fetal programming by different insults results in low birth weight and increased risk for adult development of cardiovascular and renal diseases. Among the health hazards of smoking
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Also presents the daily urinary protein excretion in 13-week-old Sk offspring (n = 6) compared to appropriated age-matched controls (n = 5) (f). The data are reported as the median and quartile deviation. * p ≤ 0.05 versus control (Mann-Whitney or Kruskal-Wallis test with post-hoc comparisons by Bonferroni’s contrast test).
Fig. 3. Representative TGF-β1 and fibronectin immunoreactivity in Co and Sk kidney. Immunofluorescence reveal a normal content of TGF-β1 in the control offspring (a and c, n = 5) compared to notable staining obtained in maternal smoking offspring groups
(b and d, n = 5). Additionally, in e and g we have the normal pattern of fibronectin distribution in the kidney from control offspring and in Sk (f and h, n = 5) we can observe the enhanced deposition of this protein in glomeruli and peritubular extracellular matrix. (For figure 3 see next page.)
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significant increase comparatively to age-matched control (n = 4) group (fig. 4). Otherwise, in the renal medullar zone, a higher but not significant collagen content was also observed in Sk offspring at 16-week of age.
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cardiovascular disease [29] and lung disease [30] have attracted considerable attention. Recently, Al-Odat et al. [31], demonstrate that maternal tobacco exposure during pregnancy, as well as lactation leads to significant renal underdevelopment and functional abnormalities in adulthood. This study shows that nephron development was delayed, with fewer nephron numbers and reduced glomeruli volume from early postnatal day persisted to adulthood. These changes were accompanied by an increased albumin/creatinine ratio in adulthood, suggesting accentuated renal dysfunction. Here, in maternal smoking offspring, we focus on adult renal function, arterial hypertension development and epithelial to mesenchymal transdifferentiation process as a novel mechanism that may promote renal fibrosis. In the present study we also investigate whether the known causes of renal fibrosis transforming growth factor β1 (TGF-β1) act through this pathway. The present study confirms the findings of an earlier report from Al-Odat et
al., showing that the birth weight of male maternal smoking offspring was significantly 13% lower, and this finding is associated with 14% reduced kidney mass compared to the control group. Furthermore, this investigation shows an early and enhanced fractional urinary sodium excretion in maternal Sk offspring at 5 weeks of age, when compared to the age-matched control group. The higher FENa was accompanied by an increased FEPPNa and unaltered FEPNa. However, 8 weeks later, the renal sodium excretion from 13-week-old Sk offspring decline significantly, becoming indistinct to control group. These results occurred despite the unchanged glomerular filtration rate and were associated with 14% reduced kidney mass, suggesting that presumable tubular dysfunction with enhanced water and sodium reabsorption might, at least in part, be responsible for programming of adult hypertension in Sk offspring. Although the present study shows a reduced glomeruli number (about 13%) in Sk, this finding as well as the glomerular volume was not different when compared to the
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tion of two critical Na+ transporters. The Na+/K+-2Cl and the Na-Cl co-transporters, but not the Na+/H+ nor the Na+ channel, were significantly increased in the rats with IUGR [35]. These alterations led to a lower rate of urinary sodium excretion, associated with sodium retention and hypertension as observed in the present study. Although the mechanism by which renal sodium excretion decline in 13-week-old Sk rats remain to be elucidated, the loss of organ function, enhanced sympathetic neural and reninangiotensin activity and renal non-control of the fluid and electrolyte balance is thought to play a dominant role on the long-term salt and water retention. Taking into account the urinary sodium excretion change and increased blood pressure in adult maternal Sk offspring, the current study may corroborates Brenner’s hypothesis, by which, the presumable hyperfiltration in LBW leads to glomerular hypertension and in the future, to sustained renal function disorder [36]. By the histochemistry and immunofluorescence staining, the present study verified in the Sk offspring, an increased glomerular expression of TGF-β1, fibronectin and type I collagen, intrinsically related to fibrotic process as compared to age-matched control group. As previously demonstrated [7, 28], this higher expression of TGF-β1, a major inducer of EMT in epithelial cells [37], could be associated with decreased expression of EMT-inducing transcriptional factor [38–40], which in turn, upregulated fibronectin and collagen renal expression, proteins being considered mesenchymal markers. The production of interstitial matrix compounds suggests that podocytes have adapted a mesenchymal phenotype in maternal tobacco exposure offspring, which could profoundly change their functions [41, 42]. Here, we also found that the glomerular structural alterations were accompanied by parallel proteinuria in Sk offspring, suggesting a decreased efficiency on the glomerular filter barrier in these rats. As observed in the current study, the process leading to tissue fibrosis, is considered a dominant mechanism induced by TGF-β1 in the kidney, largely on the basis of the observation that glomerular and tubulo-intersticial scarring are universal outcomes of renal disease progression. Indeed, a chronic hypoxia hypothesis has been proposed, stating that abnormal post-glomerular hypertension and vasoconstriction reduce peritubular capillary blood flow and cause rarefaction of peritubular capillaries, resulting in local hypoxia and tubular atrophy. Thus, in the current study, the putative renal sodium transport disorder could promote further peritubular reduction in oxygen partial pressure with the generation of oxidative stress and impaired pressure natriuresis [43]. We may not also rule out Block/Mesquita/de Lima/Boer/Gontijo
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control group data. In previous studies, we and others have demonstrated in different models of fetal programming, that even when associated with decreased glomeruli number units, the offspring maintain a normal wholekidney glomerular filtration rate, estimated by creatinine clearance, suggesting, in fact, a compensatory glomerular hyperfiltration [4–6, 10]. In the current study, the putative tubular functional changes and absolute reduction of glomeruli number could be associated with glomerular hyperfiltration/overflow that may account for the glomerular filtration barrier breakdown and early glomerulosclerosis [4–6, 10] in this smoking model. Although the data presented here do not offer any support for the kidney hemodynamic factors or humoral hypothesis, we cannot rule out the possibility that several humoral factors or renal vascular resistance and blood flow changes, induced by maternal tobacco exposure, may be involved in mediating the urinary sodium response. In Sk offspring at 5-week of age, the natriuresis occurs by a fall in sodium reabsorption in the post-proximal tubule segments of nephron as compared to the control group. The natriuretic response was suppressed in the 13-week-old Sk offspring. Cowley [32] has suggested that increased perfusion pressure and renal blood flow may enhance sodium excretion, whereas increased renal vascular resistance may have the opposite effect. Studies have demonstrated that renal prostaglandin E2 (PGE2) is necessary for full natriuretic response by decreased Na+/K+2Cl activity in thick ascending limb of loop of Henle, and increased renal interstitial hydrostatic pressure during increased renal perfusion pressure. The microsomal isoform of PGES, mPGES-1, is inducible by many factors and is widely regarded as the main contributing enzymatic tandem for PGE2 biosynthesis under pathological conditions, including active tobacco smoking [33]. On the other hand, the study has also demonstrated that tobacco exposure promoted a significant reduction in urinary excretion of 6-keto-PGF1α [34]. These observations suggest that tobacco smoking reduces PGI2 production, including in kidneys; this result could explain the antinatriuresis response verified later, in the 13-week-old Sk offspring. In the current study, we have hypothesized that natriuresis in an Sk offspring could be mediated, at least in part, by changes in interstitial hemodynamic factors, modulated in a time-dependent fashion, by imbalance between the different types of renal prostaglandin production. Additionally, the hypothesis that fetal kidney is programmed to inappropriately retain Na+ in later life has been corroborated by different studies [35]. Intrauterine growth restriction model (IUGR) in rats resulted in the upregula-
to explain the impaired pressure natriuresis in the Sk group, the renal interstitial scarring associated with the generation of reactive oxygen species and increased local angiotensin II activity [43, 44] that suppress natriuresis. Considering that the glomeruli are the first nephron structures affected by overflow and pressure overload, we may state that hypertension development beyond 13week of age following maternal smoking exposure, associated with overload in remaining nephrons, could be a preponderant factor for the development of fibrous process and altered glomerular ultrastructure in the Sk offspring. From the present data, we demonstrate that fetalprogrammed Sk offspring present pronounced glomerular TGF-β1 and fibrotic marker expression that may, subsequently, cause glomerular EMT. Although the precise mechanism responsible for the subsequently renal morphological and functional response in Sk offspring is incompletely known, the current data suggest, that changes in renal function are conducive to excess sodium tu-
bule reabsorption that associated with enhanced TGF-β1, fibronectin and collagen deposition, intrinsically related to fibrotic process, might potentiate the programming of adult hypertension. In conclusion, we may suppose that, under smoking exposure, the fetus responds with adaptations accommodating to intrauterine conditions. After birth, the maternal tobacco exposure can result in progressive nephron senescence in parallel to functional loss and higher blood pressure. Disclosure Statement We affirm that there is no conflict of interest.
Funding Grants from CNPq (No. 500868/91–3), CAPES and FAPESP (10/52696–0 and 11/17016–1) supported this work.
References
Kidney Disorders and Gestational Smoking
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16 Liu Y: New insights into epithelial-mesenchymal transition in kidney fibrosis. J Am Soc Nephrol 2010;21:212–222. 17 Lee HS: Mechanisms and consequences of TGF-β overexpression by podocytes in progressive podocyte disease. Cell Tissue Res 2012;347:129–140. 18 Kim JH, Kim BK, Moon KC, et al: Activation of the TGF-beta/Smad signaling pathway in focal segmental glomerulosclerosis. Kidney Int 2003;64:1715–1721. 19 Shankland SJ, Hugo C, Coats SR, et al: Changes in cell-cycle protein expression during experimental mesangial proliferative glomerulonephritis. Kidney Int 1996; 50: 1230– 1239. 20 Carew RM, Wang B, Kantharidis P: The role of EMT in renal fibrosis. Cell Tissue Res 2012; 347:103–116. 21 Ruiz-Ortega M, Egido J: Angiotensin II modulates cell growth-related events and synthesis of matrix proteins in renal interstitial fibroblasts. Kidney Int 1997; 52: 1497– 1510. 22 Wolf G, Zahner G, Schroeder R, Stahl RA: Transforming growth factor beta mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells. Nephrol Dial Transplant 1996;11:263–269. 23 Mello PRB, Okay TS, Dores EFGC, Botelho C: Marcadores de exposição tabágica em ratas lactantes utilizando um modelo de exposição passiva desde o início da gestação. Pulmão RJ 2005;14:289–293.
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1 Taal HR, Geelhoed JJ, Steegers EA, Hofman A, Moll HA, Lequin M, van der Heijden AJ, Jaddoe VW: Maternal smoking during pregnancy and kidney volume in the offspring: the Generation R Study. Pediatr Nephrol 2011; 26:1275–1283. 2 Schreuder M, Delemarre-van de Waal H, van Wijk A: Consequences of intrauterine growth restriction for the kidney. Kidney Blood Press Res 2006;29:108–125. 3 Hershkovitz D, Burbea Z, Skorecki K, et al: Fetal programming of adult kidney disease: cellular and molecular mechanisms. Clin J Am Soc Nephrol 2007;2:334–342. 4 Mesquita FF, Gontijo JA, Boer PA: Expression of renin-angiotensin system signalling compounds in maternal protein-restricted rats: effect on renal sodium excretion and blood pressure. Nephrol Dial Transplant 2010; 25: 380–388. 5 Mesquita FF, Gontijo JA, Boer PA: Maternal undernutrition and the offspring kidney: from fetal to adult life. Braz J Med Biol Res 2010;43:1010–1018. 6 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 2013, Epub ahead of print. 7 Sene Lde B, Mesquita FF, de Moraes LN, Santos DC, Carvalho R, Gontijo JA, Boer PA: Involvement of renal corpuscle microRNA expression on epithelial-to-mesenchymal transition in maternal low protein diet in adult programmed rats. PLoS One 2013;8:e71310.
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31 Al-Odat I, Chen H, Chan YL, Amgad S, Wong MG, et al: The impact of maternal cigarette smoke exposure in a rodent model on renal development in the offspring. PLoS One 2014; 9:e103443. 32 Cowley AW Jr: Role of the renal medulla in volume and arterial pressure regulation. Am J Physiol 1997;273:R1–R15. 33 Dilmé JF, Solà-Villà D, Bellmunt S, Romero JM, Escudero JR, Camacho M, Vila L: Active smoking increases microsomal PGE2-synthase-1/PGE-receptor-4 axis in human abdominal aortic aneurysms. Mediators Inflamm 2014;2014:316150. 34 Nadler JL, Velasco JS, Horton R: Cigarette smoking inhibits prostacyclin formation. Lancet 1983;1:1248–1250. 35 Manning J, Beutler K, Knepper MA, Vehaskari VM: Upregulation of renal BSC1 and TSC in prenatally programmed hypertension. Am J Physiol Renal Physiol 2002;283:F202–F206. 36 Brenner BM, Garcia DL, Anderson S: Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1998;1:335–347. 37 Bracken CP, Gregory PA, Kolesnikoff N, Bert AG, Wang J, et al: A double-negative feedback loop between ZEB1-SIP1 and the micro RNA-200 family regulates epithelial-mesenchymal transition. Cancer Res 2008;68:7846– 7854.
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38 Yang J, Liu Y: Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 2001;159:1465–1475. 39 Li Y, Kang YS, Dai C, et al: Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria. Am J Pathol 2008;172:299–308. 40 Xiong M, Jiang L, Zhou Y, Qiu W, Fang L, et al: The miR-200 family regulates TGF-β1induced renal tubular epithelial to mesenchymal transition through Smad pathway by targeting ZEB1 and ZEB2 expression. Am J Physiol Renal Physiol 2012;302:F369–F379. 41 Böttinger EP, Bitzer M: TGF-beta signaling in renal disease. J Am Soc Nephrol 2002; 13: 2600–2610. 42 Zhang J, Hansen KM, Pippin JW, Chang AM, Taniguchi Y, et al: De novo expression of podocyte proteins in parietal epithelial cells in experimental aging nephropathy. Am J Physiol Renal Physiol 2012;302:F571–F580. 43 Johnson RJ, Schreiner GF: Hypothesis: the role of acquired tubulointerstitial disease in the pathogenesis of salt-dependent hypertension. Kidney Int 1997;52:1169–1179. 44 Rodríguez-Iturbe B, Vaziri ND, HerreraAcosta J, Johnson RJ: Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension: all for one and one for all. Am J Physiol Renal Physiol 2004; 286:F606– F616.
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24 Gomes PR, Seraphim PM: [Effect of cigarette smoke exposure during pregnancy and lactation of rats and the offspring on the serum and morphometric parameters]. Rev Bras Ginecol Obstet 2010;32:591–596. 25 Mandarim-de-Lacerda CA: Stereological tools in biomedical research. An Acad Bras Cienc 2003;75:469–486; Erratum in 2007;79:51. 26 Gontijo JA, Garcia WE, Figueiredo JF, SilvaNetto CR, Furtado MR: Renal sodium handling after noradrenergic stimulation of the lateral hypothalamic area in rats. Braz J Med Biol Res 1992;25:937–942. 27 Furlan FC, Marshall PS, Macedo RF, Carvalheira JB, Michelotto JB, Gontijo JA: Acute intracerebroventricular insulin microinjection after nitric oxide synthase inhibition of renal sodium handling in rats. Life Sci 2003; 72: 2561–2569. 28 Pinhal CS, Lopes A, Torres DB, Felisbino SL, Rocha Gontijo JA, Boer PA: time-course morphological and functional disorders of the kidney induced by long-term high-fat diet intake in female rats. Nephrol Dial Transplant 2013;28:2464–2476. 29 Brenner BM, Chertow GM: Congenital oligonephropathy and the etiology of adult hypertension and progressive renal injury. Am J Kidney Dis 1994;23:171–175. 30 Sherman CB: The health consequences of cigarette smoking. Pulmonary diseases. Med Clin North Am 1992;76:355–375.
Received: October 31, 2014 Accepted after revision: February 1, 2015 Published online: April 18, 2015
Fetal Kidney Programming by Maternal Smoking Exposure: Effects on Kidney Structure, Blood Pressure and Urinary Sodium Excretion in Adult Offspring Daniel B. Block Flávia F. Mesquita Ize P. de Lima Patricia A. Boer José A.R. Gontijo Department of Internal Medicine School of Medicine, State University of Campinas, Campinas, Brazil
Abstract Introduction: Fetal programming by different insults results in low birth weight and reduction in nephron number increasing the risk for adult development of cardiovascular and renal diseases. Maternal smoking is an important modifiable adverse fetal exposure worldwide and leads to a decrease in the offspring’s birth weight. Thus far, the specific adverse fetal smoking exposures and mechanisms underlying these associations on renal development and functional disorder are unclear. Methods: The present study investigates, in adult male rats, the effect of smoking exposure (Sk) in uteri on blood pressure (BP) by an indirect tail-cuff method using an electrosphygmomanometer, and its association with nephron structure by stereological estimation, immunohistochemical and histological techniques, in parallel with kidney function creatinine and lithium clearance. Results: The current study showed in a 16-week old Sk offspring enhanced arterial blood pressure associated with, reduced urinary sodium excretion and higher TGF-β1 glomerular expression. Sk glomeruli also presented an upregulated collagen and fibronectin deposition intrinsically related to fibrotic process as compared to age-matched control group. Conclusion: Here, we demonstrate that fetal-programmed
© 2015 S. Karger AG, Basel 1660–8151/15/1294–0283$39.50/0 E-Mail [email protected] www.karger.com/nef
Sk offspring present pronounced glomerular TGF-β1 and fibrotic marker expression that may, subsequently, promote a glomerular epithelial-mesenchymal transition activated process in an Sk offspring. Although the precise mechanism responsible for the subsequently renal morphological and functional response in Sk offspring is incompletely known, the current data suggest that changes in renal function are conducive to excess sodium tubule reabsorption that is associated with enhanced TGF-β1, fibronectin and collagen deposition, intrinsically related to fibrotic process, might potentiate the programming of adult hypertension. © 2015 S. Karger AG, Basel
Introduction
The developmental plasticity hypothesis suggests that various adverse intrauterine exposures lead to persistent fetal developmental adaptations. These adaptations may be beneficial in the short term, but may have adverse consequences in postnatal life [1]. Fetal programming by different insults results in low birth weight and reduction in nephron number [2–7], and increases the risk for adult development of cardiovascular and renal diseases [4–6]. Low birth weight (LBW) is also associated with impaired renal growth, raised blood pressure and impaired renal function [8–11]. A reduction in nephron number and, therefore, in the whole-kidney glomerular filtration area would result in reduced sodium excretory capacity, enDr. José A.R. Gontijo Departamento de Clínica Médica Faculdade de Ciências Médicas, Universidade Estadual de Campinas Campinas, SP 13083–592 (Brazil) E-Mail gontijo @ fcm.unicamp.br
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Key Words Fetal programming · Maternal smoking exposure · Arterial hypertension · Glomerular fibrosis · TGF-β1 · Epithelial-to-mesenchymal transition
Material and Methods Animals The experiments were conducted on age-matched rats of sibling-mated Wistar HanUnib rats (250–300 g) allowed free access to water and normal rat chow. The experimental protocol and study design was approved by Institutional Animal Ethics Com-
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mittee (protocol 2419–1) and the general guidelines established by the Brazilian College of Animal Experimentation (COBEA) were followed throughout the investigation. Our local colonies originated from a breeding stock supplied by CEMIB/Unicamp (CEMIB/Unicamp, Campinas, SP, Brazil). Immediately after weaning at 3 weeks of age, animals were maintained under controlled temperature (25 ° C) and lighting conditions (7: 00 a.m.– 7:00 p.m.), with free access to tap water and standard rodent laboratory chow (Nuvital, Curitiba, PR, Brazil) and followed up to 16 weeks of age. The dams were divided into two groups as follows: (1) control group (Co) and (2) smoker group (Sk). The method of smoking exposure was adapted from studies by Mello et al. [23] and Gomes et al. [24]. Briefly, rats were placed in a Perspex chamber containing one inlet and one outlet. The inlet was connected, via a two-way valve, to a multicigarette smoking apparatus, containing five cigarettes, and to the external atmosphere. The outlet was attached to a negative-pressure pump. Under these conditions, a volume of air was sucked through the chamber, a portion of which was drawn through the cigarettes, thereby carrying smoke into the chamber. Combustion of five cigarettes occurred in approximately 30 min, by which time the chamber had been cleared of smoke by the passage of clean air. The animals of the Sk group, five animals at each time, were exposed to the inhalation of tobacco by 30 min at day, throughout the first week (adjustment period). After the adjustment period, animals were then mated and the day that the sperm were seen in the vaginal smear was designated as day 0 (zero) of pregnancy. After establishing pregnancy, the rats were subjected to basic smoke dose of two such exposures over 30min each per day (1 h total exposure), throughout the entire threeweek gestational period. It is estimated that the amount of smoke inhaled from cigarettes derived during the period of 4 weeks correspond to inhalation of 14 mg tar, 11 mg nicotine and 15 mg carbon monoxide [23, 24]. Between each exposure, rats were removed from the smoking chamber and offered fresh air, tap water and standard rodent chow, ad libitum. The smoking dams body mass was obtained at 9th, 14th and 16th days of gestation. Delivered Co or Sk male pups were weighed weekly.
Blood Pressure Measurement The systemic arterial pressure was measured in conscious offspring in 5th 10th, and 16th weeks of age by an indirect tail-cuff method using an electrosphygmomanometer (IITC Life Science – BpMonWin Monitor Version 1.33) combined with a pneumatic pulse transducer/amplifier. This indirect approach allowed repeated measurements with a close correlation (correlation coefficient = 0.975) compared to direct intra-arterial recording. The mean of three consecutive readings represented the blood pressure. Stereological Estimation of Kidney and Glomerular Volume and Glomerular Number At postnatal day 12 (post complete kidney maturation), offspring were anesthetized with a mixture of ketamine (75 mg·kg–1 body weight, i.p.) and xylasine (10 mg·kg–1 body weight, i.p.) and the left kidney was removed, weighed and volume was estimated by the Cavalieri’s principle. Then, they were longitudinally divided into two halves and a 4-mm slice, from each kidney, was weighted. The slices were placed in 10% formalin for stereological estimation of total nephron number. After paraffin inclusion, the tissue blocks were exhaustively sectioned at 5 μm and then stained with haematoxilin-eosin. We used the ‘fractionators’ method to estimate the
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hanced susceptibility to hypertension and reduced renal reserve; therefore, the limiting compensation for renal injury may, at least in part, explain the higher prevalence of hypertension and renal disease observed in populations with a high prevalence of low birth weight [4–6, 10]. Maternal smoking is a very important modifiable adverse fetal exposure worldwide and leads to a decrease of offspring’s birth weight [12]. Studies suggested that maternal smoking exposure might also be associated with higher blood pressure in the offspring, independent of birth weight [1, 13], which might be explained by adverse effects on fetal kidney development [1, 14]. Also, the classical vasoactive tobacco action has been extended due to its properties as cytokine that actively participates in the renal physiopathology. Thus far, the specific adverse fetal smoking exposures and mechanisms underlying these associations on renal development and functional disorder are not known. Irreversible renal fibrosis is a common consequence after most renal injuries [15, 16]. Incidentally, the transforming growth factor β (TGFβ) is a key regulator of extracellular matrix (ECM) protein deposition in renal tissue [17]. Podocyte increased expression of TGF-β mRNA and glomeruli ECM protein deposition have been associated to focal segmental glomerulosclerosis [18], membranous nephropathy and diabetic nephropathy [19]. Renal TGF-β1 expression is implicated in the pathogenesis of fibrosis in both glomerular and interstitial compartments [20]. It is clear that well-described phenomenon of type II epithelial-to-mesenchymal transition (EMT) plays a pivotal role in organ fibrosis progression [20, 21]. It has been demonstrated that TGF-β1 may stimulate the accumulation of matrix and inflammation [21, 22]. In the current study, we hypothesized that maternal smoking exposure may affect the body and kidney growth, the renal morphological structure and function and, the arterial blood pressure levels in adulthood rats. Thus, the present study was performed in 12-days and 16-week-old maternal smoking male offspring compared to appropriated gender and age-matched animals evaluating the kidney expression of fibrotic and EMT markers in parallel with kidney function.
Renal Function Evaluation The renal function tests were performed and estimated by creatinine and lithium clearance on the last day at 5, and 13 weeks of age in unanaesthetized, unrestrained Co (n = 15) and Sk (n = 11) male offspring. Briefly, fourteen hours before the renal test, 60 μmol CLi 100 g–1 body weight was given by gavage. After an overnight fast, each animal received a load of tap water by gavage (5% of body weight), followed by a second load of the same volume, 1 h later, and spontaneously voided urine was collected over a 120 min period into a graduated centrifuge tube. At the end of the experiment, blood samples were drawn through cardiac puncture in anesthetized rats, and urine and plasma samples were collected for analysis [26–28]. Measurement of Proteinuria In the 13th week of age, male rats from all groups (Co, n = 5 and Sk, n = 6) received a load of tap water by gavage (5% of the body weight) and twenty minutes later were housed individually in metabolic cages; spontaneously voided urine was collected over a 2 h period and immediately stored at –20 ° C until processing. The proteinuria was detected using the BIOPROT U/LCR (Bioclin).
Data Presentation, Statistical and Biochemical Analysis Data obtained from this study are expressed as the mean ± SD or median and quartile deviation when appropriated. Plasma and urinary sodium, potassium and lithium concentrations were measured by flame photometry (B262; Micronal, São Paulo, Brazil). Creatinine was determined spectrophotometrically (362; Micronal, São Paulo, Brazil) by the alkaline picrate method. Creatinine clearance was used to estimate the glomerular filtration rate (GFR) and lithium clearance (CLi) was used to assess proximal tubule output [26–28]. Fractional sodium excretion (FENa) was calculated as CNa/ CCr × 100, where CNa is sodium clearance and CCr is creatinine clearance. The fractional proximal (FEPNa) and post-proximal (FEPPNa) sodium excretion was calculated as CLi/CCr × 100 and CNa/CLi × 100, respectively. Data obtained over time were analyzed using appropriate Kruskal-Wallis one-way analysis of variance. Post-hoc comparisons between selected means were made by Bonferroni’s contrast test when initial analysis indicated statistical differences between experimental groups. Comparisons involving only two samples of independent observations within or between groups were made using the Student t-test or Mann-Whitney U test when appropriated from GraphPad Prism 5.01 (GraphPad Software, Inc., USA). The level of significance was set at p ≤ 0.05.
Results
Immuno and Histochemical Procedures Sixteen week-old adult male offspring from different mothers of the Co (n = 5) and Sk (n = 5) groups were anesthetized with a mixture of ketamine (75 mg·kg–1 body weight, i.p.) and xylasine (10 mg·kg–1 body weight, i.p.), and the level of anesthesia was controlled by monitoring the corneal reflex. The animals were then perfused with saline containing heparin (5%) for 15 min under constant pressure, followed by perfusion with 0.1 M phosphate buffer (pH 7.4) containing 4% (w/v) paraformaldehyde and 0.1 M sucrose for 25 min. After perfusion, kidneys were removed and placed in the same fixative for 2 h, followed by PBS containing 0.1% glycine for 30 min and PBS containing 15% (w/v) sucrose overnight at 4 ° C. In the following day, the tissues were placed in an OCT compound cryoprotector (Tissue-tech), cut on a Leica cryostat and mounted on silane-coated slides. For immunohistochemistry, the sections were incubated sequentially with (1) phosphate-buffered saline (PBS) containing 2% milk for 45 min to minimize non-specific reactions, (2) mouse anti-fibronectin antiserum (Santa Cruz) or rabbit anti-TGF-β1 antiserum (Santa Cruz) and (3) anti-mouse or rabbit Alexa 488 labeled antibody (molecular Probes) for 2 h at room temperature. After incubation, the sections were rinsed in 0.1 M PBS and cover-slipped with Vectashield anti-fading medium containing DAPI (Vector). The sections were examined with a fluorescence microscope (Olympus BX51). No immunoreactivity was seen in control experiments in which primary antibodies was omitted. After picrosirius staining, we evaluated the density of collagen. Ten cortical and ten medullar fields of histological sections for each experimental (Co, n = 4) or control (Sk, n = 4) animals were analyzed and the average of collagen density readings were determined. Images were captured with photomicroscope and analyzed by the Leica Qwin 3.1 for windows.
The maternal smoking exposure during pregnancy caused a significant decreased dam’s body weight over the whole gestation (fig. 1a). However, the smoking exposure did not affect the number of offspring per litter and the proportion between male and female offspring (p = 0.3245). Sk male offspring also presented significant reduction in birth body mass (7.20 ± 0.051 g in Co, n = 27 vs. 6.36 ± 0.24 g in Sk, n = 26, p < 0.0007) (fig. 1b). The body weight was unchanged in smoke offspring group (n = 11) from the 4th to 9th week of age (fig. 1c) when compared to control (n = 11) age-matched rats. However, beyond the 9th week of age, Sk offspring shows a significant decrease in body mass (fig. 1c). Beyond 16 weeks of age, the Sk offspring (n = 11) showed an increased systolic blood pressure from (Sk: 130.5 ± 2.43 mm Hg vs. 130.7 ± 2.43 mm Hg in Co at 5th week of age; NS), to (Sk: 146.1 ± 3.5 mm Hg vs. 134.2 ± 3.01 mm Hg in Co, at 16th week of age; p = 0.0001), when compared to control (n = 15) group (fig. 1d). Stereological estimations, at postnatal 12 days, immediately after complete renal maturation (n = 5 for each group), we verified that Sk offspring although present a reduction of the kidney mass (Sk: 0.1095 ± 0.006 g/100 g body weight vs. 0.1264 ± 0.021 g/100 g body weight in Co, p = 0.0278; fig. 1e), the glomeruli number per kidney (Sk: 9,908 ± 3,178.96 units vs. 10,143 ± 3,533.54 units in Co, p = 0.3452; fig. 1f), and glomeruli volume (15,761.21 ± 2,202.24 μl vs. 16,488.34 ± 4,792.07 μl in Co, p = 0.500; fig. 1g) were unchanged when compared with Co group.
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number of glomeruli and estimated the total number of renal corpuscles per kidney considering the analyzed fraction of the kidney corrected to the entire organ as described at Mandarim-de-Lacerda [25].
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0.0020. The enhanced FENa+ in Sk rats was accompanied by unchanged proximal sodium excretion and increased fractional potassium excretion (FEK+) (Sk: 0.36 ± 0.036% vs. 0.26 ± 0.022% in Co, p = 0.0169) compared with the Co age-paired control group. However, this increased FENa+ occurred in parallel to significant augmentation in FEPPNa+ (Sk: 2.85 ± 0.047% vs. 1.13 ± 0.15% in Co, p = 0.0001; fig. 2a). On the other hand, in offspring at 13-week of age, the renal tubule sodium handling parameters, estimated by CLi and CCr, did not significantly differ among the groups Co and maternal smoking exposure offspring (fig. 2b).
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Renal Function Data The data for renal function in 5- and 13-week-old male offspring of both (Co, n = 15 and Sk, n = 11) groups are summarized in figure 2. In 5-week-old rats (fig. 2a), in both studied groups, the urinary flow rates (data not show) and the glomerular filtration rates, estimated by CCr, did not significantly differ during the renal tubule sodium handling studies. In an offspring at 5 weeks of age, the fractional urinary sodium excretion (FENa+) was significantly higher in Sk rats when compared with Co age-matched group, as follows: 1.11 ± 0.19% vs. 0.45 ± 0.07% in Co, p =
Proteinuria Thirteen-week-old Sk group observed a significant increased urinary protein excretion (7.19 ± 1.16 μg/dl, n = 6) when compared to age-matched control group (4.84 ± 0.78 μg/dl) (p = 0.0087, n = 5) (fig. 2f). Immuno and Histochemical Procedures The study shows an intensive rise of the TGF-β1 immunoreactivity in 16-week-old Sk (n = 5) in both glomeruli and parietal epithelium (fig. 3a–d) compared to that found in the control (n = 5) group. Additionally, fibronectin immunoreactivity was also highly expressed in Sk (n = 5) offspring glomeruli and peritubular spaces when compared to age-matched Co (n = 5) animals (fig. 3e–h). The density of collagen fibers deposition in the renal cortical zone of 16-week-old Sk (n = 4) offspring, semi-quantitative evaluated by picrosirius staining technique, showed a
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The main purpose of the current study was to expose embryos and fetuses to smoking in conditions similar to those inherent in active smoker’s mothers. The developmental plasticity hypothesis supports that adverse intrauterine exposures lead to persistent fetal adaptations and organ disorders in adulthood. Therefore, fetal programming by different insults results in low birth weight and increased risk for adult development of cardiovascular and renal diseases. Among the health hazards of smoking
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Also presents the daily urinary protein excretion in 13-week-old Sk offspring (n = 6) compared to appropriated age-matched controls (n = 5) (f). The data are reported as the median and quartile deviation. * p ≤ 0.05 versus control (Mann-Whitney or Kruskal-Wallis test with post-hoc comparisons by Bonferroni’s contrast test).
Fig. 3. Representative TGF-β1 and fibronectin immunoreactivity in Co and Sk kidney. Immunofluorescence reveal a normal content of TGF-β1 in the control offspring (a and c, n = 5) compared to notable staining obtained in maternal smoking offspring groups
(b and d, n = 5). Additionally, in e and g we have the normal pattern of fibronectin distribution in the kidney from control offspring and in Sk (f and h, n = 5) we can observe the enhanced deposition of this protein in glomeruli and peritubular extracellular matrix. (For figure 3 see next page.)
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significant increase comparatively to age-matched control (n = 4) group (fig. 4). Otherwise, in the renal medullar zone, a higher but not significant collagen content was also observed in Sk offspring at 16-week of age.
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Sk
d 50 μm
20 μm
e
f
Fig. 4. Renal cortical (a–d) and medullar (e and f) collagen content
after picrosirius technique. The collagen is distributed in perivascular, peritubular, mesangial and subcapsular matriz and, in Sk (b, d and f, n = 4) cortex, the content is significantly (*** p ≤ 0.01)
30
20
10
0
enhanced (quantification in graphic) when compared to that observed in Co (a, c and e, n = 4) group; * Sk versus control group (Student’s t-test).
cardiovascular disease [29] and lung disease [30] have attracted considerable attention. Recently, Al-Odat et al. [31], demonstrate that maternal tobacco exposure during pregnancy, as well as lactation leads to significant renal underdevelopment and functional abnormalities in adulthood. This study shows that nephron development was delayed, with fewer nephron numbers and reduced glomeruli volume from early postnatal day persisted to adulthood. These changes were accompanied by an increased albumin/creatinine ratio in adulthood, suggesting accentuated renal dysfunction. Here, in maternal smoking offspring, we focus on adult renal function, arterial hypertension development and epithelial to mesenchymal transdifferentiation process as a novel mechanism that may promote renal fibrosis. In the present study we also investigate whether the known causes of renal fibrosis transforming growth factor β1 (TGF-β1) act through this pathway. The present study confirms the findings of an earlier report from Al-Odat et
al., showing that the birth weight of male maternal smoking offspring was significantly 13% lower, and this finding is associated with 14% reduced kidney mass compared to the control group. Furthermore, this investigation shows an early and enhanced fractional urinary sodium excretion in maternal Sk offspring at 5 weeks of age, when compared to the age-matched control group. The higher FENa was accompanied by an increased FEPPNa and unaltered FEPNa. However, 8 weeks later, the renal sodium excretion from 13-week-old Sk offspring decline significantly, becoming indistinct to control group. These results occurred despite the unchanged glomerular filtration rate and were associated with 14% reduced kidney mass, suggesting that presumable tubular dysfunction with enhanced water and sodium reabsorption might, at least in part, be responsible for programming of adult hypertension in Sk offspring. Although the present study shows a reduced glomeruli number (about 13%) in Sk, this finding as well as the glomerular volume was not different when compared to the
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c
Collagen/medullar area (%)
40
290
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tion of two critical Na+ transporters. The Na+/K+-2Cl and the Na-Cl co-transporters, but not the Na+/H+ nor the Na+ channel, were significantly increased in the rats with IUGR [35]. These alterations led to a lower rate of urinary sodium excretion, associated with sodium retention and hypertension as observed in the present study. Although the mechanism by which renal sodium excretion decline in 13-week-old Sk rats remain to be elucidated, the loss of organ function, enhanced sympathetic neural and reninangiotensin activity and renal non-control of the fluid and electrolyte balance is thought to play a dominant role on the long-term salt and water retention. Taking into account the urinary sodium excretion change and increased blood pressure in adult maternal Sk offspring, the current study may corroborates Brenner’s hypothesis, by which, the presumable hyperfiltration in LBW leads to glomerular hypertension and in the future, to sustained renal function disorder [36]. By the histochemistry and immunofluorescence staining, the present study verified in the Sk offspring, an increased glomerular expression of TGF-β1, fibronectin and type I collagen, intrinsically related to fibrotic process as compared to age-matched control group. As previously demonstrated [7, 28], this higher expression of TGF-β1, a major inducer of EMT in epithelial cells [37], could be associated with decreased expression of EMT-inducing transcriptional factor [38–40], which in turn, upregulated fibronectin and collagen renal expression, proteins being considered mesenchymal markers. The production of interstitial matrix compounds suggests that podocytes have adapted a mesenchymal phenotype in maternal tobacco exposure offspring, which could profoundly change their functions [41, 42]. Here, we also found that the glomerular structural alterations were accompanied by parallel proteinuria in Sk offspring, suggesting a decreased efficiency on the glomerular filter barrier in these rats. As observed in the current study, the process leading to tissue fibrosis, is considered a dominant mechanism induced by TGF-β1 in the kidney, largely on the basis of the observation that glomerular and tubulo-intersticial scarring are universal outcomes of renal disease progression. Indeed, a chronic hypoxia hypothesis has been proposed, stating that abnormal post-glomerular hypertension and vasoconstriction reduce peritubular capillary blood flow and cause rarefaction of peritubular capillaries, resulting in local hypoxia and tubular atrophy. Thus, in the current study, the putative renal sodium transport disorder could promote further peritubular reduction in oxygen partial pressure with the generation of oxidative stress and impaired pressure natriuresis [43]. We may not also rule out Block/Mesquita/de Lima/Boer/Gontijo
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control group data. In previous studies, we and others have demonstrated in different models of fetal programming, that even when associated with decreased glomeruli number units, the offspring maintain a normal wholekidney glomerular filtration rate, estimated by creatinine clearance, suggesting, in fact, a compensatory glomerular hyperfiltration [4–6, 10]. In the current study, the putative tubular functional changes and absolute reduction of glomeruli number could be associated with glomerular hyperfiltration/overflow that may account for the glomerular filtration barrier breakdown and early glomerulosclerosis [4–6, 10] in this smoking model. Although the data presented here do not offer any support for the kidney hemodynamic factors or humoral hypothesis, we cannot rule out the possibility that several humoral factors or renal vascular resistance and blood flow changes, induced by maternal tobacco exposure, may be involved in mediating the urinary sodium response. In Sk offspring at 5-week of age, the natriuresis occurs by a fall in sodium reabsorption in the post-proximal tubule segments of nephron as compared to the control group. The natriuretic response was suppressed in the 13-week-old Sk offspring. Cowley [32] has suggested that increased perfusion pressure and renal blood flow may enhance sodium excretion, whereas increased renal vascular resistance may have the opposite effect. Studies have demonstrated that renal prostaglandin E2 (PGE2) is necessary for full natriuretic response by decreased Na+/K+2Cl activity in thick ascending limb of loop of Henle, and increased renal interstitial hydrostatic pressure during increased renal perfusion pressure. The microsomal isoform of PGES, mPGES-1, is inducible by many factors and is widely regarded as the main contributing enzymatic tandem for PGE2 biosynthesis under pathological conditions, including active tobacco smoking [33]. On the other hand, the study has also demonstrated that tobacco exposure promoted a significant reduction in urinary excretion of 6-keto-PGF1α [34]. These observations suggest that tobacco smoking reduces PGI2 production, including in kidneys; this result could explain the antinatriuresis response verified later, in the 13-week-old Sk offspring. In the current study, we have hypothesized that natriuresis in an Sk offspring could be mediated, at least in part, by changes in interstitial hemodynamic factors, modulated in a time-dependent fashion, by imbalance between the different types of renal prostaglandin production. Additionally, the hypothesis that fetal kidney is programmed to inappropriately retain Na+ in later life has been corroborated by different studies [35]. Intrauterine growth restriction model (IUGR) in rats resulted in the upregula-
to explain the impaired pressure natriuresis in the Sk group, the renal interstitial scarring associated with the generation of reactive oxygen species and increased local angiotensin II activity [43, 44] that suppress natriuresis. Considering that the glomeruli are the first nephron structures affected by overflow and pressure overload, we may state that hypertension development beyond 13week of age following maternal smoking exposure, associated with overload in remaining nephrons, could be a preponderant factor for the development of fibrous process and altered glomerular ultrastructure in the Sk offspring. From the present data, we demonstrate that fetalprogrammed Sk offspring present pronounced glomerular TGF-β1 and fibrotic marker expression that may, subsequently, cause glomerular EMT. Although the precise mechanism responsible for the subsequently renal morphological and functional response in Sk offspring is incompletely known, the current data suggest, that changes in renal function are conducive to excess sodium tu-
bule reabsorption that associated with enhanced TGF-β1, fibronectin and collagen deposition, intrinsically related to fibrotic process, might potentiate the programming of adult hypertension. In conclusion, we may suppose that, under smoking exposure, the fetus responds with adaptations accommodating to intrauterine conditions. After birth, the maternal tobacco exposure can result in progressive nephron senescence in parallel to functional loss and higher blood pressure. Disclosure Statement We affirm that there is no conflict of interest.
Funding Grants from CNPq (No. 500868/91–3), CAPES and FAPESP (10/52696–0 and 11/17016–1) supported this work.
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![[Relationship between urinary sodium and potassium excretion and blood pressure].](/assets/img/hold.png)
