Download - Fetal Kidney Programming by Maternal Smoking Exposure: Effects on Kidney Structure, Blood Pressure and Urinary Sodium Excretion in Adult Offspring

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Experimental Nephrology and Genetics: Original Paper

Nephron 2015;129:283–292

DOI: 10.1159/000377634

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

Sk offspring present pronounced glomerular TGF-β1 and fi-

brotic 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 as-

sociated with enhanced TGF-β1, fibronectin and collagen

deposition, intrinsically related to fibrotic process, might po-

tentiate 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 con-sequences in postnatal life [1] . Fetal programming by dif-ferent 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, en-

Key Words

Fetal programming · Maternal smoking exposure ·

Arterial hypertension · Glomerular fibrosis · TGF-β1 ·

Epithelial-to-mesenchymal transition

Abstract

Introduction: Fetal programming by different insults results

in low birth weight and reduction in nephron number in-

creasing the risk for adult development of cardiovascular

and renal diseases. Maternal smoking is an important modi-

fiable adverse fetal exposure worldwide and leads to a de-

crease in the offspring’s birth weight. Thus far, the specific

adverse fetal smoking exposures and mechanisms underly-

ing these associations on renal development and functional

disorder are unclear. Methods: The present study investi-

gates, in adult male rats, the effect of smoking exposure (Sk)

in uteri on blood pressure (BP) by an indirect tail-cuff meth-

od using an electrosphygmomanometer, and its association

with nephron structure by stereological estimation, immu-

nohistochemical and histological techniques, in parallel

with kidney function creatinine and lithium clearance. Re-

sults: The current study showed in a 16-week old Sk off-

spring enhanced arterial blood pressure associated with, re-

duced urinary sodium excretion and higher TGF-β1 glomer-

ular 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

Received: October 31, 2014

Accepted after revision: February 1, 2015

Published online: April 18, 2015

Dr. 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

© 2015 S. Karger AG, Basel1660–8151/15/1294–0283$39.50/0

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284

hanced susceptibility to hypertension and reduced renal reserve; therefore, the limiting compensation for renal in-jury 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 ad-verse fetal exposure worldwide and leads to a decrease of offspring’s birth weight [12] . Studies suggested that ma-ternal 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 clas-sical 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 disor-der are not known.

Irreversible renal fibrosis is a common consequence after most renal injuries [15, 16] . Incidentally, the trans-forming growth factor β (TGFβ) is a key regulator of ex-tracellular matrix (ECM) protein deposition in renal tis-sue [17] . Podocyte increased expression of TGF-β mRNA and glomeruli ECM protein deposition have been associ-ated to focal segmental glomerulosclerosis [18] , membra-nous 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 accu-mulation of matrix and inflammation [21, 22] . In the cur-rent study, we hypothesized that maternal smoking expo-sure 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 mater-nal 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.

Material and Methods

Animals The experiments were conducted on age-matched rats of sib-

ling-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-

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 origi-nated 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 con-trolled temperature (25 ° C) and lighting conditions (7: 00 a.m.–7: 00 p.m.), with free access to tap water and standard rodent labo-ratory 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 cham-ber containing one inlet and one outlet. The inlet was connected, via a two-way valve, to a multicigarette smoking apparatus, con-taining five cigarettes, and to the external atmosphere. The outlet was attached to a negative-pressure pump. Under these condi-tions, 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 ap-proximately 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 tobac-co 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 30-min each per day (1 h total exposure), throughout the entire three-week gestational period. It is estimated that the amount of smoke inhaled from cigarettes derived during the period of 4 weeks cor-respond to inhalation of 14 mg tar, 11 mg nicotine and 15 mg car-bon 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 off-

spring 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 repeat-ed 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) , off-

spring 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 haema-toxilin-eosin. We used the ‘fractionators’ method to estimate the

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Kidney Disorders and Gestational Smoking

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number of glomeruli and estimated the total number of renal cor-puscles per kidney considering the analyzed fraction of the kidney corrected to the entire organ as described at Mandarim-de-Lacerda [25] .

Renal Function Evaluation The renal function tests were performed and estimated by cre-

atinine 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 C Li 100 g –1 body weight was given by gavage. After an over-night 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 experi-ment, 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 process-ing. The proteinuria was detected using the BIOPROT U/LCR (Bioclin) .

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 con-trolled 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 immunohisto-chemistry, the sections were incubated sequentially with (1) phos-phate-buffered saline (PBS) containing 2% milk for 45 min to min-imize non-specific reactions, (2) mouse anti-fibronectin antise-rum (Santa Cruz) or rabbit anti-TGF-β1 antiserum (Santa Cruz) and (3) anti-mouse or rabbit Alexa 488 labeled antibody (molecu-lar Probes) for 2 h at room temperature. After incubation, the sec-tions 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 pri-mary 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 deter-mined. Images were captured with photomicroscope and analyzed by the Leica Qwin 3.1 for windows.

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 mea-sured by flame photometry (B262; Micronal, São Paulo, Brazil). Creatinine was determined spectrophotometrically (362; Micronal, São Paulo, Brazil) by the alkaline picrate method. Creatinine clear-ance was used to estimate the glomerular filtration rate (GFR) and lithium clearance (C Li ) was used to assess proximal tubule output [26–28] . Fractional sodium excretion (FE Na ) was calculated as C Na /C Cr × 100, where C Na is sodium clearance and C Cr is creatinine clearance. The fractional proximal (FEP Na ) and post-proximal (FEPP Na ) sodium excretion was calculated as C Li /C Cr × 100 and C Na /C Li × 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 Soft-ware, Inc., USA). The level of significance was set at p ≤ 0.05.

Results

The maternal smoking exposure during pregnancy caused a significant decreased dam’s body weight over the whole gestation ( fig. 1 a). 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 re-duction 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. 1 b). The body weight was unchanged in smoke offspring group (n = 11) from the 4th to 9th week of age ( fig. 1 c) when compared to control (n = 11) age-matched rats. However, beyond the 9th week of age, Sk offspring shows a signifi-cant decrease in body mass ( fig. 1 c). 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. 1 d). Stereological estimations , at postnatal 12 days, immediately after complete renal maturation (n = 5 for each group), we verified that Sk offspring although pres-ent 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. 1 e), the glomeruli number per kid-ney (Sk: 9,908 ± 3,178.96 units vs. 10,143 ± 3,533.54 units in Co, p = 0.3452; fig. 1 f), and glomeruli volume (15,761.21 ± 2,202.24 μl vs. 16,488.34 ± 4,792.07 μl in Co, p = 0.500; fig. 1 g) were unchanged when compared with Co group.

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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. 2 a), in both studied groups, the urinary flow rates (data not show) and the glomerular filtration rates, estimated by C Cr , did not significantly differ during the renal tubule sodium han-dling studies. In an offspring at 5 weeks of age, the frac-tional urinary sodium excretion (FE Na+ ) 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 =

0.0020. The enhanced FE Na+ in Sk rats was accompanied by unchanged proximal sodium excretion and increased fractional potassium excretion (FE K+ ) (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 FE Na+ occurred in parallel to significant augmentation in FEPP Na+ (Sk: 2.85 ± 0.047% vs. 1.13 ± 0.15% in Co, p = 0.0001; fig. 2 a). On the other hand, in offspring at 13-week of age, the renal tubule sodium handling parameters, estimated by C Li and C Cr , did not significantly differ among the groups Co and maternal smoking exposure offspring ( fig. 2 b).

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Fig. 1. The figure shows gestational (Co, n = 7 and Sk, n = 8) dams body weight ( a ), offspring (Co, n = 27 and Sk, n = 26) birth body weight ( b ), body weight ( c ) and arte-rial blood pressure (Co, n = 11 and Sk, n = 11) ( d ) and, offspring stereological estima-tion of kidney mass ( e ), glomeruli number ( f ) and glomeruli volume ( g ) in control (Co) compared to smoking (Sk) group; n = 5 for each group * p ≤ 0.05 versus control (Student’s t-test).

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Proteinuria Thirteen-week-old Sk group observed a significant in-

creased 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. 2 f).

Immuno and Histochemical Procedures The study shows an intensive rise of the TGF-β1 im-

munoreactivity in 16-week-old Sk (n = 5) in both glom-eruli and parietal epithelium ( fig. 3 a–d) compared to that found in the control (n = 5) group. Additionally, fibronec-tin immunoreactivity was also highly expressed in Sk (n = 5) offspring glomeruli and peritubular spaces when com-pared to age-matched Co (n = 5) animals ( fig. 3 e–h). The density of collagen fibers deposition in the renal cortical zone of 16-week-old Sk (n = 4) offspring, semi-quantita-tive evaluated by picrosirius staining technique, showed a

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.

Discussion

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 develop-mental plasticity hypothesis supports that adverse intra-uterine exposures lead to persistent fetal adaptations and organ disorders in adulthood. Therefore, fetal program-ming 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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Fig. 2. Renal function studies of the creatinine clearance (C Cr , a ), fractional sodium excretion (FE Na+ , b ), proximal (FEP Na+ , c ) and post-proximal (FEPP Na+ , d ), fractional sodium excretion and frac-tional potassium excretion (FE K+ , e ) in 5-week-old and 13-week-old control (n = 15) offspring compared to Sk (n = 11) age-matched rats.

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 devia-tion. * 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 pat-tern 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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cardiovascular disease [29] and lung disease [30] have at-tracted 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 in-creased albumin/creatinine ratio in adulthood, suggest-ing accentuated renal dysfunction.

Here, in maternal smoking offspring, we focus on adult renal function, arterial hypertension development and ep-ithelial 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 smok-ing 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 excre-tion in maternal Sk offspring at 5 weeks of age, when com-pared to the age-matched control group. The higher FE Na was accompanied by an increased FEPP Na and unaltered FEP Na . However, 8 weeks later, the renal sodium excretion from 13-week-old Sk offspring decline significantly, be-coming 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 glo-merular volume was not different when compared to the

Fig. 4. Renal cortical ( a–d ) and medullar ( e and f ) collagen content after picrosirius technique. The collagen is distributed in perivas-cular, peritubular, mesangial and subcapsular matriz and, in Sk ( b , d and f , n = 4) cortex, the content is significantly ( * * * p ≤ 0.01)

enhanced (quantification in graphic) when compared to that ob-served in Co ( a , c and e , n = 4) group; * Sk versus control group (Student’s t-test).

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control group data. In previous studies, we and others have demonstrated in different models of fetal program-ming, that even when associated with decreased glomeru-li number units, the offspring maintain a normal whole-kidney 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/over-flow 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 ex-posure, 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-proxi-mal tubule segments of nephron as compared to the con-trol 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 vas-cular resistance may have the opposite effect. Studies have demonstrated that renal prostaglandin E2 (PGE2) is nec-essary 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 in-creased renal perfusion pressure. The microsomal iso-form of PGES, mPGES-1, is inducible by many factors and is widely regarded as the main contributing enzymatic tandem for PGE 2 biosynthesis under pathological condi-tions, including active tobacco smoking [33] . On the oth-er hand, the study has also demonstrated that tobacco ex-posure promoted a significant reduction in urinary excre-tion of 6-keto-PGF 1α [34] . These observations suggest that tobacco smoking reduces PGI 2 production, including in kidneys; this result could explain the antinatriuresis re-sponse 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. Addi-tionally, the hypothesis that fetal kidney is programmed to inappropriately retain Na + in later life has been cor-roborated by different studies [35] . Intrauterine growth restriction model (IUGR) in rats resulted in the upregula-

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 renin-angiotensin 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 ac-count 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 glomeru-lar hypertension and in the future, to sustained renal func-tion disorder [36] .

By the histochemistry and immunofluorescence stain-ing, the present study verified in the Sk offspring, an in-creased glomerular expression of TGF-β1, fibronectin and type I collagen, intrinsically related to fibrotic process as compared to age-matched control group. As previous-ly 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 in-terstitial 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 glomeru-lar structural alterations were accompanied by parallel proteinuria in Sk offspring, suggesting a decreased effi-ciency on the glomerular filter barrier in these rats. As observed in the current study, the process leading to tis-sue fibrosis, is considered a dominant mechanism in-duced by TGF-β1 in the kidney, largely on the basis of the observation that glomerular and tubulo-intersticial scar-ring 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 im-paired pressure natriuresis [43] . We may not also rule out

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Nephron 2015;129:283–292DOI: 10.1159/000377634

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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 13-week of age following maternal smoking exposure, asso-ciated with overload in remaining nephrons, could be a preponderant factor for the development of fibrous pro-cess and altered glomerular ultrastructure in the Sk off-spring. From the present data, we demonstrate that fetal-programmed Sk offspring present pronounced glomeru-lar TGF-β1 and fibrotic marker expression that may, subsequently, cause glomerular EMT. Although the pre-cise mechanism responsible for the subsequently renal morphological and functional response in Sk offspring is incompletely known, the current data suggest, that chang-es 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 adapta-tions accommodating to intrauterine conditions. After birth, the maternal tobacco exposure can result in pro-gressive 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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