SIRT1 reduction is associated with sex-specific dysregulation of renal lipid metabolism and stress responses in offspring by maternal high-fat diet

Rodent models of maternal obesity have been associated with kidney damage and dysfunction in offspring. However, the underlying mechanisms are yet to be elucidated. In this study, female rats were fed a high-fat diet (HFD) for 6 weeks prior to mating, throughout gestation and lactation; both male and female offspring were examined at weaning. Our results demonstrate that renal lipid deposition was increased in male offspring only, which is associated with reduced protein expression of Sirtuin (SIRT) 1, an essential regulator of lipid metabolism and stress response. Other components in its signalling network including phosphorylated 5′-AMP-activated protein kinase (pAMPKα), Forkhead box FOXO3a and Peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-alpha (PGC-1α) were also downregulated. By contrast, in female offspring, renal fat/lipid distribution was unchanged in coupling with normal SIRT1 regulation. Specific autophagy and antioxidant markers were suppressed in both sexes. On the other hand, fibronectin and Collagen type IV protein expression was significantly higher in the offspring born HFD-fed dams, particularly in the males. Collectively, these findings suggest that maternal HFD consumption can induce sex-specific changes in offspring kidney lipid metabolism and stress responses at early ages, which may underpin the risk of kidney diseases later in life.

Sirtuins (SIRTs) are a group of d NAD + -dependent deacetylases and mono-ADP-ribosyl transferases. In mammals, seven members of the family (SIRT1-7) have been identified, among which SIRT1 is the most extensively studied, having pivotal roles in multiple processes especially stress responses and glucose/fat metabolism 14 . Its expression and activities are known to be promoted during nutrient deprivation 15 and suppressed when nutrient is in excess, such as in obese individuals 16 . With regard to the kidney, activation of the SIRT1 signalling pathway has been shown to reduce renal lipotoxicity 17 , improve renal autophagy 18 and antioxidant defence 19 , thereby attenuate kidney diseases in obese and diabetic animals.
In models of maternal obesity, it has been shown that SIRT1 expression is reduced in the fetus 20 and specifically in the offspring liver 21 , suggesting a potential role of SIRT1 as a mediator of the programming effects by maternal obesity 22 . Nevertheless, the regulation of renal SIRT1 signalling in the offspring has yet to be demonstrated. In this study, we examine the hypothesis that maternal obesity induces renal lipid metabolic disorders and autophagy deficiency in the offspring and SIRT1 signalling plays a central role in this mechanism. As the risk of CKD differs between sexes 23 and so does the fetal programming of kidney disorders [24][25][26][27] , we also examined whether these mechanisms are regulated in a sex-specific manner.

Results
Maternal HFD consumption increased body weight, fat deposition, plasma glycerides and kidney weight in both male and female offspring. At weaning, HFD-fed dams had significantly higher body weight, retroperitoneal fat mass and liver weight compared to chow-fed dams (P < 0.05, Table 1). Both male and female offspring of the HFD-fed dams showed significantly greater body weight than those born to chow-fed dams (P < 0.05, Table 2). Moreover, female offspring were significantly smaller than males (P < 0.05, sex effect). Mesenteric, retroperitoneal and perigonadal fat mass in both MHF male and female offspring was significantly higher in the offspring from HFD-fed dams (P < 0.05). The difference remained significant when adjusted for body weight (P < 0.01) except for male offspring's mesenteric fat. Similar to the body weight, the overall percentage of retroperitoneal fat in female offspring was significantly smaller than in the males (P < 0.05, sex effect). The   Table 2. Body weight and organ mass of male and female offspring at weaning. Results are expressed as means ± SD. The data were analysed using two-way ANOVA with Bonferroni post hoc test. MC (offspring of dams fed chow); MHF (offspring of dams fed high-fat diet). *P < 0.05, **P < 0.01 (vs MC offspring); † P < 0.05 (vs male offspring), a (P < 0.05, Maternal diet effect), b (P < 0.5, Sex effect), c (P < 0.05, interaction effect). BW (Body weight), BGL (Blood glucose level).
net liver weight was significantly increased by MHF only in female offspring. The sex effect for liver weight was significant (P < 0.05, sex effect), as well as the interaction between sex and maternal diet (P < 0.05, interaction). Kidney weight was also significantly increased in the offspring (P < 0.05 in male and P < 0.01 in female respectively). Overall, the females have lighter kidneys than the males. Consistent with the increased body weight and adiposity, plasma triglyceride levels were also increased in both sexes in the offspring (P < 0.05 and P < 0.01 respectively), whereas the levels of non-esterified fatty acid were unchanged.
Maternal HFD consumption increased perirenal fat deposition and renal triglyceride concentration in male but not female offspring. Male offspring from HFD-fed dams demonstrated increased perirenal fat deposition ( Fig. 1), which was unchanged in female offspring. Similarly, kidney levels of triglycerides in male offspring were significantly higher when born to dams fed a HFD (P < 0.01), whereas in female offspring, no difference was observed between the groups. The sex-specific effect is reflected by a significant interaction (P < 0.05) between the two factors: sex and maternal diet.
The effects of maternal HFD consumption on lipid metabolism regulators in offspring kidney. As kidney triglyceride levels were increased in the male offspring only, we examined the mRNA expression of potential regulator(s) involved in lipid metabolism. As shown in Table 3, only mRNA expression of SIRT1 and PGC-1α was significantly downregulated in the male offspring due to maternal HFD consumption (P < 0.01 and P < 0.05 respectively). In female offspring, no differences at the transcriptional level were detected. The sex-specific regulation of SIRT1 mRNA is reflected by a significant interaction (P < 0.05) between the two factors of sex and maternal diet. Together with SIRT1 and PGC-1α, PPARα and PPARγ are also essential regulators of lipid metabolism. In this study, we showed that the mRNA expression of PPARα in the offspring kidney were generally lower in females (P < 0.05, sex effect, Table 3). By a sharp contrast, the levels of PPARγ were significantly higher in the females (P < 0.05, sex effect). The effect of maternal HFD consumption on the transcription of the two genes was minor except a slight tendency of downregulation of PPARα in the MHF males (Table 3).
Similar to PPARγ, the overall mRNA expression of FABP3 was also significantly higher in the females (P < 0.05, sex effect) and unchanged by maternal HFD consumption. No difference in the renal expression of other regulators of the lipogenesis pathway including SREBP1c and FAS were detected.
Maternal HFD consumption regulates renal SIRT1 signalling network in the offspring in a sex-specific manner. To confirm our observations, the protein expression of SIRT1 and other markers of its signalling pathway were analysed. Our results demonstrated that SIRT1 protein level was indeed reduced in the kidney of MHF male offspring (P < 0.05, Fig. 2), whereas in the females, no change was detected. The level of total AMPK, a positive regulator of the SIRT1 signalling network, was substantially reduced by approximately 50% (P = 0.052) in the MHF males. More importantly, the level of its active phosphorylated form pAMPK was also significantly suppressed correspondingly (P < 0.05). In the female offspring, the renal levels of AMPK and pAMPK were both unaffected. The sex-maternal diet interactions were near-significant (P = 0.07) and significant (P < 0.05) for SIRT1 and pAMPK respectively. Similarly, the expression of FOXO3a, a downstream regulator of SIRT1, was also regulated in such interactive fashion (P < 0.05) with a near-significant reduction in MHF male offspring (P = 0.053). Interestingly, despite the unchanged mRNA expression in MHF females, the protein levels of PGC-1α were significantly reduced by maternal HFD consumption in both sexes (P < 0.001 and P < 0.05 respectively, Fig. 2).
SIRT1 is known as a suppressor of PPARγ transcription 28 . However, in this study, the expression of PPARγ seems to be independent of SIRT1 signalling. The protein expression of both PPARγ isoforms 1 and 2 were substantially reduced (P = 0.059 and P < 0.01 respectively, Fig. 3) in MHF female offspring kidney. However, in the male offspring, no change was detected. The sex-specific effect is reflected by a significant interaction between the two factors of sex and maternal diet for the regulation of PPARγ2 (P < 0.05).

Maternal HFD consumption downregulates renal autophagy markers. Autophagy can be
regulated by SIRT1 in multiple ways: either through the action of FOXO3a to upregulate the transcription of autophagy-related genes 29 , or by direct deacetylation of Atg proteins thereby increasing their activity 30 . Our results indicate that maternal HFD consumption has significant impacts on the renal expression of essential autophagy markers including Beclin-1, Atg12-Atg5 complex, LC3-I, LC3-II and p62 (P < 0.05, maternal diet effect, Fig. 4). Notably, while Beclin-1, LC3-I and II were suppressed to the same levels in both sexes, Atg12-Atg5 and p62 were regulated in a sex-specific fashion with significant reductions of the former in the female (P < 0.001) and the latter in the male offspring (P < 0.05) respectively. There was also a trend of reduction in the expression of Atg7 only in the females (Fig. 4).
Maternal HFD consumption reduces renal levels of antioxidant marker GPx-1. SIRT1 signalling has been associated with cellular antioxidant defence. MnSOD and GPx-1 were examined as representative markers of antioxidant defence, each of which is involved in one of the two-step reaction to convert reactive oxygen species into water and oxygen . As shown in Fig. 5, while the renal levels of MnSOD were not affected by maternal HFD consumption, GPx-1 proteins were significantly and consistently reduced in the offspring in both sexes (P < 0.01).

The effects of maternal HFD consumption on renal markers of inflammation and extracellular matrix.
Obesity is often associated with a low-grade inflammation 31 . Particularly in the kidney, it is associated with the elevation of TGFβ1 as well as structural modifications including glomerulosclerosis and interstitial fibrosis 32,33 . In this study, TGFβ1 mRNA expression in the male offspring kidney was not affected by maternal HFD consumption. Higher levels of TGFβ1 were detected in the females (P < 0.05, sex effect, Table 4). By contrast, the renal expression of TGFβ receptor 1 was reduced in the kidney of MHF offspring (P < 0.05, maternal diet effect) and not different between the two sexes. Similarly, Col3A mRNA expression was gender-dependent (P < 0.05, sex effect), whereas Col1A was reduced by maternal HFD consumption (P < 0.05, maternal diet effect). Fibronectin mRNA expression showed a trend of increase by maternal HFD consumption (Table 4). In addition, immunohistochemistry staining also indicated an overall elevation of fibronectin protein in the offspring born to HFD-fed dams (Fig. 6B, P < 0.05, maternal diet effect). The renal protein level of Col4 was also significantly increased in MHF offspring (Fig. 6C, P < 0.05, maternal diet effect), especially in the males (P < 0.05).

Discussion
Maternal high-fat diet consumption and obesity have been associated with kidney damage and dysfunction in the offspring in adulthood 8,9 , which are likely the result of accumulative cellular/molecular events occurred during early developmental periods. In this study, we attempted to clarify the underlying mechanisms. We demonstrate that maternal overnutrition is associated with increased lipid deposition and the suppression of multiple stress responses including autophagy, antioxidant defence and inflammation in the offspring kidney with a significant relevance to the SIRT1 signalling network. In addition, the study also suggests that many of these early changes are sex-dependent.  Table 3. mRNA expression of lipid metabolism markers in offspring kidney. MC (offspring of dams fed chow); MHF (offspring of dams fed high-fat diet). SIRT1 (Sirtuin1); PPARγ (Peroxisome proliferator-activated receptor gamma); PPARα (Peroxisome proliferator-activated receptor alpha); PGC-1α (PPARγ coactivator 1-alpha); FAS (Fatty acid synthase); SREBP-1c (Sterol regulatory element-binding protein); Fatty acid binding protein (FABP) 3. Results are expressed as means ± SD. Data were analysed using two-way ANOVA with Bonferroni post hoc test. *P < 0.05, **P < 0.01 (vs MC offspring), † P < 0.05 (vs male offspring). a (P < 0.05, Maternal diet effect), b (P < 0.5, Sex effect), c (P < 0.05, interaction effect).
Consistent with previous studies [6][7][8]34 , maternal HFD consumption preconception, during gestation and laction significantly increased ofsspring body weight, fat deposition, plasma triglyceride level and kidney weight at weaning, suggesting increased caloric intake and cell proliferation. Male offspring showed higher body weight and fat mass compared to females, which is also in line with previous studies in rodents 26 and human 35 , suggesting Results are expressed as mean ± SD. Data were analysed by two-way ANOVA followed by Bonferroni post hoc test. (a) (P < 0.05, Maternal diet effect), (b) (P < 0.5, Sex effect), (c) (P < 0.05, interaction effect), *P < 0.05, **P < 0.01 (vs MC offspring). n = 6. a higher tendency of males for obesity development. In addition, the fact that maternal HFD consumption significantly increased the liver weight of female but not male offsrping may imply a higher capacity of female liver for growth and perhaps also lipid storage and processing, which in turn may reduce lipid spillover to other tissues such as kidney.
Indeed, the levels of perirenal fat as well as intrarenal lipid content were higher only in MHF males but not females, suggesting the effects of maternal HFD consumption on offspring kidney are also sex-specific. Sex-dependent differences in fetal programming of nephropathy are well-established in maternal undernutritional conditions 24,26,27 and to a lesser extend maternal overnutrition, for instance, a high-protein diet 25 . Increased intrarenal lipid content has been associated with glomerulopathy and tubulointerstitial sclerosis in obese patients 10 , while perirenal lipid deposition has also been suggested as an independent predictor of CKD 11 . Such early changes in lipid metabolism are likely to induce lipotoxicity and predispose to kidney dysfunction later in life, especially when a second insult to the kidney occurs, such as the development of diabetes or obesity in the offspring 5,8 . SIRT1 and its downstream factor PGC-1α are two stimuli of fatty acid oxidation (52,53). As a consequence of maternal HFD consumption, renal mRNA and protein levels of SIRT1 and PGC-1α levels were reduced specifically in the male offspring, which may explain the male-specific elevation of renal fat. Together with SIRT1 and PGC-1α, the protein levels of phosphorylated AMPK was also downregulated. AMPK can indirectly activate SIRT1 by increasing the NAD + /NADH ratio through the upregulation of Nicotinamide phosphoribosyltransferase 36 . Conversely, SIRT1 is also required for AMPK activity 37 . As renal AMPK has been shown to be reduced in obesity and diabetes 17,38 , its reduced expression and activation due to maternal obesity is consistent with the downregulation of SIRT1 signalling and increased lipid deposition in the male offspring kidney. Both SIRT1 and AMPK are activators of FOXO3a, while FOXO3a can also promote SIRT1 transcription by interfering with the suppressor p53 on the SIRT1 promoter 39 . As such, a positive feedback loop is formed within the SIRT1 signalling network.
In MHF female offspring, there was no change in renal expression of SIRT1, pAMPK or FOXO3a. Alternatively, the protein level of PPARγ2 was significantly reduced. As PGC-1α is a co-activator of PPARγ, its reduced level in MHF females supports downregulated PPARγ signalling. Although PPARγ is well-known as an indispensable controller of adipocyte differentiation and insulin sensitivity 40 , its role in the kidney is not clearly understood. It has been shown that PPARγ +/− mice showed resistance to obesity-associated kidney lipid accumulation and injuries 41 . The reduction of renal PPARγ2 reduction in MHF female offspring may reflect an adaptive response to the increased fatty acid influx due to maternal HFD consumption. The regulation of PPAR signalling by maternal overnutrition has been previously reported in a transcriptome study 42 . In this study, the protein levels of multiple autophagy markers were significantly reduced in the offspring kidney, suggesting suppression of autophagic processes. Hence, accumulation of misfolded proteins and dysfunctional organelles such as malfunctioning mitochondria is likely to occur 43 . Although autophagy was suppressed by maternal HFD consumption in both sexes, there were sex-dependent differences in the regulation of individual markers. The fact that the reduction of Atg12-Atg5 complex only occurred in the MHF females suggests that autophagosome elongation was likely to be impaired. On the other hand, p62 expression was only affected in the MHF males, indicating autophagosome degradation was interfered specifically in MHF male offspring. Prolonged deficiency in autophagy can increase renal susceptibility to glomerular and tubulointerstitial pathology 12,13 .
also suppressed, reflected in the marked reduction in GPx-1 levels. The fact that the expression of MnSOD was unchanged indicates that the impairment of antioxidant defence was only partial and may not necessarily lead to oxidative stress at such young neonatal age. However, there is a certainly higher risk for oxidative stress and inflammation to occur in the offspring kidney if the defects is prolonged. Indeed, our group has recently demonstrated that maternal obesity can exacerbate kidney oxidative stress, inflammation and fibrosis induced by type 1 diabetes in the offspring 5 . It has been shown that the SIRT1/AMPK/FOXO3a axis positively regulates autophagy 43,46,47 and antioxidant defence 48,49 , thereby preventing oxidative damage, particularly in kidney tubular cells 50,51 . As such, the reduction of these markers in male MHF offspring kidney is likely to underline the deficiency of kidney autophagy and antioxidant defence markers. In female offspring, as the renal levels of SIRT1, pAMPK and FOXO3a were normal, the alterations in autophagy and antioxidant defence are likely to be mediated through a different pathway, for example the PPARγ/PGC-1α pathway 48,52 . Further investigations in female offspring are required to confirm this hypothesis.
In the study, the renal mRNA expression of TGFβ1, Col3A and FN in the MHF offspring was unchanged, while TGFβR1 and Col1A mRNA expression was moderately decreased. On the other hand, fibronectin and Col4 protein level was significantly upregulated in MHF offspring, suggesting that maternal HFD consumption can induce renal fibrogenesis in the offspring at weaning. Such effect has been shown to extend to adulthood 9 . Interestingly, only in the male offspring was the Col4 protein expression significantly upregulated. This result further supports higher levels of kidney disorders and damage in male offspring than the female offspring caused by maternal HFD consumption, which is likely linked to increased renal lipid distribution and reduced SIRT1 signalling in the male kidney. It has been demonstrated that SIRT1 activation can suppress renal fibrogenic signalling in vitro and in vivo 53 . Whether such therapy is applicable in the offspring at early age to attenuate or reverse the programming effects of maternal HFD consumption on kidney structure requires further investigations.
In summary, the current study demonstrates that maternal HFD consumption can increase renal fat deposition in male offspring and impair renal autophagy and antioxidant defences. Importantly, there are sex-specific differences in the magnitude of effects in the regulatory pathways. In male offspring there is downregulation of the SIRT1/AMPK/FOXO3a/PGC-1α regulatory network; whereas in females, downregulation of PPARγ/ PGC-1α is evident. The more marked abnormalities in the male offspring may underpin the well documented increased risk of CKD in males than females in adulthood 23 . Such significant sex differences emphasize the need to independently study both sexes and to employ sex-specific approaches to overcome the adverse effects of maternal obesity on the development of CKD in the offspring. Further investigations with a longitudinal observation targeting SIRT1 signalling pathway are required to confirm sex specificity and the implication of SIRT1 regulation in the development of CKD in the context of maternal obesity.

Methods
Animals. The study was approved by the Animal Care and Ethics Committee of the University of Technology Sydney (ACEC# 2009-350). All methods were performed in accordance with the relevant guidelines and regulations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Female Sprague-Dawley rats (8 weeks) were fed HFD (HFD, 20 kJ/g, 43.5% calorie as fat, Specialty Feed, WA, Australia) or standard rodent chow (11 kJ/g, 14% calorie as fat, Gordon's Speciality Stockfeeds, NSW, Australia) for 6 weeks before mating, throughout gestation and lactation 54 . On postnatal day (P) 1 the litter size was adjusted to 10 pups/ litter (sex ratio controlled at approximately 1:1). Both male and female offspring were investigated. At weaning (P20), all pups were sacrificed after overnight fasting. Blood was collected via cardiac puncture after anaesthesia (Pentothal, 0.1mg/g, i.p., Abbott Australasia Pty Ltd, NSW, Australia). Fat tissues and liver were weighed. The whole kidney was dissected, snap frozen and stored at −80 °C for later analysis. For each type of analysis, 5-6 renal tissues/group were randomly selected.
Kidney histology. Kidneys were fixed in 10% formalin for 36-h and embedded in paraffin. Paraffin sections were prepared at 4 μm thickness and mount on microscope slides. The sections were stained with hematoxylin and eosin (H&E) for general renal structure and perirenal fat visualisation. Immunohistochemistry (IHC) staining was performed as previously described 55 . Briefly, the tissue sections underwent dewaxing (with xylene), rehydration (with ethanol), antigen-retrieval (99 °C for 20 min in 0.01M, pH 6.0 citric buffer), washing (with water), endogenous peroxidase deactivation with 3% H 2 O 2 (Sigma-Aldrich, Dublin, Ireland), blocking (with Protein Block Serum-Free, Dako, Glostrup, Denmark), incubation with primary antibodies against fibronectin (1:1000, Abcam, Cambridge, UK) and collagen IV (1:1000, Abcam, Cambridge, UK) followed by biotinylated secondary anti-rabbit IgG antibodies and finally horseradish peroxidase (HRP)-conjugated streptavidin (Dako, Glostrup, Denmark). The tissue specimens were examined by brightfield microscopy (Olympus, Japan). Six consecutive non-overlapping fields from each section of renal cortex were photographed under high magnification. Image J (National Institutes of Health, USA) was used for estimation of the specific staining area. IHC score was determined by log transformation of the staining area.
Renal protein and lipid extraction. The kidney was homogenized in Triton X-100 lysis buffer (pH 7.4, 150 mM NaOH, 50 mM Tris-HCl, 1% Triton X-100, Roche protease inhibitor) using TissueRuptor (Qiagen, Hilden, Germany). A part of the homogenate was used for lipid extraction according to a published protocol 56 . The lipid extract of each sample was then added to a 96-well plate, followed by incubation with Roche triglyceride reagent GPO-PAP (Roche Life Science, NSW, Australia) at 37 °C for 20 min. The absorbance was read at 490 nm. Glycerine was used in constructing the standard curve.
The remaining tissue homogenate was centrifuged at 10,000 g for 15 min. The supernatant containing total protein was collected and quantitated for protein concentration using Pierce BCA Protein Assay Kit (Thermo Scientific, VIC, Australia) according to the manufacturer's instructions. The protein concentration of all samples was standardised as 5 μg/μl. The samples were stored at −80 °C for further analyses.
Quantitative real time PCR (qRT-PCR). Total RNA was isolated from the kidney tissue using RNeasy Plus Mini Kit (Qiagen Pty Ltd, CA, USA) according to the manufacturer's instructions. The purified total RNA was