Postnatal persistence of sex-dependent renal developmental programmed structural and molecular changes in nonhuman primates

Background: Poor nutrition during development programs kidney function. No studies on postnatal consequences of decreased perinatal nutrition exist in nonhuman primates (NHP) for translation to human renal disease. Our baboon model of moderate maternal nutrient restriction (MNR) produces intrauterine growth restricted (IUGR) and programs renal fetal phenotype. We hypothesized that the IUGR phenotype persists postnatally influencing responses to a high-fat, high-carbohydrate, high-salt (HFCS) diet. Methods: Pregnant baboons ate chow Control (CON) or 70% of control intake (MNR) from 0.16 gestation through lactation. MNR offspring were IUGR at birth. At weaning, all offspring, (control and IUGR females and males n=3/group) ate chow. At ~3.5 years age, blood, urine, and kidney biopsies were collected before and after a 7-week high HFCS diet challenge. Kidney function, unbiased kidney gene expression, and untargeted urine metabolomics were evaluated. Results: IUGR female and male kidney transcriptome and urine metabolome differed from CON at 3.5 years, prior to HFCS. After the challenge, we observed sex-specific and fetal exposure-specific responses in urine creatinine, urine metabolites, and renal signaling pathways. Conclusions: We previously showed mTOR signaling dysregulation in IUGR fetal kidneys. Before HFCS, gene expression analysis indicated that dysregulation persists postnatally in IUGR females. IUGR male offspring response to HFCS showed uncoordinated signaling pathway responses suggestive of proximal tubule injury. To our knowledge, this is the first study comparing CON and IUGR postnatal juvenile NHP and the impact of fetal and postnatal life caloric mismatch. Perinatal history needs to be taken into account when assessing renal disease risk. Animal Selection and Management: All animal procedures were approved by the Institutional Animal Care and Use Committee at Texas Biomedical Research Institute and conducted in Association for Assessment and Accreditation of Laboratory Animal Care approved facilities at the Southwest National Primate Research Center. Details of housing, individual feeding cages, training, and environmental enrichment were previously published in [24]. Twelve non-pregnant female baboons (Papio species), 11.5 ± 0.51 (mean ± standard error of the mean [SEM]) years of age and of similar morphometric phenotype, were selected for study and group-housed in outdoor cages with a vasectomized male, thereby providing full social and physical activity. Animals were trained prior to pregnancy to feed in individual cages as described previously [24]. Briefly, at feeding time all baboons passed along a chute and into individual feeding cages. Each baboon's weight was obtained while crossing an electronic scale (GSE 665; GSE Scale Systems, Milwaukee, Wisconsin). Water was continuously available in the feeding cages via individual waterers (Lixit, Napa, California). Following the introduction of a fertile male and starting at 30 days of gestation (dG; equivalent to 0.16G), six females were randomly assigned to the CON group and fed Purina Monkey Diet 5038 (chow; Purina, St Louis, MO) ad libitum. The chow diet contained 12% energy from fat, 18% from protein, and 69% from carbohydrate, consisting of 0.29% glucose and 0.32% fructose. The remaining six females were assigned to the MNR group and fed 70% of the feed eaten by the CON females on a weight-adjusted basis from 0.16G through the rest of pregnancy and lactation [11]. Water was available to all animals, ad libitum. CON and MNR mothers spontaneously delivered CON and IUGR offspring, respectively, at full term. The offspring were reared with their mothers in group housing until weaning at approximately nine months of age. Then they were moved to a juvenile cage of mixed males and females and maintained on the chow diet. HFCS Diet Challenge: At approximately 4.5 years of age (human equivalent of 10.5 years of age), 6 CON offspring ( 3 females and 3 males) and 6 age-matched IUGR offspring (3 females and 3 males) were challenged with a 7-week HFCS diet and given access to a high fructose drink in addition to water, which were all available to the animals throughout the study ad libitum. The HFCS diet contained 73% Purina Monkey Chow 5038 (a grain-based meal), 7% lard, 4% Crisco, 4% coconut oil, 10.5% flavored high fructose corn syrup, and1.5% water. Vitamins and mineral preparations were added to match the micronutrient composition of the chow diet. Palatability was enhanced using non-caloric artificial fruit flavors. Details have been published [25]. The high fructose drink included water, high fructose corn syrup (2.83 Kcal/g, 76% sugar, 41.8% fructose, 34.2% dextrose, ISOSWEET 5500, Staley, Decatuer, IL), and artificial fruit flavoring [25]. During the HFCS diet challenge, all baboons from a single group cage were run once per week into individual feeding cages, passing over an electronic weighing scale. Urine was collected in a pan below each feeding cage with in a 3 hour period. Food and drink were not available during this time to minimize contamination of urine. After the urine was collected during this 3-hour period, animals were given free access to the HFCS diet, high fructose drink, and water for 13.5 hours in the individual feeding cages. They were then returned to the group social area. The high fructose drink was provided in a Lixit waterer (Lixit, Napa, California) that was connected to a gauge to measure sugar drink consumption for each animal. Food consumption was recorded for each animal. Following the 13.5-hour consumption period, animals were returned to the group cage. RNA Isolation and Microarray Hybridization: Total RNA was isolated from an approximately 5 mg section of each frozen kidney biopsy using a Power Gen Homogenizer (Omni International, Wilmington, DE) and TRI Reagent™ Solution (Invitrogen™, Carlsbad, CA) as previously described in [12]. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), and RNA concentration was determined by UV-Vis spectrophotometry using a Nanodrop™ 2000 (Thermo Fisher Scientific, Wilmington, DE). Complementary RNA (cRNA) was synthesized and biotinylated using the Illumina™ TotalPrep™-96 RNA Amplification Kit (Illumina Inc., San Diego, CA). cRNA samples were then hybridized to HumanHT-12 v3 Expression BeadChips (Illumina, Inc.). Microarray Data Analysis: Gene expression data were extracted and log2-transformed using GenomeStudio software (Illumina, Inc.) and subsequently analyzed using Partek® Genomics Suite (Partek®, St. Louis, MO). Signal intensities were all-median normalized, and differentially expressed genes were identified by Analysis of Variance (ANOVA; p < 0.05). Differentially expressed genes were overlaid onto canonical pathways and networks generated using differentially expressed genes in the dataset and the Ingenuity Pathway Analysis (IPA; QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) Knowledge Base. Right-tailed Fisher's exact test was used to calculate a significant enrichment of differentially expressed genes in pathways, p< 0.01 [29]. Networks were built using the IPA Knowledge Base, requiring direct connections between molecules based on experimental evidence. We used an end-of-pathway gene expression approach to identify coordinated pathways. We hypothesized that a pathway may only be relevant to the kidney phenotype if gene expression profiles at the end of the pathway were consistent with the overall pathway change. Therefore, pathways meeting this criterion, as well as those downstream of these pathways, were investigated. If there were no differentially expressed genes at the end of a pathway, that pathway was not considered relevant to the phenotype [12]. We performed upstream regulatory network analysis to identify potential causal networks which integrate previously observed cause-effect relationships, leveraging experimental knowledge about the direction of effects to infer upstream regulatory molecules and potential mechanisms explaining observed gene expression changes. The analysis includes prediction of activation or inhibition of the network and consistency of molecules in the network with the predicted activation state. The calculated z-score makes predictions about regulation directions and infers the activation state of a putative regulator (i.e., activated or inhibited) and can be used to determine likely regulators based on statistical significance of the pattern match. The statistical models are provided in detail in Kramer et al., 2014 [30]. Networks were considered significant for p < 0.05. Statistical Analyses: PCA and hierarchical clustering showed differences between females and males in morphometrics, clinical measures, kidney transcriptome, and urine metabolome; therefore, we analyzed all data separately for females and males. Initially, normalized transcriptome and metabolome datasets were analyzed by Principal Component Analysis (PCA) to identify sources of variation in each dataset using Partek® Genomics Suite. The greatest variance in each dataset was attributed to sex; therefore, data from females and males were analyzed separately. Pairwise comparisons were performed using two-tailed t-tests. Data from each diet group were analyzed independently for pre- and post-challenge means and then evaluated using two-tailed t-test and two-way ANOVA with significance p < 0.05.