A roadmap for the genetic analysis of renal aging

Several studies show evidence for the genetic basis of renal disease, which renders some individuals more prone than others to accelerated renal aging. Studying the genetics of renal aging can help us to identify genes involved in this process and to unravel the underlying pathways. First, this opinion article will give an overview of the phenotypes that can be observed in age‐related kidney disease. Accurate phenotyping is essential in performing genetic analysis. For kidney aging, this could include both functional and structural changes. Subsequently, this article reviews the studies that report on candidate genes associated with renal aging in humans and mice. Several loci or candidate genes have been found associated with kidney disease, but identification of the specific genetic variants involved has proven to be difficult. CUBN, UMOD, and SHROOM3 were identified by human GWAS as being associated with albuminuria, kidney function, and chronic kidney disease (CKD). These are promising examples of genes that could be involved in renal aging, and were further mechanistically evaluated in animal models. Eventually, we will provide approaches for performing genetic analysis. We should leverage the power of mouse models, as testing in humans is limited. Mouse and other animal models can be used to explain the underlying biological mechanisms of genes and loci identified by human GWAS. Furthermore, mouse models can be used to identify genetic variants associated with age‐associated histological changes, of which Far2, Wisp2, and Esrrg are examples. A new outbred mouse population with high genetic diversity will facilitate the identification of genes associated with renal aging by enabling high‐resolution genetic mapping while also allowing the control of environmental factors, and by enabling access to renal tissues at specific time points for histology, proteomics, and gene expression.     

DNA methylation analysis of murine hematopoietic side population cells during aging

Stem cells have been found in most tissues/organs. These somatic stem cells produce replacements for lost and damaged cells, and it is not completely understood how this regenerative capacity becomes diminished during aging. To study the possible involvement of epigenetic changes in somatic stem cell aging, we used murine hematopoiesis as a model system. Hematopoietic stem cells (HSCs) were enriched for via Hoechst exclusion activity (SP-HSC) from young, medium-aged and old mice and subjected to comprehensive, global methylome (MeDIP-seq) analysis. With age, we observed a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands. Just over 100 significant (FDR < 0.2) aging-specific differentially methylated regions (aDMRs) were identified, which are surprisingly few considering the profound age-based changes that occur in HSC biology. Interestingly, the polycomb repressive complex -2 (PCRC2) target genes Kiss1r, Nav2 and Hsf4 were hypermethylated with age. The promoter for the Sdpr gene was determined to be progressively hypomethylated with age. This occurred concurrently with an increase in gene expression with age. To explore this relationship further, we cultured isolated SP-HSC in the presence of 5-aza-deoxycytdine and demonstrated a negative correlation between Sdpr promoter methylation and gene expression. We report that DNA methylation patterns are well preserved during hematopoietic stem cell aging, confirm that PCRC2 targets are increasingly methylated with age, and suggest that SDPR expression changes with age in HSCs may be regulated via age-based alterations in DNA methylation.     

Gene expression changes with age in skin,adipose tissue,blood and brain

Background

Previous studies have demonstrated that gene expression levels change with age. These changes are hypothesized to influence the aging rate of an individual. We analyzed gene expression changes with age in abdominal skin, subcutaneous adipose tissue and lymphoblastoid cell lines in 856 female twins in the age range of 39-85 years. Additionally, we investigated genotypic variants involved in genotype-by-age interactions to understand how the genomic regulation of gene expression alters with age.

Results

Using a linear mixed model, differential expression with age was identified in 1,672 genes in skin and 188 genes in adipose tissue. Only two genes expressed in lymphoblastoid cell lines showed significant changes with age. Genes significantly regulated by age were compared with expression profiles in 10 brain regions from 100 postmortem brains aged 16 to 83 years. We identified only one age-related gene common to the three tissues. There were 12 genes that showed differential expression with age in both skin and brain tissue and three common to adipose and brain tissues.

Conclusions

Skin showed the most age-related gene expression changes of all the tissues investigated, with many of the genes being previously implicated in fatty acid metabolism, mitochondrial activity, cancer and splicing. A significant proportion of age-related changes in gene expression appear to be tissue-specific with only a few genes sharing an age effect in expression across tissues. More research is needed to improve our understanding of the genetic influences on aging and the relationship with age-related diseases.     

Evidence for reduced neurogenesis in the aging human hippocampus despite stable stem cell markers

Reduced neurogenesis in the aging mammalian hippocampus has been linked to cognitive deficits and increased risk of dementia. We utilized postmortem human hippocampal tissue from 26 subjects aged 18–88 years to investigate changes in expression of six genes representing different stages of neurogenesis across the healthy adult lifespan. Progressive and significant decreases in mRNA levels of the proliferation marker Ki67 (MKI67) and the immature neuronal marker doublecortin (DCX) were found in the healthy human hippocampus over the lifespan. In contrast, expression of genes for the stem cell marker glial fibrillary acidic protein delta and the neuronal progenitor marker eomesodermin was unchanged with age. These data are consistent with a persistence of the hippocampal stem cell population with age. Age‐associated expression of the proliferation and immature neuron markers MKI67 and DCX, respectively, was unrelated, suggesting that neurogenesis‐associated processes are independently altered at these points in the development from stem cell to neuron. These data are the first to demonstrate normal age‐related decreases at specific stages of adult human hippocampal neurogenesis.     

Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days). Wistar rats of the same age were used as controls. Expression levels of the genes Ext1, Ext2, and Hpse in the brain of rats of both strains were maximal during the two first weeks of life, and the contents of mRNAs of all genes in the brain of newborn and 7-day-old OXYS rats were significantly higher than in Wistar rats. By the 14th day of life the differences leveled, but at the age of 30 days on the background of a decrease in the contents of mRNAs of Ext1, Ext2, and Hpse in OXYS rats they became more pronounced (three-, four-, and twofold, respectively). Differences between the strains were absent at the age of 60 days and 14 months, and expression of all the genes was significantly lower than in the newborn animals. A strong positive correlation was found between contents of mRNAs of all the studied genes, and this suggested that heparanase should be involved in HSPG metabolism together with Ext1 and Ext2. Based on these and earlier findings, we conclude that development of the OXYS rat brain occurs on the background of significant alterations in HSPG metabolism that precede the development of neurodegenerative manifestations recently detected by magnetic resonance imaging.     

Obesity Is Accompanied by Disturbances in Peripheral Glucocorticoid Metabolism and Changes in FA Recycling

The glucocorticoid activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) is of major interest in obesity‐related morbidity. Alterations in tissue‐specific cortisol levels may influence lipogenetic and gluco/glyceroneogenetic pathways in fat and liver. We analyzed the expression and activity of 11βHSD1 as well as the expression of phosphoenolpyruvate carboxykinase (PEPCK), sterol regulatory element binding protein (SREBP), and fatty acid synthase (FAS) in adipose and liver and investigated putative associations between 11βHSD1 and energy metabolism genes. A total of 33 obese women (mean BMI 44.6) undergoing gastric bypass surgery were enrolled. Subcutaneous adipose tissue (SAT), omental fat (omental adipose tissue (OmAT)), and liver biopsies were collected during the surgery. 11βHSD1 gene expression was higher in SAT vs. OmAT (P = 0.013), whereas the activity was higher in OmAT (P = 0.009). The SAT 11βHSD1 correlated with waist circumference (P = 0.045) and was an independent predictor for the OmAT area in a linear regression model. Energy metabolism genes had AT depot–specific expression; higher leptin and SREBP in SAT than OmAT, but higher PEPCK in OmAT than SAT. The expression of 11βHSD1 correlated with PEPCK in both AT depots (P = 0.05 for SAT and P = 0.0001 for OmAT). Hepatic 11βHSD1 activity correlated negatively with abdominal adipose area (P = 0.002) and expression positively with PEPCK (P = 0.003). In human obesity, glucocorticoid regeneration in the SAT is associated with central fat accumulation indicating that the importance of this specific fat depot is underestimated. Central fat accumulation is negatively associated with hepatic 11βHSD1 activity. A disturbance in peripheral glucocorticoid metabolism is associated with changes in genes involved in fatty acid (FA) recycling in adipose tissue (AT).     

Aging mechanisms in fruit flies

Genetic analysis of Drosophil has provided evidence in support of two proposed evolutionary genetic mechanisms of aging: mutation accumulation and antagonistic pleiotropy. Both mechanisms result from the lack of natural selection acting on old organisms. Analyses of large numbers of flies have revealed that mortality rates do not continue to rise with age as previously thought, but plateau at advanced ages. This phenomenon has implications both for models and for definitions of aging, and may be explained by the evolutionary theories. The physiological processes and genes most relevant to aging are being identified using Drosophila lines selected in the laboratory for postponed senescence. Oxidative stress and insufficient metabolic reserves/capacity may be particularly important factors in limiting the fruitfly lifespan. Genes which exhibit aging-related changes in expression are now being identified. Transgenic flies are being used to analyze the mechanisms of such aging-related gene expression, and to test the effects of specific genes on aging and aging-related deterioration.     

The effect of age on protein composition of rat cerebral microvessels

The effect of age on protein composition of cerebral microvessels was investigated by examining the content of glycosylation endproducts in cerebral microvessels isolated from young (3–6 month old), intermediate age (18 month) and aged (24–26 month old) Fischer 344 male rats and by quantitating various protein spots identified with two dimensional (2D) electrophoresis. The results indicate that aging in rats is not associated with significant increase in glycosylation of microvessel proteins. Of the 26 proteins in cerebral microvessels identified on the 2-D gel, ten showed significant age-related changes (p<0.0004) and in two of these the changes were significant as early as 18-months of age. A large acidic protein with a molecular weight of 144,000 and isoelectric point (pI) of 5.4 (Spot #1) was found only in aged rats. The results indicate that aging is associated with significant quantitative changes in protein composition of cerebral microvessels. It is possible that Spot #1 may be a novel biochemical marker of aging blood-brain barrier.     

Age‐dependent changes and biomarkers of aging in Caenorhabditis elegans

Caenorhabditis elegans is an exceptionally valuable model for aging research because of many advantages, including its genetic tractability, short lifespan, and clear age‐dependent physiological changes. Aged C. elegans display a decline in their anatomical and functional features, including tissue integrity, motility, learning and memory, and immunity. Caenorhabditis elegans also exhibit many age‐associated changes in the expression of microRNAs and stress‐responsive genes and in RNA and protein quality control systems. Many of these age‐associated changes provide information on the health of the animals and serve as valuable biomarkers for aging research. Here, we review the age‐dependent changes in C. elegans and their utility as aging biomarkers indicative of the physiological status of aging.     

Expression of Obesity Markers and Persistent Organic Pollutants Levels in Adipose Tissue of Obese Patients: Reinforcing the Obesogen Hypothesis?

Introduction

Persistent Organic Pollutants (POPs) accumulate in adipose tissue and some are described to possess endocrine disrupting capacities. Therefore, it is important to evaluate their effects on key endocrine pathways in adipose tissue (AT), to further evaluate their potential role in metabolic pathologies such as obesity.

Objectives

The aim is twofold: (i) evaluate gene expression levels of obesity marker genes, i.e. the adipokines leptin (LEP), adiponectin (ADIPOQ) and Tumor Necrosis Factor α (TNFα) and the nuclear receptor, Peroxisome Proliferator Activated Receptor γ (PPARγ) in paired subcutaneous (SAT) and visceral (VAT) AT of obese subjects (n = 50) and to relate these values to serum concentrations of LEP and ADIPOQ (ii) evaluate the association of expression levels of marker genes in AT and serum with POP concentrations in AT.

Results and Conclusions

Leptin and adiponectin levels in serum were positively correlated to respectively expression levels of leptin in SAT and adiponectin in VAT. Our study shows more significant correlations between gene expression of obesity marker genes and POP concentrations in VAT compared to SAT. Since VAT is more important than SAT in pathologies associated with obesity, this suggests that POPs are able to influence the association between obesity and the development of associated pathologies. Moreover, this finding reveals the importance of VAT when investigating the obesogen hypothesis. Concerning PPARγ expression in VAT, negative correlations with polychlorinated biphenyls (PCBs) concentrations were found in non T2D patients. LEP serum concentrations correlated with several PCBs in women whereas in men no correlations were found. This strengthens the potential importance of gender differences in obesity and within the obesogen hypothesis.     

Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41?±?11 yrs of age) presenting a wide range of BMI (21.4 to 48.6?kg/m²) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p?<?.0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBα, which was reduced (p?<?.05) at the PM period in SAT and VAT of both women and men (women: ～53% lower; men: ～78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r?=??.549; p?=?.001) and SATPER2 (r?=??.613; p?=?.0001) and positively with VATCLOCK (r?=?.541; p?=?.001) and VATBMAL1 (r?=?.468; p?=?.007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)     

Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome‐wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild‐type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3–3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson–Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild‐type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.