Background
The aging process can be described as a universal, intrinsic, progressive accumulation of deleterious changes in cells and tissues that increase morbidity and lead to death [
1]. According to the recent theory of oxidation-inflammation, chronic oxidative and inflammatory stress conditions explain the aging process [
2].
Several studies have focused on the role played by receptor for advanced glycation end products (RAGE) on aging, because RAGE is known inducer of inflammation and oxidative stress.
RAGE belongs to the immunoglobulin superfamily of cell surface molecules and has an extracellular region containing one V-type immunoglobulin domain and two C-type immunoglobulin domains [
3,
4]. The extracellular portion of the receptor is followed by a hydrophobic transmembrane-spanning domain and then by a highly charged, short cytoplasmic domain that is essential for intracellular RAGE signaling [
3,
4].
RAGE has several ligands, such as, advanced glycation end products (AGEs), proinflammatory S100/calgranulin family members, and high motility group box 1 protein (HMGB1) [
5,
6]. RAGE is also a signal transduction receptor for amyloid β [
7] and endogenous phospholipids such as lysophosphatidic acid [
8].
After binding these ligands, RAGE activates an inflammation-related signaling cascade involving nuclear factor-(NF)κB, ERK (extracellular signal-regulated kinase) 1/2, p38 MAPK (mitogen-activated protein kinases), JNK (c-Jun N terminal kinases), PKC (protein kinase C), Rac/Cdc42, and TIRAP and MyD88 (adaptor proteins for TLR 2 and 4) [
5,
6].
Because AGEs accumulates in organs, such as, kidney [
9], liver [
10], brain [
11], and skeletal muscle during aging [
12], researches have tended to investigate the role played by the AGEs-RAGE pathway during aging. The AGEs-RAGE pathway is a primary contributor to kidney aging [
13]. The accumulation of AGEs and a progressive decline in renal function during aging may induce the release of inflammatory mediators and the generation of reactive oxygen species (ROS) [
10,
11]. Furthermore, this accumulation starts before a clinical decrease in kidney function is evident, and is one of the characteristic features of kidney aging [
14,
15].
Although much evidence indicates that RAGE-related inflammation and oxidative stress participate in the aging process, the majority of studies on the topic have been focused only AGEs and not on other RAGE ligands [
13‐
15]. In fact, few studies have addressed the roles of these other RAGE ligands in different organs in same animals.
Therefore, we sought to determine whether the accumulation patterns of the RAGE ligands which are AGEs, HMGB1, and S100β and the binding intensities between RAGE and its ligands in kidney, liver, and skeletal muscle are age-dependent.
Discussion
The present study shows; (1) the age-related accumulation patterns of RAGE ligands (AGEs, HMGB1, and S100β) are organ dependent; (2) binding intensities between RAGE and its ligands in kidney and liver increased with age, but binding intensities in skeletal muscle were not changed by aging; (3) infiltrations of activated macrophage into kidney and liver were increased with age, but infiltrations into skeletal muscle were not changed; (4) M1 expression was increased and M2 expression was decreased by age in kidney and liver, but M1 expression in skeletal muscle was unchanged; (5) GLO-1 expressions in kidney and liver were decreased by age, but unchanged in skeletal muscle; and (6) the activation of inflammation related signal pathway in kidney and liver increased with age, but in skeletal muscle they remained unchanged.
Several studies have shown that the AGEs-RAGE pathway is related to aging of humans and animals [
16,
17]. In addition, many studies have shown AGEs accumulates in tissues during aging. The liver is a site for clearance and catabolism of circulating AGEs but can also be a target organ for AGEs [
10,
18]. AGEs are removed and metabolized by the kidney, but the kidney is also a site for AGEs accumulation and AGEs-associated damage [
19]. It is well known that older adults exhibit increased collagen cross-linking and AGEs deposition in skeletal muscle [
12]. Thus, we considered that accumulation of AGEs in liver, kidney, and skeletal muscle would be more prominent than in other tissues during aging.
In the present study, AGEs accumulations in kidney in the middle-aged and old groups were significantly higher than in the young group, however no difference was observed between the middle-aged and old groups. These results suggest AGEs accumulation in kidney reaches a maximum level even in middle-age. AGEs are generated endogenously by glycation, and this process is enhanced by ROS or hyperglycemic conditions or by ingestion of exogenous AGEs in food [
13]. Physiological glycation state is regulated by a balance between the formation and clearance of AGEs [
20], and this balance is maintained in part by glycation state, host defense machinery, including anti-glycation enzymes (e.g., glyoxalase), and kidney filtration function (glomerular filtration rate), which excretes AGEs and AGEs precursors from the body [
13,
20].
The glomerular changes in C57B6 mice begin at 18 to 20 months of age, before recognizable tubulointerstitial changes, and these progressively increase to death at ~30 months [
21,
22]. Although glomerular function might be preserved until 20 months [
21,
22], it is known that GLO-1 levels in kidney decrease with age [
23]. Many previous studies have shown AGEs accumulation in kidney linearly increases with age. The present study shows that AGEs accumulation peaked during middle-age in our animal model. Though we did not check glomerular function in the present study, we did observe GLO-1 expression decreased with aging, which concurs with other studies [
23]. We speculate that decreasing GLO-1 activity by aging plays an important role in the renal accumulation of AGEs, because AGEs accumulation in kidney might accelerate before middle-age when glomerular filtration function is preserved [
21,
22].
We found that AGEs accumulation in liver was significantly increased by aging, whereas GLO-1activity decreased, which agrees with other studies [
24]. Age-related AGEs accumulation in skeletal muscle has been previously reported in an animal model [
25], but in the present study, AGEs accumulation was unchanged by aging. Even GLO-1 levels were not changed in skeletal muscle. Other studies have shown an increasing pattern of AGEs accumulation in skeletal muscle by aging used 33-month rats as the old groups [
25]. In the present study, we used 22-month mice, which could explain the discrepancy between results, and suggests AGEs accumulation in the skeletal muscle might accelerate after 22 months in mice.
Unlike AGEs, the age-related accumulation patterns of other RAGE ligands, such as, HMGB1 and S100β, have not been in different organs. However, the effects of the accumulations of HMGB1 and S100β during aging have been in the context of brain aging. In particular, the expression of S100 protein is increased in the aging brain [
26]. It has also been reported that the distribution of HMGB1 appears to be altered in the aged brain [
27]. More specifically, HMGB1 is downregulated in neurons in the aged brain, but it is upregulated in astrocytes, which suggests HMGB1 plays different roles in different types of brain cells and structures [
27].
In the present study, HMGB1 and S100 β accumulation in kidney increased with age. In liver, there was no difference between the middle-aged and old groups in HMGB1 and S100β accumulation. In skeletal muscle, the accumulations of both ligands were not changed by aging.
Although RAGE ligand age-related accumulation patterns differed in kidney, liver, and skeletal muscle, binding intensities between RAGE ligands and RAGE increased with age in kidney and liver. These patterns paralleled age-related inflammation signal pathway activation, activated macrophage infiltration, M1 increases, and M2 decreases.
Ligand binding with RAGE triggers ROS increases, activates NADPH oxidase, increases the expressions of adhesion molecules, and upregulates inflammation through NFκB and other signaling pathways [
18,
28]. Furthermore, activation of NFκB results in increased RAGE expression, thereby prolonging NFκB activation [
29]. In addition, RAGE expression occurs in an inducible manner and is upregulated at sites where its ligands accumulate [
29].
In the present study, we measured binding intensities between RAGE and its ligands rather than RAGE expression. Binding intensities between RAGE and its ligands in the liver and kidney were increased by aging, regardless of age-related ligand accumulation. The patterns of binding intensities between RAGE and its ligands in the liver and kidney matched NFκB and IL-1β activations, which suggests that binding intensity between RAGE and its ligands in the liver and kidney is a more important factor of age-related inflammation and oxidative stress than absolute ligand accumulations.
A previous study showed that the RAGE pathway can enhance macrophage migration [
30] and promote proinflammatory mediator production, such as, those of IL-1β, IL-6, and TNF-α [
31]. The RAGE/NF-κB pathway not only predominantly induces macrophages to secrete inflammatory cytokines but also induces M1 polarization [
32], and M1 macrophages are Th1-biased and considered to be pro-inflammatory and most notably express TNFα and IL-6 [
33]. The majority of the macrophages found in sites of inflammation in inflammatory diseases are considered to be M1 macrophages [
33]. By contrast, M2 macrophages are Th2-biased and are thought to play more diverse roles, namely in anti-inflammatory pathways, tissue remodeling and wound healing [
33].
The patterns of binding intensities between RAGE and its ligands in liver and kidney were similar to patterns of M1 expression increase and M2 expression decrease with age in liver and kidney, which suggests local tissue inflammation was increased by aging in liver and kidney and that the RAGE pathway plays an important role in the aging process. On the contrary, the aging process of skeletal muscle appears to be different from those of liver or kidney. The binding intensity between RAGE and its ligands, activation of inflammatory signal pathway
s, macrophage activation, and M1 polarization were not changed by aging in skeletal muscle, which suggests the RAGE pathway and inflammation induced by RAGE pathway do not play a main role in skeletal muscle aging. In aged animal skeletal muscle, AGEs cross-linking collagen was increased and those collagen made muscle stiff [
11,
25]. Taken together, we speculate that mechanical property changes, such as, increased muscle stiffness caused by collagen fiber cross linking by AGEs might be more important than RAGE induced inflammation during skeletal muscle aging.
The main limitation of this study was that we did not measure ROS levels in tissues directly, however many studies have been shown that ROS levels increase during aging in many tissues. Our study shows NFκB increased with age in kidney and liver, and the NFκB signal pathway is known to be importantly related to ROS generation.