The Expression Changes Of Inflammasomes In The Aging Kidneys

Objective:The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. During aging, the adaptive immunity response significantly declines (immunosenescence), and the innate immunity response is markedly activated, which leads to the aging type of low-level, chronic inflammatory phenotype (inflamm-aging). Kidneys exhibit high energy metabolism and are thus prone to aging. As an important components of innate immune system in the body, the inflammasome is a high-molecular weight, multi-protein complex that comprises pattern recognition receptors (PRRs), which can separately assemble with adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) and procaspase-1. Inflammasome formation is triggered by a range of foreign molecules that emerge during infections such as bacteria, which are designated pathogen-associated molecular patterns (PAMPs), as well as molecules native to the body that are altered during tissue damage or metabolic imbalances such as reactive oxygen species, which are designated danger-associated molecular patterns (DAMPs). As a consequence, oligomerization of pattern recognition receptors including NLRP1, NLRP3, NLRC4 and AIM2, recruits adaptor proterin ASC and effector protein procaspase-1. Once inflam-masomes have formed, they can activate procaspase-1. Activated caspase-1 can cleave proinflammatory cytokines such as pro-interleukin-1β (pro-IL-1β) and pro-IL-18, producing corresponding mature cytokines to be released into the extracellular plasma, which triggers immune response. As a key transcription factor, NF-κB regulates the expression of intracellular PRRs such as NLRP1, NLRP3, NLRC4, AIM2, and proinflammatory cytokines such as pro-IL-1β and pro-IL-18 by activation of TLR4, IL-1R and some key molecules in the NF-κB signal transduction pathway. We call this process "priming". Recently, only four kinds of pattern recognition receptors including NLRP1, NLRP3, NLRC4 and AIM2 can assemble into inflammasome to promote the conversion of procaspase-1 into enzymatically active protease caspase-1 which leads to the production of proinflammatory IL-1(3 and IL-18. NLRP3 has most number of ligands and is one of best studied inflammasome.However, the function of inflammasomes and their underlying mechanisms in renal aging remain unclear. In this study, for the first time, we systematically investigated the role of the inflammasomes and the inflammatory responses activated by inflammasomes during kidney aging. Our results are helpful in understanding the role of inflammasome in the onset and development of kidney aging and in exploring the therapeutic potential of inflammasome modulation that could delay age-related renal disease.Methods:1、2-month-old male Fischer 344 rats were randomly divided into two groups:3-month group (young group) and 24-month group (old group),10 rats each group. The kidney tissues from each group of rats were removed and perfused with ice-cold,0.9% Sodium Chloride Solution to remove any remaining blood. A portion of the tissue was placed into 10% neutral buffered formalin for immunohistochemistry staining and PAS staining. Another portion was immersed into OCT compound for immunofluorescence staining. The remaining tissue was immediately frozen in liquid nitrogen and stored at -80℃ until further using for western blot and quantitative real time PCR.2、PAS staining was used to detect the histological changes in different groups.3、Western blot analysis was performed to assess①the expression of the members of priming signal pathway including TLR4, IL-IR, and the downstream of Phospho-IKKβ、Phospho-IκBα, Phospho-NF-KB p65 (activated forms, respective) in aging renal tissues;②the level of inflammasome including NLRP1, NLRP3, NLRC4, AIM2, procaspase-1, caspase-1 and the downstream of proinflammatory cytokines IL-1β and IL-18 during kidney aging process.4、Quantitative real time-PCR was used to analysis the expression changes of MLRP3、IL-1β and IL-18 in mRNA level during the kidney aging process.5、Immunohistochemistry staining and immunofluorescence staining were performed to further explore the expression and the location of NLRP3, NLRC4 and caspase-1 in aging renal tissues.Results:1、The results of PAS staining demonstrated that, compared with young renal tissues, old renal tissues showed marked pathological features of aging, including the number of functioning glomeruli decreases while the proportion of sclerotic glomeruli increases. Progressive expansion of mesangium, atrophy of renal tubules, and interstitial fibrosis, along with some inflammatory cell infiltration.2、Our results of western blot showed that during aging kidneys,① the expression of the members of priming signal pathway including TLR4, IL-1R, Phospho-IRAK4 Phospho-IKKβ, Phospho-1κBα and Phospho-NF-κBp65 were significantly elevated;② the levels of the inflammasome components NLRP3, NLRC4, procaspase-1 and caspase-1 were prominently up-regulated; and the proinflammatory cytokines IL-1β and IL-18 were notably increased in the kidneys of 24-month-old rats.3、The results of RT-qPCR shown that the expression of NLRP3, IL-1β and IL-18 increased in aging renal tissues.4、Our results of immunohistochemistry and immunofluorescence staining results showed that the high expression of NLRP3, NLRC4 and caspase-1 mainly located in the endothelial cells of glomeruli.Conclusion:During the kidney aging process, priming signal pathway are significantly activated, NLRP3 and NLRC4 inflammasome are markedly activated, leading to the maturation and secretion of the proinflammatory cytokines IL-1β and IL-18. Our results are helpful in understanding the role of inflammasome in the onset and development of kidney aging and in exploring new therapies that could delay age-related renal disease.