INTRODUCTION
Aging is associated with significant structural and functional changes in the kidneys, including progressive nephron loss, reduced kidney volume, and marked decline in glomerular filtration rate.1) These changes occur through multiple molecular mechanisms involving altered signaling pathways, increased tissue fibrosis, oxidative stress, and chronic inflammation.2)
Age-related kidney inflammation is characterized by increased pro-inflammatory cytokine levels that correlate with renal diseases. Aging kidneys show decreased antioxidant capacity and reduced levels of protective enzymes, with age-related decline in sirtuins that normally protect against renal inflammation, fibrosis, and apoptosis.3) These inflammatory pathways can be modulated through various mechanisms, including PPARγ-mediated suppression of NF-κB activation.4) The clinical implications are substantial, as aging represents a major risk factor for chronic kidney disease, acute kidney injury, and end-stage renal failure.5)
Cellular senescence serves as a crucial factor in aging and age-related diseases. Senescent cells accumulate in aging tissues and undergo metabolic reprogramming while secreting senescence-associated secretory phenotype (SASP) factors.6) SASP is a complex program in which senescent cells secrete pro-inflammatory cytokines, chemokines, and growth factors that promote chronic inflammation both locally and systemically.7) While SASP composition may vary depending on cell type and context, certain pro-inflammatory cytokines such as interleukin-6 (IL6) and interleukin-8 (IL8) are consistently expressed by senescent cells. Additionally, these and other cytokines, including TNF-α, IL-17, and IFN-γ, can themselves induce cellular senescence, further amplifying inflammatory signaling.8)
Calorie restriction (CR) is a promising anti-aging intervention that attenuates age-related changes through multiple mechanisms, including suppression of cellular senescence, reduction of inflammatory pathways, and direct protection against kidney injury.9,10) Studies show that moderate CR extends lifespan and improves metabolic and physical health, associated with epigenetic alterations.11) A key mechanism underlying these benefits is the downregulation of inflammatory pathways, with long-term moderate CR inducing persistent inhibition of inflammation while maintaining cell-mediated immunity.12) Additionally, CR directly improves renal function and alleviates age-related renal fibrosis.13,14)
Our previous studies have extensively characterized the molecular mechanisms underlying aging-related renal inflammation and the protective effects of CR. We established key inflammatory pathways involved in renal aging, identifying transcriptomic signatures of age-dependent functional changes.15) Additionally, we demonstrated that CR effectively suppresses pro-inflammatory factors and SASP during aging, while also investigating CR effects on metabolism and epigenetic regulation.16-18) However, the specific molecular targets through which CR modulates cellular senescence in the kidney remain incompletely characterized.
Therefore, this study aimed to identify key target genes involved in aging-related renal inflammation by examining differential gene expression during kidney aging and CR using integrated transcriptomic, bioinformatics, and molecular biology approaches. We employed a 4-week short-term CR intervention, as previous studies have demonstrated that this duration is sufficient to elicit measurable molecular and metabolic changes in aged rats while avoiding potential complications of prolonged restriction. Through this systematic investigation, we discovered that Il19 and Il24 show differential expression during aging and CR and contribute to cellular senescence regulation in the kidney.
MATERIALS AND METHODS
Experimental Design
This study employed a multi-level approach combining in vivo and in vitro models to investigate the effects of aging and CR on renal gene expression and cellular senescence. The experimental design consisted of two complementary components (Supplementary Fig. S1).
1) In vivo transcriptomic profiling and validation: male Sprague Dawley rats were used to examine age-related changes in kidney gene expression and the modulatory effects of short-term CR. Kidney tissues from three experimental groups (young, old, and old-CR) were subjected to RNA-seq analysis to identify differentially expressed genes, followed by independent qRT-PCR validation and protein-protein interaction (PPI) network analysis. This component aimed to identify candidate genes associated with renal aging and CR responses at the tissue level.
2) In vitro functional validation: NRK52E rat renal epithelial cells were used to model cellular aging through H₂O₂-induced oxidative stress, which mimics key features of cellular senescence. This cellular model allowed us to examine the expression dynamics of candidate genes (Il19, Il24) and senescence markers (p16, p21) in a controlled environment, complementing the in vivo findings and providing mechanistic insights into the role of the candidates in renal cellular senescence.
By integrating transcriptomic discovery, molecular validation, and functional assessment across these two experimental platforms, we aimed to comprehensively characterize the involvement of candidate genes in aging-related renal decline and their responsiveness to CR intervention.
Animal Models
Male Sprague Dawley (SD) rats were obtained from Samtako (Osan, Korea) and maintained under controlled conditions (23°C±2°C, 60%±5% humidity, 12-hr light/dark cycle) with ad libitum access to standard rodent chow (20% protein, 4.5% fat, 6% fibre, 7% ash, 0.5% calcium, 1% phosphorus) and water. Animals were divided into three experimental groups: young (6 months, n=6, 522.1±7.9 g), old (21 months, n=6, 691.4±30.4 g), and old subjected to CR (old-CR; 21 months, n=6, 538.4±34.5 g). All procedures were approved by the Institutional Animal Care and Use Committee of Pusan National University (Approval No. PNU-2015-1044).
Calorie Restriction Protocol
Baseline food intake was determined during a 7-day acclimation period by daily weighing of offered and remaining food to calculate mean ad libitum intake per rat (young 20.83±0.64 g/day, old 21.95±1.40 g/day, old-CR 19.14±0.63 g/day before CR initiation). The CR ration was set at 60% of baseline intake (40% CR), which was adjusted weekly based on body weight trends to maintain the 60% feeding level. Food was provided once daily at 18:00 (onset of dark phase), aligned with the natural circadian feeding pattern of rats to minimize circadian confounding effects. Water was available ad libitum throughout the study. Body weight and food intake were monitored three times per week; residual chow was weighed to derive actual intake. Pair-feeding was not applied in this study, as the research focused on CR per se rather than macronutrient-matched intake. The 4-week CR duration was selected based on previous studies demonstrating that short-term CR is sufficient to induce measurable metabolic and molecular changes in aging rats while minimizing potential adverse effects of prolonged restriction.
Tissue Collection
After 4 weeks of CR intervention, rats were fasted overnight and anesthetized with diethyl ether. Blood samples were collected by abdominal aorta blood collection, and blood urea nitrogen (BUN) was measured by BUN colorimetric detection kit (EIABUN; Thermo Fisher Scientific, Waltham, MA, USA). Kidney tissues were rapidly excised, snap-frozen in liquid nitrogen, and stored at -80°C for subsequent RNA extraction and molecular analyses.
Transcriptomic Analysis
Total RNA was extracted from kidney samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Equal RNA amounts from each group (n=3) were pooled and sequenced once per group as a discovery screen. This pooled design does not allow for assessment of inter-individual variability or application of inferential statistics based on biological replicates; therefore, RNA-seq findings are considered exploratory. Libraries were prepared and sequenced on the MGI-T7 platform. Reads were quality-filtered (Cutadapt, Trimmomatic), aligned to the rat genome (rn6) using STAR, and quantified with RSEM.
Differential Expression Analysis
Gene counts were normalized and analyzed using DESeq2 (version 1.30.1). Differentially expressed genes (DEGs) were identified using the following criteria: p-value < 0.05 and absolute fold change ≥1.5 (equivalent to ±1.5-fold change). Comparisons included old vs. young and old-CR vs. old. Volcano plots visualizing the distribution of DEGs were generated using VolcaNoseR. Given the pooled RNA-seq design, interpretation focused on fold changes and directionality rather than inferential p-values. Key candidate genes identified from RNA-seq were independently validated by qRT-PCR using unpooled samples.
Gene Ontology and Pathway Enrichment Analyses
Gene Ontology (GO) enrichment analysis was conducted to identify overrepresented biological processes (BP), cellular components (CC), and molecular functions (MF) associated with the DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis was performed to investigate DEG-related signaling pathways and metabolic processes. Both GO and pathway enrichment analyses were conducted using database for annotation, visualization and integrated discovery (DAVID) v2024q2 bioinformatics platform with the following parameters: species: Rattus norvegicus (NCBI Taxonomy ID: 10116); background: all expressed genes detected in RNA-seq; enrichment threshold: p<0.05 with Benjamini-Hochberg correction for multiple testing. Pathway over-representation analyses reflect enrichment conditional on the discovery gene lists and should be interpreted as exploratory summaries rather than replicate-based inferential statistics.
qRT-PCR
Frozen kidney samples were homogenized using RiboEX (0.5 mL per 30 mg) (Geneall, Seoul, South Korea) with a few beads in a tissue homogenizer. Total RNA from rat kidney tissues (20 mg) and NRK52E cells (n=6) was reverse-transcribed using a cDNA synthesis kit (GenDEPOT, Katy, TX, USA) according to the manufacturer's instructions. qRT-PCR was performed using SYBR Green (Bioneer, Daejeon, Korea) on a CFX Connect system with the following cycling conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes. Each sample was analyzed in technical triplicates. Primer sequences are listed in Supplementary Table S1. Primer efficiency was validated to be 90%–110% for all targets. Expression levels were normalized to Gapdh, which was confirmed as a stable reference gene across experimental groups (coefficient of variation <5%). Relative expression was calculated by the 2-ΔΔCt method.
PPI Network Analysis
A human PPI database was constructed by integrating HPRD, BioGRID, IntAct, MINT, and STRING (version 12.0, confidence ≥0.9). Rat gene identifiers were mapped to human orthologs using NCBI Gene (https://www.ncbi.nlm.nih.gov/datasets/gene/) when rat-specific interaction data were limited. Network visualization and topological analysis were performed using Cytoscape (version 3.10.3), with key network parameters including degree centrality, betweenness centrality, and clustering coefficient calculated to identify hub genes.
Cell Culture Experiments
NRK52E rat renal epithelial cells (ATCC, Manassas, VA, CRL-1571) were maintained in Dulbecco's modified Eagle medium (DMEM) (LM001-07; WELGENE, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) (LS202-02; WELGENE) and 1% penicillin-streptomycin (15140; WELGENE) at 37°C in a humidified atmosphere of 5% CO₂. Cells were passaged every 2–3 days at 80%–90% confluence and used between passages 5–15. For senescence induction experiments, cells were seeded at a density of 2×105 cells per well in 6-well plates and allowed to adhere for 24 hours. Cells were then treated with 200 μM H₂O₂ (H1009; Sigma-Aldrich, Burlington, MA, USA) in serum-free DMEM for 2 hours, after which the medium was replaced with complete DMEM. Control cells received serum-free DMEM without H₂O₂. Cells were harvested 48 hours post-treatment for RNA extraction and gene expression analysis by qRT-PCR. Each experiment was performed with n=3 biological replicates, with each replicate consisting of triplicate wells (technical replicates).
Statistical Analysis
Sample sizes were determined based on previous studies: n=6 per group for animal studies and n=3 for cell culture experiments.15,19) Data are mean ± SEM. Statistical tests included one-way analysis of variance (ANOVA) with Tukey's post-hoc test. Significance was set at p<0.05. All statistical analyses were performed using the GraphPad Prism software. It should be noted that the RNA-seq data were generated from pooled samples (n=3 per group), which precludes assessment of inter-individual variability and inferential statistics. Therefore, RNA-seq findings are considered exploratory and were validated through independent qRT-PCR analyses using unpooled samples.
RESULTS
Transcriptomic Analysis of Gene Expressions Changed during Aging and CR
RNA-seq analysis of kidney tissues from young (6 months), old (21 months), and old-CR rats revealed substantial differential gene expression patterns across experimental groups. The transcriptomic profiling was conducted as a discovery screen using pooled samples; key findings were subsequently validated by independent qRT-PCR analyses.
In the old vs. young comparison, we identified 812 differentially expressed genes, with 696 genes significantly upregulated and 116 genes downregulated in aged kidneys, while the old-CR vs. old comparison showed 628 differentially expressed genes, with 161 upregulated and 467 downregulated following CR treatment (Fig. 1A). Given the exploratory nature of the pooled RNA-seq design, these patterns represent directional trends that were confirmed for key candidate genes by independent qRT-PCR validation.
To elucidate the functional significance of transcriptomic changes, we performed comprehensive pathway analysis using DAVID across five major categories: BP, KEGG, CC, MF, and Reactome, and we listed top 30 terms of each category with criteria of p<0.05 with Benjamini-Hochberg correction for multiple testing (Fig. 1B, 1C and Supplementary Fig. S2). This multi-dimensional approach revealed significant alterations in immune/inflammatory, SASP, and cell cycle-related processes.
Comprehensive GO and pathway analysis across five categories (BP, CC, MF, KEGG, and Reactome) revealed consistent enrichment of inflammation-related pathways (e.g., cytokine-cytokine receptor interaction, JAK-STAT signaling, NF-κB signaling), ECM remodeling (e.g., extracellular matrix organization, collagen fibril organization, integrin binding), and cell cycle regulation (e.g., mitotic spindle checkpoint, G1/S transition) in the top 30 terms of BP and KEGG (Fig. 1B and 1C). These exploratory pathway enrichment results, derived from pooled RNA-seq, provide hypothesis-generating insights rather than statistical inference; key pathways were confirmed through independent qRT-PCR validation of representative genes.
Taken together, these results revealed that genes associated with the SASP (immunity, inflammation, ECM, and growth factor) and the cell cycle exhibited differential expression patterns during aging and CR. This suggests that the SASP and cell cycle play critical roles in renal aging, whereas CR modulates these processes, potentially influencing renal aging.
Gene Expression Changes in Renal Aging and Modulation by CR
To further investigate the effects of CR on renal aging, GO and pathway analyses were performed on the genes that exhibited contrasting changes in expression between aging and CR. To reduce redundancy and improve interpretability, we consolidated representative GOs and pathways from GO, KEGG, and Reactome databases into five major functional categories (Table 1, Supplementary Table S2). Inflammatory signaling pathways were prominently enriched, including cytokine-cytokine receptor interaction, JAK-STAT signaling, and IL-6 family signaling, with key genes such as Il19, Il24, Ccl20, and Ccl7. Cell cycle regulation featured aging-related processes and chromosome segregation, notably including cell cycle inhibitors Cdkn2b (p15INK4B), Cdkn2a (p16INK4A), and Cdkn1a (p21). SASP components encompassed cytokine, chemokine, and growth factor activities, including Il1rn, Cntf, Bdnf, and Timp1. ECM remodeling included extracellular matrix organization and integrin interactions with genes such as Col16a1, Postn, and Adamts1/4. Immune cell activation featured interferon-gamma responses and chemotaxis pathways, including Socs3 and Fcer1g.
Additionally, analysis of fold changes in gene expression related to cytokines/chemokines, growth factors, ECM, and the cell cycle during aging and CR revealed that genes such as Il6, Il19, and Il24 (cytokines), Ccl2, Ccl7, and Ccl20 (chemokines), Tgfb1, Mmp2, and Mmp12 (growth factors), Col1a1, Col16a1, and Timp1 (ECM), and Cdkn2b and Cdkn1a (cell cycle) were upregulated in the Old group compared to the Young group, but downregulated in the Old-CR group compared to the Old group (Table 2).
Notably, among the cytokines, the IL-20 family genes Il19, Il24, and Il20rb exhibited increased expression with aging and decreased expression under CR conditions, suggesting that Il19 and Il24 are associated with renal aging and CR responses and may represent hypothesis-generating candidates for future functional validation (Supplementary Table S3). These results suggest that genes closely associated with the SASP and cell cycle increase during aging and decrease with CR, and that these genes may be considered as potential targets for further mechanistic investigation, pending functional verification. Il19 and Il24 have emerged as candidate molecules for future studies on renal aging intervention.
Regulation of SASP- and Cellular Senescence-Associated Genes in Renal Aging and CR
To validate the expression changes of genes associated with the SASP and the cell cycle identified through transcriptome analysis during the aging process and under CR, qRT-PCR was performed to measure mRNA levels (Supplementary Table S1). The analysis revealed that cytokines and chemokines, such as Il1b, Ccl3, and Ccl5 were upregulated in the old group but downregulated in the old-CR group. Similarly, IL-20 family members, including Il19, Il20ra, Il22, and Il24, demonstrated increased expression during aging and decreased expression under CR conditions (Fig. 2A). A similar trend was observed for SASP-related genes, such as Timp1, Mmp12, and Tgfb1 (Fig. 2B). Notably, some cell cycle-related genes, including p15INK4B, p16INK4A, and p21, exhibited consistent directional patterns of age-associated upregulation and CR-associated downregulation, while p15INK4B and p16INK4A did not reach statistical significance (Fig. 2C).
Additionally, to confirm successful CR implementation, baseline food intake was determined during a 7-day acclimation period. Old rats showed a baseline intake of 21.95±1.40 g/day, while the Old-CR rats consumed 19.14±0.63 g/day during acclimation (Supplementary Table S4). During the 4-week CR intervention, Old-CR rats consumed 11.48±0.38 g/day, achieving 40% CR relative to their baseline intake. The old-CR group showed a modest reduction in body weight compared to the age-matched old group (old, 691.4±30.4 g; old-CR. 538.4±34.5 g) (Supplementary Fig. S3).
Furthermore, BUN levels, a marker of renal function, increased significantly with aging (young, 16.5±0.7 mg/dL; old, 24.5±5.5 mg/dL) and were partially reversed by CR (old-CR 8.9±1.2 mg/dL; p<0.01 vs. old), confirming that the 4-week CR intervention exerted measurable physiological effects on renal function (Fig. 3A). Other renal function-related genes Kim-1 (Havcr1) and Lcn2 were also increased with age and decreased under CR (Fig. 3B). Collectively, these results indicated that aging contributes to kidney damage and impaired renal function, which are likely influenced by changes in SASP- and cell cycle-related genes. Furthermore, modulation of gene expression by CR highlights its potential to mitigate the effects of aging and cellular senescence.
The Predictive Roles of Il19 and Il24 in Cellular Senescence
To explore the relationship between SASP- and cell cycle-related genes that exhibited expression changes during aging and CR, a PPI network was constructed using all DEGs from the old vs young and old-CR vs old datasets (Supplementary Fig. S4A). Based on previous analyses and experimental data, we focused on Il24, a member of the IL-20 family, and Cdkn2b, a cellular senescence-related gene, as both showed a consistent pattern of increased expression during aging and decreased expression under CR. In the PPI network, the shortest path connecting Il24 and Cdkn2b was calculated, revealing that Il24 is linked to Cdkn2b through inflammation-related genes, such as Ccr5, Cxcr2, Cxcr4, and Lck, ultimately connecting via Cd44 (Supplementary Fig. S4B).
To further investigate the relationship between Il19, Il24, and cellular senescence, the mRNA expression levels of Il24 and other senescence-associated genes were measured in H₂O₂-treated NRK52E cells to mimic the aging process. The results showed that Il19, Il24 expression, and the senescence marker p16INK4A increased in a dose-dependent manner upon H₂O₂ treatment (Fig. 4). These findings suggest that Il19 and Il24, may contribute to the regulation of cellular senescence during renal aging; however, causal roles remain to be established through gain- and loss-of-function studies.
DISCUSSION
This study identified key gene expression changes associated with renal aging and their modulation by short-term CR in aged rats. Through integrated transcriptomic profiling and molecular validation, we demonstrated that: (1) SASP-related genes, including cytokines (Il1b, Il19, Il24), chemokines (Ccl2, Ccl3, Ccl5), and growth factors (Timp1, Mmp12), were upregulated with aging and downregulated by CR; (2) cell cycle arrest markers (p15INK4B, p16INK4A, p21) followed similar patterns; and (3) among these, IL-20 family members Il19 and Il24 showed consistent responses across in vivo aging (Fig. 5), CR intervention, and in vitro senescence models. It is important to note that our findings are associative and exploratory in nature; causal inference would require gain- and loss-of-function perturbations (e.g., recombinant protein treatment, siRNA-mediated silencing) to establish whether Il19 and Il24 directly drive cellular senescence or are downstream markers of aging-related processes.
Our transcriptomic analysis revealed widespread upregulation of inflammatory mediators in aged kidneys, including cytokines (Il1b, Il6, Il19, Il24), chemokines (Ccl2, Ccl3, Ccl5, Ccl7, Ccl20), and growth factors (Tgfb1, Mmp2, Mmp12) (Table 2, Fig. 3). These age-associated changes were attenuated by short-term CR, confirming anti-inflammatory effect of CR. Studies report upregulation of Cxcl10, Il6, and Ccl2 in aged organs,20,21) with aged kidneys exhibiting increased Ccl3, Ccl4, and Ccl5 expression.22) Functionally, blocking CCR1 signaling attenuates age-related inflammation and reduces iNOS, COX-2, and MMP13 expression,23) consistent with our findings of widespread chemokine upregulation as a hallmark of renal aging. Aging kidneys also exhibit significant growth factor alterations, with increased TGF-β1, TIMP, and MMP-12 levels.24,25) Inflammatory stress activates TGF-β1/Smad signaling, while focal VEGF increases occur in aged renal cortex.26) and downregulates systemic inflammatory gene expression by reducing IGF-1 signaling.27) Collectively, our data position SASP-related gene expression as a key molecular signature of renal aging that is responsive to CR intervention.
Among the inflammatory mediators, we observed particularly robust age-associated induction of Il19 (79-fold) and Il24 (62-fold), with both genes downregulated by CR (Table 2, Fig. 3). qRT-PCR validation confirmed these patterns, and H2O2-induced senescence in NRK52E renal epithelial cells recapitulated Il19 and Il24 upregulation alongside p16INK4A expression (Fig. 4), suggesting a link between IL-20 family cytokines and cellular senescence.
The IL-20 subfamily, part of the IL-10 cytokine family, includes IL19, IL20, IL22, IL24, and IL26, which signal through shared receptor complexes to coordinate epithelial and immune responses.28) These cytokines promote epithelial proliferation and stimulate antimicrobial proteins, pro-inflammatory cytokines, and chemokines.29) IL19 enhances IL6 and TNF-α production,30) while IL20 modulates macrophage polarization.31) Notably, IL-20 family members show increased expression with age, with elevated IL20 linked to age-related conditions.32) Their involvement in cellular senescence is supported by STAT1-mediated pathway activation.33)
IL24 regulates immune responses by inducing cytokines and increases pro-inflammatory mediators.34) Long-term IL24 exposure induces cellular senescence in multiple cell types through STAT1-mediated pathways.35) Our previous findings showed increased Il24 expression in aged rat kidneys, reversed by CR,36) and the current study extends these observations by demonstrating that both Il19 and Il24 respond to CR and correlate with senescence markers across multiple experimental models, suggesting these cytokines may serve as hypothesis-generating candidates for understanding renal aging mechanisms.
Our analysis revealed coordinated upregulation of senescence-associated cell cycle inhibitors, including p15INK4B, p16INK4A, and p21 in aged kidneys (Fig. 2C). CR attenuated the expression of these markers, consistent with its known anti-aging properties. Cellular senescence functions both as a marker and driver of renal aging. Senescent cells undergo metabolic reprogramming and secrete bioactive SASP factors that influence neighboring cells and promote chronic inflammation.37) Key senescence marker p16INK4A was significantly increased during renal aging, which is consistent with our findings.38) In addition, TGF-β1 secreted by podocytes induces senescence in glomerular endothelial cells via p21 activation and p16INK4A nuclear translocation through Smad3 signaling.39)
CR effectively reduces senescent cell accumulation in both animal models and humans.9) RNA-sequencing shows CR significantly reduces senescence-related gene expression, including p16INK4A and senescence-associated β-galactosidase.40) CR also inhibits age-related senescent cell accumulation,41) highlighting the role of cell senescence in renal aging and supporting CR as a strategy for modulating senescence and delaying renal decline.
While our study establishes an association between CR and reduced Il19/Il24 expression, the upstream regulatory mechanisms remain to be fully elucidated. Accumulating evidence suggests that CR exerts its anti-inflammatory effects through coordinated activation of the SIRT1–AMPK–PPARα axis, which in turn suppresses NF-κB transcriptional activity and reshapes the inflammatory transcriptome.42-44) Given that IL-20 family cytokines signal through IL-20 receptor complexes to activate JAK/STAT1/3 and MAPK pathways28,29)—all of which intersect with NF-κB-mediated inflammation45,46)—it is plausible that CR indirectly modulates Il19/Il24 expression by dampening upstream inflammatory programs. However, it remains unclear whether Il19/Il24 act as drivers of age-related inflammation or merely as downstream markers of broader inflammatory changes. CR may primarily target master regulators (e.g., NF-κB, SIRT1, AMPK), with Il19/Il24 downregulation occurring as a secondary consequence.
Our previous studies extensively characterized renal aging and CR-associated changes, demonstrating TNF signaling upregulation during aging,15) increased FOXO1 phosphorylation and NF-κB activation attenuated by CR.42) We showed CR mimetics reduce inflammation via FoxO1–NF-κB competition and that CR modulates fibrosis through mitochondrial metabolic pathways.43,44) These findings underscore the role of CR in suppressing SASP and emphasize CR-responsive anti-aging targets.
Anti-inflammatory effects of CR have been demonstrated across diverse experimental designs, though protocols vary in duration, restriction intensity, and target populations. Previous studies employing short-term CR (8 weeks, 30% restriction) in middle-aged rats (12 months) reported reductions in inflammatory mediators (IL6, IL1β, TNF-α, TGF-β1) in renal arteries.47) Similarly, short-term CR (8 weeks, 40% restriction) in aged rats (24 months) suppressed matrix metalloproteinase activity and limited growth factor increases in aorta.48) Also, long-term CR (20 years, 30% restriction) in adult rhesus monkeys reduced inflammation, incidence of cancer, diabetes, and cardiovascular disease, and improved survival compared to ad libitum controls.49)
Our current 4-week CR protocol (40% restriction) in aged rats (21 months) represents a shorter intervention window but still recapitulated key inflammatory gene suppression, particularly Il19 and Il24. This finding suggests these genes are among the early responders to CR, detectable before the structural and functional improvements typically observed in longer-term studies. However, whether sustained or amplified effects occur with prolonged CR, and whether short-term molecular changes translate to functional renal protection, remain open questions requiring comparative temporal studies.
The 4-week CR duration was selected based on evidence that short-term CR (4–8 weeks) can rapidly modulate inflammatory gene expression and metabolic parameters in aged rodents while minimizing confounders of prolonged restriction.50) The observed downregulation of Il19 and Il24 within this timeframe suggests these genes are early CR responders. However, longer-term studies are needed to determine whether these molecular changes translate to sustained functional improvements in renal aging.
Several limitations should be acknowledged. The pooled RNA-seq design (n=3 per group) precludes estimation of inter-individual variance and application of inferential statistics; accordingly, our transcriptomic findings are exploratory and were interpreted alongside independent qRT-PCR validation using unpooled samples. Furthermore, causal roles of Il19 and Il24 were not established; demonstrating causality will require gain- and loss-of-function perturbations to examine effects on p16INK4A/p21/SASP markers and renal injury phenotypes. Future studies will implement non-pooled RNA-seq with biological replicates to strengthen reproducibility, perform functional validation of Il19/Il24 through gain/loss-of-function experiments, and investigate upstream regulatory mechanisms linking CR to IL-20 family modulation.
In conclusion, this study demonstrates that aging and short-term CR modulate SASP- and senescence-related gene expression in kidneys. Transcriptomic profiling and experimental validation revealed age-associated upregulation of Il19 and Il24, which was attenuated by short-term CR. These findings suggest that Il19 and Il24 are associated with aging and cellular senescence pathways in kidneys and may represent hypothesis-generating candidates for future mechanistic studies. Establishing their causal roles will require targeted functional perturbation experiments.
PDF Links
PubReader
XML Download
Full text via DOI
Download Citation
agmr-25-0103-Supplementary-Fig-S1.pdf
