1. Introduction
Kidney rejection occurs when, after transplantation, the immune system recognizes the graft as foreign and mounts an immune response characterized by inflammation and tissue damage. This process involves both humoral and cellular immunity: T cells primarily mediate the cellular response, while B cells and antibodies drive the humoral responses [
1]. Key mechanisms underlying rejection include MHC incompatibility, donor-specific antibodies (DSAs), and T cell activation through MHC class I and II molecules, triggering the release of cytokines such as TNF-α, IL-6, and IL-1β [
2]. These cytokines initiate fibrogenic and inflammatory pathways, contributing to graft injury and loss [
3].
At the molecular level, the Wnt/β-catenin signaling pathway regulates the activation and differentiation of key immune cells involved in rejection, including CD4+ and CD8+ T lymphocytes, macrophages, and dendritic cells. This pathway promotes the differentiation of T cells into pro-inflammatory Th1 and Th17 subsets, directs macrophages toward the inflammatory M1 phenotype, and enhances dendritic cell antigen presentation, thereby sustaining inflammation and tissue damage in the graft.
Sirtuins—particularly SIRT1, SIRT3, and SIRT6—modulate rejection by inhibiting the NF-κB signaling pathway to reduce inflammation, controlling the release of pro-inflammatory cytokines, supporting mitochondrial function, and maintaining genomic stability to protect tissues from damage during rejection.
Additionally, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) form complex regulatory networks that fine-tune gene expression in immune cells. They regulate cytokine production and cellular differentiation, significantly contributing to the progression and severity of graft rejection.
Regulatory molecules including lncRNAs, miRNAs, and inflammatory cytokines—create complex interaction networks influencing gene expression and immune responses, thus contributing to the progression and severity of graft rejection [
4,
5]. However, limited studies have examined the interplay between sirtuins and Wnt signaling in kidney rejection, and the molecular roles of lncRNAs and miRNAs remain underexplored.
Sirtuins are NAD⁺-dependent deacetylases that are critical for the regulation of cellular homeostasis and inflammation. During kidney rejection, SIRT1, SIRT3, and SIRT6 reduce inflammation and oxidative stress, helping to protect tissues and promote recovery [
6]. SIRT1 suppresses inflammatory pathways such as NF-κB and p53, modulates immune cell activity, and controls pro-inflammatory cytokine release, thereby mitigating the severity of rejection [
7]. SIRT3 preserves mitochondrial function and limits oxidative stress, which are essential for kidney tissue integrity [
8]. SIRT6 maintains genomic stability and curbs pro-inflammatory responses, aiding in post-transplant recovery [
9].
Wnt/β-catenin signaling is pivotal in cellular proliferation, differentiation, and immune activation. Overactivation promotes inflammation and fibrosis, worsening graft injury [
10]. Pharmacologically targeting this pathway shows promise for reducing inflammation and fibrosis to improve graft survival.
Despite limited direct evidence in kidney rejection cases, studies of sirtuins and Wnt signaling in kidney disease support their involvement in modulating inflammation and tissue damage [
7,
11]. Further research on their interactions could reveal novel therapeutic targets.
LncRNAs (non-coding RNAs > 200 nucleotides) and miRNAs (~20–24 nucleotides) regulate gene expression at the transcriptional and post-transcriptional levels, influencing cellular processes such as growth, differentiation, and stress response. Their crosstalk fine-tunes immune responses and disease progression [
12].
This study aims to elucidate the roles of sirtuins, Wnt/β-catenin signaling, inflammation, and regulatory RNAs in kidney rejection, correlating molecular findings with clinical and histopathological data. Understanding these mechanisms may facilitate the development of novel biomarkers and therapeutic strategies to improve transplant outcomes.
3. Findings
The gender distribution across the groups did not show a statistically significant difference (
p = 0.150). In the control group, the percentage of males was 51.5%, while in the patient group, it was 71.4%. The percentage of females in the control group was 48.5%, while in the patient group it was 28.6% (
Table 1).
Various miRNA, lncRNA, and mRNA expression levels in the patient and control groups were compared, and significant differences between them were observed. The expression levels of SNORD61, hsa-miR-34c-1, hsa-miR-122b-5b, MALAT1, HOTAIR, LINC00473, TUG, PVT1, GAPDH2, SIRT1, SIRT3, SIRT6, WNT1, TCF-LEF, LRP, AXIN1, IL1B, IL6, and IFNB1 were significantly altered in the patient group (
p < 0.05). These differences suggest that these genes may play a role in disease pathogenesis. However, no statistically significant difference was found between the two groups for hsa-miR-21-2, hsa-miR-155-5p, hsa-miR-200b-3p, and GAPDH levels (
p > 0.05). Additionally, the mean age of the patient group was higher than that of the control group, and this difference was statistically significant (
p = 0.006) (
Table 2).
A weak positive correlation was observed between hsa-miR-155-5p and SIRT1 (p = 0.034), with an FDR-adjusted p-value of 0.045, indicating statistical significance. Similarly, a significant positive correlation was found between hsa-miR-200b-3p and SIRT1 (p = 0.027; FDR = 0.038), while HOTAIR demonstrated a significant negative correlation with SIRT1 (p = 0.014; FDR = 0.025). Furthermore, the association between SIRT1 and TCF-LEF remained significant after FDR correction (p = 0.018; FDR = 0.029). Additionally, a significant negative correlation was observed between TCF-LEF and SIRT3 (p = 0.016; FDR = 0.027).
Significant differences were observed in the expression levels of certain biomarkers between the cellular and humoral rejection groups. Notably, hsa-miR-21-2 levels were significantly higher in the cellular rejection group than in the humoral rejection group (31.28 ± 1.45 vs. 29.84 ± 1.29; multivariate analysis: OR = 0.331; 95% CI: 0.126–0.868; p = 0.025; adjusted p = 0.038).
In addition, hsa-miR-155-5p levels were markedly higher in the cellular rejection group (39.48 ± 1.33 vs. 30.5 ± 1.53), while SIRT-1 levels were significantly lower (25.75 ± 1.72 vs. 28.06 ± 2.56). Multivariate analyses revealed statistical significance for both biomarkers (
p = 0.018 and
p = 0.025); although the FDR-adjusted
p-values were slightly above the threshold (adjusted
p = 0.054 and 0.052), they support the potential biological relevance of these biomarkers in association with cellular rejection (
Table 4).
In comparisons based on the degree of interstitial inflammation, differences were observed in the expression levels of hsa-miR-21-2, TUG, SIRT-1, and TCF-LEF genes, which appeared to correlate with the severity of inflammation (raw
p-values: 0.023, 0.047, 0.048, and 0.032, respectively). After applying False Discovery Rate (FDR) correction to account for multiple comparisons, the adjusted
p-values ranged between 0.06 and 0.08. These findings suggest that the expression levels of these genes may be associated with the degree of inflammation and trend toward statistical significance (
Table 5).
Table 6 shows statistically significant differences in the expression levels of certain molecular markers in relation to the presence of tubulitis. In univariate analyses, hsa-miR-155-5p levels were significantly increased in patients with tubulitis (raw
p = 0.032), SIRT-1 expression was significantly decreased (raw
p = 0.031), and TCF-LEF transcription factor levels were significantly elevated (raw
p = 0.038). These findings remained statistically significant after correction for multiple testing using the False Discovery Rate (FDR) method: hsa-miR-155-5p (FDR
p = 0.045), SIRT-1 (FDR
p = 0.048), and TCF-LEF (FDR
p = 0.047) (
Table 6).
The average values of the TUG gene also showed significant differences between the groups (
p = 0.034); however, no significant differences were found in the pairwise comparisons. Similarly, the SIRT-6 gene expression levels also showed significant differences according to the glomerulitis groups (
p = 0.049), but the difference was not significant in the pairwise comparisons between the groups. Additionally, significant differences were found in the mean expression levels of the LRP gene (
p = 0.003), which was statistically significant between the mild and moderate glomerulitis groups, as well as between the moderate and severe glomerulitis groups. These results suggest that the SNORD61, TUG, SIRT-6, and LRP genes in particular could be biomarkers in the progressive severity levels of glomerulitis (
Table 7).
As a result of the analyses, no statistically significant difference was found between many of the genes and molecular markers (
Table 8).
IL-6 expression levels tended to increase with the severity of interstitial inflammation (i1: 20.96 ± 1.43 vs. i2: 24.78 ± 2.08; raw
p = 0.034). However, this increase did not remain statistically significant after multiple testing corrections (FDR and Bonferroni) (adjusted
p > 0.05). Although a similar upward trend was observed in IL1B gene expression, the difference was not statistically significant (raw
p = 0.184). These findings suggest an association between elevated IL-6 levels and increasing inflammation severity; however, further studies with larger sample sizes are needed to confirm these results (
Table 9) (
Figure S1)
In the analysis based on the presence of tubulitis, IL1B levels were found to be significantly elevated, and this difference remained statistically significant after multiple testing correction (FDR) (raw
p = 0.003; FDR
p = 0.009). An increase in IL6 levels was also observed in the tubulitis group; although the raw
p-value was significant, the FDR-adjusted value approached but did not reach statistical significance (raw
p = 0.046; FDR
p = 0.069). No significant difference was detected in IFNB1 gene expression (
p > 0.05) (
Table 10) (
Figure S2)
4. Discussion
This study is one of the first to comprehensively investigate the potential effects of epigenetic regulators—particularly sirtuins, microRNAs, and long non-coding RNAs—on immune response and inflammation in the context of acute kidney transplant rejection. Our findings demonstrate that these molecules are regulated in direct association with the severity of inflammation, and their interactions may play a pivotal role in the rejection process.
The expression levels of SIRT1, SIRT3, and SIRT6 were significantly decreased in patients who developed acute rejection. This suggests that the anti-inflammatory roles of sirtuins are suppressed and that their protective effects are diminished during the rejection process. In particular, SIRT1 inhibits NF-κB pathways and promotes the polarization of macrophages toward the M2 phenotype, thereby exerting an anti-inflammatory effect [
7,
13]. Our findings indicate that this regulatory effect is weakened during the rejection process [
14].
These findings are consistent with the reported immunosuppressive role of SIRT1 in the literature. Indeed, experimental studies using rat renal allograft models have also shown that SIRT1 expression decreases during acute rejection and that this reduction is associated with graft injury [
2]. Moreover, T cell-specific SIRT1 deficiency has been shown to suppress the rejection response and prolong allograft survival in murine transplantation models. These data support the immunomodulatory role of SIRT1 in transplantation. Our findings are consistent with these results and demonstrate that SIRT1 is similarly downregulated in human renal transplant tissues [
15].
However, conflicting findings have also been reported in the literature. For example, Weng et al. (2024) [
16] reported that while the expression levels of several members of the SIRT family change during rejection, SIRT1 levels remain stable. This discrepancy may be due to differences in study design, the timing of tissue sample collection, or biological variability among the models used. Nonetheless, the decrease in SIRT1 observed in our study suggests that this molecule may play a more active role in the rejection process than previously thought, highlighting the need for further research. [
16].
In the I2 group, where the severity of interstitial inflammation increased, SIRT1 expression decreased and significant increases were observed in TUG1, hsa-miR-21, and TCF-LEF levels. Similarly, in the T2 group, increased tubulitis severity was associated with decreased SIRT1 levels and increased TCF-LEF and hsa-miR-155 expression. These findings suggest that SIRT1 is downregulated as inflammation progresses, whereas genes involved in regulating inflammatory and fibrotic responses become activated. The elevation of proinflammatory cytokines such as IL-6 and IL-1B reflects an immune activation that parallels these molecular changes.
Among the microRNAs, the increased expression of miR-155 and miR-21 is particularly noteworthy. miR-155 directly targets and suppresses SIRT1, thereby promoting the excessive activation of immune cells [
16]. miR-21, on the other hand, is associated with fibrosis and cellular injury and can activate the TGF-β/Smad signaling pathways [
17]. The elevation of these microRNAs indicates that both inflammation and fibrotic remodeling are enhanced during the rejection process.
At the lncRNA level, the upregulation of HOTAIR and MALAT1 is consistent with their reported pro-inflammatory and pro-fibrotic effects in the literature [
18]. In contrast, the decrease in TUG1 levels suggests that this lncRNA loses its enhancing effect on SIRT1 expression. The TUG1–SIRT1 correlation observed in our study supports this mechanism.
Moreover, the WNT/β-catenin signaling pathway was activated during rejection; this activation was further supported by the increased expression of the TCF-LEF transcription factor. These pathways play a central role in the progression of inflammation and fibrosis, and the literature reports that SIRT6 suppresses these genes.
All these findings suggest that epigenetic regulation plays a decisive role in amplifying the immune response during acute rejection. However, the causal relationships between these molecules have not been fully established. Our findings are hypothesis-generating in nature and highlight the need for advanced in vitro and in vivo studies for mechanistic validation.
In the future, molecules that activate sirtuins, anti-miR strategies targeting miR-155 and miR-21, and antisense approaches against HOTAIR/MALAT1 may serve as promising therapeutic targets for controlling transplant rejection. In particular, therapies aimed at enhancing TUG1 expression could restore SIRT1 levels and suppress inflammation. Additionally, the potential use of these molecules as biomarkers is also worth evaluating.
In conclusion, our study highlights the critical roles of sirtuins and non-coding RNAs in acute kidney transplant rejection, demonstrating their impact on disease progression and their potential as therapeutic targets. Before these findings can be translated into clinical practice, they must be validated through functional and experimental models. Nevertheless, the molecular network we have identified provides a strong foundation for future personalized approaches in both the diagnosis and treatment of acute rejection.
5. Conclusions
5.1. Molecular Signature Specific to Rejection Type
Our findings suggest that SIRT1 may play a relevant regulatory role in kidney transplant rejection. Although the data presented are observational, they indicate that SIRT1 is not only a potential biomarker but may also be involved in molecular mechanisms underlying rejection. Its downregulation appears to coincide with the activation of Wnt/β-catenin signaling and TCF-LEF transcription factors, as well as with increased miR-21 and miR-155 expression. These molecular alterations are associated with intensified tubulointerstitial inflammation and elevated IL-6 and IL-1β levels.
Such associations imply that both cellular and humoral rejection may be influenced not only by immune-mediated injury but also by epigenetic, transcriptional, and post-transcriptional mechanisms. Notably, in advanced stages of cellular rejection (i2–t2), reduced SIRT1 expression, together with increased levels of miR-21, miR-155, and TCF-LEF, corresponds with enhanced inflammatory responses.
While these findings offer valuable insights, we acknowledge that further validation—particularly through mechanistic in vitro or in vivo studies—is required to confirm the functional relevance of these molecular interactions. Nevertheless, the present results provide an important preliminary framework for future investigations. A deeper understanding of these regulatory networks may ultimately support the development of personalized therapeutic strategies aimed at improving graft outcomes.
5.2. Study Limitations and Future Perspectives
Among the limitations of this study are its single-center design and the relatively small sample size, which may reduce the generalizability of the findings. To enhance external validity, further high-level validation studies involving larger and more heterogeneous patient groups are needed. Additionally, it is important to evaluate the molecular findings using high-throughput miRNA profiling techniques and diverse molecular analysis platforms. Confirming the role of SIRT1 and related molecules in different organ transplant models would increase the clinical relevance of this study.
Specifically, future research should elucidate the immunoregulatory effects of miRNAs and lncRNAs in more detail, assessing their potential use as biomarkers or therapeutic targets in transplant rejection. Pharmacologically targeting SIRT1 may offer an innovative strategy for preventing and managing immune-mediated graft injury.
In this study, patients receiving immunosuppressive therapy and those without biopsy samples were excluded. This exclusion was intended to ensure sample homogeneity in molecular analyses and to minimize confounding effects. Immunosuppressive drugs can directly influence gene and RNA expression levels and may mask the intrinsic biological effects of the investigated molecules. Therefore, focusing on a sample not exposed to immunosuppressive treatment is crucial for accurately assessing the independent roles of these molecules in inflammation and rejection. However, this approach may also limit the clinical applicability and generalizability of the study.
Additionally, our study reports various associations between gene and miRNA expression levels and histopathological or clinical findings; however, further studies are needed to perform mechanistic and functional validation experiments. Advanced in vitro and in vivo experimental studies should be conducted to establish causal relationships and to better understand the underlying molecular pathways.
Nevertheless, as a pilot study designed with selective inclusion criteria, the purpose of these preliminary findings is to inform future clinical and translational research. Validating the results in larger and more diverse patient populations is of great importance for enhancing their clinical relevance and applicability.