Research progress of tsRNAs in kidney diseases

Introduction

Approximately 10% of adults worldwide currently suffer from chronic kidney disease (CKD), leading to around 1.2 million deaths annually (Kalantar-Zadeh et al., 2021). In Asia, the number of CKD patients is as high as 443 million, with nearly 160 million in China alone (Liyanage et al., 2022). This disease is characterized by its irreversible progression to end-stage renal disease (ESRD), which inevitably results in patient mortality (Kalantar-Zadeh et al., 2021). It is estimated that by 2030, the number of people requiring renal replacement therapy (RRT) will reach 543.9 million worldwide (Liyanage et al., 2015), and by 2040, CKD will become the fifth leading cause of death globally (Foreman et al., 2018). Addressing kidney health in Asian countries, the Global Kidney Health Atlas (GKHA) has highlighted that China, as one of the countries with the highest number of CKD patients, faces a substantial burden (Bharati & Jha, 2022). Recent studies have suggested that transfer RNA-derived small RNAs (tsRNAs) may play a role in kidney diseases by regulating gene expression, influencing protein synthesis, and inhibiting apoptosis (Li et al., 2024). Exploring the pathogenesis of kidney diseases, identifying drug intervention targets, and discovering new biomarkers are critical for advancing research in this field.

This review is aimed at researchers, clinicians, and translational medicine researchers in the fields of nephrology and molecular biology, with the goal of providing a comprehensive reference for exploring the mechanisms of tsRNAs in kidney diseases, developing biomarkers, and promoting translational applications in clinical nephrology.

Survey Methodology

Literature retrieval was conducted through the PubMed and Web of Science databases using the keyword combination “tsRNAs OR tRFs OR tiRNAs AND kidney disease” combined with Boolean operators to screen for articles up to August 1, 2025. The inclusion criteria were: original experimental, observational, or clinical studies exploring the role of tsRNAs/tRFs/tiRNAs in kidney diseases (such as acute kidney injury, chronic kidney disease, and diabetic nephropathy). Exclusion criteria included: articles outside the time range and duplicate articles, studies not related to tsRNAs/tRFs/tiRNAs, and non-experimental studies (such as meta-analyses, and editorials), as well as studies not related to kidney diseases. Systematic screening was carried out to ensure the focus, originality, and clinical relevance of the research content.

Definition of tsRNAs

Transfer RNA-derived small RNAs (tsRNAs) are a group of small non-coding RNAs generated from precursor or mature tRNA, initially discovered in prokaryotes, but increasingly recognized and studied in eukaryotes in recent years (Zuo et al., 2021). Depending on the cleavage site, tsRNAs can be categorized into two main types: tRNA-derived stress-induced RNAs (tiRNAs), which are produced through cleavage by angiogenin and ribonuclease under stress conditions, and tRNA-derived fragments (tRFs), which are generated by nuclear cleavage during either the precursor or mature stage of tRNA. Based on their origin and characteristics, tRFs can be further divided into five subtypes (Fig. 1): tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF (Chu et al., 2022; Lu et al., 2021). tRF-1 is derived from the 3′ trailer sequence of precursor tRNA and contains a poly-U sequence, tRF-3 is generated by cleavage of the TΨC loop of mature tRNA, and tRF-5 is produced by Dicer cleavage of the D-loop of tRNA. tRF-2, on the other hand, consists of a small fragment containing the anticodon of tRNA (Wang et al., 2022). tRF-5 can also be classified into three subtypes based on its length: tRF-5a (14–16 nt), tRF-5b (22–24 nt), and tRF-5c (28–30 nt) (Di Fazio & Gullerova, 2023). tiRNAs, also known as tRNA halves, are divided into two categories: 3′ tiRNA and 5′ tiRNA (Chu et al., 2022).

Recent advances in the detection of tsRNAs have largely relied on two major technological innovations. On the computational side, the tRF2Cancer platform integrates three core modules: tRFfinder, which specializes in identifying small RNA fragments of 18–30 nucleotides, optimal for conventional sequencing; tRFinCancer, which analyzes differential expression in cancer; and tRFBrowser, which maps RNA modification sites (Zheng et al., 2016). This integrative approach has proven effective in exosome research. For example, Zhou et al. (2022) utilized a three-step strategy—aligning to a tRNA reference database, identifying differentially expressed tRFs, and predicting potential functions—to efficiently uncover disease-associated tRFs. On the experimental front, PANDORA-seq introduces a novel enzymatic treatment using T4 PNK and AlkB to remove RNA modifications that typically hinder small RNA sequencing (Shi et al., 2021). While originally developed for oncology studies, this standardized bioinformatics and biochemical workflow offers valuable methodological insight for kidney-related tsRNAs research. At present, the nephrology field lacks a dedicated tsRNAs database comparable to tRF2Cancer. Future research would benefit from developing a platform tailored to kidney disease, integrating expression profiles and functional annotations of tsRNAs. Such a resource could significantly accelerate the transition from correlative observations to mechanistic understanding in tsRNA-based diagnostics and pathophysiological studies.

Biological Functions of tsRNAs

Rather than representing a single-mode regulatory class, tsRNAs participate in diverse layers of gene regulation, engaging multiple stages of the central dogma to form a highly intricate, multidimensional regulatory network. Accumulating evidence suggests their functional repertoire encompasses at least four core domains: AGO-dependent gene silencing, translational regulation, retrotransposon suppression, and immunometabolic reprogramming. Elucidating these mechanisms and their interconnectivity is critical to understanding the role of tsRNAs in kidney disease pathogenesis.

In human embryonic kidney cells (HEK293), it was found that tRF-3 and tRF-5 exhibit stronger binding affinity to AGO1, AGO3, and AGO4 than to AGO2, the primary effector of canonical miRNA pathways (Fig. 2). Interestingly, tRF-3 and miRNAs share a similar T-to-C mutation pattern at AGO-binding sites, whereas tRF-5 displays a distinct binding profile (Kumar et al., 2014). Moreover, tRF-3b silences target genes via AGO-dependent seed pairing, yet this activity is disrupted when m¹A modification alters its seed sequence, impairing gene repression without affecting AGO association (Su et al., 2022). These findings suggest that tRFs actively engage in RNA interference pathways but do so via unique sequence features and modification-dependent mechanisms, expanding the functional complexity beyond canonical miRNA paradigms.

tsRNA-mediated regulation extends beyond the cytoplasm. In HEK293T cells, knockdown of the endoribonuclease Dicer led to a concurrent decrease in both tsRNAs and miRNAs. Additionally, fluorescently labeled tsRNAs predominantly localized to the nucleus in breast cancer cells, whereas traditional siRNAs remained cytoplasmic. Further analysis showed that certain nuclear tsRNAs preferentially bind early intronic regions of nascent transcripts, mediating AGO2-dependent pre-mRNA intron cleavage and silencing (Di Fazio et al., 2022). This Dicer-dependent nuclear RNA interference mechanism, distinct from classical cytoplasmic pathways, suggests a novel strategy for transcriptional regulation and may offer a platform for developing next-generation gene silencing therapeutics. Although tRF^GluTTC does not directly mediate DNA methylation or histone modification, it exerts epigenetic-like regulatory effects through post-transcriptional mechanisms. Specifically, it forms a RISC complex via AGO binding, targeting the mRNA stability of transcription factors KLF9, KLF11, and KLF12, thereby indirectly modulating adipogenesis-related genes such as PPARγ and C/EBPα (Shen et al., 2019). This indicates that tsRNAs can function at both transcriptional and post-transcriptional levels to orchestrate rapid, systemic gene regulation. In the context of acute kidney injury (AKI), such rapid response systems are likely particularly critical for maintaining cellular integrity under stress.

Under stress conditions, tsRNAs serve as key regulators of protein synthesis homeostasis. The 5′-tiRNA Ala, derived from mature tRNA cleavage, forms a tetramolecular G-quadruplex (RG4) through its 5′ terminal oligo-Guanine motif (5′TOG). This unique structure displaces the eIF4F complex from the 5′ cap of mRNAs in a conformation-dependent manner, effectively inhibiting cap-dependent translation initiation and leading to global translational arrest. Additionally, pseudouridine-modified tsRNAs (mTOG-Ψ) can disrupt the formation of the PABPC1–PAIP1 complex and downregulate key components of the ribosome such as RPL23, RPL29, and the assembly factor EIF6, directly interfering with ribosome biogenesis (Guzzi et al., 2022). In an in vivo hepatocellular carcinoma model, LeuCAG3′tsRNA was shown to enhance the translation of ribosomal protein S28 (RPS28) mRNA, promoting cell proliferation, whereas its inhibition reduced RPS28 expression, impaired ribosome assembly, and induced apoptosis (Kim et al., 2017) (Fig. 2). These findings demonstrate that tsRNAs can exert bidirectional control over protein synthesis—globally repressing translation under stress while selectively enhancing the production of survival-related proteins. In chronic kidney diseases such as diabetic nephropathy, such fine-tuned translational control may influence podocyte integrity and ECM deposition.

In embryonic and trophoblast stem cells lacking H3K9 trimethylation, 3′CCA tRFs play a pivotal role in the suppression of endogenous retroviruses (ERVs). The 18-nt isoform directly inhibits reverse transcription by spatially blocking primer binding sites (PBS) on retrotransposons, while the 22-nt isoform loads into RISC to mediate post-transcriptional degradation of ERV transcripts (Schorn et al., 2017). This dual mechanism—targeting the PBS region of LTR elements at both transcriptional and post-transcriptional levels—highlights the evolutionary conservation of tsRNA-mediated transposon defense, which is essential for maintaining genomic stability.

Together, tsRNAs impact both the transcriptome and proteome through a multifunctional network characterized by overlapping and synergistic modules. However, several critical challenges remain. First, the cell-type specificity of tsRNAs subtypes in the kidney—such as in tubular epithelial cells, podocytes, and mesangial cells—requires further delineation. Second, the functional consequences of diverse RNA modifications on tsRNAs activity remain poorly understood. Lastly, future studies should investigate how these pathways intersect under pathophysiological conditions, such as hypoxia, hyperglycemia, or inflammation. In such contexts, tsRNA-regulated signaling may engage in complex cross-talk and dynamic feedback loops, shaping the progression of kidney disease.

Role of tsRNAs in Acute Kidney Injury

Acute kidney injury (AKI) is a condition characterized by rapid loss of kidney function, manifested by sudden decreases in urine output or anuria, often accompanied by electrolyte imbalance. This injury is usually caused by ischemia, hypoxia, or nephrotoxic substances (Gaut & Liapis, 2021). In classical AKI models induced by renal ischemia-reperfusion or cisplatin nephrotoxicity (Mami & Pallet, 2018), oxidative stress prompts a conformational shift in tRNA molecules—from a stable folded state to an unfolded form. This structural transition represents an early molecular response to cellular stress, occurring even before the onset of DNA damage or apoptosis. The unfolded tRNAs are then selectively cleaved by endoribonucleases such as angiogenin, producing tsRNAs that are released into peripheral circulation during the subclinical phase of tubular injury. This mechanism was further validated in patients undergoing aortic surgery, where plasma levels of tsRNAs rose sharply shortly after reperfusion. Notably, the kinetics of this increase preceded changes in serum creatinine and elevations in urinary KIM-1, suggesting that tsRNAs may serve as highly sensitive biomarkers of early nephron injury. Unfolded tRNAs and their fragments were also observed in regions proximal to damaged mitochondria, indicating that tsRNAs likely originate from structurally compromised nephron units under stress conditions. The ability of these fragments to maintain stability in the circulation—likely via encapsulation in exosomes or binding to RNA-binding proteins—provides a structural basis for their utility as reliable diagnostic markers (Mishima et al., 2014). This body of evidence not only highlights the sensitivity of tsRNAs for early AKI detection but also underscores the regulatory role of tRNA structural dynamics in stress responses. These findings open new avenues for mechanistic studies and the development of targeted interventions in AKI.

Recent research further suggests that tsRNAs function as key regulators within the epitranscriptomic landscape of AKI pathogenesis (Li et al., 2022). By modulating diverse pathways such as immune-inflammatory signaling, metabolic reprogramming, and programmed cell death, tsRNAs critically influence tubular epithelial cell survival and the trajectory of renal recovery. Molecules like tiRNA-Gly-GCC-003 have been shown to exacerbate oxidative stress and apoptosis by affecting natural killer cell cytotoxicity and mitochondrial energy metabolism. Additionally, they regulate the expression of pivotal genes including Rac1 and Mapk1 (Fig. 3), implicating tsRNAs in fibrosis and reperfusion injury–associated signaling networks. These discoveries provide both mechanistic insight and theoretical rationale for tsRNAs as early biomarkers and potential therapeutic targets in AKI.

A groundbreaking study by Li et al. (2025), published in Science, identified a marked upregulation of tRNA-Asp-GTC-3′tDR in murine AKI models. Inhibition of this molecule using antisense oligonucleotides (ASOs) suppressed autophagic activity and exacerbatedtubular necrosis, inflammation, and fibrosis. Conversely, targeted delivery of exogenous tDR via cationic polymeric nanoparticles activated autophagy and significantly mitigated renal injury and fibrotic remodeling. Notably, this tDR was also upregulated in kidney tissue from chronic kidney disease (CKD) patients and in the urine of preeclampsia patients with renal complications, showing a strong inverse correlation with histone mRNA expression. These findings underscore the molecule’s central role in RNA-directed autophagy in human kidney diseases and demonstrate its therapeutic potential. The translational value of this tsRNA highlights a pivotal shift in non-coding RNA research—from mechanistic discovery toward clinical application.

tRFs in chronic kidney disease

Role of tRFs and tiRNAs in Lupus Nephritis

Lupus nephritis (LN) is an autoimmune disease caused by systemic lupus erythematosus (SLE), primarily leading to kidney damage. The severity of the damage depends on the progression of the disease. Early diagnosis and treatment are crucial for this disease. However, current LN diagnosis mainly relies on laboratory tests and kidney biopsy, and there are no specific sensitive biomarkers (Anders et al., 2020; Yu et al., 2022). Studies (Zheng et al., 2022) have found that tsRNA-21109 can inhibit M1 macrophage polarization, while tRF-His-GTG-1 is significantly upregulated in the serum exosomes of SLE patients. Additionally, tRF-3009 is overexpressed in CD4+ T cells, and it is speculated that it may be involved in the pathogenesis of SLE by regulating IFN-α-induced oxidative phosphorylation in CD4+ T cells. Moreover, overexpression of tsRNA-14783 promotes M2 macrophage polarization, which helps alleviate inflammation and promote tissue repair. In LN, the presence of M2c macrophages is often associated with disease progression (Allam et al., 2020; Wang & Hu, 2022). As a subtype of M2 macrophages, whether tsRNA-14783 could influence macrophage function and serve as a marker for LN diagnosis, or how it could regulate LN progression, remains to be studied. Therefore, tsRNAs have demonstrated potential value in SLE diagnosis and treatment by regulating immune cell function and participating in disease pathogenesis.

Another study found that the expression level of tRF-Ala-AGC-2-M4 was significantly elevated in the serum of LN patients compared to healthy individuals, and this elevation correlated with disease activity. Moreover, tRF-Ala-AGC-2-M4 did not show significant differences in other autoimmune diseases, suggesting high specificity. Bioinformatic analysis indicated that tRF-Ala-AGC-2-M4 might be involved in glomerulonephritis signaling pathways associated with LN progression (Zhang et al., 2022). Furthermore, tRF-His-GTG-1 was significantly upregulated in the serum of SLE patients but markedly decreased in LN patients. Although the underlying mechanism remains unclear, Yang et al. (2021) speculated that renal damage might cause increased leakage of tRF-His-GTG-1 into the urine. Another study conducted tsRNAs sequencing of urine exosomes from SLE patients with or without LN, revealing that tRF3-Ile-AAT-1 and tiRNA5-Lys-CTT-1 were elevated in the urine exosomes of LN patients. Specifically, tRF3-Ile-AAT-1 might modulate autoimmunity by affecting signaling pathways related to the Th17/Treg balance, while tiRNA5-Lys-CTT-1 might bind to platelet-derived growth factor (PDGF) and contribute to renal fibrosis. These findings suggest that tsRNAs may contribute to LN progression via these pathways, indicating that tsRNAs could serve as non-invasive markers for distinguishing SLE patients with or without LN (Chen et al., 2023; Yang et al., 2021). ROC curve analysis demonstrated that tRF-His-GTG-1 exhibited higher sensitivity and specificity compared to current urinary protein markers used to differentiate SLE patients with glomerulonephritis. This study was the first to highlight the clinical potential of serum tsRNAs in identifying SLE patients with glomerulonephritis, providing a valuable reference for future research on serum tsRNAs as novel non-invasive biomarkers (Yang et al., 2021).

Studies conducted in China identified several abnormally expressed tRFs in CD4+ T cells of SLE patients, with tRF-3009 expression showing a positive correlation with the SLE disease activity index, renal damage, and serum IFN-α levels. Transfection of tRF-3009 mimics into CD4+ T cells, followed by IFN-α stimulation, demonstrated that overexpression of tRF-3009 activated SLE-related signaling pathways and induced oxidative phosphorylation in CD4+ T cells, whereas knockdown of tRF-3009 inhibited IFN-α-induced oxidative phosphorylation. These findings suggest that tRF-3009 may contribute to SLE pathogenesis by regulating CD4+ T cell metabolic pathways, thus providing a novel target for SLE treatment (Geng et al., 2021).

The Role of tsRNAs in Other Kidney Diseases

Limitation

To date, no peer-reviewed studies have directly profiled or functionally characterized tsRNAs (including tRFs/tiRNAs) in polycystic kidney disease (PKD/ADPKD). Existing small RNA studies in PKD have focused primarily on miRNAs and a few piRNAs. For example, several reports have documented altered miR-192/194/30 family members in urinary extracellular vesicles (uEVs) from ADPKD patients, yet tsRNAs were neither analyzed nor reported (Ali et al., 2024; Magayr et al., 2020). Meanwhile, in other kidney diseases such as AKI and CKD, tsRNAs have been reliably detected in urine-derived exosomes, suggesting their feasibility as biomarkers. This discrepancy may partially stem from technical biases: conventional small RNA sequencing often underdetects tsRNAs due to RNA base modifications (e.g., methylation), unless demethylation-enhanced methods like ARM-seq or DM-tRNA-seq are applied (Padhiar et al., 2024). Hence, the current absence of tsRNAs data in PKD may reflect technical underestimation rather than biological absence.

Beyond this, the clinical application of tsRNAs still faces important methodological barriers. tsRNAs frequently undergo extensive post-transcriptional modifications (e.g., m⁵C, Ψ), which hinder their capture by standard sequencing protocols and complicate assay standardization. In addition, the field relies heavily on bioinformatic predictions of target genes and regulatory pathways, with few studies validating these interactions through classic biochemical techniques such as RIP, RNA pull-down, or dual-luciferase reporter assays.

To realize their translational potential, future research must rigorously verify tsRNAs target networks and reduce dependency on computational inference. Well-designed prospective studies are urgently needed to compare tsRNAs markers directly with traditional indicators like serum creatinine, evaluating diagnostic accuracy via ROC analysis in real-world settings.

Summary and Outlook

Supplemental Information