Diabetic kidney disease (DKD) is a frequent microvascular complication of type 1 and type 2 diabetics. Approximately 40% of diabetic patients eventually develop DKD, which has been associated with an increased incidence of pain, falls, and reduced quality of life (1). It is also the most common cause of end-stage renal disease requiring renal replacement therapy, which is associated with high mortality and morbidity (2–4). The mortality risk in patients with DKD is 31.1% higher compared to diabetic patients. The mortality risk is even higher in incipient DKD patients, imposing substantial public health and economic burdens (5, 6). DKD is often undiagnosed until the manifestations of serious complications, inhibiting timely medical management to control disease progression (4, 7). Persistently elevated albumin excretion [albumin-to-creatine ratio (ACR) ≥ 30 mg/g] and low estimated glomerular filtration rate (eGFR < 60 ml/min/1.73 m²) are standard diagnostic indicators for DKD in a clinical setting. Still, these indicators have limited specificity and predictive power (8, 9). Kidney biopsy is superior in differentiating DKD from non-DKD and provides better risk stratification of DKD than the routine measurement of ACR and eGFR (1, 10). However, due to its invasiveness and patient burden, kidney biopsy is not feasible for the routine practice of DKD management.

Exosomes are membranous extracellular vesicles with a nanostructure and diameters ranging from 30 to 150 nm (11). Studies have found that exosomes act as messengers in cell–cell communication by transferring content to the target cells’ cytoplasm and altering the recipient cells’ physiological state (12). The generation of exosomes begins from endocytosis to form early endosomes by inward budding of the plasma membrane triggered by external stimuli or microbial attacks. After that, exosomes are shed into various body fluids and widely distributed in almost all kinds of body fluids, suggesting an irreplaceable role of exosomes in physiological and pathological conditions (13). During exosome biogenesis and release, selective cargo loading occurs, and particular cellular constituents are shuttled into exosomes containing various microRNAs (miRNAs), mRNAs, DNAs, lipids, and many other cellular components (14). Exosomes transfer autocrine or paracrine signals by a cell–cell crosstalk between kidney resident cells. High concentrations of glucose and the stimulated renal cells can lead to changes in composition and communication, further changing and damaging intact cells, which suggests that exosomes packaged with functional cargo have a vital role in diverse cellular processes and diseases, including DKD (15). Exosomes can be isolated from body fluid, including blood, urine, and saliva, making them ideal candidates for the non-invasive diagnosis of DKD (16, 17).

A thorough literature search identified three published review studies investigating the exosome biomarkers in DKD, particularly miRNA (9, 18, 19). Several dysregulated miRNAs were identified in DKD, and it was shown that specific miRNAs were significantly associated with clinical indicators of DKD, including HbAc1, ACR, and eGFR, suggesting important diagnostic and pathogenetic implications. However, the existing reviews failed to include standard components in evaluating exosomes other than functional miRNA (i.e., mRNA, long non-coding RNA, proteins) (16) or to investigate the diagnostic value of exosomes using meta-analysis.t Considering that it is challenging to inform clinical decisions without evidence related to the accuracy and sensitivity of the diagnostic tests, we conducted a systematic review and meta-analysis to synthesize evidence on clinical outcomes of all exosome types in DKD. We also analyzed the role of exosomes as biomarkers of DKD. Our main aim was to further elucidate the role of exosomes in the pathogenesis and management of DKD.

After duplicates were removed, 1,065 records were identified. Among these, 413 were excluded (conference proceedings, reviews, animal studies, etc.), and the remaining 652 studies were further screened against the inclusion and exclusion criteria. After excluding 606 studies with irrelevant outcomes, 11 with outside participants, and 3 with no full-text available, 32 eligible publications with 1,119 patients with DKD and 1,328 without DKD were included in the systematic review (16, 22–52). The PRISMA flowchart is presented in Figure 1 . The characteristics of the included studies are summarized in Table 1 . Most included studies (30/32; 93.8%) were case–control studies assessing the levels of exosomes in DKD and controls. In their study, Almquist et al. investigated the effects of simvastatin alone or with ezetimibe on microparticles in patients with or without DKD using a randomized controlled design (31). Sun et al. described a two-stage randomized controlled study, and in the second stage, they evaluated the potential role of urine exosomes as early diagnostic biomarkers for DKD (16). The majority of the included studies were conducted in China (15/32; 46.9%), followed by India (3/32; 9.4%), Poland (2/32; 6.3%), the Netherlands (2/32; 6.3%), and Italy (2/32; 6.3%). Twenty-three studies (71.9%) recruited patients with type II DKD only, three studies (9.4%) recruited patients with type I DKD only, and one study (3.1%) included both types I and II. In comparison, five studies (15.6%) did not specify the etiology of included DKD patients. With regard to the source of the sample, 23 studies (71.9%) used urine samples, 9 (28.1%) involved plasma or serum, and 1 study (3.1%) used lipids. qRT-PCR was the most frequently used method for detecting and measuring exosomes (18/32; 56.3%). Most included studies used P <0.05 as the cutoff value (20/32; 62.5%), while others used fold change values as the cutoff criteria (10/32; 31.3%).

This systematic review and meta-analysis identified 110 unique exosomes (i.e., miRNAs, mRNAs, proteins, and lipids) that were significantly dysregulated in DKD patients. In the meta-analysis of DKD diagnostic tests, exosomes had good sensitivity (0.70) and specificity (0.78). The AUC was 0.82 for the SROC curve, indicating excellent overall diagnostic accuracy.

Approximately half of the included studies investigated the role of miRNA in DKD management, and a total of 78 upregulated and 22 downregulated miRNAs were identified in our review. Four miRNA families were consistently significantly dysregulated in at least two included studies. Three studies found that miR-126 was upregulated in extracellular vesicles but downregulated in total plasma (26, 33, 50), which is consistent with Park’s systematic review (2018). This finding could be explained by the loss of kidney reabsorption and subsequent excretion, leading to the depletion of circulating miR-126 (19). miR-216 participates in maintaining endothelial cells and vascular hemostasis. It enhances vascular endothelial growth factor (VEGF) signaling (53, 54). The dysregulation of miR-126 indicates that it might be related to the progression of diabetes and altered during kidney damage (32, 33). Two studies found the upregulation of miR-145, the glomerular marker of mesangial cells (33). However, the role of this miR-145 in renal tissue damage remains unclear (33). miR-150 promotes renal fibrosis, and it was upregulated in both serum and urine samples in DKD patients compared to controls (36, 48). miR-21-5p was upregulated in urinary exosomes in DKD patients and correlated with creatinine and eGFR (49, 52), which is consistent with the review by Gholaminejad et al. (9). This mRNA participates in activating transforming growth factor (TGF)-β, which works in glomerular cell proliferation and matrix expansion, contributing to renal failure (55). Five studies analyzed the diagnostic value of miRNA (28, 42, 47, 49, 51). Compared to single miRNA biomarkers (i.e., miR-192, miR-194, miR-215, miR-615-3p, miR-3147, miR-4534), a combination of miRNAs (i.e., let-7i-5p, miR-15b-5p, miR-24-3p, and miR-27b-3p) or miRNA in combination with other clinical characteristics (i.e., miR-615-3p and ACR; miR-30b-5p, miR-21-5p, age, gender, and HDL-C) seemed to have higher accuracy in predicting DKD. Our review identified different miRNAs in DKD studies; future studies should confirm the most accurate and stable miRNA biomarker for DKD, and the diagnostic value of miRNA in combination with other clinical indicators should also be further explored.

For mRNA, two upregulated and four downregulated mRNAs were identified in the included studies, and the upregulation of WT1 in DKD was demonstrated in two studies. WT1 is the transcriptional regulator of genes related to growth and apoptosis and is vital in embryogenesis during kidney development (56). Regarding diagnostic value, WT1 seems to have a higher accuracy in predicting overt DKD (AUC: 0.705–0.83) than incipient DKD (AUC: 0.63). Feng et al. found that CCL21 mRNA was an efficient inflammatory marker to differentiate DKD patients without eGFR reduction from non-DKD diabetic patients. Also, its predictive ability was better than standard indicators (i.e., ACR and eGFR). Furthermore, CCL21 mRNA (AUC: 0.888–1.0) seemed to have better accuracy than other mRNAs (i.e., CDH2, MCP-1, and ACE; AUC: 0.61–0.75). Our review identified seven dysregulated proteins, and the diagnostic value was investigated in four of them. The WT1 protein in urine exosomes can effectively predict an early reduction in GRF (AUC: 0.92) (29). As previously mentioned, WT1 has been associated with podocyte malfunction and can be used as a marker for podocyte damage (57). Additionally, the WT1 protein seemed more accurate and sensitive in diagnosing DKD than WT1 mRNA; future studies with head-to-head comparisons are needed to confirm this finding. CALM1 is a regulatory protein for cell motility, differentiation, and proliferation. It was also found to have an excellent diagnostic value for DKD in combination with serum albumin levels (38, 58). The same research team identified the upregulated PAK6 and EGFR as diagnostic biomarkers of DKD (39). While the relationship between PAK6 and DKD is not well understood, the role of EGFR in the pathogenesis of DKD has been extensively studied. Elevated glucose levels activate EGFR and contribute to multicellular dysfunction, which triggers and accelerates kidney injury (59, 60). EGFR in combination with PAK6 has good predictive value and sensitivity (AUC: 0.897) (39).

An increasing number of studies have revealed a significant interest in exosomes for diagnosing and treating DKD, which presents both opportunities and challenges. First, the development of exact and non-invasive diagnostic methods is still of great importance. Due to the complexity of sources and cargoes, one obstacle to applying exosomes in DKD diagnosis is the discrepancy between the sensitivity and specificity of cargoes in diagnosing various kidney-related diseases. Finding reliable and specific exosomal RNAs and/or proteins may be beneficial for the widespread application of exosomes in diagnosing DKD, especially for urinary exosomes. In general, plasma exosomes may not pass through the glomerular filtration barrier. Moreover, the exosomes are protected by their bilayer membrane structure. Thus, urinary exosomes reflect the physiopathological state of the kidney other than the serum or circulation (61). Second, exosomes involving “long-distance” intercellular communication underlying pathogenesis may provide some novel clues to reveal the pathological mechanisms of DKD.

The present study has some limitations. First, the sample sizes of cases and controls were not always matched, and there were some baseline differences between groups, including age and blood pressure. Although hypertension is associated with DKD, it could be a confounding factor that was seldom adjusted in the included studies (62). Second, the heterogeneity of the included studies was high as they reported many different exosomes using diverse samples and methods. Future meta-analysis studies with more homogeneous studies (i.e., the outcome of the same exosome) are needed to confirm the reliability of the diagnostic results. Third, not all the included studies reported the sensitivity and specificity of the diagnostic test. Nonetheless, this review demonstrated that exosomes, especially in combination with other exosomes or clinical indicators, may be suitable as diagnostic biomarkers of DKD. More clinical data are required in the future to verify this finding.

This is the first study that reviewed the role of exosomes in the pathogenesis and management of DKD and the first meta-analysis on the diagnostic values of exosomes in DKD. The included exosomes had an AUC of 0.70 (95% CI: 0.59–0.80), sensitivity of 0.79 (95% CI: 0.70–0.85), and specificity of 0.82 (95% CI: 0.78–0.85), indicating that exosomes, as a non-invasive method, may be appropriate for use as diagnostic biomarkers of DKD. Moreover, panels of exosomes or the combination of exosomes with other clinical indicators seemed more accurate than single exosomes.