Expression and diagnostic value evaluation of urinary exosomal miR-142-3p in diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is one of the most severe microvascular complications of diabetes mellitus and a common cause of end-stage renal disease. Although kidney biopsy is the gold standard for diagnosing DN, it carries risks of complications such as infection and bleeding. Urinary microalbumin is currently used as a clinical indicator for diagnosing DN, but its early predictive ability is limited. Therefore, identifying non-invasive biomarkers for diagnosing DN has become a recent research focus. This study aimed to screen and identify differentially expressed miRNAs in diabetes and DN and evaluate their clinical diagnostic value to provide new directions for DN diagnosis. This study included 42 patients with diabetes and 57 patients with DN. Differentially expressed miRNAs in DN and type 2 diabetes mellitus were screened using the GEO database. General clinical data and urine samples were collected from both groups of patients. Urinary exosomes were extracted using ultracentrifugation, and qRT-PCR was used to detect changes in urinary exosomal miRNA expression between the two groups of patients. Receiver operating characteristic (ROC) curve analysis and Spearman correlation analysis were performed to evaluate the clinical diagnostic value of miRNAs. Urinary exosomal miR-142-3p was upregulated in DN patients compared to T2DM patients. ROC curve analysis showed that miR-142-3p had good diagnostic value for the disease and was positively correlated with UACR and risk stratification indicators for the progression of chronic kidney disease. Additionally, KEGG enrichment analysis revealed that urinary exosomal miR-142-3p may be involved in disease progression through pathways such as fatty acid metabolism, fatty acid biosynthesis, Hippo signaling pathway, cGMP-PKG signaling pathway, and Wnt signaling pathway. These results suggest that urinary exosomal miR-142-3p has good diagnostic performance in DN and may serve as a potential biomarker for diagnosing DN.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-06002-z.

Introduction

Diabetic nephropathy (DN), as one of the most severe microvascular complications of diabetes, has evolved into a leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) in Western countries. It also ranks globally as a major reason for renal replacement therapy, characterized by high morbidity and mortality rates^1–3. Currently, renal biopsy remains the gold standard for DN diagnosis in clinical practice. However, due to its invasive nature, renal biopsy carries risks such as infection, bleeding, and arteriovenous fistula formation, making it impractical for widespread use among patients. Therefore, renal biopsy is not suitable as a universal monitoring method for this disease. In clinical practice, DN diagnosis primarily relies on persistent albuminuria or estimated glomerular filtration rate (eGFR), with the urinary microprotein creatinine ratio being the most widely applied⁴. Yet recent studies have found that albuminuria, a routine biomarker for disease diagnosis, lacks sufficient sensitivity and specificity. Some patients exhibit advanced glomerular lesions even in the presence of microalbuminuria or without proteinuria^5,6. Additionally, emerging research has introduced the concept of non-proteinuric DN⁷. Thus, the search for novel non-invasive biomarkers for DN diagnosis has become a recent research focus. The discovery of such biomarkers may enable early diagnosis and treatment, thereby delaying disease progression.

Exosomes are extracellular vesicles with diameters typically ranging from 40 to 160 nm, formed through a continuous process involving the inward budding of the plasma membrane and the formation of multivesicular bodies (MVBs) containing intraluminal vesicles. These mature MVBs can either fuse with lysosomes or autophagosomes for degradation or fuse with the plasma membrane, releasing their intraluminal vesicles as exosomes into the extracellular space. These exosomes are subsequently taken up by recipient cells and mediate intercellular communication through various mechanisms⁸. The specific surface markers on exosomes (such as CD9, CD63, CD81, Alix, and TSG101) also imbue them with potential as biomarkers. Furthermore, exosomes have been identified in various body fluids, including breast milk, blood, cerebrospinal fluid, semen, saliva, ascites, and urine. They carry a diverse array of bioactive substances such as proteins, lipids, and genetic materials (e.g., DNA, RNA, miRNA), participating in the biological regulation of target cells^9,10. Moreover, multiple studies in recent years have demonstrated that exosomes secreted by various renal intrinsic cells play crucial roles in the diagnosis, treatment, and drug delivery processes of several diseases, including DN¹¹. Therefore, exosomes as non-invasive biomarkers involved in the progression of DN have attracted widespread attention.

MicroRNAs (miRNAs) are endogenous non-coding RNAs approximately 18–22 nucleotides in length, which regulate gene expression post-transcriptionally by targeting mRNA for degradation^12,13. In addition, compared with other types of non-coding RNAs (such as LncRNA, CircRNA, etc.), miRNAs have become a focus of research in recent years due to their high nucleotide sequence conservation and spatio-temporal specificity¹⁴. Previous studies indicate that the pathogenesis of kidney disease and the progression of DN are closely related to dysregulated exosomal miRNAs^15–19. In Jia et al.²⁰ confirmed in a study that urinary exosomal miR-192 was upregulated in patients with type 2 diabetes mellitus (T2DM) accompanied by early microalbuminuria compared to pure T2DM patients and healthy controls. Concurrent in vitro experiments indicated increased expression of miR-192 in exosomes secreted by human renal tubular epithelial cells under high glucose conditions, suggesting urinary exosomal miR-192 as a potential non-invasive biomarker for early DN diagnosis. In the same year, another study²¹ demonstrated elevated expression levels of urinary exosomal miR-133b-3p, miR-342-3p, and miR-30a-5p in T2DM patients with microalbuminuria or macroalbuminuria compared to pure T2DM patients or healthy controls. Moreover, compared to healthy controls, these miRNAs also exhibited an increasing trend in expression among pure T2DM patients, indicating that follow-up of these cases may aid in early detection of DN before microalbuminuria occurs, thereby facilitating early DN diagnosis. In Liu et al.²², through animal models and in vitro cell experiments, respectively confirmed increased expression of miR-483-5p in exosomes secreted by renal tubular epithelial cells stimulated with high glucose in diabetic mice and upregulated expression of MAPK1 and TIMP2, thereby inducing renal interstitial fibrosis and extracellular matrix deposition, providing new insights into the pathogenic mechanisms of DN. These studies collectively suggest that exosomal miRNAs not only serve as novel biomarkers for DN diagnosis but also play critical roles as key factors in disease progression, holding significant clinical implications. Furthermore, due to their renal tissue origin and simple non-invasive extraction methods, urinary exosomal miRNAs possess inherent advantages over other exosomes, making them ideal biomarkers for diagnosing renal diseases, which is currently a research hotspot.

In this study, we aim to screen and identify differentially expressed urinary exosomal miRNAs in patients with DN and T2DM, evaluate the diagnostic value of urinary exosomal miRNAs in DN, explore their correlations with clinical indicators and risk stratification parameters of chronic kidney disease and provide new insights into finding novel non-invasive biomarkers for DN diagnosis.Biological Characteristics of Extracellular Vesicles.

Materials and methods

Screening of differentially expressed miRNAs in DN

Datasets GSE51674 (including 6 DN patients, 6 T2DM patients with membranous nephropathy, and 4 normal kidney tissue samples) and GSE114477 (including 20 DN patients and 20 healthy controls’ urine samples) were selected for differential miRNA expression analysis. Differential gene expression between DN patients and T2DM patients or healthy controls was identified using the GEO2R online tool. An adjusted P < 0.01 and |logFC|≥ onefold change were set as the cutoff criteria for differential gene expression.

Patient characteristics

This study enrolled 42 hospitalized T2DM patients and 57 DN patients diagnosed in the Nephrology and Endocrinology Departments of the First Affiliated Hospital of Shandong First Medical University from December 1, 2020, to December 31, 2023. All included T2DM patients met the diagnostic criteria of the American Diabetes Association’s 2020 ADA Standards of Medical Care in Diabetes. All the selected DN patients, in addition to meeting the above criteria, were accompanied by pathological biopsy of renal aspiration or urinary albumin/creatinine ratio (UACR) > 30 mg/g, and the UACR reached or exceeded the critical value twice within 3 to 6 months after repeated examination. Exclusion criteria were: (1) refusal to participate, (2) type 1 diabetes patients, (3) special types of diabetes patients, (4) recent infections or other urological diseases, (5) transplant recipients, patients with malignant tumors, or immune-related diseases, (6) recent cerebrovascular disease patients, (7) patients with acute complications of diabetes or other critically ill conditions, and (8) patients excluded by laboratory tests such as ANA/dsDNA, ENA/fluid immunoelectrophoresis, anti-PLA2R antibodies, anti-glomerular basement membrane antibodies, ANCA, complement C3, C4, urinary light chain κ, λ, markers of infections such as hepatitis B, hepatitis C, HIV, syphilis, thyroid function, or renal biopsy to exclude other renal and systemic diseases.

General clinical data and laboratory test results of patients were collected through the medical record system, and urine samples were collected. Subsequently, according to the renal biopsy pathological results, 3 DN-A group patients (DN-A group) were selected and matched with 3 T2DM patients (T2DM-A group); then, according to the inclusion and exclusion criteria, 54 patients with diabetic nephropathy with proteinuria (DN-B group) and 39 patients with T2DM (T2DM-B group) were further selected for the verification queue. The collection of all samples was approved by the Medical Ethics Committee of the First Affiliated Hospital of Shandong First Medical University (Clinical Trial Registration Number: NCT06097351). And the informed consent was obtained from all subjects and/or their legal guardian(s). We also confirmed that all experiments were performed in accordance with relevant guidelines and regulations.

Collection and processing of urine samples

Patients were instructed to consume a light diet the day before sample collection. Morning urine (50 ml) from enrolled patients was collected in RNase-free centrifuge tubes, centrifuged at 3000 × g for 10 min at 4 °C, and the supernatant was collected to remove debris and dead cells. The cleared supernatant was then transferred to new centrifuge tubes, centrifuged at 13,000 × g for 2 min at 4 °C to further remove cellular fragments. After discarding the supernatant, the cleared supernatant was transferred to new RNase-free centrifuge tubes, labeled, and stored at − 80 °C for subsequent use.

Isolation and extraction of urinary exosomes

Processed urine samples were transferred to ultracentrifuge tubes and centrifuged at 13,500 × g for 20 min at 4 °C, followed by centrifugation at 200,000 × g for 2 h at 4 °C. After removing the supernatant, 1 ml of PBS buffer was added to resuspend the pellet, centrifuged at 200,000 × g for 1 h at 4 °C. The supernatant was discarded, and 200 μl of PBS buffer was added to resuspend the pellet, which was stored at − 80 °C for subsequent use.

Identification of urinary exosomes

Transmission electron microscopy (TEM)

The exosome suspension was mixed 1:1 with 4% PFA and placed on copper grids. After incubation for 20 min, the grids were stained with uranyl acetate solution and dried for 5 min. Exosome morphology was observed using transmission electron microscopy.

Nanoparticle tracking analysis (NTA)

Exosome samples were diluted according to the manufacturer’s instructions and analyzed using the Flow NanoAnalyzer U30 instrument (FloNovo Biosciences). NanoFCM software (NanoFCM Profession V1.0) was used to record particle trajectories, calculate the diameter distribution of all samples, and determine the exosome concentration of the original solution based on the dilution.

Western blotting

Western blotting was performed to further identify surface marker proteins of exosomes. Human-immortalized podocyte total protein was used as a control. RIPA buffer (Beyotime Biotechnology, China) with Protease Inhibitor Cocktail (APExBIO, America) at a ratio of 1:100 was used to extract exosome and podocyte proteins. Protein samples were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, blocked with 5% skim milk for 1 h, washed 3 times with TBST (10 min each), and then incubated overnight at 4 °C with primary antibodies against common exosome proteins CD63 (Abcam, America), CD9 (Abcam, America), and negative control protein Calnexin (Abcam, America). The membranes were washed and incubated with secondary antibodies at room temperature for 1 h. Protein bands were visualized using Immobilon TM Western Chemiluminescent HRP Substrate (Millipore, America) and imaged using the ChemiDoc Imaging System (BIO-RAD, America).

Real-time quantitative PCR (qPCR)

Exosome RNA was extracted using the Exosomal RNA Isolation Kit (Norgen Biotek, Canada) according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA using the Hifair®III 1st Strand cDNA Synthesis Kit (YISHENG, China) and subjected to gene expression analysis using the SYBRGreen method. Relative gene expression was calculated using the − ∆Ct method, and relative quantitative analysis of target miRNA was performed using the worm miR-39-3p (miDETECTTM miRNA External Control, RIBOBIO, China) expression as an external reference gene.

Primers for miR-142-3p, miR-301a-3p, miR-301a-5p, and miR-39-3p were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

Statistical analysis

SPSS® 25.0 software was used for statistical analysis. Results were presented as mean ± standard deviation, frequency, rate, median, or quartile. Spearman rank correlation analysis was used to analyze the correlation between clinical parameters of chronic kidney disease and its progression risk stratification. The sensitivity and specificity of exosomal miRNA were evaluated by receiver operating characteristic (ROC) curve analysis. P < 0.05 was considered statistically significant. Target genes of dysregulated miRNAs were predicted using the miRTarBase database (https://mirtarbase.cuhk.edu.cn/), and functional prediction of target genes was obtained through KEGG enrichment analysis using the R software clusterProfiler (version 3.14.3) package.

Results

Differential expression of exosomal miRNAs in diabetic nephropathy

Using the GEO database (http://www.ncbi.nlm.nih.gov/geo), we identified 19 differentially expressed miRNAs by analyzing DN patients compared to healthy controls or diabetic patients with membranous nephropathy. After a thorough literature review, miR-142-3p, miR-301a-3p, and miR-301a-5p were selected as potential candidate biomarkers for further validation (Fig. 1).

Fig. 1.

Differentially expressed miRNAs in DN: Differentially expressed genes between DN patients and diabetic patients or healthy controls were identified by GEO2R online tool, and the final screening data and differentially expressed miRNAs were miR-142-3p, miR-301a-3p, miR-301a-5p (|logFC|≥ 1 P < 0.01).

Identification of urinary exosomes

In this study, we extracted exosomes from urine samples of DN and T2DM patients using ultracentrifugation. We characterized the exosomes by structure, size and surface marker proteins. Transmission electron microscopy (TEM) revealed that exosomes exhibited a cup-shaped or vesicular structure (Fig. 2A). The diameter of exosomes predominantly ranged between 50 and 150 nm (Fig. 2B). Furthermore, Western blot analysis confirmed the presence of exosomal markers CD9 and CD63 in both groups’ urine-derived vesicles, whereas Calnexin, a negative control protein, was only detected in podocyte lysates (Fig. 2C).

Fig. 2.

Identification of exosomes: (A) The structure of exosomes was detected by transmission electron microscopy; (B) The size distribution of exosomes was measured by a single particle nanobiodetector; (C) Western blot assay was used to detect exosome surface marker proteins CD9 and CD63 and negative control protein Calnexin.

Clinical characteristics of screening queue patients

The screening queue included 3 T2DM patients (T2DM-A group) and 3 DN patients with definite renal pathologic findings (DN-A group). Statistical analysis revealed no significant differences between DN-A and T2DM-A groups in terms of age, gender, diabetes histor, systolic blood pressure, diastolic blood pressure, glycosylated hemoglobi, fasting blood glucose, urea nitrogen, serum creatinine, serum albumin, total protein, cholesterol, triglycerides, and LDL cholesterol. However, the DN-A group showed significantly lower eGFR (P = 0.020) and higher UACR (P = 0.034) compared to T2DM-A group (Table 1).

Table 1.

Clinical parameters of patients in T2DM-A and DN-A groups.

	T2DM-A(3)	DN-A(3)	P
Age	63.00 ± 15.39	55.67 ± 3.71	0.456
Male (%)	2(100)	3(100)	1.00
Diabetes history (years)	10.00(1, 10.00)	3.00(3.00, 3.00)	0.814
Systolic blood pressure (mmHg)	129.67 ± 111.71	153.67 ± 42.59	0.400
Diastolic blood pressure (mmHg)	79.67 ± 6.66	80.33 ± 26.65	0.968
Glycosylated hemoglobin (%)	10.57 ± 4.21	7.53 ± 1.33	0.300
Fasting blood glucose (mmol/L)	6.93 ± 1.71	6.84 ± 2.78	0.955
eGFR (ml/min1.73 m2)	102.93 ± 16.82	29.23 ± 29.68	0.020*
Serum creatinine (umol/L)	85.33 ± 40.53	420.33 ± 427.32	0.248
Urea nitrogen (mmol/L)	6.83 ± 4.15	18.17 ± 12.22	0.203
Cystatin C (mg/L)	1.01 ± 0.17	2.93 ± 1.90	0.223
Serum albumin (g/L)	35.90(35.90, 35.90)	31.60(28.60, 31.60)	0.507
Serum Total protein (g/L)	61.57 ± 4.63	54.00 ± 8.67	0.253
Cholesterol (mmol/L)	4.86 ± 1.70	5.68 ± 1.93	0.612
Triglyceride (mmol/L)	0.68(0.66, 0.68)	1.33(1.27, 1.33)	0.513
Low density lipoprotein (mmol/L)	2.74(1.82, 2.74)	2.93(2.88, 2.93)	0.275
UACR (mg/g)	19.53(0.00, 19.53)	7450.03 (4716.71, 7450.03)	0.034*

(*P < 0.05).

qRT-PCR validation of exosomal miRNAs in screening queue patients

Based on the initial miRNA screening results, we extracted RNA from purified exosomes obtained from patient urine and validated the expression of miR-142-3p, miR-301a-3p, and miR-301a-5p using qRT-PCR in the screening queue. The study found that miR-142-3p expression was significantly higher in the DN-A group compared to the T2DM-A group (P < 0.05), whereas miR-301a-3p and miR-301a-5p showed no significant differences between groups (Fig. 3).

Fig. 3.

Changes in the expression of urine exosomes miR-142-3p, miR-301-3a, and miR-301a-5p in the screening cohort: The expression of urine exosomes miR-142-3p, miR-301-3a, and miR-301a-5p in the screening cohort was detected by qRT-PCR. (*P < 0.05).

Clinical characteristics of validation queue patients

In the validation queue, we collected 54 DN patients (DN-B group) and 39 T2DM patients (T2DM-B group) to confirm the results from the screening queue. Comparison of clinical parameters between the two groups showed no statistical differences in gender, systolic blood pressure, diastolic blood pressure, glycosylated hemoglobin, fasting blood glucose, fasting C-peptide, fasting insulin, urea nitrogen, serum albumin, total protein, cholesterol, triglycerides, and LDL cholesterol. However, DN-B patients were older (P = 0.007), had a longer duration of diabetes (P = 0.048), lower eGFR (P = 0.001), and higher serum creatinine (P = 0.001) and UACR (P = 0.000) compared to T2DM-B group (Table 2).

Table 2.

Clinical parameters of T2DM-B group and DN-B group.

	T2DM-B(39)	DN-B(54)	P值
Age	52.67 ± 12.10	59.57 ± 11.59	0.007**
Male (%)	30(76.90)	36(66.70)	0.282
Diabetes history (years)	5(1, 14)	10(5.75, 15.25)	0.048*
Systolic blood pressure (mmHg)	135.15 ± 14.80	142.11 ± 20.04	0.070
Diastolic blood pressure (mmHg)	81.62 ± 11.48	82.59 ± 11.23	0.683
Glycosylated hemoglobin (%)	8.00(7.30, 9.50)	8.15(6.80, 10.10)	0.981
Fasting blood glucose (mmol/L)	7.44(6.50, 8.32)	7.56(6.00, 10.18)	0.794
Fasting C-peptide (ng/mL)	2.00(1.37, 2.63)	2.70(1.41, 3.65)	0.060
Fasting insulin (UIu/mL)	8.16(3.80, 10.30)	8.25(3.77, 15.45)	0.538
eGFR (ml/min1.73 m2)	106.53(99.07, 113.97)	94.92(69.91, 109.13)	0.001**
Serum creatinine (umol/L)	69.50(58.25, 79.00)	76.00(60.75, 94.25)	0.125
Urea nitrogen (mmol/L)	5.20(4.30, 6.10)	5.85(4.70, 7.50)	0.062
	T2DM-B(39)	DN-B(54)	P值
Cystatin C (mg/L)	0.75(0.68, 0.81)	0.87(0.74, 1.10)	0.001**
Serum albumin (g/L)	43.51 ± 4.40	41.80 ± 4.57	0.077
Serum Total protein (g/L)	69.17 ± 7.05	67.87 ± 5.67	0.333
Cholesterol (mmol/L)	4.73(4.03, 5.18)	4.75(4.05, 5.69)	0.413
Triglyceride (mmol/L)	1.51(1.07, 2.22)	1.54(1.05, 1.95)	0.569
Low density lipoprotein (mmol/L)	2.86(2.16, 3.17)	2.85(2.29, 3.25)	0.548
UACR (mg/g)	5.78(0.00, 13.89)	183.87(52.90, 719.52)	0.000**

(*P < 0.05; ** P < 0.01).

qRT-PCR validation of exosomal miRNAs in validation queue patients

In the validation queue, qRT-PCR was performed to validate the expression of miR-142-3p. Results showed that miR-142-3p expression was significantly higher in the DN-B group compared to the T2DM-B group (P < 0.05), which was consistent with the screening queue and earlier validation results (Fig. 4). To adjust for potential confounders such as age and duration of diabetes, multiple logistic regression analysis was conducted, revealing that even after adjusting for these factors, miR-142-3p expression remained statistically significant in DN patients (P = 0.040) (Table 3).

Fig. 4.

Changes in the expression of urine exosome miR-142-3p of patients in the validation cohort: qRT-PCR was used to detect the expression of urine exosome miR-142-3p in the validation cohort. (*P < 0.05).

Table 3.

Multi-factor logistic regression.

	B	Waldx2	P	Exp(B)	95% CI
miR-142-3p	0.140	4.201	0.040*	1.150	1.006	1.314
Age	0.049	4.569	0.033*	1.050	1.004	1.097
Diabetes history	0.008	0.060	0.807	1.009	0.944	1.077

(*P < 0.05).

Clinical diagnostic value of urinary exosomal miR-142-3p in DN

The diagnostic value of urinary exosomal miR-142-3p in DN was assessed by constructing ROC curves. The area under the curve (AUC) for miR-142-3p was 0.629, with a sensitivity of 41.4% and specificity of 87.2%. The optimal cutoff value was -10.944, with a positive predictive value of 81.5% and a negative predictive value of 51.5%. The Youden index was 0.287, and the P < 0.05, indicating statistical significance. These results suggest that miR-142-3p may serve as a novel biomarker for diagnosing DN (Table 4, Fig. 5).

Table 4.

Diagnostic value evaluation of urinary exosome miR-142-3p in DN.

	AUC	Cutoff	Sensitivity %	Specificity %	Positive predictive %	Negative predictive %	Youden index	P	95% CI
miR-142-3p	0.632	− 10.944	41.4	87.2	81.5	51.5	0.287	0.035*	0.513	0.745

(*P < 0.05) Jorden index = sensitivity + specificity − 1, cut-off value was determined by the Youden index.

Fig. 5.

ROC curve of urinary exosome miR-142-3p.

Correlation analysis of urinary exosomal miR-142-3p with clinical parameters and risk stratification indicators of chronic kidney disease progression

Spearman correlation analysis revealed that urinary exosomal miR-142-3p expression was positively correlated with UACR (ρ = 0.257, P = 0.013) and risk stratification indicators of chronic kidney disease progression (ρ = 0.231, P = 0.027) (Table 5, Fig. 6).

Table 5.

Correlation analysis of risk stratification between urinary exosome miR-142-3p and progression of chronic kidney disease.

Spearman	Risk stratification indicators of chronic kidney disease progression
	ρ	P
miR-142-3p	0.231	0.027*

(*P < 0.05).

Fig. 6.

Correlation between urinary exosome miR-142-3p and clinical parameters: Spearman correlation analysis was used to analyze the correlation between urinary exosome miR-142-3p and UACR. (ρ = 0.257 P = 0.013).

Functional prediction of urinary exosomal miR-142-3p

In this study, we used the miRTarBase database for bioinformatics analysis to predict the target and pathway of the regulation of miR-142-3p in urinary exosomes. The results showed that there were 179 target genes related to miR-142-3p in the database, and 48 pathways including fatty acid metabolism, fatty acid biosynthesis, Hippo signaling pathway, and cyclic guanylate protein kinase G (cGMP-PKG) signaling pathway were identified through KEGG enrichment analysis of these genes^23,24. Through extensive literature review, we believe that miR-142-3p may regulate the disease progression of DN through several signaling pathways, including fatty acid metabolism, fatty acid biosynthesis, Hippo signaling pathway, cGMP-PKG signaling pathway, AMPK and Wnt signaling pathway (Fig. 7).

Fig. 7.

KEGG enrichment pathway analysis circle of urine exosome miR-142-3p.

Discussion

Type 2 diabetes mellitus (T2DM) is a heterogeneous systemic metabolic disease characterized by various clinical and pathological features, including insulin deficiency, insulin resistance (IR), or both, leading to vascular complications that cause varying degrees of damage to vital organs such as the heart, brain, kidneys, and eyes. Diabetic nephropathy (DN) is the most common microvascular complication of T2DM, with approximately 40% of T2DM patients developing DN annually⁹. Currently, DN is a leading cause of end-stage renal failure and mortality in diabetic patients, characterized pathologically by mesangial matrix proliferation, podocyte injury, thickening of the glomerular basement membrane, interstitial fibrosis, and extracellular matrix accumulation, ultimately resulting in irreversible kidney damage that significantly affects patients’ quality of life and imposes substantial economic burden on society. In this study, we enrolled a total of 42 T2DM patients and 57 DN patients. Statistical analysis of patients’ general data and clinical indicators revealed that most DN patients had a diabetes history of over 10 years, with higher cystatin C levels compared to the T2DM group, along with significantly decreased eGFR and markedly increased UACR. This aligns with the traditional understanding of the natural progression of DN. However, with advancing research, differences between the clinical presentation and pathological damage of DN have been noted. It is well known that microalbuminuria is currently the best non-invasive marker for assessing DN. Nevertheless, recent studies have indicated that approximately 15–65% of diabetic patients with microalbuminuria may revert to normal levels of proteinuria, and 20–60% of diabetic patients progress to DN with reduced eGFR but without microalbuminuria. Additionally, some patients have been diagnosed with advanced glomerular lesions despite presenting with either microalbuminuria or no proteinuria²⁵. Therefore, a thorough understanding of the pathogenesis of DN and the search for novel non-invasive biomarkers beyond albuminuria are crucial for early prevention and treatment of DN.

Exosomes are heterogeneous membrane vesicles released by cells. Initially, exosome production was believed to be a mechanism for clearing metabolic waste during reticulocyte maturation²⁶. However, further research has revealed that exosomes can carry various bioactive substances, such as mRNA and miRNA, and play a role in mediating intercellular communication, making exosomes a recent focus of research. miRNAs are a class of small non-coding RNAs, approximately 22 nucleotides in length, that regulate transcription and translation processes by binding to the 3’ untranslated region of mRNA, thereby controlling the expression of target genes²⁷. Previous studies have shown that miRNAs are involved in the pathogenesis of kidney diseases and the progression of DN, closely related to processes such as autophagy, fibrosis, inflammation, and epithelial-mesenchymal transition²⁶. Furthermore, since miRNAs can stably exist in urine exosomes and multiple studies have confirmed that exosomes are secreted by various types of renal cells and participate in the pathophysiological mechanisms of DN, urinary exosome miRNAs may serve as novel biomarkers for diagnosing DN.

In recent years, along with the rapid development of high-throughput sequencing technology, various miRNAs involved in disease progression have been identified, making genetic-level disease diagnosis and treatment a crucial research approach. In this study, we screened differentially expressed miRNAs in DN and T2DM patients from the GEO database. Using qRT-PCR, we first identified increased expression of urinary exosomal miR-142-3p in both screening and validation cohorts of DN patients. We evaluated the clinical diagnostic value of urinary exosomal miR-142-3p in DN by plotting ROC curves. Consistent with previous research, miR-142-3p has been implicated in the development and prognosis of various cancers, including lung adenocarcinoma, breast cancer, prostate cancer, pancreatic cancer, and colorectal cancer^28–31. Studies have also linked miR-142-3p expression to the occurrence and progression of renal cell carcinoma. In Peng et al.³² evaluated the prognostic value of miR-142-3p in renal cell carcinoma, finding elevated expression associated with poorer overall survival, suggesting miR-142-3p as not only a biomarker for diagnosing renal cell carcinoma but also an independent predictor of adverse prognosis. Besides oncological diseases, research indicates miR-142-3p’s association with the progression of renal diseases. In Zhao et al.³³ observed decreased miR-142-3p expression in high-glucose-treated renal tubular epithelial cells, which, through targeted action on the BOD1 molecule, elevated its expression levels, thereby alleviating high-glucose-induced renal tubular epithelial cell damage. The proposed miR-142-3p/BOD1 axis offers new insights into DN diagnosis and treatment, although further research is needed to confirm whether miR-142-3p from different sources can exert varying effects on DN through different target points. Additionally, in another study, researchers noted in a type 1 diabetes model that miR-142-3p interferes with pancreatic islet autoimmunity via the miR142-3p/Tet2/Foxp3 axis³⁴, providing important insights into the pathogenesis of diabetes with clinical implications. Hence, these findings collectively suggest urinary exosomal miR-142-3p as a potential novel biomarker for diagnosing DN.

To further evaluate the clinical application value of miR-142-3p, we conducted correlation analyses with clinical indicators and risk stratification indices for the progression of chronic kidney disease. The results revealed a significant correlation between the expression of urinary exosomal miR-142-3p and the severity of clinical indicators of renal injury, as well as the risk stratification for chronic kidney disease progression. This indicates that urinary exosomal miR-142-3p may not only be a potential biomarker for diagnosing DN but also play a role in its progression, providing new avenues for disease prevention and treatment.

The pathogenesis of DN is highly intricate and remains incompletely understood to date. Current perspectives widely acknowledge that abnormalities in glucose metabolism, renal hemodynamics due to glomerular hyperperfusion, high transmembrane pressure, and hyperfiltration, along with oxidative stress, inflammation, and genetic factors, are closely interlinked with DN. In this study, to delve deeper into the potential role of miR-142-3p in DN’s pathological mechanisms, we identified 179 potential target genes of miR-142-3p through the miTarBase database. Subsequent enrichment analysis using the KEGG pathway revealed 48 pathways, including fatty acid metabolism, the Hippo signaling pathway, the cGMP-PKG signaling pathway, and the Wnt signaling pathway. Based on bioinformatics analysis and existing research, we propose that urinary exosomal miR-142-3p may contribute to DN’s pathological damage through pathways such as fatty acid metabolism, fatty acid biosynthesis, Hippo signaling, cGMP-PKG signaling, AMPK signaling, and Wnt signaling. Fatty acids are known as the primary energy source for metabolically active organs like the liver, muscles, and kidneys. Hence abnormalities in fatty acid metabolism can lead to varying degrees of organ damage. Recent studies have also highlighted abnormal fatty acid metabolism as a significant contributor to DN³⁵. In Nagai et al.³⁶ demonstrated that impaired fatty acid utilization leads to mitochondrial dysfunction and reactive oxygen species (ROS) production in DN. They also reported that inhibiting ROCK1 improves renal glomerular fatty acid utilization disorder by upregulating the AMPK/PGC-1a axis, underscoring the critical roles of fatty acid metabolism and AMPK signaling in DN. Another study using proteomic data from diabetic mice found reduced fatty acid biosynthesis and increased ketone body production, which contributes to atherosclerosis in mice, suggesting that reduced fatty acid biosynthesis may serve as a potential marker for diabetes-related vascular diseases³⁷. The Hippo signaling pathway, cGMP-PKG signaling pathway, AMPK signaling pathway, and Wnt signaling pathway have also been shown to play pivotal roles in the progression of DN. For example, Sun et al.³⁸ reported that YAP, a downstream effector of the Hippo pathway, is closely associated with renal interstitial fibrosis in diabetes. Under hyperglycemic conditions, Hippo signaling promotes YAP interaction with TEAD, resulting in cell proliferation and epithelial-mesenchymal transition, accelerating DN onset and progression. Chen et al.³⁹ found that sodium-glucose cotransporter inhibitors (DAP), oral medications for controlling blood glucose levels in diabetic patients, significantly downregulate genes associated with the cGMP-PKG signaling pathway in DN, such as CACNA1D, CACNA1C, ATP1B2, and IGF2, thereby inhibiting inflammation and fibrosis, and delaying DN progression. Lu et al.⁴⁰ indicated that AMPK phosphorylation is significantly reduced in renal tubular epithelial cells under high glucose conditions and in kidney tissues of T2DM mice, increasing cellular and tissue iron death, suggesting that AMPK may be a crucial pathway involved in DN iron death. Studies have confirmed that the Wnt signaling pathway plays a crucial role in kidney cell injury and repair through processes such as inflammation, fibrosis, vascular regeneration, and insulin secretion, with Wnt/β-catenin being extensively studied as the most typical Wnt signaling pathway^41,42. According to recent research, complement fragment C3a and C5a inhibitors can improve fibrosis and endothelial-mesenchymal transition in diabetic rats and high-glucose-treated glomerular endothelial cells by downregulating α-SMA, TGF-β, and increasing β-catenin expression through the Wnt/β-catenin pathway, indicating that C3a and C5a may participate in fibrosis processes in DN⁴³. Therefore, based on KEGG pathway analysis results and previous literature, we suggest that fatty acid metabolism, fatty acid biosynthesis, Hippo signaling pathway, cGMP-PKG signaling pathway, AMPK signaling pathway and Wnt signaling pathway may be the potential downstream mechanisms of miR-142-3p’s involvement in the occurrence and development of DN.

However, this study also has certain limitations: (1) The lack of follow-up means that the relationship between changes in urinary exosomal miR-142-3p levels and the progression of DN remains unclear. (2) Additionally, this study is single-center and has a limited sample size, necessitating further multicenter large-sample randomized controlled trials for validation.

Conclusion

In conclusion, in this study, we found for the first time that the expression of urinary exosome miR-142-3p was increased in DN patients through clinical samples, which has good clinical diagnostic value, and found that urinary exosome miR-142-3p was significantly correlated with the severity of risk stratification of proteinuria and even chronic kidney disease progression. The involvement of miR-142-3p in the disease was further predicted.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

YL contributed to the conception and design of the study. SL and CS wrote and reviewed the manuscript. SZ, SL, HL,JW,YT,XG and FQ revised the manuscript. SL and HL reviewed this article.

Funding

This study was supported by grants from the Young Taishan Scholars Program (tsqn202408366), and the National Natural Science Foundation of China (82170742).

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.