Urinary exosomes aggravate diabetic kidney disease by inducing podocyte ferroptosis via the miR-217/SIRT1/Nrf2 pathway.

Markers of and Risk Factors for the Development and Progression of Diabetic Kidney Disease

Background and Aims Diabetic kidney disease (DKD), occurs in 20–40% of patients with diabetes mellitus (DM), is the leading cause of end-stage renal disease (ESRD). DKD is a clinical diagnosis mainly based on the persistent albuminuria and reduced estimated glomerular filtration rate (eGFR) [1]. However, increased UACR and reduced eGFR are the final consequences due to DKD, novel biomarkers are critical for predicting DKD development. Osteopontin (OPN) is a profibrotic adhesion phosphoprotein that participates in cell chemotaxis, adhesion, migration, and proliferation, as well as extracellular matrix (ECM) hyperplasia. Hyperglycemia could enhance OPN gene expression through the activation of the renin-angiotensin system (RAS), mTOR pathway, NF-κB, and TGF-β pathway and then cause podocyte injury and ECM hyperplasia. Therefore, OPN upregulation is not only the result of various pathophysiological processes in DKD, but also results in kidney injury. Osteopontin (OPN) could predict incident DKD in DM patients [2], N-terminal OPN (ntOPN) has a stronger profibrotic adhesion effect than full-length OPN. This study aims to reveal the clinical benefit of ntOPN as a potential marker to identify DM patients at high risk of DKD, and establish ntOPN-based diagnostic and forecast models for renal outcomes in DM patients. Method We performed a cross-sectional study of 316 adults with Type 1 DM≥ 5 years or Type 2 DM, then followed by a prospective observational cohort study of 143 adult DM patients without renal involvement at baseline and follow-up for at least one year. During the follow-up period, the primary endpoint was “DKD occurrence”, defined as the presence of one of the following conditions in DM patients [3]: (1) repeat UACR ≥30 mg/g at least 2 of 3 measurements within 3 to 6 months; (2) eGFR <60 mL/min/1.73 m2 for more than three months; (3) renal pathological findings were consistent with DKD. The secondary endpoint was “DKD progression”, which included: (1) eGFR sustained decreased by at least 25%; (2) development of ESRD, and/or need for renal replacement therapy; (3) death from the renal cause. Logistic regression analysis was performed to analyze the relationship between parameters and the events of DKD occurrence and progression. Receiver operator characteristic (ROC) analysis was used to assess the predictive ability of established models for clinical endpoints. Results The median value of urinary ntOPN (UntOPN) was 44.15 ng/ml in the cross-sectional cohort, DKD prevalence was significantly higher in the high UntOPN group than in the low UntOPN group. The ROC curves of UntOPN, urinary neutrophil gelatinase-associated lipocalin (UNGAL), and their combination indicated that both combination and ntOPN alone perform better than UNGAL for DKD diagnosis (Fig. 1A). In the prospective cohort, UntOPN was an independent risk factor and further improved the predictive ability for DKD occurrence and DKD progression than UNGAL (Figs. 1B and 1C). Based on the parameters detected as risk factors for DKD occurrence and progression, we set up a series of multi-biomarker panels for DKD prediction using UntOPN, UNGAL, serum cystatin C, serum creatinine (Scr), UACR, and TCH/HDL-C ratio. Compared with the model of Scr + UACR, the area under ROC curve (AUC) of the six-biomarker model was higher, and also ranked the highest among the six ROC curves in predicting 1-year risk of DKD occurrence and DKD progression (Figs. 1B and 1C). Conclusion Our results showed that urinary ntOPN is associated with DKD development, and elevated urinary ntOPN is an independent predictor for DKD occurrence and progression. Compared with the traditional biomarkers of Scr + UACR, our multi-biomarker models based on urinary ntOPN performed better in predicting DKD development, which could provide more accurate tools for DKD risk prediction, thereby improving the renal prognosis in DM patients.

Diabetic Kidney Disease (c. 2018).

Background Diabetic kidney disease (DKD) is one of the most common chronic complications of type 2 diabetes mellitus (T2DM), and it is particularly important to identify a high-quality method for evaluating disease progression. Urinary exosomes contain microRNA that might promise early diagnostic and monitoring markers of DKD. The present study aimed to identify novel exosome-related markers associated with inflammation and fibrosis to assess the progression of DKD. Method Exosomes were extracted from the urine of 83 participants to determine the expression levels of miRNA-615-3p and miRNA-3147 in 20 healthy people, 21 patients with T2DM and 42 patients with DKD, as determined by RT-qPCR. The circulating expression level of TGF-β1 was detected by ELISA. Serum Cystatin C was measured by a latex-enhanced immunoturbidimetric method. The correlation analyses were performed for all clinical and laboratory parameters. Result The expression level of urinary exosomal miRNA-615-3p in DKD patients was significantly higher than that in the control group and the T2DM group by RT-qPCR. The expression of miRNA-3147 showed an upward trend in the three groups of subjects, but it was not statistically significant. The urinary exosomal miRNA-615-3p was positively correlated with serum Cystatin C, plasma TGF-β1, creatinine, BUN, PCR and 24-h urine protein, and negatively correlated with eGFR and albumin. The diagnostic efficacy of urinary exosomal miRNA-615-3p combined with the ACR was higher than that of ACR alone. Conclusions Urinary exosomal miRNA-615-3p may be used as a novel biomarker for evaluating the progression of DKD, and may be involved in the process of inflammation and fibrosis in DKD. The combined diagnosis of urinary exosomal miRNA-615-3p and ACR may be used as more stable and sensitive diagnostic criteria for DKD.

Understanding, and Reversing, Metabolic Memory Is Within Reach

Rationale and Strategies for Early Detection and Management of Diabetic Kidney Disease

Clinical Challenges in Diagnosis and Management of Diabetic Kidney Disease

BACKGROUND AND AIMS Urinary exosomes are small extracellular vesicles mainly secreted by epithelial cells of urinary system, reflecting the composition and characteristics of bioactive molecules in physiology and disease conditions of kidney. Diabetic nephropathy (DN) has become the leading cause of CKD and ESRD in the world. The purpose of this study was to describe the proteomic characteristics by LC-MS/MS, and clarify the protein composition and functional changes of urinary exosomes in DN. METHOD A total of 50 mL of first morning urine were collected from 24 subjects, including healthy controls group (Ctrl, n = 8), type 2 diabetes mellitus (T2DM, n = 8) and renal biopsy confirmed DN (n = 8). Exosomes were isolated by differential centrifugation and proteins were extracted and analyzed by LC-MS/MS. The types and expression abundance of proteins in urinary exosome were identified. Differential expression proteins (DEPs) were screened, and gene ontology (GO) and Reactome pathway enrichment analysis were performed to reveal the composition and function changes of DEPs in urinary exosomes of DN. RESULTS Under transmission electron microscope, scattered vesicles with diameters of 40–100 nm were observed in the urine. Nanoparticle tracking analysis showed that the number of urine exosomes was about 10^ 11 to 10^ 12 in 24-h urine with a peak diameter of about 100 nm. Western blot showed the expression of CD63, TSG101 and CD9, which were regarded as the markers of exosome. An average of 3110 (2506∼3380) proteins were identified in the urinary exosomes of the 24 subjects. In each group, the numbers of co-expressed proteins in urinary exosomes of eight subjects were 1680, 1309 and 1416, respectively. GO Cellular Component (CC) analysis showed that extracellular exosomes ranked first in the functional annotation of co-expressed proteins. Subcellular localization showed that the proportions of proteins annotated as extracellular exosome in the three groups were 77%, 81% and 76%, respectively. In upregulated proteins of urinary exosome in DN, GOCC annotation showed that ‘Extracellular exosome’ ranks first. GO molecular function (MF) enrichment analysis showed that the top 10 terms identified that pathways associated with complement activation such as ‘Regulation of Complement activation’, ‘Complement activation,’ ‘Complement activation, classical Pathway’ and ‘Complement activation, alternative Pathway’. Reactome pathway analysis showed that ‘Complement cascade’ and ‘Regulation of complement cascade’ were significantly activated in the top 10 pathways. CONCLUSION Exosomes can be steadily isolated from urine of healthy adults, T2DM and DN, and about 2506 to 3380 proteins can be identified by LC-MS/MS technique, of which about 76%–83% are related to the secretion and composition of exosomes. DEP analysis showed significant enrichment of exosome production-related annotations in urinary exosomes in DN patients compared with HC or T2DM; GOMF and Reactome pathway enrichment analysis showed that pathways related to complement activation were significantly enriched in the urinary exosomes of DN. This study provides proteomics characters of urinary exosome for healthy adults, T2DM and DN patients, suggesting that the production of urinary exosome is increased during DN, and complement activation may be one of the main characteristics of urinary exosome in DN, of significance of reference for studying the pathogenesis and developing new biomarkers of DN.

504-P: TGF-ß1 and VCAM-1 as Early Diagnostic Biomarkers of Diabetic Kidney Disease Progression

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.

Diabetic kidney disease (DKD) is a severe complication of diabetes mellitus. Urinary exosomal miRNAs play a prominent regulatory role in the pathogenesis of DKD, but the potential mechanisms remain largely unknown. Our research was designed to explain the pathogenesis of urine-derived exosomal microRNA-516b-5p (miR-516b-5p) in the DKD development. Urine-derived exosomes were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Immunofluorescence staining was used to detect cellular internalization. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis was performed to measure the levels of miR-516b-5p and SIRT3. The secretion of inflammatory cytokines and Caspase-1 activity were evaluated via ELISA and flow cytometry, respectively. Expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome markers and genes associated with the SIRT3/AMPK signaling pathway were measured using Western blot. Bioinformatics tools and dual-luciferase reporter gene assay were used to confirm the correlation between miR-516b-5p and SIRT3. Blood glucose and renal function indexes were determined by the corresponding commercial kits. Hematoxylin and eosin (H&E) staining was exploited to examine the renal pathological changes. MiR-516b-5p was memorably upregulated in HKB-20 cells exposed to DKD-Exo. DKD-Exo introduction led to an increase in Caspase-1 activity, promoted inflammatory response and NLRP3 inflammasome activity, and inactivation of SIRT3/AMPK signaling pathway, which was partially reversed by silencing miR-516b-5p. SIRT3 was identified as a target gene of miR-516b-5p. SIRT3 overexpression reversed the influences of DKD-Exo and miR-516b-5p mimic. In the in vivo model, DKD-Exo exacerbated streptozotocin (STZ)-induced kidney injury through promoting inflammatory response and activating the NLRP3 inflammasome. Urinary exosomal miR-516b-5p plays a key role in DKD by promoting inflammatory response and activating the NLRP3 inflammasome through the SIRT3/AMPK pathway.NEW & NOTEWORTHY Urinary exosomal miR-516b-5p plays a key role in diabetic kidney disease (DKD) by promoting inflammatory response and NOD-like receptor family pyrin domain containing 3 (NLRP3)inflammasome activation through the SIRT3/AMPK pathway.

FTO-mediated m6 A modification of SOCS1 mRNA promotes the progression of diabetic kidney disease.

Although diabetic kidney disease is the leading cause of ESKD in the United States, identifying those patients who progress to ESKD is difficult. Efforts are under way to determine if plasma biomarkers can help identify these high-risk individuals. In our case-cohort study of 894 Chronic Renal Insufficiency Cohort Study participants with diabetes and an eGFR of <60 ml/min per 1.73 m2 at baseline, participants were randomly selected for the subcohort; cases were those patients who developed progressive diabetic kidney disease (ESKD or 40% eGFR decline). Using a multiplex system, we assayed plasma biomarkers related to tubular injury, inflammation, and fibrosis (KIM-1, TNFR-1, TNFR-2, MCP-1, suPAR, and YKL-40). Weighted Cox regression models related biomarkers to progression of diabetic kidney disease, and mixed-effects models estimated biomarker relationships with rate of eGFR change. Median follow-up was 8.7 years. Higher concentrations of KIM-1, TNFR-1, TNFR-2, MCP-1, suPAR, and YKL-40 were each associated with a greater risk of progression of diabetic kidney disease, even after adjustment for established clinical risk factors. After accounting for competing biomarkers, KIM-1, TNFR-2, and YKL-40 remained associated with progression of diabetic kidney disease; TNFR-2 had the highest risk (adjusted hazard ratio, 1.61; 95% CI, 1.15 to 2.26). KIM-1, TNFR-1, TNFR-2, and YKL-40 were associated with rate of eGFR decline. Higher plasma levels of KIM-1, TNFR-1, TNFR-2, MCP-1, suPAR, and YKL-40 were associated with increased risk of progression of diabetic kidney disease; TNFR-2 had the highest risk after accounting for the other biomarkers. These findings validate previous literature on TNFR-1, TNFR-2, and KIM-1 in patients with prevalent CKD and provide new insights into the influence of suPAR and YKL-40 as plasma biomarkers that require validation.

Role of Kidney Biopsies for Biomarker Discovery in Diabetic Kidney Disease.

MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.