Expression analysis of urinary exosome miR-136-5p in type 2 diabetic nephropathy and evaluation of its clinical diagnostic value

1. Preliminary screening of miRNAs: Through GEO database and reading related literature, miRNAs differentially expressed in type 2 diabetes and diabetic nephropathy were selected as potential candidate biomarkers for follow-up verification.
2. Collection and treatment of clinical samples: Urine of diabetic patients was collected from the First Affiliated Hospital of Shandong First Medical University in strict accordance with the drainage standard, and the basic information of patients was registered. Meanwhile, the collected samples were divided into type 2 diabetes group and diabetic nephropathy group according to whether the urine was accompanied by UACR≥300/g. Subsequently, the collected samples were centrifuged and retained for supernatant.
3. Isolation and identification of urinary exosomes: Ultrafast centrifugation method was used to separate the treated urine samples of exosomes, and then identified by transmission electron microscopy, nanoparticle analysis and Western blot respectively.
4. Real-time PCR was used to detect the changes in urinary exosomal miRNA expression levels of the two groups of patients, and statistical analysis and correlation analysis of clinical indicators of the differentially expressed mirnas were performed.
5. Target gene prediction and enrichment pathway analysis of differentially expressed mirnas were performed to screen out pathways that might be related to the occurrence and development of DKD, and then the relevant pathways were verified by immunofluorescence, immunocoprecipitate, Western blot, Real- time PCR and other methods.

Inclusion Criteria:

1. Age ≥18 years old;
2. Previously diagnosed with type 2 diabetes;
3. Typical diabetic symptoms (polydipsia, polydipsia, polyuria and weight loss) plus random blood glucose ≥11.1mmol/l;
4. Fasting blood glucose ≥7.0mmol/l, fasting is defined as at least 8 hours without intake of calories;
5. Oral glucose tolerance test (OGTT) 2-hour blood glucose ≥11.1mmol/ l, using a glucose load equivalent to 75 grams of anhydrous glucose dissolved in water (venous blood was drawn in all patients;
6. With or without UACR≥300mg/g.

Exclusion Criteria:

1. Refused to enroll patients;
2. Type 1 diabetic nephropathy and other special types of diabetic nephropathy; 3.Patients with kidney stones and urinary system infection;

4.Patients with autoimmune system diseases, malignant tumors, and blood system diseases; 5.Patients with severe chronic cardiopulmonary disease, chronic liver and kidney disease; 6.Patients with severe infectious diseases; #7#Use of glucocorticoids, immunosuppressants or cytotoxic drugs.

Patients with a clinically confirmed diagnosis of type 2 diabetes during hospitalization in the Department of nephrology and Endocrinology from December 2020 to June 2022.