Diabetic kidney disease is a growing health burden that lacks specific early non-invasive diagnostic procedures. To approach a solution for this clinical need, we sequenced microRNAs of urinary extracellular vesicles and performed biomarker discovery by small RNA sequencing in a type 1 diabetes cohort including males with and without albuminuria. The results were replicated by sequencing or qPCR in two independent cohorts and four previously published datasets including type 1 and 2 diabetes as well as both sexes. Non-diabetic and prostate cancer cohorts were used as additional controls and miRNAs changed due to preanalytical urine collection variables were excluded. Using these data, we additionally validated previously identified reference candidate miRNAs. Correlations with clinical parameters, receiver operating characteristic analysis, targeted mRNAs and pathways including integrated single cell data, and targeted circulating proteins from type 1 and 2 diabetes cohorts were analyzed. We pinpointed 6 stable microRNAs, 11 differentially expressed microRNAs, 9 target proteins and 16 DKD-associated pathways in individuals with diabetic kidney disease. Replication showed that the differentially expressed miRNAs in DKD were partly shared between diabetes subtypes and sexes with overall strongest evidence for miR-192-5p, miR-146a-5p, miR-486-5p, and miR-574-5p. Combination of these miRNAs with clinical variables showed potential to classify individuals with the fastest kidney function decline (sensitivity 0.75-1.00 and specificity 0.83-1.00) even in the normoalbuminuria group, thus holding the potential as early diagnostic markers. Altogether, the candidate microRNAs show specificity for diabetic kidney disease, identify declining kidney function, and target key kidney cell types, mRNAs, proteins, and pathogenic mechanisms.

P-HG is an advisory board member for AstraZeneca, Bayer, Boehringer Ingelheim, Merck Sharp & Dohme, Nestle, Novo Nordisk, and has received lecture fees from Astellas, AstraZeneca, Bayer, Berlin Chemie, Boehringer Ingelheim, Menarini, Merck Sharp & Dohme, Medscape, Novo Nordisk, and Sanofi. AG is founder of Real World Genetics Oy.

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115974. The JU receives support from the European Union Horizon 2020 research and innovation programme and EFPIA and JDRF. Any dissemination of results reflects only the author's view; the JU is not responsible for any use that may be made of the information it contains. The DIREVA Study (A.K) has been supported by the Vasa Hospital District, State Research Funding via the Turku University Hospital, Vasa Central Hospital, Jakobstadsnejdens Heart Foundation, the Medical Foundation of Vaasa and the Finnish Medical Foundation. The FinnDiane study has been supported by Folkhalsan Research Foundation, Wilhelm and Else Stockmann Foundation, Liv och Halsa Society, Sigrid Juselius Foundation (220027), State funding for university-level health research by Helsinki University Hospital, Novo Nordisk Foundation (NNF23OC0082732), and Diabetes Research Foundation. This study has also been financially supported (TT, OD) by grants from Folkhalsan Research Foundation, the Sigrid Juselius Foundation, The Academy of Finland (grants no. 263401, 267882, 312063, 336822, 312072,336826, 317599), University of Helsinki, Ollqvist Foundation, the Health Care Center in Vasa, and the Finnish Cultural Foundation personal grant (#240316) to KB.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study protocol of the FinnDiane Study has also been approved by the Ethical Committee of the Helsinki and Uusimaa Hospital District (FinnDiane 491/E5/2006; 238/13/03/00/15; HUS/3313/2018), and that of DIREVA, by the Ethics Committees of the Vaasa Central Hospital (6/2007) and Turku University Hospital (48/1801/2014; 116/1805/2016).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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The raw count data for miRNA sequencing data used in this manuscript (discovery phase - T1D FinnDiane and DIREVA studies) is provided in the Supplementary Table S3a. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request compatible with the study consents.