Supplementary MaterialsS1 File: Additional procedures and information related to sequencing data analysis. injection of streptozotocin (STZ, 50 mg/kg body weight). Non-diabetic control (CTRL) rats were injected with vehicle. Insulin (INS) treatment (5U/d, s.c.) was provided to 50% of the DM rats. Urine samples were collected at weeks 3, 6, and 9 following injections and UE prepared. An increase in miR-451-5p and miR-16, observed by pilot small RNA sequencing of UE RNA, was confirmed by quantitative real-time polymerase chain reaction (qPCR) and selected for further study. Subsets of rats were euthanized after 3, 6, and 9 weeks of diabetes for renal pathology analysis, including determination of the tubulointerstitial fibrotic index (TFI) and glomerulosclerotic index (GI) scores. qPCR showed a substantial rise in miR-451-5p in GSK1120212 novel inhibtior UE from DM rats during the course of diabetes, with a significant rise (median fold switch 1000) between 3 and 6 weeks. Moreover, UE miR-451-5p at 6 weeks predicted urine albumin at 9 weeks (r = 0.76). A delayed but significant rise was also observed for miR-16. In contrast, CDC2 mean urine albumin only increased 21% between 3 and 6 weeks (non-significant rise), and renal TFI and GI were unchanged till 9 weeks. Renal expression of miR-451-5p and miR-16 (at 10 weeks) did not correlate with urine levels, and moreover, was negatively associated with indices of renal pathology (r-0.70, p = 0.005 for TFI and r-0.6, p0.02 for GI). Overall, a relative elevation in renal miR-451-5p and miR-16 in diabetes appeared protective against diabetes-induced kidney fibrosis; while UE miR-451-5p may hold prognostic value as an early and GSK1120212 novel inhibtior sensitive non-invasive indication of renal disease. Introduction Over one third of diabetic patients develop serious complications including nephropathy [1, 2]. A test for the rise in urine albumin levels (albuminuria) is routinely used for GSK1120212 novel inhibtior non-invasive serial monitoring of renal injury in these patients [3, 4]. However, it has limited ability to predict the earliest stages of diabetic nephropathy . Early signatures such as microRNAs (miRNAs) have the potential to identify patients at risk and may improve disease prognosis. MiRNAs control mRNA expression of multiple genes and are thus, critical for many physiological processes including cell proliferation, cell differentiation, and cell death [6, 7]. MicroRNA are small (21C25 nucleotides) non-coding, single-stranded RNA molecules which are highly conserved . They are endogenously produced and play significant role in the regulation of genes at a post-transcriptional level. They bind to the 3-untranslated region (UTR) of the target mRNA, inducing its degradation and thereby, resulting in translational repression . Due to their capability to regulate gene expression at the mRNA level, they serve as important upstream players in various cellular and physiological activities, including cell development, differentiation, proliferation, and apoptosis, and also in a variety of human diseases . Moreover, miRNA mis-expression has been implicated in the pathogenesis of both diabetic, as well as, non-diabetic kidney diseases [11C18]. However, studies to examine the value of miRNA signatures for early diagnosis of diabetic nephropathy and to categorize subsets of diabetic patients that go on to develop overt nephropathy, a major clinical challenge, are lacking . Moreover, kidney biopsy would not be considered the method of choice to serially monitor altered microRNA signatures in patients. In this light, exosomes in urine could show helpful, as they have been shown to encapsulate biomolecules of renal origin including miRNAs [19, 20]. Exosomes are 30C100-nm intraluminal vesicles of multivesicular body (MVB). These are released upon exocytic fusion of the MVB with the plasma membrane and are increasingly recognized as a novel mode of cell-independent communication . These tiny vesicles were recently discovered in urine by  and named, urinary exosomes (UE). Other than the presence of proteins, these vesicles are also enriched in mRNAs, microRNAs, and other non-coding RNAs . Isolation of UE from total urine aids in enrichment of less-abundant biomolecules, including miRNAs, with a potentially high diagnostic value relative to the physiological and pathological state of the renal system [19, 23, 24]. Data on human UE reported by us as well as others, have suggested the usefulness of these vesicles as early non-invasive markers for diabetic nephropathy [23, 25]. Overall, microRNA analysis in urinary exosomes could lead to the discovery of new non-invasive biomarkers for early kidney disease [26, 27], and provide us with a better understanding.