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      Urinary Exosomal microRNA-451-5p Is a Potential Early Biomarker of Diabetic Nephropathy in Rats

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          Abstract

          Non-invasive renal signatures can help in serial monitoring of diabetic patients. We tested whether urinary exosomal (UE) microRNA (miR) analysis could non-invasively predict renal pathology in diabetic rats during the course of diabetes. Diabetes mellitus (DM) was induced in male Wistar rats by a single intraperitoneal 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 UE from DM rats during the course of diabetes, with a significant rise (median fold change >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, 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, p≤0.02 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 sensitive non-invasive indicator of renal disease.

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          Most cited references 36

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          MicroRNAs in development and disease.

          MicroRNAs (miRNAs) are a class of posttranscriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. There are currently over 10,000 miRNAs that have been identified in a range of species including metazoa, mycetozoa, viridiplantae, and viruses, of which 940, to date, are found in humans. It is estimated that more than 60% of human protein-coding genes harbor miRNA target sites in their 3' untranslated region and, thus, are potentially regulated by these molecules in health and disease. This review will first briefly describe the discovery, structure, and mode of function of miRNAs in mammalian cells, before elaborating on their roles and significance during development and pathogenesis in the various mammalian organs, while attempting to reconcile their functions with our existing knowledge of their targets. Finally, we will summarize some of the advances made in utilizing miRNAs in therapeutics.
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            Diabetic nephropathy: diagnosis, prevention, and treatment.

            Diabetic nephropathy is the leading cause of kidney disease in patients starting renal replacement therapy and affects approximately 40% of type 1 and type 2 diabetic patients. It increases the risk of death, mainly from cardiovascular causes, and is defined by increased urinary albumin excretion (UAE) in the absence of other renal diseases. Diabetic nephropathy is categorized into stages: microalbuminuria (UAE >20 microg/min and or =200 microg/min). Hyperglycemia, increased blood pressure levels, and genetic predisposition are the main risk factors for the development of diabetic nephropathy. Elevated serum lipids, smoking habits, and the amount and origin of dietary protein also seem to play a role as risk factors. Screening for microalbuminuria should be performed yearly, starting 5 years after diagnosis in type 1 diabetes or earlier in the presence of puberty or poor metabolic control. In patients with type 2 diabetes, screening should be performed at diagnosis and yearly thereafter. Patients with micro- and macroalbuminuria should undergo an evaluation regarding the presence of comorbid associations, especially retinopathy and macrovascular disease. Achieving the best metabolic control (A1c 1.0 g/24 h and increased serum creatinine), using drugs with blockade effect on the renin-angiotensin-aldosterone system, and treating dyslipidemia (LDL cholesterol <100 mg/dl) are effective strategies for preventing the development of microalbuminuria, in delaying the progression to more advanced stages of nephropathy and in reducing cardiovascular mortality in patients with type 1 and type 2 diabetes.
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              Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.

              Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                21 April 2016
                2016
                : 11
                : 4
                Affiliations
                [1 ]Department of Molecular Medicine & Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
                [2 ]Department of Medicine, Georgetown University, Washington, DC, United States of America
                [3 ]Structural Biology and Bioinformatics Division, CSIR - Indian Institute of Chemical Biology, Kolkata, India
                Emory University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AM DG S. Tiwari. Performed the experiments: AM RSS DG MK AU. Analyzed the data: AM RSS DG CME S. Tripathy S. Tiwari. Contributed reagents/materials/analysis tools: RSS S. Tripathy S. Tiwari. Wrote the paper: AM CME S. Tripathy S. Tiwari.

                Article
                PONE-D-15-48249
                10.1371/journal.pone.0154055
                4839711
                27101382
                © 2016 Mohan et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 7, Tables: 0, Pages: 18
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001501, University Grants Commission;
                Award ID: F 2-89/98; SA I
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001411, Indian Council of Medical Research;
                Award ID: 3/1/3JRF-2009/HRD-108(21088)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001407, Department of Biotechnology, Ministry of Science and Technology;
                Award ID: BT/PR2698/AGR/36/705/2011
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001412, Council of Scientific and Industrial Research;
                Award ID: 22/12/2013(ii)EU-V
                Award Recipient :
                This work was supported by grant to S. Tiwari from Department of Biotechnology, India (BT/PR2698/AGR/36/705/2011) ( http://www.dbtindia.nic.in/). AM was supported by a Senior Research Fellowship from University Grant Commission, India (F 2-89/98; SA I) ( http://www.ugc.ac.in/). RSS was supported by a Senior Research Fellowship from Indian Council of Medical Research, India ( http://www.icmr.nic.in/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Endocrinology
                Endocrine Disorders
                Diabetes Mellitus
                Medicine and Health Sciences
                Metabolic Disorders
                Diabetes Mellitus
                Biology and Life Sciences
                Anatomy
                Renal System
                Kidneys
                Medicine and Health Sciences
                Anatomy
                Renal System
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                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
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                Anatomy
                Body Fluids
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                Biochemistry
                Proteins
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                Endocrinology
                Diabetic Endocrinology
                Insulin
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                Insulin
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                Molecular biology
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