Introduction: Diabetic nephropathy (DN) is the most common cause of endstage renal disease worldwide and new therapies are urgently needed. The studies focusing in new mechanistic insights into the (patho)physiology of renal injury in diabetes, as well as potentially leading to the development of novel therapeutic interventions in DN, through targeting non-coding RNA-regulated epigenetic mechanisms, are very important. This proposal builds on a recent work from Dr. Advani´s group, in which they showed that histone methyltransferase enhancer of zeste homolog 2 (EZH2) upregulation through inhibition of microRNA, miR-101, downregulated the expression of the endogenous antioxidant inhibitor, thioredoxin interacting protein (TxnIP), and attenuated oxidative stress in DN. Aims: The overall hypothesis to be tested is that miR-101 promotes renal injury in diabetes. The two specific aims are: 1) to determine the effect of miR-101 inhibition on renal structure and function in diabetic rats and 2) to examine the mechanisms through which miR-101 inhibition prevents high glucose-induced podocyte injury. Methods: AIM 1 - Male Sprague Dawley rats (8 weeks of age, n=12/group) will be randomized to receive a tail-vein injection of streptozotocin (50mg/kg, in 0.1 mol/L sodium citrate buffer) or buffer alone; 24-hours later, rats will be further randomized to receive LNA-anti-microRNA-101 (2mg/kg, twice weekly, s.c.) or scramble control. Rats will receive a thrice weekly s.c. injection of insulin (1-4 units). Metabolic (body weight, blood glucose, HbA1c, systolic blood pressure) and renal function (urine protein and albumin excretion, glomerular filtration rate, urinary 8-OHdG excretion and plasma creatinine) parameters will be continuously evaluated. After 8 weeks, rats will be sacrificed and renal structure will be analyzed: 1) miR-101 expression by real-time PCR (qPCR); 2) EZH2 protein expression by immunoblotting; 3) TxnIP expression by qPCR and immunoblotting; 4) podocyte ultrastructure and number by transmission electron microscopy (TEM); 5) mesangial matrix expansion and basement membrane thickening by TEM and by semiquantitive scoring system of PAS-stained kidney sections. AIM 2 - Differentiated mouse podocytes will be depleted of EZH2 through transfection of a dominant negative Flag-tagged EZH2 mutant using a retroviral vector or pharmacologically using DZNep (5¼M). TxnIP expression (qPCR and immunoblot) and intracellular reactive oxygen species (ROS, by CFDA method and flow cytometry) will be determined. Having confirmed that augmented EZH2 expression is implicated in the prevention of oxidative stress, we will next determine whether miR-101 inhibition results in increased association of EZH2 with the TxnIP promoter, by exposing podocytes to LNA-anti-microRNA-101 in the presence or absence of high glucose. Association of the EZH2 with the TxnIP promoter will be determined by chromatin immunoprecipitation (ChIP) and trimethylation of histone 3 lysine 27 at this site will be determined using an anti-H3K27me3 antibody. To determine whether increased thioredoxin (Trx) activity is responsible for the prevention of ROS accumulation, podocytes will be transfected with shRNA adenovirus directed against Trx (shADV- 275378) before CFDA-loading and to determine whether miR-101 inhibition regulates ROS- generation through regulation of NADPH oxidase, we will determine NOX4 expression (immunoblotting and qPCR) and activity (lucigenin-enhanced chemiluminescence method. Overall significance: With these series of experiments we expect to gain mechanistic insights into the role that miR-101 plays in the diabetic kidney at a time when microRNA inhibition is receiving increasing attention as a novel therapeutic approach.
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