Alpha Omega Alpha Honor Medical Society

2014 Research Abstract

Role of hsa-miR-4747-5p and hsa-miR-4313 in the regulation of CYP2B1 and CYP4F4 activity in diabetic nephropathy-induced kidney injury: An in-vivo and in-vitro model of Type I Diabetes

Investigator: Rami Diab, American University of Beirut School of Medicine

Mentor(s): Assad E. Eid, PhD, and Hanna Abboud, MD

Abstract

Diabetic nephropathy is amongst the most serious complications of diabetes affecting up to 25% and 40% of all type 1 and type 2 diabetics respectively. Increased oxidative secondary to heightened levels of Reactive Oxygen Species (ROS) have been correlated with worsening outcomes in diabetic nephropathy (DN). The renally active CYP P450 arachidonic acid metabolites, HETEs and EETs, have also been implicated in DN, with HETEs showing a deterioration and EETs showing a reversal in objective markers of kidney injury paralleled by an increase and decrease in ROS production respectively. MicroRNAs (miRNAs) are small (22 nucleotide long) non-coding RNAs active in the down regulation of gene expression through their binding of 3’UTRs of mRNA targets, and inhibition their translation and/or facilitating their degradation. Little research has been pursued bridging miRNA regulation of renal CYP enzyme expression with concomitant ROS production, and resulting outcomes of DN. In our study, male Sprague Dawley rats were given an intravenous injection of streptozotocin (STZ) (55mg/kg) in mimicking a model of Type I diabetes. MicroRNA were dissolved in propylene glycol/Tween 80 (4/1) and injected subcutaneously into Sprague Dawley male rats, yet no effect was seen neither on CYP4F4 nor on CYP2B1 protein expression or mRNA expression. Alternatively, sense and antisense oligonucleotides targeting both HETE-generating CYP4F4 and EET-generating CYP2B1 were administered using ALZET mini pumps for four weeks (n=10). Down regulation of CYP4F4 (antisense CYP4F4 treatment arm) showed reversal of diabetic kidney injury whereas down regulation of CYP2B1 (antisense CYP2B1 treatment arm) showed a worsening pattern of DN in comparison with sense CYP4F4 treated, sense CYP2B1 treated, and untreated diabetic rats respectively. This was evidenced by an increase and decrease in all of extracellular matrix fibronectin expression, ROS production, podocyte loss, and albuminuria respectively. To our knowledge these are the first in vivo observations suggesting an antagonistic role of both the hydroxylase (HETE producing) and epoxygenase (EET producing) pathways of arachidonic acid metabolism by renal CYP P-450 enzymes in a rat model. We concluded that subcutaneously administered microRNA did not reach the rat kidney and that oligonucleotide-based down regulation of CYP4F4 and CYP2B1 was successful in altering objective markers of diabetic kidney injury, supporting its further investigation as a novel approach for the treatment of DN.

Last modified: 10/20/15

Updated on October 20, 2015.


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