RNA Next Gen Sequencing of Kidneys from Diabetic Rats Provides Insights Into Reno‐protective Effects Of Vitamin D Receptor Activation

Abstract

IntroductionDiabetic nephropathy (DN) is the leading cause of end stage renal disease. The current therapy of DN remains suboptimal despite many currently available options. Recent experimental studies including from our laboratory and clinical reports support the therapeutic potential of vitamin D receptor activators, particularly paricalcitol. However molecular mechanisms underlying such protective effects are unclear. The goal of this study was to evaluate global gene expression changes using next‐generation sequencing‐based transcriptome analysis following paricalcitol treatment in obese ZSF1 rats, a murine model of metabolic syndrome that develops DN.MethodsAll rats were fed with a high fat diet to maintain hyperglycemia from 8 to 31 weeks. The test group of rats was injected subcutaneously twice a week with 0.2 μg of paricalcitol from 21 to 31 weeks. Following treatment, kidneys were harvested and total RNA was isolated and processed for transcriptome analysis using Illumina HiSeq 2500. Paricalcitol‐ treated ZSF1 rats were compared with obese untreated rats. The differential gene list from this analysis was imported into Ingenuity® Pathway Analysis (IPA®) for global transcriptome analysis and pathway mapping.ResultsWe detected 650 genes that had a significant change of at least 2 fold based on log 2 fluorescence intensity values. Of these 568 transcripts were up‐regulated whereas 82 transcripts were down regulated. Established pathways that were dominantly regulated include the Wnt signaling pathway that is primarily associated with cell cycle regulation. Specific molecules that were regulated with this pathway are Wnt3 Wnt9B and Wnt11 that were up regulated. Wnt 10b was also down regulated. OX40 signaling that involves regulation of the NF kB pathway was also regulated. Vitamin D signaling molecules that significantly changed following paricalcitol include the transcription factor enhancer binding protein α (EBPα), interleukin‐12 (IL‐12), interferon γ (IFNγ) and IP‐10. Several pathways that have previously been linked to diabetic nephropathy were differentially regulated by paricalcitol. These include endothelial nitric oxide synthase (eNOS) signaling, vascular endothelial growth factor (VEGF), protein kinase C and NF kB.ConclusionsParicalcitol treatment induces significant changes of gene expression in the kidney and most of these changes involve up regulation of several genes. Wnt signaling as well as changes in vitamin D signaling are regulated by paricalcitol. Thus the reno‐protective effects by paricalcitol treatment possibly involve inhibition of signaling pathways that have previously been associated with the progression of DN.Support or Funding InformationSupported by Woirhaye Research Endowment