Completion of the human genome sequencing project highlighted the richness of the cellular RNA world, and opened the door to the discovery of a plethora of short and long non‐coding RNAs (the dark transcriptome) with regulatory or structural potential, which shifted the balance of pathological gene alterations from coding to non‐coding RNAs. Thus, disease risk assessment currently has to also evaluate the expression of new RNAs such as small micro RNAs (miRNAs), long non‐coding RNAs (lncRNAs), circular RNAs (circRNAs), competing endogenous RNAs (ceRNAs), retrogressed elements, 3′UTRs of mRNAs, etc. We are interested in the pathogenic mechanisms of atherosclerosis (ATH) progression in patients suffering Chronic Kidney Disease, and in this review, we will focus in the role of the dark transcriptome (non‐coding RNAs) in ATH progression. We will focus in miRNAs and in the formation of regulatory axes or networks with their mRNA targets and with the lncRNAs that function as miRNA sponges or competitive inhibitors of miRNA activity. In this sense, we will pay special attention to retrogressed genomic elements, such as processed pseudogenes and Alu repeated elements, that have been recently seen to also function as miRNA sponges, as well as to the use or miRNA derivatives in gene silencing, anti‐ATH therapies. Along the review, we will discuss technical developments associated to research in lncRNAs, from sequencing technologies to databases, repositories and algorithms to predict miRNA targets, as well as new approaches to miRNA function, such as integrative or enrichment analysis and their potential to unveil RNA regulatory networks.

Our interest in the mechanisms of atherosclerosis progression (ATHp) has led to the recent identification of 13 miRNAs and 1285 mRNAs whose expression was altered during ATHp. Here, we deepen the functional relationship among these 13 miRNAs and genes associated to oxidative stress, a crucial step in the onset and progression of vascular disease. We first compiled a list of genes associated to the response to oxidative stress (Oxstress genes) by performing a reverse Gene Ontology analysis (rGO, from the GO terms to the genes) with the GO terms GO0006979, GO1902882, GO1902883 and GO1902884, which included a total of 417 unique Oxstress genes. Next, we identified 108 putative targets of the 13 miRNAs among these unique Oxstress genes, which were validated by an integrated miRNA/mRNA counter-expression analysis with the 1285 mRNAs that yielded 14 genes, Map2k1, Mapk1, Mapk9, Dapk1, Atp2a2, Gata4, Fos, Egfr, Foxo1, Ccr7, Vkorc1l1, Rnf7, Kcnh3, and Mgat3. GO enrichment analysis and a protein–protein-interaction network analysis (PPI) identified most of the validated Oxstress transcripts as components of signaling pathways, highlighting a role for MAP signaling in ATHp. Lastly, expression of these Oxstress transcripts was measured in PBMCs from patients suffering severe coronary artery disease, a serious consequence of ATHp. This allowed the identification of FOXO1 and CCR7 as blood markers downregulated in CAD. These results are discussed in the context of the interaction of the Oxstress transcripts with the ATHp-associated miRNAs.

Background: Chronic kidney disease (CKD) is associated with endothelial dysfunctions thus prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors. Purpose of the study: We have previously reported that siRNA-silencing of CD40 (siCD40) reduced atherosclerosis (ATH) progression. Here, we have deepened on the effects of the siCD40 treatment by evaluating retrospectively, in stored kidneys from the siCD40 treated ApoE^−/− mice, the renal microcirculation (measured as the density of peritubular capillaries), macrophage infiltration and NF-κB activation. Methods: Kidneys were isolated after 16 weeks of treatment with the anti-CD40 siRNA (siCD40), with a scrambled control siRNA (siSC) or with PBS (Veh. group). Renal endothelium, infiltrating macrophages and activated NF-κB in endothelium were identified by immunohistochemistry, while the density of stained peritubular capillaries was quantified by image analysis. Results: ATH was associated with a reduction in renal MC, an effect reversed by the anti-CD40 siRNA treatment (3.8 ± 2.7% in siCD40; vs. 1.8 ± 0.1% in siSC; or 1.9 ± 1.6% in Veh.; p < 0.0001). Furthermore, siCD40 treatment reduced the number of infiltrating macrophages compared to the SC group (14.1 ± 5.9 cells/field in siCD40; vs. 37.1 ± 17.8 cells/field in siSC; and 1.3 ± 1.7 cells/field in Veh.; p = 0.001). NF-κB activation also peaked in the siSC group, showing lower levels in the siCD40 and Veh. groups (63 ± 60 positive cells/section in siCD40; vs. 152 ± 44 positive cells/section in siSC; or 26 ± 29 positive cells/section in veh.; p = 0.014). Lastly, serum creatinine was also increased in the siCD40 (3.4 ± 3.3 mg/dL) and siSC (4.6 ± 3.0 mg/dL) groups when compared with Veh. (1.1 ± 0.9 mg/dL, p = 0.1). Conclusions: Anti-CD40 siRNA therapy significantly increased the density of peritubular capillaries and decreased renal inflammation in the ATH model. These data provide a physiological basis for the development of renal diseases in patients with ATH. Furthermore, our results also highligth renal off-target effects of the siRNA treatment which are discussed. Graphical