Parallel Analysis of mRNA and microRNA Microarray Profiles to Explore Functional Regulatory Patterns in Polycystic Kidney Disease: Using PKD/Mhm Rat Model

<div><p>Autosomal polycystic kidney disease (ADPKD) is a frequent monogenic renal disease, characterised by fluid-filled cysts that are thought to result from multiple deregulated pathways such as cell proliferation and apoptosis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of many genes associated with such biological processes and human pathologies. To explore the possible regulatory role of miRNAs in PKD, the PKD/Mhm (cy/+) rat, served as a model to study human ADPKD. A parallel microarray-based approach was conducted to profile the expression changes of mRNAs and miRNAs in PKD/Mhm rats. 1,573 up- and 1,760 down-regulated genes were differentially expressed in PKD/Mhm. These genes are associated with 17 pathways (such as focal adhesion, cell cycle, ECM-receptor interaction, DNA replication and metabolic pathways) and 47 (e.g., cell proliferation, Wnt and Tgfβ signaling) Gene Ontologies. Furthermore, we found the similar expression patterns of deregulated genes between PKD/Mhm (cy/+) rat and human ADPKD, PKD1^L3/L3, PKD1^−/−, Hnf1α-deficient, and Glis2^lacZ/lacZ models. Additionally, several differentially regulated genes were noted to be target hubs for miRNAs. We also obtained 8 significantly up-regulated miRNAs (rno-miR-199a-5p, −214, −146b, −21, −34a, −132, −31 and −503) in diseased kidneys of PKD/Mhm rats. Additionally, the binding site overrepresentation and pathway enrichment analyses were accomplished on the putative targets of these 8 miRNAs. 7 out of these 8 miRNAs and their possible interactions have not been previously described in ADPKD. We have shown a strong overlap of functional patterns (pathways) between deregulated miRNAs and mRNAs in the PKD/Mhm (cy/+) rat model. Our findings suggest that several miRNAs may be associated in regulating pathways in ADPKD. We further describe novel miRNAs and their possible targets in ADPKD, which will open new avenues to understand the pathogenesis of human ADPKD. Furthermore they could serve as a useful resource for anti-fibrotic therapeutics.</p></div

TaqMan assays for miRNAs.

<p>The figure shows high abundance of transcripts of rno-miR-146b, -199a-5p, -214 and -31 in PKD/Mhm (cy/+) rat model as observed on microarrays. ‘***’ indicates for p-value <0.0001. The black and white box plots represent cystic and healthy kidneys.</p

Systematic workflow to explore functional regulatory patterns in PKD.

<p>Systematic workflow to explore functional regulatory patterns in PKD.</p

Differential expression of top 30 genes in PKD and control animals.

<p>The heatmap was produced by clustering the data matrix of top 30 genes using Pearson correlation. The gene clustering tree is shown on the left and the sample clustering tree is shown on the top. The other information such as fold change expression, p-value, gene symbol and involvement are given on the right. The samples are broadly divided into two groups, healthy (control) and PKD. The color scale shown at the top illustrates the relative expression level of the indicated genes across all samples.</p

Overview of binding site predictions of 8 miRNAs within the representative members of deregulated pathways.

<p>Overview of binding site predictions of 8 miRNAs within the representative members of deregulated pathways.</p

Differentially regulated GO biological processes associated with up- and down-regulated genes.

<p>Differentially regulated GO biological processes associated with up- and down-regulated genes.</p

Overview of fold changes of miRNAs versus a measure of statistical significance.

<p>The volcano plot shows the -log₁₀ (p-value) on the y-axis and the fold change (log₂) on the x-axis. A cut-off [-log₁₀ (p-value) = 1.86] was considered to determine differentially expressed miRNAs between diseased and health (control) animals. Seven miRNAs i.e. rno-miR-146b, -132, -21, -503, -199a-5p, -214 and -34a were found to be significantly up-regulated in diseased animals with a fold change ≥0.5 with significant p-value ≤0.01.</p

Differentially regulated pathways on significantly up- and –down-regulated genes.

<p>Differentially regulated pathways on significantly up- and –down-regulated genes.</p

Presentation1.PDF

<p>Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell (SMC) activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous SMCs such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans.</p

Survey of the need for experimental facilities including a Journal and dissertations evaluation

R code of TTCA (EUPL). (TXT 816 kb