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

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

Quantitative Characterization of Tissue Globotetraosylceramides in a Rat Model of Polycystic Kidney Disease by PrimaDrop Sample Preparation and Indirect High-Performance Thin Layer Chromatography–Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight-Mass Spectrometry with Automated Data Acquisition

Glycosphingolipids (GSL) have been associated with a variety of diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). In contrast to glucosylceramide and gangliosides, alterations in complex neutral GSLs such as globotetraosylceramide (Gb4Cer) have not been investigated in ADPKD yet, and mass spectrometry analysis of Gb4Cer from tissue extracts remains challenging. To this end, we introduce PrimaDrop as an improved and widely applicable sample preparation method for automated matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of lipid extracts, which promotes homogeneous cocrystallization and enables relative quantification by indirect thin layer chromatography (TLC)–MALDI-time-of-flight (TOF)-MS against an internal bradykinin standard. Application of the method for detailed investigation of Gb4Cer isoforms in kidneys of an ADPKD rat model revealed increased levels of sphingoid base-containing isoforms in cystic kidneys, whereas changes were subtle for Gb4Cer-containing phytosphingoid bases. We furthermore established an absolute LC–ESI-MS/MS quantification method and demonstrate that absolute quantities of Gb4Cer correlate well with relative quantities obtained by indirect TLC–MALDI-TOF-MS. Taken together, our study proposes an effective sample preparation method for automated analysis of lipid extracts and TLC eluates and suggests that indirect high-performace (HP)­TLC–MALDI-TOF-MS with automated data acquisition is a viable option for analysis of neutral glycosphingolipids and that Gb4Cer may play a role in the pathogenesis of ADPKD

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

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

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

Highly Sensitive Raman Spectroscopy with Low Laser Power for Fast In-Line Reaction and Multiphase Flow Monitoring

In process analytics, the applicability of Raman spectroscopy is restricted by high excitation intensities or the long integration times required. In this work, a novel Raman system was developed to minimize photon flux losses. It allows specific reduction of spectral resolution to enable the use of Raman spectroscopy for real-time analytics when strongly increased sensitivity is required. The performance potential of the optical setup was demonstrated in two exemplary applications: First, a fast exothermic reaction (Michael addition) was monitored with backscattering fiber optics under strongly attenuated laser power (7 mW). Second, high-speed scanning of a segmented multiphase flow (water/toluene) with submicroliter droplets was achieved by aligning the focus of a coaxial Raman probe with long focal length directly into a perfluoroalkoxy (PFA) capillary. With an acquisition rate of 333 Raman spectra per second, chemical information was obtained separately for both of the rapidly alternating phases. The experiment with reduced laser power demonstrates that the technique described in this paper is applicable in chemical production processes, especially in hazardous environments. Further potential uses can be envisioned in medical or biological applications with limited power input. The realization of high-speed measurements shows new possibilities for analysis of heterogeneous phase systems and of fast reactions or processes