microRNA Expression of Renal Proximal Tubular Epithelial Cells and Their Extracellular Vesicles in an Inflammatory Microenvironment In Vitro

Abstract

Renal proximal tubular epithelial cells (PTCs) are central players during renal inflammation. In response to inflammatory signals, PTCs not only self-express altered mRNAs, microRNAs (miRNAs), proteins, and lipids, but also release altered extracellular vesicles (EVs). These EVs also carry inflammation-specific cargo molecules and are key players in cell–cell-communication. Understanding the precise molecular and cellular mechanisms that lead to inflammation in the kidney is the most important way to identify early targets for the prevention or treatment of acute kidney injury. Therefore, highly purified human PTCs were used as an in vitro model to study the cellular response to an inflammatory microenvironment. A cytokine-induced inflammatory system was established to analyze different miRNA expression in cells and their EVs. In detail, we characterized the altered miR expression of PTCs and their released EVs during induced inflammation and showed that 12 miRNAs were significantly regulated in PTCs (6 upregulated and 6 downregulated) and 9 miRNAs in EVs (8 upregulated and 1 downregulated). We also showed that only three of the miRNAs were found to overlap between cells and EVs. As shown by the KEGG pathway analysis, these three miRNAs (miR-146a-5p, miR-147b, and miR-155-5p) are functionally involved in the regulation of the Toll-like receptor signaling pathway and significantly correlated with the inflammatory mediators IL6 and ICAM1 released by stimulated PTCs. Especially with regard to a possible clinical use of miRs as new biomarkers, an accurate characterization of the miR expression altered during inflammatory processes is of enormous importance.

miRNA-Mediated Fine Regulation of TLR-Induced M1 Polarization

“…We observed that Akt1 mRNA levels are downregulated following TLR4 or TLR2 stimulation [94]. This is in line with the description of Akt1 as a target gene of the miRNA miR-155-5p [66,97], whose expression has been consistently reported to be upregulated by various members of the TLR family [4,5,7,11,12,20,24,25,34,42,47,58,[70][71][72][73]83,[90][91][92][98][99][100][101][102][103]. In addition, Akt1 activity is subject to post-transcriptional modulation.…”

“…Changes in the expression levels of the so-called early miRNAs occur immediately after ligand binding. For example, miR-155-5p was reported to be upregulated within 2 h of stimulation [57,72,[91][92][93]. As a higher-ranking miRNA, miR-155-5p then regulates the expression of further miRNAs of the late response [57,74,89].…”

“…The differential effect of miR-155-5p is another prominent example of the yin-yang effect of miRNAs in macrophage polarization. MiR-155-5p is upregulated following TLR stimulation mediated by the transcription factors AP-1 and NFκB [83,90,98,99,113] and is considered critical for the polarization of macrophages to the M1 phenotype [4,11,18,20,58,91]. Indeed, knocking out miR-155-5p in macrophages is associated with reduced clearance of Streptococcus pneumoniae or Mycobacterium bovis [70,71].…”

“…Besides these proinflammatory effects, miR-155-5p also acts as an anti-inflammatory effector. Among others, miR-155-5p causes the translational repression of the TLR cascade proteins MyD88 and TAB2 and mediates the mRNA degradation of IKK-epsilon [4,5,7,20,24,25,35,47,57,72,76,[90][91][92][93][100][101][102]113,114,116]. Furthermore, Akt1 and PKI-alpha, an inhibitor of protein kinase A, are targeted by miR-155-5p [71].…”

In Vitro Models of Tissue and Organ Regeneration

“…Marhuenda and coworkers show that culturing primary alveolar epithelial cells on lung extracellular matrix-derived hydrogels facilitated the prolonged culturing of these cells and enhanced the preservation of the type II phenotype [7]. Baer and coworkers characterised the mRNA expression of renal proximal tubular epithelial cells and the cargo in extracellular vesicles in an inflammatory microenvironment [8]. This study demonstrates the altered miRNA expression of epithelial cells and their released vesicles during induced inflammation, with only three miRNAs overlapping between cells and vesicles.…”