Review Article MicroRNA in Diabetic Nephropathy: Renin Angiotensin, AGE/RAGE, and Oxidative Stress Pathway

MicroRNAs (miRNA) are a novel class of small, noncoding RNA molecules that have gained the attention of many researchers in recent years due to their ability to posttranscriptionally regulate the expression of families of genes simultaneously. Their role in normal physiology and pathobiology is intriguing and their regulation in normal and disease states is fascinating. That the cells can return to a state of homeostasis when these small molecules are perturbed is truly remarkable given the multiple cellular targets of each miRNA and that many mRNAs are targeted by multiple miRNAs. Several reviews have covered aspects of miRNA function in biology and disease. Here, we review the role of miRNA in regulating the renin-angiotensin system, AGE/RAGE signalling, and under conditions of oxidative stress in the context of diabetic nephropathy

Ramipril inhibits AGE-RAGE-induced matrix metalloproteinase-2 activation in experimental diabetic nephropathy

Background: Advanced glycation end products (AGE)-receptor for AGE (RAGE) axis and renin-angiotensin system (RAS) play a role in diabetic nephropathy (DN). Matrix metalloproteinase-2 (MMP-2) activation also contributes to DN. However, the pathological interaction among AGE-RAGE, RAS and MMP-2 in DN remains unknown. We examined here the involvement of AGE and RAS in MMP-2 activation in streptozotocin (STZ)-induced diabetic rats and in AGE-exposed rat renal proximal tubular cells (RPTCs).Methods. Experimental diabetes was induced in 6-week-old male Sprague-Dawley (SD) rats by intravenous injection of STZ. Diabetic rats received ramipril (3 mg/kg body weight/day) or vehicle for 32 weeks. AGE-modified rat serum albumin (AGE-RSA) or RSA was intraperitoneally administrated to 6-week-old male SD rats for 16 weeks. RPTCs were stimulated with 100 μg/ml AGE-modified bovine serum albumin (AGE-BSA) or BSA in the presence or absence of 10 M ramiprilat, an inhibitor of angiotensin-converting enzyme or 100 nM BAY11-7082, an IκB- phosphorylation inhibitor.Results: AGE and RAGE expression levels and MMP-2 activity in the tubules of diabetic rats was significantly increased in association with increased albuminuria, all of which were blocked by ramipril. AGE infusion induced tubular MMP-2 activation and RAGE gene expression in SD rats. Ramiprilat or BAY11-7082 inhibited the AGE-induced MMP-2 activation or reactive oxygen species generation in RPTCs. Angiotensin II increased MMP-2 gene expression in RPTCs, which was blocked by BAY11-7082.Conclusions: Our present study suggests the involvement of AGE-RAGE-induced, RAS-mediated MMP-2 activation in experimental DN. Blockade of AGE-RAGE axis by ramipril may protect against DN partly via suppression of MMP-2

miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

OBJECTIVE: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2. RESEARCH DESIGN AND METHODS: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. CONCLUSIONS: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes

The Use of Targeted Next Generation Sequencing to Explore Candidate Regulators of TGF-β1’s Impact on Kidney Cells

Aims/Hypothesis: Transforming growth factor-beta (TGF-β1) plays an important regulatory role in the progression of chronic kidney failure. Further, damage to kidney glomerular mesangial cells is central to the progression of diabetic nephropathy. The aim of this study was to explore the genetic associations between mRNA, microRNA, and epigenetics in mesangial cells in response to TGF-β1.Methods: The regulatory effects of TGF-β1 on mesangial cells were investigated at different molecular levels by treating mesangial cells with TGF-β1 for 3 days followed by genome-wide miRNA, RNA, DNA methylation, and H3K27me3 expression profiling using next generation sequencing (NGS).Results: Our results provide the first comprehensive, computationally integrated report of RNA-Seq, miRNA-Seq, and epigenomic analyses across all genetic variations, confirming the occurrence of DNA methylation and H3K27me3 in response to TGF-β1. Our findings show that the expression of KLF7 and Gja4 are involved in TGF-β1 regulated DNA methylation. Our data also provide evidence of the association between epigenetic changes and the expression of genes closely related to TGF-β1 regulation.Conclusion: This study has advanced our current knowledge of mechanisms that contribute to the expression of TGF-β1-regulated genes involved in the pathogenesis of kidney disease. The molecular underpinnings of TGF-β1 stimulation of kidney cells was determined, thereby providing a robust platform for further target exploration

Absolute Quantitation of MDR1 Transcripts Using Heterologous DNA Standards—Validation of the Competitive RT-PCR (CRT-PCR) Approach

The multidrug resistance (MDR1) gene product, P-glycoprotein (Pgp), is a 170-kDa ATP-dependent pump that expels a variety of anticancer drugs out of malignant cells, reducing drug accumulation and thus antitumor activity. In recent years, considerable data has been presented that indicates the need to standardize detection methods for Pgp and MDR1. Reverse transcription (RT)-PCR is one of the most sensitive and specific techniques used to detect MDR1. Nevertheless, there is the need to address working criteria for quantitation by RT-PCR. In this study, we describe a flexible assay used to quantify MDR1 gene expression using heterologous (nonhomologous) standards for use in competitive RT-PCR (CRT-PCR). Our guidelines were to use a RT-PCR quantitation method that was independent of exponential phase kinetics, sensitive (detect low levels of gene measurement in clinical samples) and did not require radiolabel. Furthermore, the method would need to be flexible enough for the user to express quantitation as either the number of cells or amount of cDNA used in CRT-PCR. Using low-stringency amplification, heterologous DNA competitors were constructed for MDR1 and as an internal reference, the ubiquitously expressed human histone variant 3.3 (H3.3). The benefits of this approach are threefold: (i) amplification kinetics of target and competitor molecules are identical, (ii) low-stringency PCR is a simple way of constructing heterologous DNA competitors that do not require special storage conditions and (iii) heterologous competitors avoid the formation of heteroduplex molecules. We conclude that CRT-PCR is an extremely flexible and sensitive assay that can quantify MDR1 based on competitive amplification of a heterologous competitor. This might complement future efforts to standardize MDR1 detection methods using RT-PCR

Precipitous Release of Methyl-CpG Binding Protein 2 and Histone Deacetylase 1 from the Methylated Human Multidrug Resistance Gene (MDR1) on Activation

Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) were used to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter. We have established that methyl-CpG binding protein 2 (MeCP2) is involved in methylation-dependent silencing of human MDR1 in cells that lack the known transcriptional repressors MBD2 and MBD3. In the repressed state the MDR1 promoter is methylated and assembled into chromatin enriched with MeCP2 and deacetylated histone. TSA induced significant acetylation of histones H3 and H4 but did not activate transcription. 5aC induced DNA demethylation, leading to the release of MeCP2, promoter acetylation, and partial relief of repression. MDR1 expression was significantly increased following combined 5aC and TSA treatments. Inhibition of histone deacetylase is not an overriding mechanism in the reactivation of methylated MDR1. Our results provide us with a clearer understanding of the molecular mechanism necessary for repression of MDR1