The Role Of MRTF-A In Renal Tubular Fibrosis During Diabetic Nephropathy

Objectives:Diabetes mellitus is a metabolic disorder as a result of poorly controlled hyperglycemia and associated with a number of complications. Diabetic nephropathy is one of the most common complications of diabetes mellitus. Patients with diabetic nephropathy eventually develop kidney fibrosis, leading to end-stage renal disease. Interstitial fibrosis is characterized by excessive deposition and/or reduced turnover of extracellular matrix components (ECM) such as collagen type I and III. In recent years, various studies have focused on the pathophysiology of diabetic nephropathy associated renal fibrosis. The definitive molecular mechanisms involved in the development of diabetic kidney, however, are not well understood.As a transcriptional mediator, myocardin-related transcription factor A (MRTF-A, also known as MKL1) have been proved to mediate transcriptional regulation of vascular smooth muscle cells (VSMC)-specific genes associated with contractile phenotype of SMC. Little is known regarding the role of MRTF-A in other cell types such as renal tubular epithelial cells. In the present study, we examined the role of MRTF-A in high glucose induced indution of collagen type I genes in both rodent and human renal tubular epithelial cells. Overexpression of MRTF-A synergistically enhanced the stimulation of collagen type I transcription by high glucose. In contrast, silencing MRTF-A by small interfering RNA abolished collagen induction, indicating MRTF-A might play an important role in the progression of renal fibrosis. P300functions as histone acetyltransferase and regulates transcription via acetylating histones and non-histone factors. Histone acetylation gives an epigenetic tag for transcriptional activation. Recent investigations have suggested that MRTF-A mediates transcriptional regulation by epigenetic mechanisms. Therefore, we hypothesized that MRTF-A might mediate the upregulation of collagen type I by high glucose through recruiting p300. The purpose of the current investigation is to demonstrate the role of MRTF-A in the development of kidney fibrosis.Methods:mRNA levels of collagen type I and MRTF-A after high glucose treatment were examined by real-time quantitative PCR (qPCR) in rat renal tubular epithelial cell (NRK-52E) and human renal tubular epithelial cell (HK-2). Luciferase reporter assays were performed to assess the effect of high glucose on the promoter activities of these genes. Western blotting was performed to detect the protein levels of collagen type I and MRTF-A in the presence of high glucose. To assess whether high glucose augmented the accumulation of acetylated histone H3on the collagen type I gene promoter, chromatin immunoprecipitation (ChIP) assayswere performed. To evaluate whether MRTF-A was necessary for the upregulation of collagen transcription by high glucose, cells were transfected with small interfering RNA (siRNA) specifically targeting MRTF-A. Expression of collagen and H3acetylation after high glucose treatment were evaluated as described earlier.Results:high glucose simultaneously stimulated mRNA levels of collagen type I and MRTF-A. MRTF-A synergistically activated collagen type I transcription by high glucose. Meanwhile, high glucose augmented the acetylation levels of histone H3and in particular promoted the accumulation of AcH3K9, AcH3K27, and AcH3K18on the collagen promoter. Conversely, when MRTF-A was silenced, collagen type I expression and histone H3acetylation levels were down-regulated. Similarly, when p300was silenced, the upregulation of collagen expression by high glucose was also down-reglulated. Conclusion:high glucose induces the expression of MRTF-A, which in turn promotes collagen type I expression by increasing the enrichment of acetylated H3on the collagen promoter. Therefore, our study provides a potential target for clinical intervention of renal fibrosis.