The Regulatory Mechanism Of The UCH-L1Gene In Glomerulus Podocytes And Its Significance In Nephropathy

The ubiquitin carboxy terminal hydrolase1(UCH-L1) is a member of the deubiquitinating enzymes family that has an important role in regulation of the ubiquitin-proteasome system (UPS), which is a principal pathway for the metabolism of intracellular proteins and ensuring the proper execution of cellular processes such as cell cycles, signal transduction, growth, differentiation and receptor function, etc. The expression of UCH-L1normally exhibits a prominent specificity of tissues, including brain, genital system(including testis and oocyte) and kidney. Increasing evidence has demonstrated that the mutation or the abnormal expression of UCH-L1is tightly associated with some diseases in these tissues and some tumours, such as neurodegenerative diseases, ischemic and traumatic brain injury and cancers metastasis. Recently, it was reported that the aberrant expression of UCH-L1is related to podocyte injury and proteinuria. Our earlier work demonstrated that UCH-L1is up-expressed in podocytes in a variety of immunocomplex-mediated nephrites. However, the mechanism underlying the abnormal expression of UCH-L1in podocytes with nephropathy is poorly understood. Activation of NF-κB is thought to be the predominant risk factor for kidney disease and can regulate many molecules involved in inflammatory reaction. Increasing information indicates activation of NF-κB is the critical response for kidney diseases. Therefore, it is postulated that UCH-L1may be one of the NF-κB target genes. In this study, we investigated whether the NF-κB activation is involved in the regulation of UCH-L1expression and its detail mechanism. We found that there is positive regulation between NF-κB and UCH-L1. Stimulation of podocytes with the cytokines TNF-α and IL-1β activated NF-κB, and then up-regulated UCH-L1expression rapidly at the mRNA and protein level. The up-regulation of UCH-L1is dose and time-dependent. However stimulation with the NF-KB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation of UCH-L1. Analysis of the promoter region of the UCH-L1gene using MatInspector2.2software revealed that the5’-flanking region contains three putative regulatory elements for NF-κB. We confirmed by luciferase reporter gene analysis that two of them are specific binding sequences of NF-κB under stimulation. NF-κB up-regulates UCH-L1via binding the-300bp and-109bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA-nuclear protein binding. Over-expression of p65can up-regulate UCH-L1expression at the mRNA and protein level. Conversely, interference of p65can cause the down-regulation. In the renal biopsy from lupus nephritis, IgA nephropathy, membranous glomerulonephritis, the expressions of NF-κB and UCH-L1increased in immunohistochestry staining and were positively correlated. But in minimal change disease, there is no obvious expression of NF-κB and UCH-L1.Further, activation of NF-κB/UCH-L1changes other podocytes molecules, such as nephrin and snail. However, interference of NF-κB/UCH-L1can cause opposite change of the corresponding proteins. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes.In conclusion, in glomerulonephritis NF-κB signal pathway is activated, then up-regulate the expression of UCH-L1, and following changes other important podocytes molecules, such as nephrin and snail, which is the important mechanism to affect the podocyte injury in glomerulonephritis.This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB-UCH-L1interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.