Nuclear receptor NR5A2 is involved in the calreticulin gene regulation during renal fibrosis.

BACKGROUND: Renal fibrosis is a common histological finding present in many pathologies; however, key signaling pathways and molecular determinants involved in the development of fibrosis are not fully known yet. Previous findings have established a causative role of calreticulin's up-regulation during the development of renal fibrosis while its down-regulation exhibited a protective effect against fibrosis. Therefore, the mechanism of its up-regulation needs to be explored. METHODS: Bioinformatics analyses of the calreticulin gene promoter combined with transcriptional assays and in vivo chromatin immunoprecipitation experiments in the Unilateral Ureteric Obstruction (UUO) model of renal fibrosis, indicated that NR5A2 is a critical regulator of calreticulin expression. To confirm this finding, and further study post-translational modifications of NR5A2, real time RT-qPCR, immunohistochemistry and Western blotting experiments were performed. RESULTS: NR5A2 is up-regulated at both mRNA and protein level during kidney fibrosis in the UUO model. The post-translational modification of SUMOylation was identified as a critical parameter in this phenomenon and SUMOylation was observed to be up-regulated during the development of renal fibrosis. The enzyme Ubc9, critical for the process of SUMOylation was also upregulated at mRNA and protein level during the process. CONCLUSION: These data establish for the first time a role for NR5A2 and its SUMOylation on the transcriptional regulation of the calreticulin gene in a rodent model of renal fibrosis and raise the possibility that NR5A2 might be a novel target for future anti-fibrotic interventions.

Kallikrein-related peptidase 6 (KLK6) expression in the progression of colon adenoma to carcinoma.

KLK6 is a secreted trypsin-like serine protease. KLK6 mRNA expression and its association with colon cancer (CC) progression was studied using quantitative real-time PCR. We examined the expression of KLK6 in 232 colon tissues (cancerous, non-cancerous, and adenomatous). We proved that KLK6 expression in CC behaves as a continuous variable, as its expression correlates significantly with increasing tumor stage (p=0.004) and histological grade (p=0.007). Interestingly, the expression of KLK6 in adenomas was significantly higher than that in the cancerous or non-cancerous tissues examined (p<0.001). Cox proportional hazard regression model using univariate analysis revealed that positive KLK6 expression is a significant factor for disease-free survival (DFS) (p=0.017) and overall survival (OS) (p=0.002) of patients. Kaplan-Meier survival curves demonstrated that KLK6-negative expression is significantly associated with longer DFS (p=0.009) and OS (p=0.001). ROC analysis showed that KLK6 expression has significant discriminatory power in distinguishing cancerous from non-cancerous colon tissues (p<0.001), or cancerous from adenoma tissues (p=0.001), or adenoma from non-cancerous colon tissues (p<0.001). Additionally, strong KLK6 immunostaining was seen in the cancer cells of selected CC sections, as well as in glandular cells and inflammatory cells of adenomas. In conclusion, KLK6 may represent a potential unfavorable prognostic biomarker for CC.

Whole-transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases.

Transcriptome analysis by RNA-seq technology allows novel insights into gene expression and regulatory networks in health and disease. To better understand the molecular basis of renal fibrosis, we performed RNA-seq analysis in the Unilateral Ureteric Obstruction (UUO) mouse model. We analysed sham operated, 2- and 8-day post-ligation renal tissues. Thousands of genes with statistical significant changes in their expression were identified and classified into cellular processes and molecular pathways. Many novel protein-coding genes were identified, including critical transcription factors with important regulatory roles in other tissues and diseases. Emphasis was placed on long non-coding RNAs (lncRNAs), a class of molecular regulators of multiple and diverse cellular functions. Selected lncRNA genes were further studied and their transcriptional activity was confirmed. For three of them, their transcripts were also examined in other mouse models of nephropathies and their up- or down-regulation was found similar to the UUO model. In vitro experiments confirmed that one selected lncRNA is independent of TGFß or IL1b stimulation but can influence the expression of fibrosis-related proteins and the cellular phenotype. These data provide new information about the involvement of protein-coding and lncRNA genes in nephropathies, which can become novel diagnostic and therapeutic targets in the near future.