Renin plays a crucial role in the control of various physiological processes such as blood pressure and body fluid homeostasis. Here, we show that a splice variant of the Wilms' tumor protein lacking three amino acids WT1(-KTS) suppresses renin gene transcription. Using bioinformatics tools, we initially predicted that a WT1-binding site exists in a regulatory region about 12 kb upstream of the renin promoter; this was confirmed by reporter gene assays and gel shift experiments in heterologous cells. Co-expression of Wt1 and renin proteins was found in rat kidney sections, mouse kidney blood vessels, and a cell line derived from the juxtaglomerular apparatus that produces renin. Knockdown of WT1 protein by siRNA significantly increased the cellular renin mRNA content, while overexpression of WT1(-KTS) reduced renin gene expression in stable and transiently transfected cells. A mutant WT1(-KTS) protein found in Wilms' tumors failed to suppress renin gene reporter activity and endogenous renin expression. Our findings show that renin gene transcription is regulated by the WT1(-KTS) protein and this may explain findings in patients with WT1 gene mutations of increased plasma renin and hypertension.

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of ∼775 bp in size, ∼14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score.

The aim of the study was to identify markers for the early diagnosis of endoprosthesis loosening, for the differentiation between wear particle-induced and septic loosening and to gather new insights into the pathogenesis of endoprosthesis loosening. Gene expression profiles were generated from five periprosthetic membranes of wear particle-induced and five of infectious (septic) type using Affymetrix HG U133A oligonucleotide microarrays. The results of selected differentially expressed genes were validated by RT-PCR (n ¼ 30). The enzyme activity and the genotype of chitinase-1 were assessed in serum samples from 313 consecutive patients hospitalized for endoprosthesis loosening (n ¼ 54) or for other reasons, serving as control subjects (n ¼ 259). Eight hundred twenty-four genes were differentially expressed with a fold change greater than 2 (data sets on http://www.ncbi.nlm.nih.gov/geo/ GSE 7103). Among these were chitinase 1, CD52, calpain 3, apolipoprotein, CD18, lysyl oxidase, cathepsin D, E-cadherin, VE-cadherin, nidogen, angiopoietin 1, and thrombospondin 2. Their differential expression levels were validated by RT-PCR. The chitinase activity was significantly higher in the blood from patients with wear particle-induced prosthesis loosening ( p ¼ 0.001). However, chitinase activity as a marker for early diagnosis has a specificity of 83% and a sensitivity of 52%, due to a high variability both in the disease and in the control group.

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