Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation

Neurokinin 1 (NK(1)) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms downstream from NK(1) receptor (NK(1)R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK(1)R(-/-) and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP(4))-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP(4)-ovalbumin, and control mice were challenged with saline. At 1, 4, and 24 h after instillation, bladders were removed for 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), and 3) morphological analysis (n = 6). For cDNA array experiments, three bladders from each group were homogenized, and total RNA was obtained. DNase-treated RNA was reverse-transcribed to cDNA, labeled with [alpha-(32)P]dATP and hybridized to Atlas Mouse 1.2 Arrays (Clontech). After calculating the mean and SD for background spots, each experimental value was assigned a normalized score S using the formula S' = (S - Av)/SD, where S' is the original pixel value, and Av and SD are the mean and standard deviation of background spots, respectively. Only genes that expressed 3 SD values above background were used. Hypervariable genes were sorted by cluster analysis. Matrices of correlation coefficients were calculated and represented in a connectivity mosaic. As results, we found that in WT mice the most prominent gene cluster had neprilysin in a central position and positively correlated to a group of activator protein-1 (AP-1)-responsive genes, including laminin-alpha3, tissue plasminogen activator 11, fos-B, and TNF-beta. In WT mice, antigen-induced bladder inflammation led to a downregulation in neprilysin expression. In contrast, NK(1)R(-/-) mice failed to mount an inflammatory reaction and presented neprilysin negatively correlated with the same genes described in WT. In conclusion, this work indicates an overriding participation of NK(1)R and neprilysin in bladder inflammation, provides a working model for the involvement of AP-1 transcription factor, and evokes testable hypotheses regarding the role of NK(1)R and neprilysin in inflammation.