Mapping and quantifying neuropeptides in the enteric nervous system.

Neuropeptides are a highly diverse group of signaling molecules found in the central nervous system (CNS) and peripheral organs, including the enteric nervous system (ENS). Increasing efforts have been focused on dissecting the role of neuropeptides in both neural- and non-neural-related diseases, as well as their potential therapeutic value. In parallel, accurate knowledge on their source of production and pleiotropic functions is still needed to fully understand their implications in biological processes. This review will focus on the analytical challenges involved in studying neuropeptides, particularly in the ENS, a tissue where their abundance is low, together with opportunities for further technical development.

(Pro)renin receptor activation increases profibrotic markers and fibroblast-like phenotype through MAPK-dependent ROS formation in mouse renal collecting duct cells.

Recent studies suggested that activation of the PRR upregulates profibrotic markers through reactive oxygen species (ROS) formation; however, the exact mechanisms have not been investigated in CD cells. We hypothesized that activation of the PRR increases the expression of profibrotic markers through MAPK-dependent ROS formation in CD cells. Mouse renal CD cell line (M-1) was treated with recombinant prorenin plus ROS or MAPK inhibitors and PRR-shRNA to evaluate their effect on the expression of profibrotic markers. PRR immunostaining revealed plasma membrane and intracellular localization. Recombinant prorenin increases ROS formation (6.0 ± 0.5 vs 3.9 ± 0.1 nmol/L DCF/µg total protein, P < .05) and expression of profibrotic markers CTGF (149 ± 12%, P < .05), α-SMA (160 ± 20%, P < .05), and PAI-I (153 ± 13%, P < .05) at 10-8  mol/L. Recombinant prorenin-induced phospho ERK 1/2 (p44 and p42) at 10-8 and 10-6  mol/L after 20 minutes. Prorenin-dependent ROS formation and augmentation of profibrotic factors were blunted by ROS scavengers (trolox, p-coumaric acid, ascorbic acid), the MEK inhibitor PD98059 and PRR transfections with PRR-shRNA. No effects were observed in the presence of antioxidants alone. Prorenin-induced upregulation of collagen I and fibronectin was blunted by ROS scavenging or MEK inhibition independently. PRR-shRNA partially prevented this induction. After 24 hours prorenin treatment M-1 cells undergo to epithelial-mesenchymal transition phenotype, however MEK inhibitor PD98059 and PRR knockdown prevented this effect. These results suggest that PRR might have a significant role in tubular damage during conditions of high prorenin-renin secretion in the CD.

Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct.

Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10(-6) M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD.