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 nM DCF/μg total protein, P<0.05) and expression of profibrotic markers CTGF (149 ± 12%, P<0.05), α-SMA (160 ± 20%, P<0.05), and PAI-I (153 ± 13%, P<0.05) at 10-8 M. Recombinant prorenin induced phospho ERK 1/2 (p44 and p42) at 10-8 and 10-6 M after 20 min. 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 h 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.

The binding of prorenin to the (pro)renin receptor (PRR) triggers the activation of MAPK/ERK1/2 pathway, induction of cyclooxygenase-2 (COX-2), NOX-4-dependent production of reactive oxygen species (ROS), and the induction of transforming growth factor β (TGF-β) and profibrotic factors connecting tissue growth factor (CTGF) and plasminogen activator inhibitor (PAI-I) in collecting duct (CD) cells. However, the role of COX-2 and the intracellular pathways involved are not clear. We hypothesized that the PRR activation increases profibrotic factors through COX-2-mediated PGE2 activation of E prostanoid receptor 4 (EP4), upregulation of NOX-4/ROS production, and activation of Smad pathway in mouse CD cells. Recombinant prorenin increased ROS production and protein levels of CTGF, PAI-I, and TGF-β in M-1 CD cell line. Inhibition of MAPK, NOX-4, and COX-2 prevented this effect. Inhibition of MEK, COX-2, and EP4 also prevented the upregulation of NOX-4. Because TGF-β activates Smad pathway, we evaluate the phosphorylation of Smad2 and 3. COX-2 inhibition or EP4 antagonism significantly prevented phosphorylation of Smad 2/3. Mice that were infused with recombinant prorenin showed an induction in the expression of CTGF, PAI-I, TGF-β, fibronectin, and collagen I in isolated collecting ducts as well as the expression of alpha smooth muscle actin (α-SMA) in renal tissues. COX-2 inhibition prevented this induction. These results indicate that the induction of TGF-β, CTGF, PAI-I, and ROS occurs through PRR-dependent activation of MAPK and NOX-4; however, this mechanism depends on COX-2-derived PGE2 production and the activation of EP4 and Smad pathway.

Background Prostaglandin E2 (PGE2) regulates renin expression in renal juxtaglomerular cells. PGE2 acts through E-prostanoid (EP) receptors in the renal collecting duct (CD) to regulate sodium and water balance. CD cells express EP1 and EP4, which are linked to protein kinase C (PKC) and protein kinase A (PKA) downstream pathways, respectively. Previous studies showed that the presence of renin in the CD, and that PKC, and PKA pathways activate its expression. The (pro)renin receptor (PRR) is also expressed in CD cells and its activation enhances cyclooxygenase-2 (COX-2) through extracellular signal–regulated kinase (ERK). We hypothesized that PGE2 stimulates prorenin and renin synthesis leading to subsequent activation of PRR and upregulation of COX-2. Methods We used a mouse M-1 CD cell line that expresses EP1, EP3, and EP4 but not EP2. Results PGE2 (10−6 M) treatment increased prorenin and renin protein levels at 4 and 8 h. No differences were found at 12 h post PGE2 treatment. Phospho-ERK was significantly augmented after 12 h. COX-2 expression was decreased after 4 h of PGE2 treatment, but increased after 12 h. Interestingly, the full-length form of the PRR was upregulated only at 12 h. PGE2 mediated phospho-ERK and COX-2 upregulation was suppressed by PRR silencing. Conclusions Our results suggest that PGE2 induces biphasic regulation of COX-2 through renin-dependent PRR activation via EP1 and EP4 receptors. PRR-mediated increases in COX-2 expression may enhance PGE2 synthesis in CD cells serving as a buffer mechanism in conditions of activated renin angiotensin system.

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Sepsis syndrome is a highly lethal uncontrolled response to an infection, which is characterized by sepsis-induced coagulopathy (SIC). High-density lipoprotein (HDL) exhibits antithrombotic activity, regulating coagulation in vascular endothelial cells. Sepsis induces the release of several proinflammatory molecules, including reactive oxygen species, which lead to an increase in oxidative stress in blood vessels. Thus, circulating lipoproteins, such as HDL, are oxidized to oxHDL, which promotes hemostatic dysfunction, acquiring prothrombotic properties linked to the severity of organ failure in septic-shock patients (SSP). However, a rigorous and comprehensive investigation demonstrating that oxHDL is associated with a coagulopathy-associated deleterious outcome of SSP, has not been reported. Thus, we investigated the participation of plasma oxHDL in coagulopathy-associated sepsis pathogenesis and elucidated the underlying molecular mechanism. A prospective study was conducted on 42 patients admitted to intensive care units, (26 SSP and 16 non-SSP) and 39 healthy volunteers. We found that an increased plasma oxHDL level in SSP was associated with a prothrombotic phenotype, increased mortality and elevated risk of death, which predicts mortality in SSP. The underlying mechanism indicates that oxHDL triggers an endothelial protein expression reprogramming of coagulation factors and procoagulant adhesion proteins, to produce a prothrombotic environment, mainly mediated by the endothelial LOX-1 receptor. Our study demonstrates that an increased plasma oxHDL level is associated with coagulopathy in SSP through a mechanism involving the endothelial LOX-1 receptor and endothelial protein expression regulation. Therefore, the plasma oxHDL level plays a role in the molecular mechanism associated with increased mortality in SSP.