Continuously increased generation of ROS in human plasma after cardiac arrest as determined by Amplex Red oxidation.

Oxidative stress is believed to be a major cause of injury after cardiac arrest (CA). While the effects of ROS generated within tissues have been extensively investigated, the potential of plasma-generated ROS in contributing to CA pathology has not been examined. We utilized Amplex Red (AR) to measure the real time-generation of ROS in isolated plasma from human CA patients. We first used post-CA rat plasma to identify interfering factors for AR oxidation, and then applied this knowledge to analyze human plasma samples, accounting for the identified confounders. We found significantly increased AR oxidation rates lasting for 4 h in post-CA rat plasma compared to baseline. AR oxidation was unchanged with removal of horseradish peroxidase or addition of catalase. However, adding carboxylesterase inhibitors significantly decreased AR oxidation in rat plasma, which implicated increased carboxylesterase activity, not ROS leading to increased AR oxidation. AR oxidation rates were also significantly increased in human CA patient plasma compared to control and this increase persisted even with carboxylesterase inhibition, suggesting continuously increased ROS-generation within plasma post-CA in humans. The increased ROS generation may be one major source of injury post-CA that may be mitigated with antioxidative therapeutic strategies that can manage the ROS systemically generated in plasma over time.KEY POLICY HIGHLIGHTSWe examined the potential of plasma as a source of ROS generation post-cardiac arrestRat cardiac arrest was used to guide the application of Amplex Red in human plasmaROS generation in plasma is significantly increased after cardiac arrest in humansScavenging excessive ROS in post-resuscitation plasma may improve outcomes of patients.

Hedgehog pathway plays a vital role in HIV-induced epithelial-mesenchymal transition of podocyte.

HIV-associated nephropathy (HIVAN) is characterized by heavy proteinuria, rapidly progressive renal failure, and distinct morphological features in the kidney. HIV-induced epithelial-mesenchymal transition (EMT) is critically important for the progression of kidney injury. In this study, we tested the role of hedgehog pathway in the HIV-induced EMT and fibrosis of kidney. We used the Tg26 mice, the abundantly used HIVAN mouse model, to investigate the activation of hedgehog pathway by HIV. Western blotting and real time PCR results showed that renal tissue expression of hedgehog pathway related molecules, including hedgehog homologous (Shh, Ihh, Dhh), PTCH, and Gli1, were increased in HIVAN (Tg26) mice; while immunofluorescent staining displayed localization PTCH expression in podocytes. For in vitro studies, we used recombinant sonic hedgehog (Shh) and HIV for their expression by podocytes. Both the methods activated the hedgehog pathway, enhanced the expression of EMT markers, and decreased impermeability. Overexpression of Gli1 by human podocytes also augmented their expression of EMT markers. On the other hand, the blockade of hedgehog pathway with Gant 58, a specific blocker for Gli1-induced transcription, dramatically decreased HIV-induced podocyte EMT and permeability. These results indicate that hedgehog pathway plays an important role in HIV-induced podocyte injury. The present study provides mechanistical insight into a new target for therapeutic strategy.