The Role of Protein Kinase C Epsilon in Hydrogen Peroxide and Nitric Oxide Release During Oxidative Stress Caused by Extracorporeal Shockwave Lithotripsy

Clinical extracorporeal shock wave lithotripsy (ESWL) treatment to ablate kidney stones can cause acute damage to the renal microvasculature. Accumulation of continued treatment with shockwave therapy can lead to chronic damage to the kidney, and lead to clinical hypertension. Shockwaves have been shown to stimulate endothelial cells to release superoxide (SO), which is converted to hydrogen peroxide (H2O2), and reacts with nitric oxide (NO) to produce peroxynitrite anion (OONO-), creating a powerful oxidant that increases oxidative stress while simultaneously reducing NO bioavailability. Increased oxidative stress during events such as ESWL, also uncouples NO production reaction in endothelial nitric oxide synthase (eNOS), causing eNOS to produce SO instead of NO, exacerbating the oxidative insult. NO is an essential signaling molecule responsible for vasodilation, which also functions in inhibiting platelet adhesion and reducing leukocyte-endothelial interactions. This increased oxidative stress, decreased NO bioavailability, and the direct physical force of the shock wave causes prolonged renal vasodilation and eventually vascular endothelial dysfunction in the renal vasculature. Protein kinase C epsilon (PKC-ε) positively regulates eNOS, increasing its activity regardless of whether eNOS is producing SO or NO. We hypothesized that the PKC-ε peptide inhibitor (N-Myr-EAVSLKPT, MW = 1054) would attenuate ESWL-induced increased H2O2 release and increase NO release compared to ESWL-saline control rats, while the PKC-ε peptide activator (N-Myr-HDAPIGYD, MW 1097) would increase ESWL-induced H2O2 release and reduce NO release. H2O2 and NO was measured in real-time by inserting a H2O2 or NO microsensor (100 um diameter) into the left renal vein in anesthetized male Sprague-Dawley rats. ESWL treatment was administered with 16 kV shock waves for 13 minutes in a period of 500 shocks at 60 beats/min then 500 shocks at 120 beats/min by an Epos Ultra lithotripter. Immediately post-ESWL treatment, saline or drug was infused through the external jugular vein. Infusion of PKC-ε inhibitor in ESWL-treated rats significantly reduced H2O2 release (n = 5) from 5 minutes (p \u3c 0.05) to 30 minutes (p \u3c 0.01) compared to ESWL-saline controls (n = 5). PKC-ε inhibitor also significantly increased NO release (n = 5) from 5 minutes (p \u3c 0.01) to 30 minutes (p \u3c 0.01) compared to ESWL-saline controls (n = 5). Contrary to the hypothesis that PKC-ε activator would increase H2O2 release and reduce NO release, results from PKC-ε activator showed a similar but not statistically significant different trend to ESWL-saline controls in H2O2 release (n = 5) and NO release (n = 5). The data shows that inhibition of PKC-ε effectively decreases ESWL-induced increased H2O2 release and significantly restores NO release, which suggests that uncoupled eNOS is a significant source of oxidative stress during ESWL treatment. This results in decreased oxidative stress, attenuating endothelial dysfunction and further damage to the renal microvasculature

The Effects of Modulating Endothelial Nitric Oxide Synthese (eNOS) Activity and Coupling in Extracorporeal Shock Wave Lithotripsy (ESWL)

Introduction: ESWL is a clinical therapy to break down kidney and uretal stones into smaller fragments that are more easily eliminated through the urinary tract. High-energy shock waves are focused on the stone to cause shear stress and cavitation bubbles which synergistically ablate the stones. While ESWL is the preferred treatment for kidney stones over invasive surgeries, the repetitive shock waves necessary to break up the stones may also cause damage to the renal vasculature endothelium and that can lead to chronic hypertension [1]. Previous studies have found that ESWL can cause endothelial dysfunction which is characterized decreased nitric oxide (NO) bioavailability and increased production of reactive oxygen species (ROS) such as superoxide (O2-) [2]. Normally, endothelial nitric oxide synthase (eNOS) is in a coupled state which forms NO in the presence of essential cofactor tetrahydrobiopterin (BH4) and molecular oxygen. Oxidative stress, such as that caused by ESWL-induced ROS, can cause BH4 to be oxidized to dihydrobiopterin (BH2). When the BH2:BH4 ratio is increased, eNOS becomes uncoupled and produces O2- instead of NO [2, 3] (Figure 1). O2- is short-lived and converted to hydrogen peroxide (H2O2) in blood by superoxide dismutase. Protein kinase C epsilon (PKCε) has previously been found to regulate eNOS activity via phosphorylation at serine-1177. Cell-permeable PKCε peptide activator (PKCε+) increases eNOS activity while PKCε inhibitor (PKCε-) reduces eNOS activity [2]. Using a combination of eNOS cofactors BH4 or BH2 with eNOS activity regulators PKCε+ or PKCε-, we can explore the role of modulating eNOS to reduce oxidative stress and endothelial dysfunction caused by ESWL

The Role of Endothelial Nitric Oxide Synthase (eNOS) Uncoupling on Leukocyte-Endothelial Interactions in Rat Mesenteric Postcapillary Venules

BACKGROUND: Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte-endothelial interactions associated with vascular injury. Endothelial NO synthase (eNOS) is coupled to L-arginine in the presence of tetrahydrobiopetrin (BH4) to produce NO. However, when BH4 is oxidized to dihydrobiopetrin (BH2) under conditions of oxidative stress, the ratio of BH2 to BH4 is increased causing the uncoupling of eNOS to use molecular oxygen as a substrate, instead of L-arginine, to produce superoxide

The Role of Protein Kinase C Epsilon in the Regulation of Endothelial Nitric Oxide Synthase (eNOS) during Oxidative Stress caused by Extracorporeal Shock Wave Lithotripsy (ESWL)

BACKGROUND: Clinical ESWL treatment to ablate kidney stones can cause acute to chronic damage in renal microvasculature leading to decreased renal blood flow and hypertension. Shockwaves can stimulate endothelial cells to release superoxide resulting in decreased nitric oxide (NO) bioavailability and increased oxidative stress, causing vascular endothelial dysfunction in the kidney. When the dihydrobiopterin:tetrahydrobiopterin ratio is increased during oxidative stress such as ESWL, eNOS becomes uncoupled and produces superoxide instead of NO. Superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase. Protein kinase C epsilon (PKC-ε) is known to positively regulate endothelial NO synthase (eNOS) activity. In order to establish controls for the effects of PKC-ε activator and inhibitor, the effect of ESWL was tested by the comparison of ESWL-treated rats to those with no ESWL exposure, both with a saline infusion. We hypothesized that the PKC-ε peptide inhibitor (Myr-EAVSLKPT, MW = 1054.6) would decrease ESWL-induced H2O2 release and decreased the attenuation of NO release compared to ESWL-saline control rats. PKC-ε activator (Myr-NDAPIGYD, MW = 1098.5) was expected to show no effect on H2O2 or NO release, displaying a similar trend to ESWL-saline control rats

The Effects of Dihydrobiopterin and Tetrahydrobiopterin on Hydrogen Peroxide and Nitric Oxide Release During Extracorporeal Shockwave Lithotripsy

Extracorporeal shockwave lithrotripsy (ESWL) is an effective, non-invasive clinical therapy utilized to break up stones in the kidney and urinary tract. A lithotripter generates high-energy acoustic pulses and propagates those shock waves through a lens on a region that focuses on the location of the stone, in turn breaking up the stone. The successive pulses generate shearing forces and cavitation bubbles. Cavitation bubbles are the formation and implosion of liquid free zones. The cavitation bubbles implode rapidly to create their own shockwaves that also put pressure on the stone. After treatment, fragmentation of the stone allows the debris to be cleared by the flow of the urinary tract. The problem is that to break up the kidney stone, it requires many repetitive shock waves that not only hit the kidney stone but also the surrounding tissue. Although lithotripsy provides a safer alternative to invasive treatments for removing harmful stones, ESWL may cause prolonged vasoconstriction after ESWL treatment, reducing renal blood flow, and subsequent endothelial dysfunction, which may cause kidney damage leading to acute to chronic hypertension clinically. ESWL-induced vascular oxidative stress and further endothelial dysfunction may be mediated by reduced levels of endothelial-derived nitric oxide (NO) and/or increased reactive oxygen species. Previous studies have shown that ESWL can induce oxidative stress, which can cause an increase in blood hydrogen peroxide (H2O2) and a decrease in endothelial-derived NO bioavailability release. Under normal conditions, tetrahydrobiopterin (BH4) is the cofactor to promote eNOS coupling, and endothelial-derived NO is produced. When the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is increased during oxidative stress, such as ESWL, BH2 promotes eNOS uncoupling and produces superoxide (SO) instead of NO. (1,2) (Figure 1) SO is then later converted to H2O2 by superoxide dismutase. BH4 and BH2 bind to eNOS with equal affinity, therefore the ratio will determine whether eNOS principally produces NO or SO

The Effects of Modulating eNOS Activity and Coupling on Leukocyte-endothelial Interactions in Rat Mesenteric Postcapillary Venules

Background: Leukocyte-endothelial interactions associated with vascular injury are attenuated by endothelial-derived nitric oxide (NO). Endothelial NO synthase (eNOS) in the presence of tetrahydrobiopterin (BH4) produces NO from L-arginine and is termed eNOS coupling. However, when the ratio of dihydrobiopterin (BH2) to BH4 is increased, eNOS becomes uncoupled and produces superoxide instead of NO. Protein kinase C epsilon (PKC ε) positively regulates eNOS activity. This study examined modulating eNOS activity and coupling by superfusing BH2 (100 μM) by itself, combined with PKC ε activator (10μM) or PKC ε inhibitor, or combined with BH4 (100μM) and PKC ε activator in rat mesenteric venules