Hydrogen Peroxide and Salt-Sensitive Hypertension

Too much salt will raise your blood pressure

This statement has been repeated with enough frequency that it has become a colloquialism. It has sparked intense debate amongst researchers in the science community as well as in the general public, with many swearing by the statement and with others pointing out that the effects are minimal at best. This wealth of interest has spurred decades of research into the matter, with mounting evidence suggesting that salt does in fact contribute to hypertension- for some. These individuals, so-described as salt-sensitive, will indeed experience elevated blood pressure that correlates with their dietary salt intake. This poses a problem as long term hypertension can lead to a variety of health concerns such as coronary artery disease, stroke and heart failure. With cardiovascular disease still the leading cause of death in the United States, researchers have turned a closer eye towards salt-sensitive hypertension in the hopes of understanding its pathology and developing future therapies.

Early work on salt-sensitive hypertension suggested that it affects a heterogeneous population of individuals, though possibly weighted towards certain demographic factors such as age and gender. It has also been postulated that there is a genetic component to saltsensitive hypertension as it seemed more frequent in individuals with a family history of high blood pressure. In terms of actual physiology, a variety of candidates have been suggested as contributing to salt sensitivity, from the renin-angiotensin-aldosterone system to blood insulin levels. More recently, researchers have narrowed in on a system of epithelial sodium channels in the renal cortex, hypothesizing that the function of these channels are at the crux of the matter.

Epithelial sodium channels (ENaC) are membrane-bound ion-channels found in the kidneys and are responsible for reabsorption of sodium ions. It has been suggested that excess reabsorption by these channels is directly linked to the expression of salt-sensitive hypertension. For example, in a paper published in 2000 by Gariepy et al., it was found that the lack of tonic inhibition of ENaC led to severe hypertension in rats with high sodium diets while rats with low sodium diets did not develop the condition. Many papers published since have also alluded to the development of salt-sensitive hypertension stemming from increased ENaC activity.

More recently, several papers have been published indicating that the upregulation of ENaC is a result of oxidative stress, mediated by the cellular secondary messenger hydrogen peroxide (H₂O₂). For a long time, H₂O₂ was viewed as merely toxic waste resulting from aerobic metabolism. It was considered damaging to cells because of its ability to induce breaks in single stranded and double stranded DNA amongst other things. Further investigation, however, has uncovered that H₂O₂ is actually involved in a complex array of redox pathways involving NOX (NADPH oxidase complex) and glutathione peroxidase. These redox mechanisms not only manage oxidative stress in cells, but also serve to regulate many signal transduction pathways through phosphorylation of key signaling proteins. With regards to salt-sensitive hypertension, many researchers believe H₂O₂ is the key to understanding the regulation of ENaC.

There are several reasons to believe that H₂O₂ may modulate ENaC activity. First, H₂O₂ is already known to regulate the function of several transmembrane proteins such as ATP-sensitive potassium channels. Second, studies in transgenic mice have found that increased catalase activity, which decomposes H₂O₂ into water and oxygen, is correlated with a decrease in sensitivity to hypertension inducing agents. Finally, studies on Tempol, an antioxidant which reduces renal oxidative stress, suggest that long term treatment prevents expression of hypertension. In more direct testing, it was found that H₂O₂ might regulate ENaC activity through phosphatidylinositide 3-kinase (PI3K). In A6 distal nephron cells, the addition of H₂O₂ led to increased ENaC open probability, which allowed for increased Na+ reuptake. However, upon inhibition of PI3K, ENaC open probability as well as Na+ uptake reverted to pre-H₂O₂ levels. This would seem to suggest that H₂O₂ not only plays a critical role in Na+ reabsorption, but does so through a kinase (PI3K) which is consistent with previous studies on H₂O₂ mechanism as a secondary messenger. If H₂O₂ is the mediator for ENaC activity, it is worth doing further investigation in the hopes of developing treatments for saltsensitive hypertension.

Our Tools

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We offer several tools to help researchers accurately study H₂O₂ and its related compounds. For example, in a study published in the Journal of Biological Chemistry, our Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit was used to quantify H₂O₂concentration in A6 distal nephron cells. This assay kit uses our Amplite^® Red peroxidase substrate to quantify H₂O₂ in solution and cell extracts through enzyme-coupled reactions. It is a one step assay that can detect as little as 3 pmol of H₂O₂ in a 100 uL assay volume. It can be conveniently applied in a 96-well or 384-well plate and be read in a fluorescence microplate reader. We offer this kit in red, near red and green fluorescence for experimental flexibility and easy multi- plexing. Because these kits are a simple mix-and-read assay, they can be easily adaptable to HTS liquid handling instruments and usable for automation.

We additionally offer Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kits for live cell applications. These kits use our OxiVision™ peroxide sensor. This is a cell-permeable probe that will generate a fluorescence signal upon reaction with H₂O₂. The probe comes in two versions, blue fluorescence and green fluorescence. Our blue fluorescence probe excites at 405 nm and emits at 450 nm, while our green fluorescence probe excites at 490 nm and emits at 530 nm. We also have kits optimized for flow cytometry applications.



In addition to our hydrogen peroxide detectors, we also provide glutathione assay kits for life science researchers. This category of kits includes assays for the quantification of GSH/GSSG ratio as well as total glutathione. This allows for easy characterization of cellular oxidative stress. Our kits are all mix-and-read, meaning that no separation step is required. This enables quick application with minimal hands-on-time. These assays are ultra-sensitive, with the ability to detect as little as 1 pmol of GSH in a 100 uL of assay volume. Our kits are compatible with standard microplate readers (Ex/Em = 490/520 nm).