Real-time electrochemical detection of ATP and H₂O₂release in freshly isolated kidneys

Abstract

Extracellular nucleotides such as adenosine-5'-triphosphate (ATP) and reactive oxygen species are essential local signaling molecules in the kidney. However, measurements of changes in the interstitial concentrations of these substances in response to various stimuli remain hindered due to limitations of existing experimental techniques. The goal of this study was to develop a novel approach suitable for real-time measurements of ATP and H₂O₂ levels in freshly isolated rat kidney. Rats were anesthetized and the kidneys were flushed to clear blood before isolation for consequent perfusion. The perfused kidneys were placed into a bath solution and dual simultaneous amperometric recordings were made with the enzymatic microelectrode biosensors detecting ATP and H₂O₂. It was found that basal levels of H₂O₂ were increased in Dahl salt-sensitive (SS) rats fed a high-salt diet compared with SS and Sprague-Dawley rats fed a low-salt diet and that medulla contained higher levels of H₂O₂ compared with cortex in both strains. In contrast, ATP levels did not change in SS rats when animals were fed a high-salt diet. Importantly, angiotensin II via AT₁ receptor induced rapid release of both ATP and H₂O₂ and this effect was enhanced in SS rats. These results demonstrate that ATP and H₂O₂ are critical in the development of salt-sensitive hypertension and that the current method represents a unique powerful approach for the real-time monitoring of the changes in endogenous substance levels in whole organs.

Reactive oxygen species as important determinants of medullary flow, sodium excretion, and hypertension

“…Relative (150). These studies not only confirmed that H 2 O 2 levels were increased in SS rats fed a high-salt diet compared with low salt but also that medullary H 2 O 2 levels were significantly higher than in the cortex.…”

Purinergic signalling in the kidney in health and disease

“…Nucleotide release from renal epithelia is both constitutive (suggestive of a 'purinergic tone') and activated by mechanical or agonistinduced stimuli. Microelectrodes have measured steady-state ATP concentrations of~400 nM in the rat kidney cortex and showed that infusion of Ang II caused a rapid and transient increase, consistent with regulated release of nucleotide [277].…”

A NOX4/TRPC6 Pathway in Podocyte Calcium Regulation and Renal Damage in Diabetic Kidney Disease

“…To test the contribution of NOX4 to H 2 O 2 release in glomeruli, we employed here a novel approach featuring enzymatic biosensors capable of detecting specific substances and dynamic changes in their concentrations in fresh tissues. 32,33 In order to test H 2 O 2 release, we isolated glomeruli from the wild-type SS rats and age-matched SS Nox42/2 animals and washed them by spinning; next, glomeruli were placed into a 100-ml tube. A biosensor was then placed into the solution (see Figure 4A) and, after allowing time for equilibration, stable basal levels of H 2 O 2 were recorded for several minutes.…”