A microelectrode for continuous monitoring of redox activity in isolated perfused tubule segments

Abstract

The design and application of a micro-plantinum electrode for continuous monitoring of reducing activity in the isolated tubule preparation is described. The electrodes response to H₂O₂ up to 0.1 mmol/l, to uric acid up to 0.3 mmol/l, ascorbic acid up to 1.0 mmol/l and cysteine up to 2.0 mmol/l is almost linear. The electrode is insensitive to extracellular ions, to changes of pH (5.5–8.0), CO₂ (1–10%) and O₂ (1–100%). The reading of the electrodes is almost doubled when the temperature is increased from 20–40°C.

When reducing substances are omitted from the perfusate for isolated perfused proximal tubules of the mouse, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce redox substances in sufficient amount to interfere with the electrode reading at flow rates ≈ 10 nl/min. When the tubule is perfused with solutions containing 0.3 mmol/l uric acid, the uric acid concentration in the collected fluid is 0.16±0.01 mmol/l after a contact time of 1.36±0.1 s, revealing net uric acid reabsorption. Adding probenecid to the luminal perfusion fluid, leads to a 37.5±1.0% increase of uric acid concentration in collected fluid, disclosing the inhibitory effect of probenecid on uric acid reabsorption. If 0.3 mmol/l uric acid is added to the bath, 0.017±0.002 mmol/l uric acid is detected in the luminal fluid. The entry of uric acid into the lumen is abolished by 10⁻⁴ mol/l pyrazinamide.