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The complexity of the cellular response, induced even by the simplest experimental stimulus, requires an increased number of cellular parameters to be simultaneously monitored. An all electrochemical system allowing the simultaneous and real-time monitoring of both cell adherence and superoxide release into the extracellular space was developed to address this challenge. Cell adherence (to neighboring cells and to substrate) was monitored using non-faradaic impedance spectroscopy while the superoxide release was monitored using a cytochrome c-based amperometric biosensor. The system was used to observe for the first time how these two cellular parameters are changing in real-time for renal cells exposed to calcium oxalate, a calculus-forming salt. It was discovered that calcium oxalate crystals decrease cell adherence and in the same time induce oxidative stress by an overproduction of superoxide. Subconfluent cells, without fully developed tight junctions, appear to be more vulnerable than confluent cells with tight junctions indicating the important protective role of these junctions.

Calcium oxalate is one of the main constituents of kidney stones and has a proved deleterious effect on renal cells that is mediated by oxidative stress. However, the subcellular source of this oxidative stress, and whether it is extending to the extracellular space or not, is still disputed. Therefore, an electrochemical superoxide biosensor was constructed, positioned above A6 renal cells, and used to measure in real-time the extracellular oxidative burst following addition of calcium oxalate crystals. It was observed that A6 cells do secrete superoxide into their extracellular space in few minutes after encountering calcium oxalate crystals. The amount of released superoxide peaks at about 20 min. Superoxide is cleared away from the extracellular space after approximately 3 h. Superoxide secretion depends on the presence of superoxide-scavenging enzyme superoxide dismutase, the age of the cells, the amount of calcium oxalate crystals, and the temperature. Moreover, the effect of calcium oxalate crystals was mimicked by phorbol 12-myristate 13-acetate. The developed sensing system can be a useful tool for biologists investigating nephrolithiasis at cellular level.