[Micromethods for the determination of metabolic characteristics in individual nephron segments]

Since a single nephron is a functional unit of the kidney, individual microdissected segments from the nephron would be ideal tissue samples for investigations on renal pharmacology. Several micromethods have enabled researchers to analyze the biochemical and pharmacological characteristics of these nephron segments. Miniaturized cuvettes containing microliter volumes of samples can be applied for general procedures of photometry like Lowry's protein determination. Fluorometry becomes a more sensitive method when enzymatic cycling systems of NAD/NADH or NADP/NADPH are combined, which have been used for assays of enzyme activities or substrate contents in minute biological samples having tissue proteins less than 1 microgram. The microchemiluminescence procedure has been successfully utilized for cellular ATP content or oxygen radical generation. Radioimmunoassay can be used to determine endogenous components such as cyclic nucleotides, eicosanoids, etc. Continuous gradient polyacrylamide microgels prepared in 5- to 10-microliters capillaries have made it possible to quantify the intranephron distribution of cytochrome P-450, xanthine oxidase and superoxide dismutase. As an example of the modern techniques, microscopic fluorometry using Fura-2AM has been established to identify agonist-induced cytosolic free calcium transients.