Quantification of renal Na-K-ATPase activity by image analysing system

The localisation of renal Na-K-ATPase activity along the rat nephron by a cytochemical method, and its quantification by an image analysis system, are described in this paper. Frozen kidney sections were exposed to a trapping agent, the lead ammoniac-citrate-acetate complex (LACA), and to all the substrates necessary to the enzyme activity. The absorbance of the histochemical reaction product (precipitated in situ), proportional to the enzymatic activity, was then measured through the analysis of the grey levels of the transmitted image of the kidney section. This method was both sufficiently sensitive and technically simple to permit measurements of the enzyme in large numbers of tubules and to determine its activity in each region of the nephron. The Na-K-ATPase activity has been determined in the proximal convoluted tubule (PCT), the medullary thick ascending limb of the Henle's loop (mTAL), and the distal convoluted tubules (DCT) of the rat nephron. The Na-K-ATPase distribution shows an activity per millimeter tubule length higher in the DCT than in the mTAL and the PCT: 1,406 +/- 33, 823 +/- 64, and 350 +/- 71 pmoles Pi/tubule mm/h, respectively. In conclusion, the described method allows the segmental quantification of Na-K-ATPase activity at a cellular level and offers a precise approach to the analysis of this enzyme along the length of nephrons.