A modified cytochemical assay for [Na‐K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na‐K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na‐K]ATPase activity in several distal sites. The concentration of oubain necessary for maximal inhibition of activity was 3·0 mM and half‐maximal inhibition was obtained in all regions with 30–100μM ouabain. In distal sites, [Na‐K]ATPase formed a higher proportion of total ATPase activity (60–80 per cent) than in proximal sites(20–40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na‐K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na‐K]ATPase per length of tubule. The highest activities over the basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin > MALout > PCT > PD > PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na‐K]ATPase activity in the major functional regions of the rat nephron.
All Science Journal Classification (ASJC) codes
- Clinical Biochemistry
- Cell Biology