Inorganic phosphate (Pi) filtered by the kidney is reabsorbed primarily in the proximal tubule. The initial and possibly rate-limiting step in this process is Na-dependent transport of Pi across the luminal brush border membrane (BBM) of the proximal tubule. The capacity of the BBM for Pi transport is altered in response to changes in the dietary Pi intake and to various hormones and drugs. While other factors may contribute to the regulation of renal Pi reabsorption, a specific deficiency in the adaptive mechanism of the Pi transport system in the BBM has serious consequences for Pi homeostasis. The long-term objective of the current proposal is to understand the cellular regulation of Na-dependent Pi transport across the renal BBM. Treatment of rats with nicotinamide leads to an increase in the NAD level in the proximal tubule and, accompanying this change, there is specific inhibition of Na-dependent Pi transport across the BBM and an increase in urinary Pi excretion.
The specific aim of this proposal is to investigate whether NAD is used for ADP-ribosylation of the cytosolic surface of the BBM, by what mechanism, whether this process causes specific inhibition of Na-dependent Pi transport across the BBM, and whether there are enzyme-catalysed reactions for reversal of ADP-ribosylation. The studies which will address these questions will be carried out largely in vitro using isolated BBM preparations. The BBM will be ADP-ribosylated with either NAD or ADP-ribose and Pi transport will be assessed as uptake of radiolabelled Pi in the presence of a transmembrane sodium gradient. Uptake of other solutes, such as glucose and amino acids, will be monitored to establish the specificity of any changes in Pi transport. Reversal of ADP-ribosylation will be studied using BBM ribosylated with radiolabelled NAD. The BBM will be incubated with various cell fractions and the radiolabelled material which is released will be measured and identified.
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