An in vitro method for the determination of protein turnover in incubated proximal tubule segments

Understanding the growth of a tissue ultimately requires that one understand the mechanisms whereby protein mass may be changed, the nature of the signal for change, and the effector pathways mediating the growth in response to the signal. Protein mass may be altered by modulating the determinants of protein turnover, namely the rates at which proteins are synthesized (PS) and degraded (PD). These determinants are incompletely characterized in kidney tissue, especially in states of hypertrophy. In other tissues, release of an unmetabolized amino acid has been used as a marker for quantifying the net degradation of intracellular proteins [1]. This strategy has yielded valuable insights concerning the overall rate of muscle proteolysis in several models of increased catabolism [2–5]. One of the impediments to the study of protein breakdown in kidney has been the lack of identification of an analogous amino acid which could serve as a suitable marker for net protein degradation in this tissue. Hence, prior attempts at estimating protein turnover in kidney have been largely qualitative in scope [6–8]. Nevertheless, it has been estimated that 30% of renal cortical proteins turn over each day [6] highlighting the potential impact that even slight changes in protein turnover might have on renal cortical mass. We report a method for simultaneously quantitating the determinants of protein turnover in incubated proximal tubule segments. Net PD is measured as the change in media lysine while PS is measured as the rate of incorporation of L-[U-14C] phenylalanine into protein. The utility of this method is underscored by applying this method to the state of renal growth induced by high-protein feeding