A variety of physiological studies provides compelling evidence that angiotensin II (Ang II) has important actions in the renal proximal convoluted tubule. Biochemical and molecular biologic techniques have permitted the identification of renin, angiotensinogen (ang-n), and angiotensin converting enzyme (ACE) proteins and mRNAs within the kidney. Several lines of evidence suggest that a major site of the intrarenal renin angiotensin system (RAS) is the proximal tubule (PT). Within the PT, ang-n and ACE mRNAs are synthesized, whereas renin is more likely reabsorbed. As there are also angiotensin receptors within PT as well as endogenous local Ang II production, there is in vivo a complete PT. Furthermore, persuasive data support the concept that a complete RAS within the proximal tubule plays important roles in a variety of tubular functions including sodium and water reabsorption, protein processing, and, perhaps, cellular hypertrophy. The present project is designed to study regulation and function of the proximal tubule (PT) renin angiotensin system (RAS) utilizing PT cells in culture. Much information lends support to the concept that the proximal tubular RAS subserves autocrine, paracrine, and intracrine functions. Available established PT-like cell lines such as OK cells are not truly analogous to PT cells in vivo; primary PT cells in culture have proven difficult to grow and passage, and also de-dedifferentiate rapidly. Thus, developing new ways to study PT cells in primary culture and concomitantly establishing transformed, immortalized PT cell lines or cell lines cloned from transgenic animals would be of great benefit to further study of the PT RAS. Thus, our aims are: 1. To deduce functions of the PT RAS by developing immortalized PT cell lines and studying cellular and subcellular regulation and localization of RAS components in these cells compared to primary cultures; 2. To study the autocrine and paracrine mechanisms of action of the PT RAS, hypothesizing that local PT angiotensinogen provides-local substrate for tubular angiotensins and that PT ACE subserves multiple functions; 3. To investigate the effects of renin on PT cells by (a) co-culture of PT cells with renin-producing cells, (b) exposure of PT cells to media into which renin has been added and (c) by transfecting renin into immortalized PT cells. Thus, the present application seeks to examine the regulation and functions of the PT RAS on a cellular and subcellular level. We anticipate that the data obtained will be novel and have implications for the ways in which the PT RAS is involved in both renal and cardiovascular homeostasis.
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