The objective of this investigation is to develop human renin producing cell lines. The ultimate goal will be to obtain a cell line which is identical to the renal juxtaglomerular cell in mechanisms of renin gene regulation and prorenin processing. Thus, the following phenotypic characteristics for a cell line include, 1) expression of renin mRNA, 2) conversion of prorenin to renin, and 3) release of renin by the regulated secretory pathway. Major caveats to the production of such a cell line include selection of renin producing cell types from primary culture and maintenance of differentiated function consistent with the phenotypic characterization. We will approach these problems by employing the -3000 upstream promotor of the human renin gene for selection based on tissue specific gene expression and by immortalization with the temperature sensitive t antigen (tsA) which will allow growth and differentiation to be maintained by altering the tissue culture temperature. We will also create an external milieu conducive to renin production; such alterations involve growing the cells on laminin and adding converting enzyme inhibition to prevent the generation of angiotensin II which is known to suppress renin gene expression. Immortalized renin producing cell lines will be developed from human kidney, uterine lining and a renin secreting tumor, since these tissues produce large quantities of renin and are readily available to us. The development of such a cell line is vital for recognition of transcription factors involved in renin gene expression and for elucidation of signal transduction involved in regulation of renin secretion. Another approach we will use to obtain renin producing cell lines is to transfect the human renin gene into established cell lines. Since recent studies indicate that cathepsin B is the human renal prorenin processing enzyme, responsible for conversion of prorenin to renin, we will also transfect an expression vector for human cathepsin B into renin producing cells. Established cell lines include Chinese hamster ovary (CHO) cells, AtT-20, mouse pituitary cell line, GH3, a rat pituitary cell line and PC12, a rat adrenal medullary tumor cell line. These cell lines, have different capability of sorting and processing human prorenin. The pathway of prorenin and renin secretion by AtT-20 cells resembles that in the kidney, while in CHO cells, it resembles that in human uterine lining. Co-transfection of expression vectors for human renin and human cathepsin B will be a key approach to 1) develop a cell line with the phenotypic characteristics defined above, 2) determine the precise role of cathepsin B in renin production, 3) investigate the cell biology of prorenin (as well as other protein) sorting and processing, and 4) create models of defective prorenin processing which resemble that in extrarenal tissues and pathological conditions.
