Figure 8. Transient expression and functional characterization of REN variants in Human Embryonic Kidney 293 cells.

The wild type renin (WT); signal peptide, prosegment and mature renin mutations; African-American (AA_REN) variants and empty vector were analyzed. (A, B) Western blot analysis of (A) cell lysates and (B) culture media. Molecular weights of immuno-reactive proteins present in the WT lysates correspond with expected molecular weights of prorenin (47 kDa), preprorenin (45 kDa) and renin (43 kDa). (C, D) Immunoradiometric assay (IRMA) of prorenin and renin amounts in (C) cell lysates and (D) culture media employing a radio-labeled antibody that specifically recognizes active site of renin. The concentration of prorenin was calculated as the difference between the renin concentration measured before and after trypsin treatment, which activates renin by proteolytic cleavage of the prosegment from prorenin. Amounts of mutated renin (grey bars) and prorenin (black bars) were normalized to the amount of wild type renin. The values represent means ± SD. Measurements were performed in three independent clones for each of the constructs. The individual measurements were carried out in triplicate. The statistical significance of the differences between the WT and renin variants protein amounts was tested by t test. *p < 0.05; **p > 0.01; ***p > 0.001; n.d. not done. (E, F) Enzymatic activity of (E) mature renin secreted into culture media and (F) prorenin secreted into culture media. The values were normalized to the WT (100%) and represent means ± SD of relative fluorescent unit (RFU) generated by renin mediated cleavage of the 5-FAM and QXL520 conjugated renin substrate. Measurements were performed in three independent clones for each of the constructs. The individual measurements were carried out in triplicate. The statistical significance of the differences between activity of the wild type renin (WT) and renin variants was tested by t test. *p < 0.05; **p > 0.01; ***p > 0.001; n.d. not done.