Spotlight on Renin

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. This heterogeneity is largely caused by differential glycosylation.6 Recently, a second product of the renin gene was identified.7 It is synthesized from a transcript that contains an alternative exon 1. It lacks the signal peptide and part of the prosegment and thus gives rise to a truncated prorenin that remains intracellular and displays enzymatic activity. The latter relates to the fact that a prosegment of insufficient length will not fully cover the enzymatic cleft. Evidence for intracellular angiotensin generation is, however, lacking,8,9 and truncated prorenin has also been demonstrated extracellularly.10,11 Prorenin Activation Prorenin can be activated in 2 ways: proteolytic or nonproteolytic. Proteolytic activation involves actual removal of the propeptide, eg, by (endogenous) kallikrein or (exogenous) trypsin or plasmin. Kallikrein is generated from prekallikrein in plasma after destruction of the natural inhibitors of contact activation, by exposure to low pH or low temperature.12 An unidentified aspartic protease activates prorenin proteolytically in acidified amniotic fluid.13 When using trypsin to activate prorenin in vitro, care must be taken to prevent destruction of prorenin, eg, by applying brief trypsin exposure times and by terminating the activation with a trypsin inhibitor. Another, more elegant way of trypsin-induced prorenin activation without destruction is incubation at 4°C with trypsin linked to Sepharose.14 This is easily removed by centrifugation. Plasmin (purified or recombinant) also activates prorenin, although the presence of endogenous plasmin inhibitors in plasma limits the use of plasmin to prorenincontaining samples other than plasma. In vivo, proteolytic activation of prorenin occurs in the kidney. Various renal processing enzymes have been proposed, including proconvertase 1 and cathepsin B.15,16 No evidence exists for in vivo prorenin activation by kallikrein, even though patients with prekallikrein deficiency (Figure 1) or high-molecular-weight kininogen deficiency have relatively low levels of renin. Bolus infusions of recombinant human prorenin in monkeys did not provide evidence for prorenin–renin conversion in the circulation.17 Proteolytic prorenin activation, possibly involving a serine protease, has however been demonstrated in isolated cardiac and vascular cells.18,19 Nonproteolytic activation of prorenin is a reversible process. It can best be imagined as an unfolding of the propeptide from the enzymatic cleft (Figure 2). This unfolding consists of at least 2 steps. In the first step, the propeptide moves out of the enzymatic cleft, and in the second step the renin part of the molecule assumes its enzymatically active conformation.20 Nonproteolytic activation can be induced by exposure to low pH (with an optimum at pH 3.3) and cold, called acid activation and cryoactivation, respectively.21,22 Acid activation leads to complete activity of prorenin, cryoactivation to partial ( 15%) activity. Note that acidification of plasma will