Background
Hypertension is a global public health issue and contributes to the burden of heart disease, stroke, kidney failure and premature mortality (13 % of total deaths worldwide)[
1]. The kidney serves as a major organ for maintaining normal blood pressure (BP) and the local renal renin angiotensin system (RAS) pathway acts as the master regulator of renal function during hypertension [
2‐
4]. The renin-angiotensin-aldosterone system (RAAS) is a signaling pathway responsible for regulating the body's blood pressure [
5‐
8]. Stimulated by low BP the kidney releases renin, this triggers a signal transduction pathway generating eventually angiotensin II that causes vasoconstriction, leading to increase in BP. Several cardiovascular therapies for high BP, target the RAAS system and these therapies are now being explored for their efficacy in treating PTSD [
9,
10].
The juxtaglomerular (JG) cells in the kidney express renin a member of the aspartyl protease family. It is the limiting enzyme in RAS pathway that converts angiotensinogen to angiotensin I (Ang I) [
11]. Renin production is tightly regulated at the transcriptional level and the active renin is released into the circulation through regulated exocytosis [
11,
12]. About 80 % of the renin present in plasma is in an enzymatically inactive form called pro-renin. Kidney processes inactive pro-renin to renin and is the major source of circulating active renin in humans. The plasma renin concentration contributes significantly to cardiovascular and renal diseases like hypertension, coronary heart disease, and chronic kidney disease [
13]. Thus the conversion of pro-renin to renin is a potential regulatory site for therapeutic intervention.
We studied the effect of the
KLKB1 (located on chromosome 4) missense variant rs3733402 (Asn124Ser) on circulating levels of active renin and observed that homozygous carriers of the minor allele (Ser/Ser) displayed lower levels of active renin [
14]. In vitro proteolysis and cell biology indicated that pro-renin was a substrate for plasma kallikrein (KAL). The KAL-activated renin in turn, was able to cleave substrate angiotensinogen to angiotensin 1 the precursor for vasoconstrictor angiotensin II. Situated on chromosome 5, the coagulation factor
F12 5’-UTR variant rs1801020 also showed significant association with plasma levels of active renin. The
F12 locus encodes for the FXIIa protease responsible for converting pre-kallikrein to KAL. The possible implication of the intrinsic coagulation system and the fibrinolytic system in renin activation has been discussed. In both the independent cohorts a strong association was observed between levels of active renin and occurrence of the minor alleles.
Discussion
In vitro studies have demonstrated that proteases such as trypsin, plasmin, pepsin, kallikrein and several others activate zymogen pro-renin to active renin [
35‐
38]. Studies before the era of mass spectroscopy suggested involvement of KLKB1 and FXIIa in pro-renin processing [
39‐
41]. Genetic variation at the
KLKB1 locus (encoding for plasma pre-kallikrein or Fletcher factor; EC 3.4.21.34) was previously most widely investigated for its roles in coagulation and allergy. We demonstrate using in vitro enzymatic assay the ability of active protease KAL in processing pro-renin → renin. A second association of renin activity and the protease
F12 locus (encoding for Factor XII or Hageman factor; EC 3.4.21.38) suggests a cascade of enzymatic events (FXIIa → KAL) in control of pro-renin activation. Generation of active renin by the cascade thus provides evidence of a site for BP regulation.
The
KLKB1 locus lies directly beneath a previously described LOD peak (LOD = 3.2) for BP on chromosome 8 in the genetically hypertensive strain of mice (BPH) [
42]. We therefore explored the effects of
KLKB1 genetic variation upon formation of active renin. While most of the
KLKB1 single nucleotide polymorphisms (SNPs) reported are located in the non-coding regions, rs3733402 in exon 5 results in an amino acid substitution Asn124Ser [
14,
33]. This mutation in the apple domain 2 of heavy chain reduces the binding of KAL to its substrate HMWK, and therefore this SNP was chosen to investigate its association with prorenin processing. Indeed, an immunoassay specific for active renin revealed that Ser/Ser homozygotes had lower circulating active renin (Fig.
1), consistent with diminished pro-renin cleavage by a less active Ser allele. Previously, rs3733402 has shown strong association with pre-pro-endothelin-1 and pre pro-adrenomedullin in the Prevention of Renal and Vascular End stage disease (PREVEND) study [
43]. In the recent study by Lieb et al. the top SNPs identified were rs12374220, an intronic variant in the
TENM3 gene, rs5030062 in the intron 6 of kininogen 1 gene and rs4253311 in intron 11 of the kallikrein B (
KLKB1) gene. The intronic SNP rs4253311 provided no evidence for association with renin concentrations and explained 0.87 % of plasma renin activity variance [
44]. In our study MALDI mass spectrometry documented the formation of active renin and the pro-peptide after digestion of pro-renin with KAL (Fig.
2 & Fig.
3). Furthermore the sub-cellular co-localization of renin with KAL suggests molecular interaction between these two proteins (Fig.
5a &b). Renin immunoreactivity has previously been shown in the cytoplasmic granules of cultured JG cells and in kidney sections [
45]. The cleavage sites involved in pro-renin processing include lysine-arginine, which is the recognition site of plasma kallikrein [
46]. Our genetic and biochemical data suggests an enzyme-substrate relation between KAL and prorenin. This suggests the possible existence of feedback regulation at the molecular level in the events leading to active renin generation by KAL and BP regulation.
KAL is a glycoprotein that takes part in the surface dependent activation of blood coagulation, fibrinolysis and kinin generation. It is synthesized in the liver and secreted into the blood as prekallikrein, which is then converted to active plasma kallikrein by factor FXIIa [
47]. The C46T 5’-UTR polymorphism associated with Hageman factor has been described to be associated with its plasma concentration and thrombotic risk [
48,
49]. The KAL protease might catalyzes the conversion of HMWK to bradykinin in one hand, and the active renin on other hand. The downstream target angiotensin converting enzyme (ACE) then modulates the concentration of angiotensin II, the key player of the RAAS system, and bradykinin, a component of the kallikrein-kinin system in opposite direction, therefore establishing a direct interaction between kallikrein-kinin and renin-angiotensin system [
50,
51].
The genetic variation in the
F12 and
KLKB1 loci directly affecting their amino acid sequence could ultimately influenced the processing, secretion or circulation of the active renin protein, which in turn mediates the BP phenotype. Allelic effects might also act on the cluster of characteristics associated with cardiovascular risk for which plasma renin is a biomarker. In the coagulation system, it has been reported that even the homozygous deficiency of the
KLKB1 loci results in no discernible coagulopathy [
52]. In treatment of hereditary angioedema inhibition of KAL does play a beneficial role, perhaps by inhibition of bradykinin formation [
53].
Advantages and limitations
Here we report a comprehensive GWAS showing correlation between polymorphisms at two independent loci (KLKB1 rs3733402 and F12 rs1801020) and plasma renin activity. Cellular and biochemical evidence is provided to establish that correlation. To our knowledge this is the first report of SNPs in two independent loci with significant trait association with activation of renin-angiotensin system. This study focused on the best characterized SNP (rs3733402) in the exon 5 of KLKB1 gene. Although association of kallikrein with renin activation has previously been described, adequate information on direct in vitro protease biochemistry was lacking. Therefore we used a mass spectrometry approach to characterize in vitro digestion of prorenin by KAL to reestablish kallikrein association with prorenin processing. In the scenario of this genetic association, the efficacy of digestion of prorenin by mutant KAL (Asp124Ser) needs to be compared with that of the wild type KAL. We have not addressed in these populations the active plasma renin association with the previously described intronic variant at KLKB1 (rs4253311) and other SNPs. Future studies will explore the association of these two SNPs with BP, renal and/or metabolic traits.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NB carried out the biochemical and cell biology studies & wrote the manuscript; AXM, FR,KZ did the genetic and statistical analysis; SAM did immunofluorescence and manuscript preparation; SK,MM,KZ &CMH did the biochemical assays; RSF did the transcriptome studies; SKM helped in the experimental studies. DGB and CMN did the human studies and helped write the manuscript. SV helped in study design, experimentation and writing of the manuscript; DTOC conceived of the study, its design and coordination and helped to draft the manuscript.