The APA peptide has been directly implicated in the degradation of angiotensinogen II to angiotensinogen III in the brain and the kidney (Wolf, et al., 1997
; Zini, et al., 1996
). Antibodies directed against APA can induce acute proteinuria in BALB/c wild type mice (Assmann, et al., 1992
; Mentzel, et al., 1996a
). This proteinuria has been shown to be independent on angiotensin II (Gerlofs-Nijland, et al., 2001
; Mentzel, et al., 1999
). Mice with a targeted deletion of the angiotensinogen (AGT
) gene also develop heavy albuminuria to comparable levels to the BALB/c wild type mice after injection of ASD37/41 (Gerlofs-Nijland, et al., 2001
). This has lead to the notion that structural alterations of the APA peptide can cause the acute albuminuria. We were therefore interested in investigating whether humans with the proteinuria podocyte disorder FSGS can carry ENPEP
mutations that may increase their susceptibility to glomerular injury.
From the sequencing of 188 patients with FSGS and 48 controls we identified 15 alleles that caused amino acid changes. Five of these (Q435E (1305C>G), D622N (1866G>A), A676T (2028G>A), E686K (2058G>A) and E687D (2061(G>T)) were found in cases and in no controls (5/188, 3%). We also found two private non-conservative alleles in two normals sequenced (2/48, 4.2%). Private non-conservative alleles were not more common in cases compared to normals (p= 0.583, Chi square analysis), although such a comparison is underpowered. In addition, the frequency of non-synonymous alleles did not differ between familial and sporadic cases with FSGS (p= 0.355, Chi-square analysis).
Two SNPs were found to have a higher allele frequency in cases (Q213R) or controls (I32V). When corrected for the multiple variants examined, these differences are not statistically significant. Nevertheless, these differences suggest that larger studies designed to determine if certain alleles of ENPEP confer susceptibility to or protection from proteinuric kidney disease may be worthwhile. Also of note, W413X was found at higher allele frequency in FSGS cases compared to normals (1.1% compared to 0.3% respectively) but, again, this difference was not statistically significant. This variant produces a truncated APA peptide that leads to loss of a GFP signal from western blotting and a loss of APA cell surface activity in vivo. Genotyping these I32V, Q213R and W413X ENPEP SNPs in large well-controlled groups with and without proteinuric kidney disease seems warranted.
We were interested to explore the enzymatic activity of alleles harboring the non-synonymous variants we identified. Six of the seven “private” variants studied lead to altered enzyme activity of APA (86%), compared to four of the eight SNPs (50%). These included both variants leading to increased as well as decreased APA activity. Two of the ENPEP variants, W413X and E686K lead to nearly complete loss of APA activity.
It is possible that private non-conservative variation in ENPEP may cause kidney dysfunction in combination with alleles in other genes that are known to cause inherited forms of FSGS, or other alleles that disrupt related pathways. None of the families we screened had disease attributed to mutations in either of the NPHS2, ACTN4, CD2AP, or TRPC6 genes from prior analysis. The identified ENPEP non-synonymous alleles demonstrated did not segregate with disease in the respective families ( and ). As would be expected, the private non-conservative ENPEP alleles we identified affected APA activity more frequently than more common polymorphisms (or SNPs). These variants can lead to either increased or decreased activity.
Several lines of evidence indicate that the APA protein is involved in experimental acute proteinuria via antibodies directed to particular regions of the peptide (Assmann, et al., 1992
; Dijkman, et al., 2003
; Gerlofs-Nijland, et al., 2001
; Gerlofs-Nijland, et al., 2003
; Mentzel, et al., 1996a
; Mentzel, et al., 1999
), and perhaps even the acceleration of slowly progressive FSGS in mice that ectopically express the Thy1.1 antigen on podocytes (Assmann, et al., 2002
). Yet other studies have shown that the APA peptide may be involved in salt-induced renal damage in male Dahl salt-sensitive rats (which have a higher renal mRNA expression for the protein compared to male Dahl salt-resistant controls mice that have elevated APA mRNA and this is thought to protect them against the hypertension and glomerulosclerosis that develops in the salt-sensitive rats) (Nomura, et al., 2005
Along these same lines, homozygous mice that have targeted deletion of ENPEP
(Lin, et al., 1998
) have elevated baseline blood pressure, which can be increased by continual infusion of angiotensinogen II to a greater extent compared to wild type mice (Mitsui, et al., 2003
). In addition, high blood pressure caused by hyperactive brain RAAS in the DOCA-salt sensitive rat can be reduced for up to 24 hours by a selective inhibitor of APA that is able to transgress the blood-brain barrier (Fournie-Zaluski, et al., 2004
). Thus, the APA protein has been implicated in proteinuria and salt-induced hypertension. There appears to be a non-trivial incidence of human ENPEP
nucleotide changes that lead to private non-conservative alleles that have functional effects. Although we were unable to show a clear relationship between the presence of a functional ENPEP variant and FSGS, such functional variants remain strong candidate for having phenotypic effects in humans. It is still unclear whether private non-conservative alleles are more common in patients with end-stage renal disease or diseases that cause cardiovascular or hypertension events, but we believe that further resequencing of ENPEP
in a cohort of cases with well defined renal and cardiovascular diseases may be justified.