Limits...
In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress.

Anderson BR, Howell DN, Soldano K, Garrett ME, Katsanis N, Telen MJ, Davis EE, Ashley-Koch AE - PLoS Genet. (2015)

Bottom Line: However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G).Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects.Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.

No MeSH data available.


Related in: MedlinePlus

In vivo modeling of human APOL1 variants associated with disease.apol1 MO injected larvae were complemented with the respective human mRNA corresponding to APOL1 G1 (S342G/I384M) (100pg/nl) and G2 (100pg/nl) risk variants and scored for edema formation at 5 dpf (n = 26–65 embryos/injection; repeated three times). (A, B) Neither risk variant of APOL1 rescues significantly the edema phenotype observed in apol1 morphants. However, when human APOL1 G2 mRNA was injected alone (B), a significant number of embryos develop edema compared to sham-injected controls, suggesting a possible dominant-negative effect of the G2 altered protein. (C, D) apol1 morpholino injected larvae were complemented with human mRNA corresponding to either (C) APOL1 G1 I384M or (D) APOL1 G1 S342G and scored for edema formation at 5 dpf (n = 48–93 embryos/injection; repeated two times). Each individual variant comprising APOL1 G1 risk rescues significantly edema formation in apol1 morphant embryos, suggesting that both G1 variants must be present to confer loss of APOL1 function. (E-F) apol1 morphants co-injected with human APOL1 G1 or G2 mRNA fail to rescue filtration defects as indicated by dextran clearance, while larvae injected with G2 mRNA alone display increased clearance over time. (G) Titration of G2 injected embryos with increasing concentrations of human WT APOL1 mRNA show a significant reduction in edema formation of developing embryos at 5 dpf. (H) Zebrafish embryos injected with APOL1 G2 mRNA (100pg/nl) alone display glomerular aberrations similar to that of myh9 suppressed larvae, with microvillus protrusions present (open arrowheads), although the glomerular basement membrane appears normal (filled arrowheads). Podocyte foot processes (* asterisk) are apparent, although sparsely present. (I) Embryos injected with APOL1 G1 mRNA (100pg/nl) alone display normal glomerular ultrastructure. Scale bar, 500nm. White bars, normal; black bars, edema. C, sham-injected control; NI, non-injected control. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492502&req=5

pgen.1005349.g004: In vivo modeling of human APOL1 variants associated with disease.apol1 MO injected larvae were complemented with the respective human mRNA corresponding to APOL1 G1 (S342G/I384M) (100pg/nl) and G2 (100pg/nl) risk variants and scored for edema formation at 5 dpf (n = 26–65 embryos/injection; repeated three times). (A, B) Neither risk variant of APOL1 rescues significantly the edema phenotype observed in apol1 morphants. However, when human APOL1 G2 mRNA was injected alone (B), a significant number of embryos develop edema compared to sham-injected controls, suggesting a possible dominant-negative effect of the G2 altered protein. (C, D) apol1 morpholino injected larvae were complemented with human mRNA corresponding to either (C) APOL1 G1 I384M or (D) APOL1 G1 S342G and scored for edema formation at 5 dpf (n = 48–93 embryos/injection; repeated two times). Each individual variant comprising APOL1 G1 risk rescues significantly edema formation in apol1 morphant embryos, suggesting that both G1 variants must be present to confer loss of APOL1 function. (E-F) apol1 morphants co-injected with human APOL1 G1 or G2 mRNA fail to rescue filtration defects as indicated by dextran clearance, while larvae injected with G2 mRNA alone display increased clearance over time. (G) Titration of G2 injected embryos with increasing concentrations of human WT APOL1 mRNA show a significant reduction in edema formation of developing embryos at 5 dpf. (H) Zebrafish embryos injected with APOL1 G2 mRNA (100pg/nl) alone display glomerular aberrations similar to that of myh9 suppressed larvae, with microvillus protrusions present (open arrowheads), although the glomerular basement membrane appears normal (filled arrowheads). Podocyte foot processes (* asterisk) are apparent, although sparsely present. (I) Embryos injected with APOL1 G1 mRNA (100pg/nl) alone display normal glomerular ultrastructure. Scale bar, 500nm. White bars, normal; black bars, edema. C, sham-injected control; NI, non-injected control. *p<0.05.

Mentions: Initial reports associating APOL1 variants with kidney disease in African Americans identified two independent sequence variants, termed G1 and G2, which reside in a 10-kb region in the last exon of the gene[5–7, 10]. The G1 allele consists of two nonsynonymous coding variants in perfect LD, rs73885319 and rs60910145, while the G2 variant consists of a six base pair deletion that removes amino acids N388 and Y389 (~21% and ~13% allele frequency in African Americans, G1 and G2 respectively; Fig 1D). Therefore, we evaluated the ability of each of the G1 and G2 alleles to rescue apol1-MO injected zebrafish larvae. APOL1 G1 (I384M/S342G) and G2 allelic constructs were generated from a WT APOL1 human cDNA clone, transcribed, and co-injected with apol1-MO in zebrafish embryos (100pg/nl). Importantly, each APOL1 allelic construct produces a stable protein detectable by immunoblotting when co-injected with apol1-MO (S2 Fig). apol1 morphants co-injected with either APOL1 G1 (I384M/S342G) or G2 human mRNA did not display significant rescue of edema formation in developing embryos compared to apol1-MO injected embryos alone (Fig 4A and 4B). In addition, we also co-injected each individual G1 variant (I384M and S342G) into apol1 morphant embryos. APOL1 message encoding either p.I384M or p.S342G were individually able to rescue significantly the edema caused by apol1 suppression (Fig 4C and 4D) suggesting that the cis effect of both variants in the same haplotype is required to confer pathogenicity. When APOL1 G2 mRNA was injected alone, a significant number of embryos developed edema in comparison to sham-injected controls (n = 52–63 embryos/injection; repeated three times; p = 0.012; Fig 4B); no edema was observed with injection of 100pg APOL1 G1 mRNA alone (Fig 4A). Additionally, dextran clearance assays demonstrated that neither APOL1 G1 or G2 mRNA were able to rescue glomerular filtration defects caused by apol1 suppression, while APOL1 G2 mRNA injected alone caused significant filtration defects compared to controls (n = 12–21; p = 0.003, Control vs. G2 mRNA; Fig 4E and 4F). Finally, when we injected embryos with APOL1 G2 titrated with increasing concentrations of APOL1 WT mRNA, we observed a significant reduction of edema formation in developing embryos (Fig 4G) suggesting that this allele is conferring a dominant negative effect on protein function.


In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress.

Anderson BR, Howell DN, Soldano K, Garrett ME, Katsanis N, Telen MJ, Davis EE, Ashley-Koch AE - PLoS Genet. (2015)

In vivo modeling of human APOL1 variants associated with disease.apol1 MO injected larvae were complemented with the respective human mRNA corresponding to APOL1 G1 (S342G/I384M) (100pg/nl) and G2 (100pg/nl) risk variants and scored for edema formation at 5 dpf (n = 26–65 embryos/injection; repeated three times). (A, B) Neither risk variant of APOL1 rescues significantly the edema phenotype observed in apol1 morphants. However, when human APOL1 G2 mRNA was injected alone (B), a significant number of embryos develop edema compared to sham-injected controls, suggesting a possible dominant-negative effect of the G2 altered protein. (C, D) apol1 morpholino injected larvae were complemented with human mRNA corresponding to either (C) APOL1 G1 I384M or (D) APOL1 G1 S342G and scored for edema formation at 5 dpf (n = 48–93 embryos/injection; repeated two times). Each individual variant comprising APOL1 G1 risk rescues significantly edema formation in apol1 morphant embryos, suggesting that both G1 variants must be present to confer loss of APOL1 function. (E-F) apol1 morphants co-injected with human APOL1 G1 or G2 mRNA fail to rescue filtration defects as indicated by dextran clearance, while larvae injected with G2 mRNA alone display increased clearance over time. (G) Titration of G2 injected embryos with increasing concentrations of human WT APOL1 mRNA show a significant reduction in edema formation of developing embryos at 5 dpf. (H) Zebrafish embryos injected with APOL1 G2 mRNA (100pg/nl) alone display glomerular aberrations similar to that of myh9 suppressed larvae, with microvillus protrusions present (open arrowheads), although the glomerular basement membrane appears normal (filled arrowheads). Podocyte foot processes (* asterisk) are apparent, although sparsely present. (I) Embryos injected with APOL1 G1 mRNA (100pg/nl) alone display normal glomerular ultrastructure. Scale bar, 500nm. White bars, normal; black bars, edema. C, sham-injected control; NI, non-injected control. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492502&req=5

pgen.1005349.g004: In vivo modeling of human APOL1 variants associated with disease.apol1 MO injected larvae were complemented with the respective human mRNA corresponding to APOL1 G1 (S342G/I384M) (100pg/nl) and G2 (100pg/nl) risk variants and scored for edema formation at 5 dpf (n = 26–65 embryos/injection; repeated three times). (A, B) Neither risk variant of APOL1 rescues significantly the edema phenotype observed in apol1 morphants. However, when human APOL1 G2 mRNA was injected alone (B), a significant number of embryos develop edema compared to sham-injected controls, suggesting a possible dominant-negative effect of the G2 altered protein. (C, D) apol1 morpholino injected larvae were complemented with human mRNA corresponding to either (C) APOL1 G1 I384M or (D) APOL1 G1 S342G and scored for edema formation at 5 dpf (n = 48–93 embryos/injection; repeated two times). Each individual variant comprising APOL1 G1 risk rescues significantly edema formation in apol1 morphant embryos, suggesting that both G1 variants must be present to confer loss of APOL1 function. (E-F) apol1 morphants co-injected with human APOL1 G1 or G2 mRNA fail to rescue filtration defects as indicated by dextran clearance, while larvae injected with G2 mRNA alone display increased clearance over time. (G) Titration of G2 injected embryos with increasing concentrations of human WT APOL1 mRNA show a significant reduction in edema formation of developing embryos at 5 dpf. (H) Zebrafish embryos injected with APOL1 G2 mRNA (100pg/nl) alone display glomerular aberrations similar to that of myh9 suppressed larvae, with microvillus protrusions present (open arrowheads), although the glomerular basement membrane appears normal (filled arrowheads). Podocyte foot processes (* asterisk) are apparent, although sparsely present. (I) Embryos injected with APOL1 G1 mRNA (100pg/nl) alone display normal glomerular ultrastructure. Scale bar, 500nm. White bars, normal; black bars, edema. C, sham-injected control; NI, non-injected control. *p<0.05.
Mentions: Initial reports associating APOL1 variants with kidney disease in African Americans identified two independent sequence variants, termed G1 and G2, which reside in a 10-kb region in the last exon of the gene[5–7, 10]. The G1 allele consists of two nonsynonymous coding variants in perfect LD, rs73885319 and rs60910145, while the G2 variant consists of a six base pair deletion that removes amino acids N388 and Y389 (~21% and ~13% allele frequency in African Americans, G1 and G2 respectively; Fig 1D). Therefore, we evaluated the ability of each of the G1 and G2 alleles to rescue apol1-MO injected zebrafish larvae. APOL1 G1 (I384M/S342G) and G2 allelic constructs were generated from a WT APOL1 human cDNA clone, transcribed, and co-injected with apol1-MO in zebrafish embryos (100pg/nl). Importantly, each APOL1 allelic construct produces a stable protein detectable by immunoblotting when co-injected with apol1-MO (S2 Fig). apol1 morphants co-injected with either APOL1 G1 (I384M/S342G) or G2 human mRNA did not display significant rescue of edema formation in developing embryos compared to apol1-MO injected embryos alone (Fig 4A and 4B). In addition, we also co-injected each individual G1 variant (I384M and S342G) into apol1 morphant embryos. APOL1 message encoding either p.I384M or p.S342G were individually able to rescue significantly the edema caused by apol1 suppression (Fig 4C and 4D) suggesting that the cis effect of both variants in the same haplotype is required to confer pathogenicity. When APOL1 G2 mRNA was injected alone, a significant number of embryos developed edema in comparison to sham-injected controls (n = 52–63 embryos/injection; repeated three times; p = 0.012; Fig 4B); no edema was observed with injection of 100pg APOL1 G1 mRNA alone (Fig 4A). Additionally, dextran clearance assays demonstrated that neither APOL1 G1 or G2 mRNA were able to rescue glomerular filtration defects caused by apol1 suppression, while APOL1 G2 mRNA injected alone caused significant filtration defects compared to controls (n = 12–21; p = 0.003, Control vs. G2 mRNA; Fig 4E and 4F). Finally, when we injected embryos with APOL1 G2 titrated with increasing concentrations of APOL1 WT mRNA, we observed a significant reduction of edema formation in developing embryos (Fig 4G) suggesting that this allele is conferring a dominant negative effect on protein function.

Bottom Line: However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G).Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects.Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.

No MeSH data available.


Related in: MedlinePlus