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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: Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein.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

myh9 expression in the context of apol1/APOL1 modulation.Zebrafish embryos were injected with either apol1-MO (1.0ng/nl dose), APOL1 G1 (S342G:I384M) mRNA (100pg), or APOL1 G2 (100pg) mRNA alone, in the absence (white bars) or presence (black bars) of atpif1α-MO. Total RNA at 5 dpf or 3 dpf (APOL1 G2/atpif1α-MO embryos did not survive to 5 dpf) was extracted and reverse-transcribed with random primers to obtain whole-embryo cDNA. myh9 expression was determined by quantitative real-time PCR and relative expression was calculated against actb1. (A) apol1-MO injected embryos do not display any significant changes in myh9 expression compared to sham-injected control embryos. Additionally, APOL1 G1 expression does not alter myh9 expression alone, however, under atpif1α-induced anemia, we observe an increase in myh9 expression. (B) APOL1 G2 expression results in a significant decrease in myh9 expression compared to sham-injected control embryos, suggesting that the altered APOL1 protein may regulate myh9 in vivo. (C) Co-injection of APOL1 G2 (100pg) and human WT MYH9 (n = 31–60; repeated two times), does not rescue edema formation caused by APOL1 G2 expression in 5 dpf larvae, suggesting that the interaction between APOL1 G2 and MYH9 may be indirect. Relative expression values are mean ± SE in triplicate with two biological replicates. * = p<0.05; ** = p<0.01.
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pgen.1005349.g006: myh9 expression in the context of apol1/APOL1 modulation.Zebrafish embryos were injected with either apol1-MO (1.0ng/nl dose), APOL1 G1 (S342G:I384M) mRNA (100pg), or APOL1 G2 (100pg) mRNA alone, in the absence (white bars) or presence (black bars) of atpif1α-MO. Total RNA at 5 dpf or 3 dpf (APOL1 G2/atpif1α-MO embryos did not survive to 5 dpf) was extracted and reverse-transcribed with random primers to obtain whole-embryo cDNA. myh9 expression was determined by quantitative real-time PCR and relative expression was calculated against actb1. (A) apol1-MO injected embryos do not display any significant changes in myh9 expression compared to sham-injected control embryos. Additionally, APOL1 G1 expression does not alter myh9 expression alone, however, under atpif1α-induced anemia, we observe an increase in myh9 expression. (B) APOL1 G2 expression results in a significant decrease in myh9 expression compared to sham-injected control embryos, suggesting that the altered APOL1 protein may regulate myh9 in vivo. (C) Co-injection of APOL1 G2 (100pg) and human WT MYH9 (n = 31–60; repeated two times), does not rescue edema formation caused by APOL1 G2 expression in 5 dpf larvae, suggesting that the interaction between APOL1 G2 and MYH9 may be indirect. Relative expression values are mean ± SE in triplicate with two biological replicates. * = p<0.05; ** = p<0.01.

Mentions: To dissect further the possible genetic interactions between myh9 and apol1, we tested whether suppression of endogenous apol1 or ectopic expression of mutant human APOL1 could alter expression of myh9 in zebrafish embryos. We monitored myh9 expression in zebrafish larvae using quantitative real-time PCR in the context of apol1 suppression, and G1 or G2 expression, as well as apol1/APOL1 modulation in conditions of anemia induced by atpif1α-MO injection at 5 dpf (Fig 6A) and 3 dpf (Fig 6B). We observed a significant decrease in myh9 expression when zebrafish embryos were injected with the proposed dominant-negative APOL1 G2 allele alone (21% reduction; p = 0.043; Fig 6B), suggesting that the mutant protein may be suppressing myh9, either directly or indirectly, to induce nephropathy. Furthermore, zebrafish embryos co-injected with APOL1 G2 mRNA and atpif1α-MO display an even greater reduction in myh9 expression compared to controls (46% reduction; p = 0.0013; Fig 6B), and a significant reduction of myh9 expression compared to APOL1 G2 mRNA alone (p = 0.0297; Fig 6B), suggesting that the altered APOL1 (p.Asn388_Tyr389del) protein has a more pronounced effect on myh9 expression in the context of anemic stress. We also observed a significant increase in myh9 expression in APOL1 G1/atpif1α-MO vs. APOL1 G1 injected embryos (Fig 6A), however, neither of these conditions induced nephropathy. To determine whether this effect was specific to myh9 or was a general effect on transcripts expressed in the glomerulus, we also assessed expression levels of other nephropathy-associated genes during apol1/APOL1 modulation and atpif1α induced anemia. We observed no significant differences in expression of genes implicated in familial focal segmented glomerulosclerosis, including anln[45], trpc6b[46], and wt1a[47] upon apol1/APOL1 modulation (S7 Fig), suggesting that APOL1 G2 regulation may be specific to myh9.


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)

myh9 expression in the context of apol1/APOL1 modulation.Zebrafish embryos were injected with either apol1-MO (1.0ng/nl dose), APOL1 G1 (S342G:I384M) mRNA (100pg), or APOL1 G2 (100pg) mRNA alone, in the absence (white bars) or presence (black bars) of atpif1α-MO. Total RNA at 5 dpf or 3 dpf (APOL1 G2/atpif1α-MO embryos did not survive to 5 dpf) was extracted and reverse-transcribed with random primers to obtain whole-embryo cDNA. myh9 expression was determined by quantitative real-time PCR and relative expression was calculated against actb1. (A) apol1-MO injected embryos do not display any significant changes in myh9 expression compared to sham-injected control embryos. Additionally, APOL1 G1 expression does not alter myh9 expression alone, however, under atpif1α-induced anemia, we observe an increase in myh9 expression. (B) APOL1 G2 expression results in a significant decrease in myh9 expression compared to sham-injected control embryos, suggesting that the altered APOL1 protein may regulate myh9 in vivo. (C) Co-injection of APOL1 G2 (100pg) and human WT MYH9 (n = 31–60; repeated two times), does not rescue edema formation caused by APOL1 G2 expression in 5 dpf larvae, suggesting that the interaction between APOL1 G2 and MYH9 may be indirect. Relative expression values are mean ± SE in triplicate with two biological replicates. * = p<0.05; ** = p<0.01.
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Related In: Results  -  Collection

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pgen.1005349.g006: myh9 expression in the context of apol1/APOL1 modulation.Zebrafish embryos were injected with either apol1-MO (1.0ng/nl dose), APOL1 G1 (S342G:I384M) mRNA (100pg), or APOL1 G2 (100pg) mRNA alone, in the absence (white bars) or presence (black bars) of atpif1α-MO. Total RNA at 5 dpf or 3 dpf (APOL1 G2/atpif1α-MO embryos did not survive to 5 dpf) was extracted and reverse-transcribed with random primers to obtain whole-embryo cDNA. myh9 expression was determined by quantitative real-time PCR and relative expression was calculated against actb1. (A) apol1-MO injected embryos do not display any significant changes in myh9 expression compared to sham-injected control embryos. Additionally, APOL1 G1 expression does not alter myh9 expression alone, however, under atpif1α-induced anemia, we observe an increase in myh9 expression. (B) APOL1 G2 expression results in a significant decrease in myh9 expression compared to sham-injected control embryos, suggesting that the altered APOL1 protein may regulate myh9 in vivo. (C) Co-injection of APOL1 G2 (100pg) and human WT MYH9 (n = 31–60; repeated two times), does not rescue edema formation caused by APOL1 G2 expression in 5 dpf larvae, suggesting that the interaction between APOL1 G2 and MYH9 may be indirect. Relative expression values are mean ± SE in triplicate with two biological replicates. * = p<0.05; ** = p<0.01.
Mentions: To dissect further the possible genetic interactions between myh9 and apol1, we tested whether suppression of endogenous apol1 or ectopic expression of mutant human APOL1 could alter expression of myh9 in zebrafish embryos. We monitored myh9 expression in zebrafish larvae using quantitative real-time PCR in the context of apol1 suppression, and G1 or G2 expression, as well as apol1/APOL1 modulation in conditions of anemia induced by atpif1α-MO injection at 5 dpf (Fig 6A) and 3 dpf (Fig 6B). We observed a significant decrease in myh9 expression when zebrafish embryos were injected with the proposed dominant-negative APOL1 G2 allele alone (21% reduction; p = 0.043; Fig 6B), suggesting that the mutant protein may be suppressing myh9, either directly or indirectly, to induce nephropathy. Furthermore, zebrafish embryos co-injected with APOL1 G2 mRNA and atpif1α-MO display an even greater reduction in myh9 expression compared to controls (46% reduction; p = 0.0013; Fig 6B), and a significant reduction of myh9 expression compared to APOL1 G2 mRNA alone (p = 0.0297; Fig 6B), suggesting that the altered APOL1 (p.Asn388_Tyr389del) protein has a more pronounced effect on myh9 expression in the context of anemic stress. We also observed a significant increase in myh9 expression in APOL1 G1/atpif1α-MO vs. APOL1 G1 injected embryos (Fig 6A), however, neither of these conditions induced nephropathy. To determine whether this effect was specific to myh9 or was a general effect on transcripts expressed in the glomerulus, we also assessed expression levels of other nephropathy-associated genes during apol1/APOL1 modulation and atpif1α induced anemia. We observed no significant differences in expression of genes implicated in familial focal segmented glomerulosclerosis, including anln[45], trpc6b[46], and wt1a[47] upon apol1/APOL1 modulation (S7 Fig), suggesting that APOL1 G2 regulation may be specific to myh9.

Bottom Line: Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein.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