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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

apol1 interacts with myh9 in an anemic context.To test for epistatic effects of apol1 and myh9 in zebrafish, we first co-injected both apol1-MO (1.0ng/nl dose) and myh9-MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between apol1 and myh9 in the context of atpif1a suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of apol1 and myh9. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19); atpif1a MO injected (n = 14); apol1-MO+myh9-MO (n = 12); apol1-MO+myh9-MO+atpif1a-MO (n = 11); Butafenacil (n = 48); But+myh9-MO+apol1-MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.
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pgen.1005349.g005: apol1 interacts with myh9 in an anemic context.To test for epistatic effects of apol1 and myh9 in zebrafish, we first co-injected both apol1-MO (1.0ng/nl dose) and myh9-MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between apol1 and myh9 in the context of atpif1a suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of apol1 and myh9. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19); atpif1a MO injected (n = 14); apol1-MO+myh9-MO (n = 12); apol1-MO+myh9-MO+atpif1a-MO (n = 11); Butafenacil (n = 48); But+myh9-MO+apol1-MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.

Mentions: Although recent studies have provided statistical evidence implicating APOL1 variation in nondiabetic nephropathies[7, 33, 34], MYH9 risk variants are still associated with chronic kidney disease (CKD) in non-African American populations[35] and in sickle cell disease nephropathy[5]. As such, our group and others have hypothesized that these genes may be co-regulated to induce nephropathy risk; in fact, when we modeled glomerular filtration rate in sickle cell patients as a function of the previously reported MYH9 risk haplotype and an APOL1 recessive model, we observed a significant interaction between the two genes[5]. Therefore, we tested for functional interaction effects between apol1 and myh9 in zebrafish, an experimentally tractable model for investigating additive and synergistic effects[36–40]. First, we co-injected both apol1-MO and myh9-MO into embryos and we scored for gross morphological defects at 5 dpf. Under this co-suppression model, we observed no significant differences in edema formation when compared to batches injected with either MO alone (Fig 5A), even when individual MO concentrations were reduced to subeffective doses (Fig 5B). Next, we tested the possibility that suppression of either apol1 or myh9 in zebrafish could be rescued significantly by the co-injection of the reciprocal human mRNA. myh9-MO was co-injected with human APOL1 WT mRNA (100pg/nl) and apol1-MO was co-injected with human MYH9 WT mRNA (100pg/nl). However, we were unable to rescue the suppression phenotypes of either apol1 or myh9 with the human mRNA of the reciprocal gene (S6 Fig).


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)

apol1 interacts with myh9 in an anemic context.To test for epistatic effects of apol1 and myh9 in zebrafish, we first co-injected both apol1-MO (1.0ng/nl dose) and myh9-MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between apol1 and myh9 in the context of atpif1a suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of apol1 and myh9. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19); atpif1a MO injected (n = 14); apol1-MO+myh9-MO (n = 12); apol1-MO+myh9-MO+atpif1a-MO (n = 11); Butafenacil (n = 48); But+myh9-MO+apol1-MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492502&req=5

pgen.1005349.g005: apol1 interacts with myh9 in an anemic context.To test for epistatic effects of apol1 and myh9 in zebrafish, we first co-injected both apol1-MO (1.0ng/nl dose) and myh9-MO (6.0ng/nl dose) into zebrafish larvae and scored for edema formation at 5 dpf. (n = 39–89 embryos/injection; repeated three times). However, under this co-suppression model (A, B), we observed no significantly increased edema formation compared to each MO alone. We next tested for an interaction between apol1 and myh9 in the context of atpif1a suppression, predicting that the added stress of anemia would mimic our initial observations in sickle cell disease patients. 70kDa dextran-FITC conjugate was injected into the cardiac venous sinus of 48 hpf zebrafish larvae and fluorescence intensity in the eye vasculature was measured at 24 and 48 hours later. (C) Representative eye image series of zebrafish embryos for each injection group show relatively stable or decreased fluorescence intensity over time. (E) Bar graphs summarize the changes observed for each injection group. Zebrafish embryos injected with all three MOs show a significant increase in dextran clearance from the vasculature compared to co-suppression of apol1 and myh9. (D, F) These data are reproduced using butafenacil induced anemia (0.195 μM in embryo media, treated at 48 hpf). Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19); atpif1a MO injected (n = 14); apol1-MO+myh9-MO (n = 12); apol1-MO+myh9-MO+atpif1a-MO (n = 11); Butafenacil (n = 48); But+myh9-MO+apol1-MO (n = 18). hpf, hours post-fertilization; hpi, hours post-injection. *p<0.001.
Mentions: Although recent studies have provided statistical evidence implicating APOL1 variation in nondiabetic nephropathies[7, 33, 34], MYH9 risk variants are still associated with chronic kidney disease (CKD) in non-African American populations[35] and in sickle cell disease nephropathy[5]. As such, our group and others have hypothesized that these genes may be co-regulated to induce nephropathy risk; in fact, when we modeled glomerular filtration rate in sickle cell patients as a function of the previously reported MYH9 risk haplotype and an APOL1 recessive model, we observed a significant interaction between the two genes[5]. Therefore, we tested for functional interaction effects between apol1 and myh9 in zebrafish, an experimentally tractable model for investigating additive and synergistic effects[36–40]. First, we co-injected both apol1-MO and myh9-MO into embryos and we scored for gross morphological defects at 5 dpf. Under this co-suppression model, we observed no significant differences in edema formation when compared to batches injected with either MO alone (Fig 5A), even when individual MO concentrations were reduced to subeffective doses (Fig 5B). Next, we tested the possibility that suppression of either apol1 or myh9 in zebrafish could be rescued significantly by the co-injection of the reciprocal human mRNA. myh9-MO was co-injected with human APOL1 WT mRNA (100pg/nl) and apol1-MO was co-injected with human MYH9 WT mRNA (100pg/nl). However, we were unable to rescue the suppression phenotypes of either apol1 or myh9 with the human mRNA of the reciprocal gene (S6 Fig).

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