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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 morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.Representative live images of (A) sham-injected control larvae, and (B) apol1 morpholino (MO) injected larvae at 5 dpf. apol1 morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of apol1-MO demonstrate dose-dependent effects when scored for generalized edema (n = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. apol1 morpholino injected embryos were complemented with the respective human mRNA to APOL1 (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of APOL1 rescues significantly the edema phenotype observed in apol1 morphants (1.0 ng/nl dose). We observed no significant phenotypes when APOL1 human mRNA is injected alone. 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 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for apol1 morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; apol1 morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) apol1 morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (n = 29); MO, apol1 morpholino injected (n = 26); apol1-MO+mRNA (n = 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.
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pgen.1005349.g002: apol1 morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.Representative live images of (A) sham-injected control larvae, and (B) apol1 morpholino (MO) injected larvae at 5 dpf. apol1 morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of apol1-MO demonstrate dose-dependent effects when scored for generalized edema (n = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. apol1 morpholino injected embryos were complemented with the respective human mRNA to APOL1 (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of APOL1 rescues significantly the edema phenotype observed in apol1 morphants (1.0 ng/nl dose). We observed no significant phenotypes when APOL1 human mRNA is injected alone. 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 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for apol1 morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; apol1 morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) apol1 morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (n = 29); MO, apol1 morpholino injected (n = 26); apol1-MO+mRNA (n = 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.

Mentions: To test the effects of apol1 suppression, we designed a translation-blocking morpholino (MO; Gene Tools, LLC) targeting the candidate zebrafish apol1 locus (apol1-MO) and we injected increasing doses into embryos at the one to four cell stage (n = 49–65 embryos/injection; repeated three times). Masked scoring for morphological defects at 5 dpf revealed a dose-dependent increase of the percent of larvae displaying pericardial and yolk sac edema, a phenotype that has been implicated previously in glomerular filtration defects[24, 30] (Fig 2A–2C). Co-injection of WT APOL1 human mRNA (GenBank Accession: BC112943.1; 100 pg/nl) rescued significantly the edema caused by apol1 suppression (p<0.0001; Fig 2D), arguing not only that the phenotype was unlikely to be a non-specific toxic effect of the MO, but also that the zebrafish locus we targeted is the ortholog of the human transcript. Importantly, co-injection of human mRNA encoding other human apolipoprotein L members (APOL2, APOL3, APOL4, APOL5, and APOL6) with apol1 MO did not rescue the edema formation of apol1 morphants (S1 Fig). Additionally, we observed a significant decrease in endogenous APOL1 protein expression in apol1-MO injected zebrafish embryos (p = 0.026), which is restored to normal levels upon co-injection with wild-type human APOL1 mRNA (S2 Fig). Furthermore, as an additional test of the specificity of apol1 perturbation to edema formation, we induced microdeletions in exon 3 of apol1 using the CRISPR/Cas9 system[31, 32] (Fig 3A–3C). Injection of guide RNA and CAS9 protein into one-cell stage embryos reproduced the edema phenotype (scored in founders, F0) seen in apol1 morphants (n = 26–38 embryos/injection, repeated three times; p<0.001; Fig 3D).


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 morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.Representative live images of (A) sham-injected control larvae, and (B) apol1 morpholino (MO) injected larvae at 5 dpf. apol1 morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of apol1-MO demonstrate dose-dependent effects when scored for generalized edema (n = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. apol1 morpholino injected embryos were complemented with the respective human mRNA to APOL1 (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of APOL1 rescues significantly the edema phenotype observed in apol1 morphants (1.0 ng/nl dose). We observed no significant phenotypes when APOL1 human mRNA is injected alone. 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 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for apol1 morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; apol1 morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) apol1 morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (n = 29); MO, apol1 morpholino injected (n = 26); apol1-MO+mRNA (n = 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.
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pgen.1005349.g002: apol1 morphant zebrafish embryos display generalized edema and glomerular filtration defects indicative of nephropathy.Representative live images of (A) sham-injected control larvae, and (B) apol1 morpholino (MO) injected larvae at 5 dpf. apol1 morphants display pericardial and yolk sac edema. (C) Injection of increasing doses of apol1-MO demonstrate dose-dependent effects when scored for generalized edema (n = 35–65 embryos/injection; repeated three times) compared to control larvae at 5 dpf. apol1 morpholino injected embryos were complemented with the respective human mRNA to APOL1 (100pg/nl) and scored for generalized edema at 5 dpf. (D) Ectopic expression of APOL1 rescues significantly the edema phenotype observed in apol1 morphants (1.0 ng/nl dose). We observed no significant phenotypes when APOL1 human mRNA is injected alone. 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 hpi. (E) Representative eye image series of zebrafish larvae for each injection group show a relatively stable or a decrease in fluorescence intensity over time compared to sham-injected controls. (F) Bar graphs summarize the fluorescence changes observed for each injection group for apol1 morphant larvae. Reduction in fluorescence intensity over the pupil was calculated relative to the 24 hpi time point; apol1 morphants display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, indicative of compromised glomerular filtration and proteinuria. These defects were rescued significantly when MO was co-injected with orthologous human mRNA. (G-I) Compared to (G) sham-injected controls, the glomerular ultrastructure of (H) apol1 morphant zebrafish display partial effacement of podocyte foot process (* asterisks), although the glomerular basement membrane (filled arrowheads) appears normal. Microvillus protrusions (open arrowheads) are also apparent in the urinary space. (I) Ultrastructure defects are rescued upon co-injection of human wild-type mRNA (100pg). Scale bar, 500nm. White bars, normal; black bars, edema. MO concentrations are in μg/μl, with 1nl injected into each embryo. C, sham-injected control; NI, non-injected control. Dextran values are in relative fluorescent intensity, mean ± SE. Control, sham-injected control (n = 29); MO, apol1 morpholino injected (n = 26); apol1-MO+mRNA (n = 28). h.p.f., hours post-fertilization; h.p.i., hours post-injection. *p<0.001.
Mentions: To test the effects of apol1 suppression, we designed a translation-blocking morpholino (MO; Gene Tools, LLC) targeting the candidate zebrafish apol1 locus (apol1-MO) and we injected increasing doses into embryos at the one to four cell stage (n = 49–65 embryos/injection; repeated three times). Masked scoring for morphological defects at 5 dpf revealed a dose-dependent increase of the percent of larvae displaying pericardial and yolk sac edema, a phenotype that has been implicated previously in glomerular filtration defects[24, 30] (Fig 2A–2C). Co-injection of WT APOL1 human mRNA (GenBank Accession: BC112943.1; 100 pg/nl) rescued significantly the edema caused by apol1 suppression (p<0.0001; Fig 2D), arguing not only that the phenotype was unlikely to be a non-specific toxic effect of the MO, but also that the zebrafish locus we targeted is the ortholog of the human transcript. Importantly, co-injection of human mRNA encoding other human apolipoprotein L members (APOL2, APOL3, APOL4, APOL5, and APOL6) with apol1 MO did not rescue the edema formation of apol1 morphants (S1 Fig). Additionally, we observed a significant decrease in endogenous APOL1 protein expression in apol1-MO injected zebrafish embryos (p = 0.026), which is restored to normal levels upon co-injection with wild-type human APOL1 mRNA (S2 Fig). Furthermore, as an additional test of the specificity of apol1 perturbation to edema formation, we induced microdeletions in exon 3 of apol1 using the CRISPR/Cas9 system[31, 32] (Fig 3A–3C). Injection of guide RNA and CAS9 protein into one-cell stage embryos reproduced the edema phenotype (scored in founders, F0) seen in apol1 morphants (n = 26–38 embryos/injection, repeated three times; p<0.001; Fig 3D).

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