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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-CRISPR F0 zebrafish embryos reproduce phenotypes observed in apol1 morphants.(A) Schematic of the zebrafish apol1 locus and location of the guide RNA (gRNA) target used for apol1-CRISPR experiments; the primers used to PCR-amplify the target region are shown (arrowheads). (B) At 1 dpf, a representative sampling of 8 founders and 8 non-injected controls were selected and subjected to T7 endonuclease 1 (T7E1) assay. The appearance of T7E1 fragments at ~180bp indicate positive gRNA targeting of exon 3 in the apol1 locus. No T7E1 fragments were detected in non-injected control embryos. In total, 25 out of 41 founders subjected to T7E1 assay showed the presence of T7E1 fragments, indicating that ~61% of founders have insertion/deletions (indels) in the exon 3 region of apol1. (C) Multiple sequence alignment of apol1 reference sequence (ENSDARG00000007425) to apol1-CRISPR variants generated from PCR amplification and subsequent TA cloning and sequencing of two representative apol1-gRNA/CAS9 injected founders. 13 PCR-cloned sequences are shown, representing four wild-type variants (c1-4) and all indel types detected among 50 PCR-clones (c5-13). Of 50 total PCR-clones sequenced, 31 showed detectable indels, representing an estimated 62% mosaicism in apol1-CRISPR/CAS9 injected founders. Lines mark the specific sequence targeted by the apol1-gRNA (exon3) and the location of the PAM recognition motif (i.e. TGG). (D) apol1-gRNA and CAS9 co-injected embryos were scored for edema formation at 5 dpf (n = 26–31 embryos/injection, repeated three times; *p<0.001). (E) apol1-gRNA and CAS9 co-injected embryos display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, similar to that of apol1-MO injected embryos (*p<0.001). Bar graphs summarize the changes for each injection group. Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19–21); apol1-gRNA+CAS9 (n = 11–17); apol1-gRNA alone (n = 13–14), repeated 2 times. (F) apol1-CRISPR/CAS9 injected embryos display podocyte foot process effacement at 5 dpf, similar to that of apol1 morphant larvae. Ultrastructural defects appear less severe when compared to apol1-MO injected embryos, however, including less foot process effacement and the absence of microvilli in the urinary space. Filled arrowheads, glomerular basement membrane. Scale bar, 500nm.
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pgen.1005349.g003: apol1-CRISPR F0 zebrafish embryos reproduce phenotypes observed in apol1 morphants.(A) Schematic of the zebrafish apol1 locus and location of the guide RNA (gRNA) target used for apol1-CRISPR experiments; the primers used to PCR-amplify the target region are shown (arrowheads). (B) At 1 dpf, a representative sampling of 8 founders and 8 non-injected controls were selected and subjected to T7 endonuclease 1 (T7E1) assay. The appearance of T7E1 fragments at ~180bp indicate positive gRNA targeting of exon 3 in the apol1 locus. No T7E1 fragments were detected in non-injected control embryos. In total, 25 out of 41 founders subjected to T7E1 assay showed the presence of T7E1 fragments, indicating that ~61% of founders have insertion/deletions (indels) in the exon 3 region of apol1. (C) Multiple sequence alignment of apol1 reference sequence (ENSDARG00000007425) to apol1-CRISPR variants generated from PCR amplification and subsequent TA cloning and sequencing of two representative apol1-gRNA/CAS9 injected founders. 13 PCR-cloned sequences are shown, representing four wild-type variants (c1-4) and all indel types detected among 50 PCR-clones (c5-13). Of 50 total PCR-clones sequenced, 31 showed detectable indels, representing an estimated 62% mosaicism in apol1-CRISPR/CAS9 injected founders. Lines mark the specific sequence targeted by the apol1-gRNA (exon3) and the location of the PAM recognition motif (i.e. TGG). (D) apol1-gRNA and CAS9 co-injected embryos were scored for edema formation at 5 dpf (n = 26–31 embryos/injection, repeated three times; *p<0.001). (E) apol1-gRNA and CAS9 co-injected embryos display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, similar to that of apol1-MO injected embryos (*p<0.001). Bar graphs summarize the changes for each injection group. Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19–21); apol1-gRNA+CAS9 (n = 11–17); apol1-gRNA alone (n = 13–14), repeated 2 times. (F) apol1-CRISPR/CAS9 injected embryos display podocyte foot process effacement at 5 dpf, similar to that of apol1 morphant larvae. Ultrastructural defects appear less severe when compared to apol1-MO injected embryos, however, including less foot process effacement and the absence of microvilli in the urinary space. Filled arrowheads, glomerular basement membrane. Scale bar, 500nm.

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-CRISPR F0 zebrafish embryos reproduce phenotypes observed in apol1 morphants.(A) Schematic of the zebrafish apol1 locus and location of the guide RNA (gRNA) target used for apol1-CRISPR experiments; the primers used to PCR-amplify the target region are shown (arrowheads). (B) At 1 dpf, a representative sampling of 8 founders and 8 non-injected controls were selected and subjected to T7 endonuclease 1 (T7E1) assay. The appearance of T7E1 fragments at ~180bp indicate positive gRNA targeting of exon 3 in the apol1 locus. No T7E1 fragments were detected in non-injected control embryos. In total, 25 out of 41 founders subjected to T7E1 assay showed the presence of T7E1 fragments, indicating that ~61% of founders have insertion/deletions (indels) in the exon 3 region of apol1. (C) Multiple sequence alignment of apol1 reference sequence (ENSDARG00000007425) to apol1-CRISPR variants generated from PCR amplification and subsequent TA cloning and sequencing of two representative apol1-gRNA/CAS9 injected founders. 13 PCR-cloned sequences are shown, representing four wild-type variants (c1-4) and all indel types detected among 50 PCR-clones (c5-13). Of 50 total PCR-clones sequenced, 31 showed detectable indels, representing an estimated 62% mosaicism in apol1-CRISPR/CAS9 injected founders. Lines mark the specific sequence targeted by the apol1-gRNA (exon3) and the location of the PAM recognition motif (i.e. TGG). (D) apol1-gRNA and CAS9 co-injected embryos were scored for edema formation at 5 dpf (n = 26–31 embryos/injection, repeated three times; *p<0.001). (E) apol1-gRNA and CAS9 co-injected embryos display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, similar to that of apol1-MO injected embryos (*p<0.001). Bar graphs summarize the changes for each injection group. Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19–21); apol1-gRNA+CAS9 (n = 11–17); apol1-gRNA alone (n = 13–14), repeated 2 times. (F) apol1-CRISPR/CAS9 injected embryos display podocyte foot process effacement at 5 dpf, similar to that of apol1 morphant larvae. Ultrastructural defects appear less severe when compared to apol1-MO injected embryos, however, including less foot process effacement and the absence of microvilli in the urinary space. Filled arrowheads, glomerular basement membrane. Scale bar, 500nm.
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pgen.1005349.g003: apol1-CRISPR F0 zebrafish embryos reproduce phenotypes observed in apol1 morphants.(A) Schematic of the zebrafish apol1 locus and location of the guide RNA (gRNA) target used for apol1-CRISPR experiments; the primers used to PCR-amplify the target region are shown (arrowheads). (B) At 1 dpf, a representative sampling of 8 founders and 8 non-injected controls were selected and subjected to T7 endonuclease 1 (T7E1) assay. The appearance of T7E1 fragments at ~180bp indicate positive gRNA targeting of exon 3 in the apol1 locus. No T7E1 fragments were detected in non-injected control embryos. In total, 25 out of 41 founders subjected to T7E1 assay showed the presence of T7E1 fragments, indicating that ~61% of founders have insertion/deletions (indels) in the exon 3 region of apol1. (C) Multiple sequence alignment of apol1 reference sequence (ENSDARG00000007425) to apol1-CRISPR variants generated from PCR amplification and subsequent TA cloning and sequencing of two representative apol1-gRNA/CAS9 injected founders. 13 PCR-cloned sequences are shown, representing four wild-type variants (c1-4) and all indel types detected among 50 PCR-clones (c5-13). Of 50 total PCR-clones sequenced, 31 showed detectable indels, representing an estimated 62% mosaicism in apol1-CRISPR/CAS9 injected founders. Lines mark the specific sequence targeted by the apol1-gRNA (exon3) and the location of the PAM recognition motif (i.e. TGG). (D) apol1-gRNA and CAS9 co-injected embryos were scored for edema formation at 5 dpf (n = 26–31 embryos/injection, repeated three times; *p<0.001). (E) apol1-gRNA and CAS9 co-injected embryos display increased glomerular clearance of 70kDa dextran-FITC compared to control embryos over time, similar to that of apol1-MO injected embryos (*p<0.001). Bar graphs summarize the changes for each injection group. Dextran values are in relative fluorescence intensity, mean ± SE. Control, sham-injected control (n = 19–21); apol1-gRNA+CAS9 (n = 11–17); apol1-gRNA alone (n = 13–14), repeated 2 times. (F) apol1-CRISPR/CAS9 injected embryos display podocyte foot process effacement at 5 dpf, similar to that of apol1 morphant larvae. Ultrastructural defects appear less severe when compared to apol1-MO injected embryos, however, including less foot process effacement and the absence of microvilli in the urinary space. Filled arrowheads, glomerular basement membrane. Scale bar, 500nm.
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