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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

Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; S, secretory domain, PFD, pore-forming domain, B, BH3 domain, MAD, membrane-addressing domain, SRA, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in APOL1, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of pod::NTR-mCherry zebrafish were flow-sorted and evaluated for apol1 RNA expression through RT-PCR. apol1 is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish podocin (nphs2) but a purkinje-cell marker, wdr81[29], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from pod::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of pod::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.
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pgen.1005349.g001: Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; S, secretory domain, PFD, pore-forming domain, B, BH3 domain, MAD, membrane-addressing domain, SRA, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in APOL1, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of pod::NTR-mCherry zebrafish were flow-sorted and evaluated for apol1 RNA expression through RT-PCR. apol1 is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish podocin (nphs2) but a purkinje-cell marker, wdr81[29], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from pod::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of pod::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.

Mentions: The apolipoprotein L family of genes evolved rapidly in humans and some non-human primates[26, 27]. However, using BLAST and reciprocal BLAST searches against the D. rerio and H. sapiens genomes, we identified a single D. rerio locus encoding a protein of unknown function (chr2:37,674,122–37,676,731 Zv9; NCBI Ref: NP_001025309.1; 38% identity, 46% similarity on the amino acid level) as a possible unique functional ancestral ortholog to the human apolipoprotein L family (Fig 1A–1D). To explore the function of this transcript in developing zebrafish, we first asked whether the candidate apol1 ortholog is expressed in a temporal manner amenable to transient assays of renal development and function. RT-PCR analysis of cDNA generated from wild-type (WT) whole-larval total RNA collected at three days post-fertilization (dpf) and 5 dpf showed expression at time points corresponding to the formation of the pronephros. Additionally, we detected apol1 expression in flow-sorted podocyte fractions harvested from glomeruli of pod::NTR-mCherry adult zebrafish (Fig 1E) [28].


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)

Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; S, secretory domain, PFD, pore-forming domain, B, BH3 domain, MAD, membrane-addressing domain, SRA, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in APOL1, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of pod::NTR-mCherry zebrafish were flow-sorted and evaluated for apol1 RNA expression through RT-PCR. apol1 is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish podocin (nphs2) but a purkinje-cell marker, wdr81[29], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from pod::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of pod::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.
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Related In: Results  -  Collection

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

pgen.1005349.g001: Comparison of APOL1 human and zebrafish protein sequences and relevance to the zebrafish kidney.Protein domain schematic of (A) zebrafish APOL1 and (B) human APOL1 is shown, with zebrafish domains (NP_001025309) aligned to the human protein (NP_001130012) and coded based on summarized consensus scores (Gonnet PAM 250 matrix, Clustal Omega, Cambridge, UK; S, secretory domain, PFD, pore-forming domain, B, BH3 domain, MAD, membrane-addressing domain, SRA, serum resistance-associated binding domain). Prominent regions of the human and zebrafish alignments are expanded, including the (C) BH3 domain and (D) SRA binding domain, and consensus symbols are displayed (* (asterisk), fully conserved;: (colon), >0.5 in the Gonnet PAM 250 matrix;. (period), = <0.5 in the Gonnet PAM 250 matrix). The leucine zipper domain (codons 365–392 in APOL1, underline), and the location of the G1 and G2 risk alleles in CKD in African Americans (S342G/I384M and ΔN388Y389) are highlighted in red. (E) Podocytes from adult glomeruli of pod::NTR-mCherry zebrafish were flow-sorted and evaluated for apol1 RNA expression through RT-PCR. apol1 is expressed in fluorescence-activated cell sorted (FACS) podocytes and the adult liver. FACS podocytes also express zebrafish podocin (nphs2) but a purkinje-cell marker, wdr81[29], was undetectable. NT = non-template reverse transcription control; L = dissected adult liver cells from pod::NTR-mCherry zebrafish; P = fluorescence-activated cell sorted podocytes from dissected glomeruli of pod::NTR-mCherry zebrafish; Em = 5 dpf whole-zebrafish embryo cDNA.
Mentions: The apolipoprotein L family of genes evolved rapidly in humans and some non-human primates[26, 27]. However, using BLAST and reciprocal BLAST searches against the D. rerio and H. sapiens genomes, we identified a single D. rerio locus encoding a protein of unknown function (chr2:37,674,122–37,676,731 Zv9; NCBI Ref: NP_001025309.1; 38% identity, 46% similarity on the amino acid level) as a possible unique functional ancestral ortholog to the human apolipoprotein L family (Fig 1A–1D). To explore the function of this transcript in developing zebrafish, we first asked whether the candidate apol1 ortholog is expressed in a temporal manner amenable to transient assays of renal development and function. RT-PCR analysis of cDNA generated from wild-type (WT) whole-larval total RNA collected at three days post-fertilization (dpf) and 5 dpf showed expression at time points corresponding to the formation of the pronephros. Additionally, we detected apol1 expression in flow-sorted podocyte fractions harvested from glomeruli of pod::NTR-mCherry adult zebrafish (Fig 1E) [28].

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