Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Abstract: Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Publisher Copyright: © 2019, The Author(s).Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria.Peer reviewe

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Effect of conditional deletion of cytoplasmic dynein heavy chain DYNC1H1 on postnatal photoreceptors.

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1F/F;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1F/F;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1F/F;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration

Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (retArl2−/−) or after ciliogenesis specifically in rods (rodArl2−/−). retArl2−/− retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2−/− inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (retARL2−/−) or DYNC1H1 (retDync1h1−/−), the dynein heavy chain, and found that both the retArl2−/− and retDync1h1−/− retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development

Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (retArl2−/−) or after ciliogenesis specifically in rods (rodArl2−/−). retArl2−/− retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2−/− inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (retARL2−/−) or DYNC1H1 (retDync1h1−/−), the dynein heavy chain, and found that both the retArl2−/− and retDync1h1−/− retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development

OS protein localization in adult ^ret<i>Rab11a</i>^<i>-/-</i> mice injected with Rab11b(S25N) AAV.

<p><b>(A</b>) Alignment of Rab11a and Rab11b protein sequences. One critical GTP binding domain is located in exon 2. Position of S25 in Rab11b is indicated in Rab11b. Both proteins contain geranylgeranylation motifs at the C-terminus. (<b>B</b>) Immunostaining results show that cMyc-Rab11b(S25N) localizes mainly at IS and OS (red channel, top left panel). Rhodopsin, PDE6, GC1, CNGA1/A3, ML- and S-opsin localize correctly to OS, and SV2 localizes correctly to the OPL. Green channel in all panels detect szGreen fluorescence. Scale bar, 20 μm.</p

Rhodopsin correctly targets to the OS in adult Rab8a retina cKO mice injected with GFP-Rab8b(T22N), GFP-Rab10(T23N), cMyc-Rab11a(S25N) and cMYc-Rab11b(S25N) AAV.

<p>One month old ^ret<i>Rab8a</i>^<i>-/-</i> mice were injected subretinally with mixed Rab11a(S25N), 11b(S25N), 8b(T22N) and 10(T23N) AAVs or Rab10(T23N) only-AAV. Three months post-injection, rhodopsin, PDE6, GC1, CNGA1/A3 and ML-opsin all localize correctly in injected animals. Scale bar, 30 μm.</p

Electroretinography (ERG) and OS protein localization in adult ^<i>ret</i><i>Rab11a</i>^<i>-/-</i> mice.

<p><b>(A)</b> Representative scotopic ERG traces at -10, 0 and 10 db from 5M-old ^ret<i>Rab11a</i>^<i>+/-</i> and ^ret<i>Rab11a</i>^<i>-/-</i> mice. (<b>B</b>) and (<b>C</b>) Quantification of scotopic a- and b-wave amplitude at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.15, n = 5 for each group, one-way ANOVA). (<b>D</b>) Representative photopic ERG trace at 0, 5 and 10 db from 5M-old control and KO mice. (<b>E</b>) Quantification of photopic b-wave amplitude at multiple light intensities. No significant difference was observed between control and KO animals (p> 0.15, n = 5 for each group, one-way ANOVA). (<b>F</b>) Immunostaining of rhodopsin, GC1, CNGA1/A3, PDE6, S-opsin, ML-opsin, ribeye and SV2 using 3M-old animals, showing that all proteins localize correctly in KO as in heterozygous control. Scale bar, 20 μm.</p