Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease

Abstract

Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-β pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.

Figure 1:

Summary of kidney compartment-based eQTL analysis. a, Diagram indicating the work flow (and results) by which eGenes were identified in the tubular and glomerular compartment of human kidneys. b, Diagram illustrating the number of tubule-specific (top) and glomerulus-specific (bottom) eGenes identified by meta-analysis of eQTLs from 46 human tissues (44 GTEx tissues plus 2 kidney compartments). c, Overlap of tubule (top) and glomerulus (bottom) eQTLs with hits of various GWA studies from GWAS catalogue. The x-axis shows the proportion of GWAS variants of each GWAS trait overlapped with tubule and glomerulus eQTLs (when the GWAS varuants and eVariants are located in the same LD, r² > 0.8). The y-axis shows enrichment of tubule and glomerulus eGenes by genes around the hits of each GWAS trait. –log₁₀ (P) was calculated by two-sided Fisher’s exact test (height was used for background estimation). Red dots, metabolite traits; blue dots, kidney-related traits; grey dots, other traits. Orange line, statistical significance threshold. d, The 15 GWAS traits showing the strongest enrichment (y-axis) for tubule- and glomerulus-specific eGenes (left) or shared eGenes (right). Tubule-specific eGene: n = 589; tubule-shared eGene: n = 7050; glomerulus-specific eGene: n = 594; glomerulus-shared eGene: n = 7090. –log (P) was calculated by two-sided Fisher’s exact test (x-axis).

Figure 2:

Compartment-specific eQTLs show greater cell-type specificity and distal regulatory element enrichment. a, Diagram describing the integration of kidney eQTLs, GWAS, single-cell expression and regulatory region. b, Heatmap of cell-type-specific expression of identified CKD target genes. The blue/yellow color corresponds to the level of expression (z-score). Endo, endothelial; Podo, podocyte; PT, proximal tubule; LOH, loop of Henle; DCT, distal convoluted tubule; CD-PC, collecting duct principal cell; CD-IC, collecting duct intercalated cell; Fibro, fibroblast; Macro, macrophage; Neutro, neutrophil; NK, natural killer cell. c, Density plots of best eVariants in tubule (top) and glomerulus (bottom) and the relationship to transcription start site (TSS). d, Distance of top eVariants from TSS (-log₁₀) by groups. e, Odds ratios of the top eVariants on kidney promoter by groups. The groups were compared to randomly selected variants matched by MAF and distance to TSS (n = 5,000 randomly selected times). Center lines show the medians; box limits indicate the 25^th and 75^th percentiles; whiskers extend to the 5^th and 95^th percentiles, outliers are represented by dots (d, e). f, Odds ratios (y-axis) of eGenes from each group enriched by kidney-specific cell type expression. P was calculated by two-sided Fisher’s exact test. RTEC: PT, LOH and DCT, Myeloid: Fibro, Macro and Neutro; Lymphoid: B cell, T cell and NK. Tubule, tubule-specific-eGenes: n = 417; Share, compartment-shared-eGenes: n = 3,493; Glom, glomerulus-specific-eGenes: n = 674 (d-f). g, Genome scale integrated analysis of gene network (GIANT) visualization of tubule-specific candidate genes. The color of each link shows the relationship confidence, from green (0.01) to red (1).

Figure 3:

CKD associated GWAS SNP (rs11959928) specifically influences DAB2 levels in human kidney tubules. a, LocusZoom plots of CKD GWAS or eQTL around the region of rs11959928. The y-axis shows -log (P) of association tests (by linear regression). Top, GWAS association (genotype and eGFR as outcome); Bottom, eQTLs (genotype and expression of DAB2 (left) or C9 (right)) in different compartments (whole kidney, tubule or glomerulus). Note the overlapping genetic region associated with kidney function and DAB2 expression. n = 133,814 individuals for CKD GWAS; n = 121 individuals for tubule eQTLs. n = 119 individuals for glomerulus eQTLs; n = 96 individuals for whole kidney eQTLs. Dash lines, significance thresholds (GWAS, P = 5 × 10⁻⁸ ; eQTL, nominal P threshold for individual gene calculated by permutation). b, Genotype (rs11959928) and gene expression (DAB2 or C9) association in human tubules (n = 121) and glomeruli (n = 119). Center lines show the medians; box limits indicate the 25^th and 75^th percentiles; whiskers extend to the 5^th and 95^th percentiles, outliers are represented by dots. P was calculated by linear regression.

Figure 4:

Functional annotation of the genomic area around rs11959928. a, The effect size (β) of eQTL association between rs11959928 and DAB2, in tubular (n = 121) glomerular (n = 119) compartments and 44 human tissues from GTEx (v6p). The x-axis shows effect size (β) with 95% CI. b, Meta-analysis result of association relationship between rs11959928 and DAB2, using eQTLs of kidney compartments and 44 human tissues from GTEx. Each dot represents one tissue data point. The y-axis shows the nominal P of association in each single tissue. The x-axis shows m, the posterior probability calculated by METASOFT, for each tissue. c, Top, LocusZoom plots of GWAS (genotype and eGFR association) and kidney tubule DAB2 eQTLs (genotype and DAB2 expression association). The y-axis show -log (P) of association tests (by linear regression). n = 133,814 individuals for CKD GWAS; n = 121 individuals for tubule eQTLs. Bottom, histone H3K27ac enrichment of the genomic region (analyzed by chromatin immunoprecipitation) in human kidney samples, blood macrophages and the 7 ENCODE cell lines.

Figure 5:

Tubule-specific expression of Dab2 influences kidney disease development in mice. a, Relative mRNA amount of Fibronectin 1, Fn1; Collagen1a1, Col1a1; and Collagen3a1, Col3a1 in C9^+/+ and C9^+/− mice with or without FA injection. b, Representative images of PAS-stained and Sirius Red-stained kidney sections from C9^+/+ and C9 ^+/− mice with or without FA injection. Scale bar: 20μm. c, Quantification of Sirius Red-stained kidney sections from C9^+/+ and C9 ^+/− mice with or without FA injection. d, Relative mRNA amount of Fn1, Col1a1 and Col3a1 in control and Ksp^cre/Dab2^flox/+ mice with or without FA injection. e, Representative images of PAS-stained and Sirius Red-stained kidney sections from control and Ksp^cre/Dab2^flox/+ mice with or without FA injection. Scale bar: 20μm. f, Quantification of Sirius Red-stained kidney sections from control and Ksp^cre/Dab2^flox/+ mice with or without FA injection. g, h, Representative western blots of p-Smad2, Smad2, p-Smad3, Smad3, p-JNK, JNK, Fibronectin in TGFβ-treated Dab2^+/+ and Dab2^+/− primary TECs from n = 4 independently repeated experiments. β-actin was used as a loading control. P values were calculated by One-way ANOVA with post-hoc Tukey test (a, c, d, f). C9^+/+ Control: n = 8, C9^+/− Control: n = 8, C9^+/+ FA: n = 5, C9^+/− FA: n = 6 (a-c). WT/Dab2^flox/+ Control: n = 5, Ksp^cre/Dab2^flox/+ Control: n = 4, WT/Dab2^flox/+ FA: n = 8, Ksp^cre/Dab2^flox/+ FA: n = 5 (d-f). Data are represented as mean ± SD.