Genome-Wide Association Studies of Metabolites in Patients with CKD Identify Multiple Loci and Illuminate Tubular Transport Mechanisms

Abstract

Background The kidneys have a central role in the generation, turnover, transport, and excretion of metabolites, and these functions can be altered in CKD. Genetic studies of metabolite concentrations can identify proteins performing these functions.Methods We conducted genome-wide association studies and aggregate rare variant tests of the concentrations of 139 serum metabolites and 41 urine metabolites, as well as their pairwise ratios and fractional excretions in up to 1168 patients with CKD.Results After correction for multiple testing, genome-wide significant associations were detected for 25 serum metabolites, two urine metabolites, and 259 serum and 14 urinary metabolite ratios. These included associations already known from population-based studies. Additional findings included an association for the uremic toxin putrescine and variants upstream of an enzyme catalyzing the oxidative deamination of polyamines (AOC1, P-min=2.4×10^-12), a relatively high carrier frequency (2%) for rare deleterious missense variants in ACADM that are collectively associated with serum ratios of medium-chain acylcarnitines (P-burden=6.6×10^-16), and associations of a common variant in SLC7A9 with several ratios of lysine to neutral amino acids in urine, including the lysine/glutamine ratio (P=2.2×10^-23). The associations of this SLC7A9 variant with ratios of lysine to specific neutral amino acids were much stronger than the association with lysine concentration alone. This finding is consistent with SLC7A9 functioning as an exchanger of urinary cationic amino acids against specific intracellular neutral amino acids at the apical membrane of proximal tubular cells.Conclusions Metabolomic indices of specific kidney functions in genetic studies may provide insight into human renal physiology.

Keywords: chronic kidney disease; genome-wide association studies; metabolites; renal tubular epithelial cells.

Figure 1.

Regional association plot illustrates the presence of serum putrescine-associated SNPs upstream of AOC1 and downstream of TMEM176A on chromosome 7. y axis, negative log₁₀(P value) from GWAS analysis; x axis, genomic position. Linkage disequilibrium with the index SNP rs4725949 (purple) in the 1000 Genomes Project EUR reference population is color-coded as detailed in the legend.

Figure 2.

Rare coding variants in ACADM are cumulatively associated with the serum concentrations of acylcarnitine ratios. The upper panel contains a schematic representation of the ACADM gene and the mapping of the identified deleterious variants to exons. The table contains P values for the cumulative effect of the variants on the concentrations of the listed acylcarnitine ratios, as well as the association P values for the single variants that contribute to the gene-based tests (burden test and sequence kernel association test, see Methods). The allele frequencies of these variants in European individuals in gnomAD database are: 0.0032 for rs147559466, 0.00099 for rs121434280, 0.0061 for rs77931234, and 0.00049 for rs150710061. C10, decanoylcarnitine; C12, dodecanoylcarnitine; C14:1, Tetradecenoylcarnitine; cMAF, cumulative MAF; MAC, minor allele count; SKAT, Sequence Kernel Association Test.

Figure 3.

Counter-transport of amino acids by SLC7A9 in the renal proximal tubule cell is revealed by the analysis of urinary metabolite ratios. The upper part (A) contains a schematic representation of SLC7A9 function in the proximal tubule, reabsorbing dibasic urinary amino acids such as lysine in exchange for intracellular neutral amino acids. Individuals homozygous for the minor C allele at the index SNP rs12460876 show higher lysine/neutral amino acid (AA⁰) ratios in the urine, indicating less efficient lysine reuptake at the brush border. Listed amino acids are restricted to the ones measured in this study. The lower panel (B) represents a heatmap of the strength of the association between genotype at intronic rs12460876 (scaled chi square statistics) and the evaluated pairwise metabolite ratios in urine. It highlights the AA⁰ counter-transported against lysine in vivo. For comparison, lysine by itself has a scaled chi square statistic of 0.39, indicated by light orange color. ala, alanine; asn, asparagine; asp, aspartate; gln, glutamine; glu, glutamate; gly, glycine; his, histidine; ile, isoleucine; lys, lysine; orn, ornithine; phe, phenylalanine; pro, proline; ser, serine; thr, threonine; trp, tryptophan; tyr, tyrosine; val, valine.