Genome-wide association and functional follow-up reveals new loci for kidney function

Abstract

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD.

Figure 1. Genetic association and LD distribution of the MPPED2 gene locus in European and African ancestry populations.

Regional association plots in the CKDGen European ancestry discovery analysis (N = 74,354) (A) and in the CARe African ancestry discovery analysis (N = 8,110) (B). LD structure: comparison between the HapMap release II – CEU and YRI samples in the region included within +/−100 kb from the target SNP rs3925584 identified in the CKDGen GWAS. The green circle highlights a stream of high LD connecting the two blocks, indicating the presence of common haplotypes (C).

Figure 2. Mpped2 and casp9 knockdowns result in defective kidney development.

(A–E) Whole mount in situ hybridization in control embryos demonstrates normal expression of the global kidney marker pax2a (A: lateral view; B: dorsal view), the glomerular marker nephrin (C), and the tubular markers slc20a1a (proximal tubule, D), and slc12a3 (distal tubule, E) at 48 hours post fertilization (hpf). (F–J) Mpped2 morpholino (MO) knockdown embryos develop glomerular gene expression defects (F–H, arrowheads), but tubular marker expression is normal (I, J). (K–O) Casp9 MO knockdown embryos demonstrate reduced glomerular gene expression (K–M, arrowheads) and shortened distal tubules (O). (P) Quantification of observed abnormalities per number of embryos reveal significant differences in expression of pax2a and nephrin in response to knockdown of both mpped2 and casp9 (Fisher's exact test). (Q–V) Embryos were injected with control, mpped2, or casp9 MO at the one-cell stage and subsequently injected with 70,000 MW fluorescent rhodamine dextran at 80 hpf. Dextran fluorescence was monitored over the next 48 hours. All dextran-injected embryos show equal loading into the cardiac sinus venosus at 2 hours post-injection (2 hpi/82 hpf; Q, S, U). Compared to control MO-injected embryos (R) and mpped2 knockdown embryos (T), knockdown of casp9 resulted in reduced dextran clearance at 48 hpi as shown by increased trunk fluorescence (V). (W) Casp9 knockdown results in increased susceptibility to edema formation both spontaneously (−dex) (P value = 0.0234, Fisher's exact test) and after dextran challenge (+dex) (P value<0.0001). Embryos injected with both MO and dextran did not survive to 6 dpf (N/A). (X) Edema develops earlier and with higher frequency in casp9 morphants following injection of the nephrotoxin gentamicin.