Kidney disease genetic risk variants alter lysosomal beta-mannosidase (MANBA) expression and disease severity

Abstract

More than 800 million people in the world suffer from chronic kidney disease (CKD). Genome-wide association studies (GWAS) have identified hundreds of loci where genetic variants are associated with kidney function; however, causal genes and pathways for CKD remain unknown. Here, we performed integration of kidney function GWAS and human kidney-specific expression quantitative trait analysis and identified that the expression of beta-mannosidase (MANBA) was lower in kidneys of subjects with CKD risk genotype. We also show an increased incidence of renal failure in subjects with rare heterozygous loss-of-function coding variants in MANBA using phenome-wide association analysis of 40,963 subjects with exome sequencing data. MANBA is a lysosomal gene highly expressed in kidney tubule cells. Deep phenotyping revealed structural and functional lysosomal alterations in human kidneys from subjects with CKD risk alleles and mice with genetic deletion of Manba Manba heterozygous and knockout mice developed more severe kidney fibrosis when subjected to toxic injury induced by cisplatin or folic acid. Manba loss altered multiple pathways, including endocytosis and autophagy. In the absence of Manba, toxic acute tubule injury induced inflammasome activation and fibrosis. Together, these results illustrate the convergence of common noncoding and rare coding variants in MANBA in kidney disease development and demonstrate the role of the endolysosomal system in kidney disease development.

Fig. 1.. Common (GWAS) and rare variant (PheWAS) analysis identifies an association between MANBA, kidney function, and kidney disease.

(A and B) LocusZoom plots of chromosome 4 region of CKD eGFR GWAS(15), MANBA eQTL and CISD2 eQTL in whole kidneys(5), glomeruli, and tubules(8). X-axis shows the chromosomal location of SNPs. Y-axis shows the strength of association or recombination rate. (C) Distribution of non-CKD, CKD without ESRD, and ESRD subjects amongst the identified MANBA heterozygous LOF alleles compared to subjects in the Biome and PMBB biobanks. Y-axis shows the percentage of the disease distributions.

Fig. 2.. Manba deficiency exacerbates fibrosis in a folic acid-induced kidney injury model

(A) Manba expression of wild-type, Manba^+/−, and Manba^−/− mice determined by real-time PCR (n= 5 to 9 animals per group). (B) Beta-Mannosidase activity was assayed in kidney tissues of wild-type, Manba^+/−, and Manba^−/− mice (n=5 to 9 animals per group). (C) Serum beta-Mannosidase expression in wild-type mice, Manba^+/−, and Manba^−/− mice (n=5 to 7 animals per group). (D) Representative images of PAS-stained kidney sections from wild-type and Manba^−/− mice 7 days after sham or folic acid (FA) injection. Scale bar, 20μm. (E) Representative images of Sirius Red-stained kidney sections from wild-type and Manba^−/− mice 7 days after sham or FA injection. Scale bar, 50μm. Quantification of Sirius Red-staining (n=3 per group). (F) Relative mRNA abundance of Vim, Fn, Colα1, and Col3α1, in wild-type and Manba^−/− mice kidneys 7 days after sham or FA injection (n=4 to 7 animals per group). (G) Representative Western blots of fibronectin andα-SMA of kidney tissues of wild-type and Manba^−/− mice 7 days after sham or FA injection (n = 3 per group). Densitometry analysis was performed to quantify protein expression. Data shown are means ± SEM. Statistical analysis by (A to C, and F to G) One-way ANOVA with Tukey’s post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, **** P< 0.0001.

Fig. 3.. Genetic deletion of Manba exacerbates cisplatin-induced kidney injury.

(A) Representative images of H&E-stained kidney sections from wild-type, Manba^+/− and Manba^−/− mice 3 days after sham or cisplatin injection. Scale bar, 20μm. Tubular injury scores of kidney sections of wild-type, Manba^+/−, and Manba^−/− mice 3 days after sham or cisplatin injection. (B) BUN and serum creatinine of wild-type, Manba^+/−, and Manba^−/− mice 3 days after sham or cisplatin injection. (C and D) Relative mRNA abundance of Lcn2, Hacvr1, Vim, Tgfβ1, Colα1, and Col3α1 in kidneys of wild-type, Manba^+/−, and Manba^−/− mice 3 days after sham or cisplatin injection. (E) Representative Western blots of fibronectin and α-SMA of kidney tissues of wild-type and Manba^−/− mice 3 days after sham or cisplatin injection (n = 3 per group). Densitometry analysis was performed to quantify protein expression. Data are means ± SEM. Statistical analysis by (A) Kruskal-Wallis followed by Conover-Iman test with Bonferroni adjustment, n=4 to 9 animals per group, #, P<0.0001, control group versus corresponding cisplatin group. ****P < 0.0001; (B to D) Two-way ANOVA test, n=4 to 9 animals per group, &, P<0.01, $, P<0.001, #, P<0.0001, control group versus corresponding cisplatin group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; (E) One-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.. Manba deficiency induces structural and functional lysosomal changes and blocks autophagy and endocytosis in cultured kidney tubule cells.

(A) Expression of Manba, Nfkb1, Ube2d3, and Cisd2 in mouse kidney single cells (18). Blue/yellow corresponds to the gene expression Z-score. Endo, containing endothelial, vascular; Podo, podocyte; PCT, proximal convoluted tubule; LOH, loop of Henle; DCT, distal convoluted tubule; CD-PC, collecting duct principal cell; CD-IC, collecting duct intercalated cell; Fib, fibroblast; Macro, macrophage; Neutro, neutrophil; B lymph, B lymphocyte; T lymph, T lymphocyte; NK, natural killer cell. (B) Representative images of MANBA immunohistochemistry staining of control and Manba knock-out mouse kidney samples. (C) Representative confocal and 3D reconstruction images of TECs from wild-type and Manba^−/− mice in fed or starved medium labeled with LAMP1 (red) and DAPI (blue). (D) Representative images of kidney cells from wild-type and Manba^−/− mice stained with Lysotracker (red) and DAPI (blue). Quantification of numbers of Lysotracker+ structures per cell. *P < 0.05, t test. (E and F) Representative confocal and 3D reconstruction images of TECs from wild-type and Manba^−/− mice incubated with 10 kD Dextran labelled with Alexa-488 for 24h and 488-Alexa conjugated albumin for 4h, respectively. (G and H) Representative Western blots of LC3B protein expression in wild-type and Manba knockout cultured renal tubule cells in fed or starved medium with or without 50μM chloroquine (CQ) or 50 nM BafilomycinA1(BafA1) for 2h or 4h. (I and J) LC3B immunofluorescence staining of wild-type and Manba^−/− tubule cells (LC3B [red] and DAPI [blue]) in fed or starved medium with or without 50μM chloroquine (CQ) or 50 nM BafilomycinA1(BafA1) for 2h or 4h. (B) Scale bar, 20 μm; (C to F and I to J) Scale bars, 10 μm.

Fig. 5.. Manba deficiency leads to impaired autophagy flux.

(A) Representative images of LAMP1 immunostaining of kidney sections of wild-type and Manba^−/− aging mice. (B) Representative electron microscopic images showing lysosomes in wild-type and Manba^−/− aging mice. Quantification of numbers and size of lysosomes in tubular epithelial cells of aging wild-type and Manba^−/− mice. Black squares contain images at higher magnification. Yellow arrowhead indicates mitochondria; red, lysosomes; blue, autophagic vacuoles. (C) Representative Western blots of SQSTM1 and LC3B from kidney tissues of wild-type and Manba^−/− mice 7 days after sham or folic acid (FA) injection (n = 3 to 6 animals per group). (D) Representative Western blots of SQSTM1 and LC3B from kidney tissues of wild-type and Manba^+/− mice 3 days after sham or cisplatin injection (n = 3 per group). (E and F) Representative images of LAMP1 and LC3B immunostaining of kidney sections from wild-type and Manba^−/− mice 3 days after sham or cisplatin injection (LAMP1 (red), LC3B(red) and DAPI (blue)). (G) Representative Western blots of SQSTM1 from kidney tissues of wild-type and Manba^−/− mice 3 days after sham or cisplatin injection (n = 3 per group). Densitometry analysis was performed to quantify protein expression. Data shown are means ± SEM. Y-axes differ between barplots. Statistical analysis by (B) t test, (C, D and G) One-way ANOVA–Tukey’s post hoc test. *P < 0.05, **P < 0.01. (B) scale bar, 1μm; (A, E and F) Scale bars, 10 μm.

Fig. 6.. Genetic Manba loss induces NLRP3 inflammasome signaling.

(A) Representative Western blots of NLRP3 and Cleaved Caspase1 from kidney tissues of wild-type and Manba^−/− mice 7 days after sham or folic acid (FA) injection (n = 3 per group). (B) Relative mRNA abundance of Il1β, Tnfα, Ccl2 in kidneys of wild-type and Manba^−/− mice 7 days after sham or FA injection (n=4 to 7 animals per group). (C) Representative Western blots of NLRP3 and Cleaved Caspase1 in kidneys of wild-type and Manba^−/− mice 3 days after sham or cisplatin injection (n = 3 per group). (D and E) Relative mRNA abundance of Il1β, Nlrp3, Tnfα, Ccl2, Cd68 and Lyz2 in wild-type, Manba^+/−, Manba^−/− mice kidneys 3 days after sham or cisplatin. (n=4 to 9 animals per group). (F) Relative mRNA abundance of Manba, Il1β, Nlrp3, Tnfα, Ccl2 of wild-type, Manba^+/−, Manba^−/− tubular epithelial cell. (n=3 per group). Densitometry analysis was performed to quantify protein expression. Data shown are means ± SEM. Statistical analysis by (A and C) One-way ANOVA–Tukey’s post hoc test. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P< 0.0001; (B and D to F) Two-way ANOVA test, ^, P<0.05, &, P<0.01, $, P<0.001, #, P<0.0001, control group versus corresponding cisplatin group. * P<0.05, ** P<0.01, *** P<0.001, **** P< 0.0001.

Fig. 7.. Phenotypic characterization of kidneys obtained from subjects with CKD associated genetic-risk variants

(A) Representative images of LAMP1 staining in human kidney tissues of subjects with AA, AG and GG alleles, respectively. The A allele is the risk allele. White squares indicate images shown at higher magnification. Scale bar, 10 μm. (B) Gene expression analysis of microdissected kidney tubule samples from risk allele vs. reference allele. Pathway enrichment analysis (DAVID) of top differentially expressed genes. The bar plot shows the significance (log₁₀(p) and log₁₀(odds ratio)) of the enrichment of specific pathways. (C) Relative mRNA expression of C1QL1 in reference and risk genotype kidney tubule samples.