Abstract
The lifetime risk of kidney disease in people with diabetes is 10–30%, implicating genetic predisposition in the cause of diabetic kidney disease (DKD). Here we identify an expression quantitative trait loci (QTLs) in the cis-acting regulatory region of the xanthine dehydrogenase, or xanthine oxidoreductase (Xor), a binding site for C/EBPβ, to be associated with diabetes-induced podocyte loss in DKD in male mice. We examine mouse inbred strains that are susceptible (DBA/2J) and resistant (C57BL/6J) to DKD, as well as a panel of recombinant inbred BXD mice, to map QTLs. We also uncover promoter XOR orthologue variants in humans associated with high risk of DKD. We introduced the risk variant into the 5′-regulatory region of XOR in DKD-resistant mice, which resulted in increased Xor activity associated with podocyte depletion, albuminuria, oxidative stress and damage restricted to the glomerular endothelium, which increase further with type 1 diabetes, high-fat diet and ageing. Therefore, differential regulation of Xor contributes to phenotypic consequences with diabetes and ageing.
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The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files. Source data are provided with this paper.
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Acknowledgements
We thank the Genome Engineering and iPSC Center (GEiC) at Washington University Department of Genetics for providing support in the generation of small guide RNAs. We acknowledge Dr. K Kelly (The Institutional Mouse Genetics and Gene Targeting CoRE at Mount Sinai) for his valuable technical help in generation of B6-Xorem1(rs50771495-A>C/rs50017899-T>A)Isd knock-in mice. We thank A. Bajpai for his assistance with mice genetics. Parts of this research have been conducted using the UK Biobank Resource under application number 53074. This work was supported by grants from National Institutes of Health (NIH) grant R01DK097253 and Department of Defense CDMRP grant E01 W81XWH2010836 to I.D., and Leducq Foundation Award Number 21CVD01 to Y.I.
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Q.W., H.Q., E.B. and I.D. conceived the idea, designed the research and wrote the first version of the manuscript. Q.W., H.Q., G.C., S.S., R.B., E.L., S.L., I.D. and L.Y. collected samples and performed the in vitro and in vivo experiments and performed the validation experiments. Z.Y. and W.Z. were responsible for the bioinformatic analyses of the microarray data. Y.W. and Y.I. performed human PheWAS. L.L. and R.W.W. performed the mouse QTL and eQTL analysis. J.D. performed in silico assessment of TFBS. K.S. performed and analysed electron paramagnetic resonance spectroscopy experiments. K.E., F.S. and R.G. performed TEM and provided pathology insights. Q.W., H.Q. and I.D. prepared the figures. I.D. developed the experimental strategy, supervised the project and wrote and edited the manuscript, which was then read by, commented on and approved by all authors.
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Extended data
Extended Data Fig. 1 Fasting blood glucose (FBG), body weight (BW), urine albumin:creatinine uACR and podocyte numbers per glomerulus in non-diabetic and diabetic D2 mice + /- XOi.
Bar graph shows the mean + /- SD of fasting blood glucose (FBG; mg/dl) in a) and BW (g) b) of D2-WT control ± allopurinol (XOi; n = 5/group) or STZ treated D2-WT mice ± XOi (n = 8/group) at 6 and 12 weeks of diabetes as indicated. c) uACR (ug/mg) of D2-WT ± XOi (n = 5/group) or STZ treated D2-WT only (n = 5) or + XOi (n = 10) at 6, 12 weeks of diabetes, and d) podocyte number (WT1 + cells) per glomerular section of control D2-WT ± XOi, or STZ treated D2-WT ± XOi at 6, 12 weeks of diabetes in >50 glomerular profiles/mouse (n = 5 mice/group). All data are presented as mean values ±SEM. Statistical significance in was calculated by one-way ANOVA with Tukey’s multiple comparisons test.
Extended Data Fig. 2 Transcription Factor Binding Sites (TFBS) in the promoter of XOR.
Mulan sequence conservation profile for the XOR promoter locus from human, cow and mouse genomes a). CEBP binding sites show high regulatory potential as indicated by a purple block depicted as colored tick marks above the conservation profile. Coding exons are in blue, UTRs in yellow, intergenic elements in red, and intronic in pink. b) multiTF visualization of XOR transcription factor binding site detected in the XOR locus constructed with mouse sequences (black rectangle). Identified TFBSs are depicted as colored blocks above the conservation profile.
Extended Data Fig. 3 Genotyping for XOR variants in mGECs and FACS gating strategy for ROS.
a) Representative agarose gel showing bands for B6-XorWT (top) and D2-WT (bottom) variants. Molecular weight markers in lane 1, lane 2: D2-WT, lane 3: B6-XorWT mice and in lane 4: mGECs. Image is representative of 4 independent tests. b) Genotyping strategy for detecting SNP variant through mismatch with AS primers F1 and F2 using a common R primer as described in Methods. c) FACS gating strategy showing a representative plot of negative unlabeled control mGECs % population of CM-H2-DCFDA, and d) % population of cells positive for CM-H2-DCFDA after 24 hr HG treatment.
Extended Data Fig. 4 Fasting blood glucose, body weights, serum suPAR and triglycerides in mice made diabetic by STZ, or genetic predisposition (Akita), or high fat diet (HFD).
a) Bar graph shows the fasting blood glucose in male B6-XorWT, B6-Xorem1 and D2-WT mice (n = 6/group) and female B6-XorWT (n = 4), B6-Xorem1 (n = 5) and D2-WT mice (n = 6) at 12 weeks of diabetes. b) Serum suPAR levels in diabetic B6-XorWT, B6-Xorem1 (n = 5/group) and D2-WT mice (n = 4). c) Fasting blood glucose and d) body weight (g) of Akita B6 (n = 4) and Akita B6-Xorem1 mice (n = 5) at 12 weeks of diabetes. e) Shows the fasting blood glucose, f) the body weight, g) triglycerides (mg/dL) and h) serum suPAR levels in B6-XorWT and B6-Xorem1 after 16 weeks of high fat diet (HFD, n = 5 mice/group). All data are presented as mean values ±SEM. In a) a two-way analysis of variance and Tukey’s multiple comparisons test was performed (*P < 0.05). In b-h) unpaired two-tailed Student’s t-tests was performed and showed no statistical significance between the groups.
Extended Data Fig. 5 C/EBPβ in high glucose or oxidized LDL treated primary GECs and podocytes.
a) Representative immunofluorescence staining for C/EBPβ (green) and CD31 (magenta) in primary glomerular endothelial cells from B6-XorWT and B6-Xorem1 mice (n = 3), treated with control normal glucose (NG), or high glucose (HG, 30 mM) for 24 hours showing nuclear translocation. Bar=20μm. b) C/EBPβ (green) and synaptopodin (red) in primary podocytes from B6-XorWT and B6-Xorem1 mice (n = 3), treated as in a). Bar=20μm. These are representative images from 3 independent experiments performed in primary cells grown from 3 separate B6-XorWT and 3 separate B6-Xorem1 mice.
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Unprocessed gel for Fig. 2b.
Source Data Fig. 2
Unprocessed western blot for Fig. 2g
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Unprocessed western blot for Fig. 3e
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Wang, Q., Qi, H., Wu, Y. et al. Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR. Nat Metab 5, 607–625 (2023). https://doi.org/10.1038/s42255-023-00776-0
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DOI: https://doi.org/10.1038/s42255-023-00776-0
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