Discovery of genetic risk variants for Diabetic Kidney Disease in susceptible mice.

A large proportion of diabetic patients develop diabetic kidney disease (DKD), while others do not, despite having similar levels of hyperglycemia. We discovered variants that regulate Xor expression to be causal for kidney disease in diabetes in mice, and associated with high risk of DKD in humans.
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Discovery of genetic risk variants for Diabetic Kidney Disease in susceptible mice.
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In diabetes, genetic and environmental factors can interact to alter pathophysiological processes, resulting in multiple consequences. The prevalence of diabetic complications is reaching epidemic proportions worldwide, and are a major cause of morbidity and mortality among diabetics. Among the most serious are the microvascular complications including DKD that is linked to a high-risk of early death. While the molecular basis for the pathology of kidney complications of diabetes is incompletely understood, it is clear that both genetic and environmental factors contribute, and that new therapies averting DKD are an extraordinary unmet need and of paramount importance given the diabetes epidemic and increased aging population throughout the world, especially in developing countries.

 Mounting evidence supports a role for reactive oxygen species as a significant contributor to the pathophysiology of DKD, including our work showing that oxidative damage in glomeruli was characteristic in DKD susceptible mice and diabetic patients with confirmed nephropathy 1. In that study, we showed that glomerular endothelial cell (GEC) dysfunction (associated with oxidative stress and increased mitochondrial injury) precedes podocyte injury and depletion in experimental mice models of DKD susceptibility and not in resistant mice. We also demonstrated that GEC mitochondrial stress and dysfunction induced by exposure to a diabetic milieu can promote the secretion of crosstalk factors that mediate podocyte cell death in vitro 2. However, we did not know “why” this would occur only in susceptible mice. So, we built a team and took on the scientific endeavor to understand why certain strains are resilient, while others are susceptible for developing kidney disease with diabetes, and whether our findings in mice could have relevance to human genetic risk for DKD.

 In our study 3 we searched for loci that influence DKD susceptibility in mice using a unique and powerful panel of recombinant inbred mouse lines known as the BXD, descended from the C57BL/6J (B6; DKD resistant) and DBA/2J (D2; DKD susceptible) laboratory strains. BXD recombinant inbred lines represent a genetic reference population, that has been maintained for up to 50 years on a known genetic background 4. Parental strains B6 and D2, as well as 38 BXD mice strains were made diabetic, and after 6 months we measured the number of podocytes per glomerulus in all mice, and mapped out quantitative trait loci (QTLs) to identify the naturally occurring polymorphisms that could influence podocyte depletion, as a key phenotype in DKD. Our study identified genome wide significant cis-acting regulatory variants of the Xdh gene encoding xanthine oxidoreductase (Xor) to be strongly associated with susceptibility to podocyte depletion in diabetes.

 We partnered with Dr. Itan’s group to delve deep into the human genetics, and confirmed that XOR orthologs in human were indeed associated with risk for diabetic complications including DKD. By examining the regulatory region of XOR with an unbiased interrogation across multiple phenotypes in EHR-based cohorts, 25 variants were identified. A phenome-wide association study in Mount Sinai Hospital’s BioMe BioBank and UK BioBank revealed that aggregation of proximal variants showed significant and relevant phenotypes including “type 1 and type 2 diabetes mellitus” and “chronic kidney disease”. These results showed that XOR variants are likely to underlie DKD or related phenotypes in human patients.

 We next knocked in the risk variants linked to DKD susceptibility, into the resistant mice using CRISPR/Cas9 gene editing. These newly engineered B6-Xorem1 mutant mice had significantly increased levels of Xor, developed glomerular lesions and albuminuria with either type 1 or type 2 diabetes 3!. We confirmed the specificity of the mechanism by blocking the phenotype using a small molecule non purine XOR inhibitor. What makes our discovery even more intriguing is that even in the absence of a diabetic state, 24-month-old aged B6-Xorem1 mice developed aging associated glomerular disease with greater podocyte depletion compared to aged matched wild type B6 mice. We were fortunate to be able to maintain these aging mice line through the long lab closures in 2020 during the peak of the COVID-19 pandemic.

 While exploring the molecular mechanisms involved, we determined that the risk variants were a transcription factor binding site for C/EBPβ in diabetes. An illustration of the XOR promoter variants with the predicted binding site for C/EBPβ is shown (Fig 1). C/EBPβ protein expression and nuclear localization was detected in cells in the glomeruli from DKD susceptible mice, and in vitro, it could increase Xor expression. We next asked, could C/EBPβ binding to XOR promoter variant in diabetes be involved in abnormal regulation of different cellular redox systems? To do this we captured in vivo lipid-derived free radical production in diabetic kidneys from susceptible mice, and we detected an increase in DNA 8-oxoG associated with TFAM1 in mitochondria in GECs. These results support the notion that diabetes in susceptible mice activates different redox systems; XOR and mitochondrial, and thus the sustained oxidative stress could lead to GEC dysfunction and podocyte depletion and promote DKD progression.

 

Figure 1 

Schematic of the C/EBPβ binding site in the sequence of the XOR promoter.

 We have uncovered a novel and previously unrecognized role for XOR in DKD, where Xor regulation through risk variants and signaling in diabetes result in glomerular injury. Given that endothelial cell stress responses in diabetes and aging are likely generalizable to other vascular beds such as the retina, the diagnostic and translational potential of our studies is extremely high. Future studies will examine the extent of injury with diabetes and determine the influence of low-risk XOR variants on DKD pathology, allowing us to define the biological basis of resilient traits. The future outcomes from these studies could move the field toward etiologic discovery and treatments that can drive resilient responses in high-risk patients.

 

REFERENCES

1          Qi, H. et al. Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility. Diabetes 66, 763-778, doi:10.2337/db16-0695 (2017).

2          Casalena, G. A. et al. The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes. Cell Commun Signal 18, 105, doi:10.1186/s12964-020-00605-x (2020).

3          Wang, Q. et al. Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR. Nature Metabolism, doi:10.1038/s42255-023-00776-0 (2023).

4          Peirce, J. L., Lu, L., Gu, J., Silver, L. M. & Williams, R. W. A new set of BXD recombinant inbred lines from advanced intercross populations in mice 1. BMC.Genet. 5, 7 (2004).

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