Abstract: FR-PO272
Loss of Diabetic Nephropathy Phenotype and Higher Esm-1 Map to Germline Mutation in Splicing Factor Sfswap in Inbred Mice
Session Information
- Diabetic Kidney Disease: Basic - 1
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 701 Diabetic Kidney Disease: Basic
Authors
- Narang, Keshav, Stanford University School of Medicine, Stanford, California, United States
- Guan, Haochen, Stanford University School of Medicine, Stanford, California, United States
- Luo, Xiao Ran, Stanford University School of Medicine, Stanford, United States
- Nandal, Randeep S., Stanford University School of Medicine, Stanford, California, United States
- Maxim, Demetri, Stanford University School of Medicine, Stanford, California, United States
- Zheng, Xiaoyi, Stanford University School of Medicine, Stanford, California, United States
- Bhalla, Vivek, Stanford University School of Medicine, Stanford, California, United States
Background
Only a subset of diabetic DBA/2 DN-susceptible mice develop albuminuria and glomerular leukocyte infiltration. Plasma levels of endothelial cell-specific molecule-1, Esm-1, an anti-inflammatory, secreted proteoglycan inversely correlate with albuminuria in mice and humans. Moreover, over-expression of Esm-1 in diabetic DN-susceptible mice prevents albuminuria and podocyte injury independent of blood glucose. Given an inverse correlation of Esm-1 and features of DN that we observed across generations of inbred mice, we explored the genetic regulation of plasma Esm-1 and susceptibility to DN.
Methods
Genomic DNA was extracted from frozen kidney tissue of N=4 mice / generation from before and after a loss of the DN phenotype and rise in plasma Esm-1. Next-generation sequencing (NGS) of 2x150 bp PCR-free paired-end libraries was performed on a NovaSeq 6000 S4 sequencer to a coverage depth of 30x. NGS data were analyzed using the Genome Analysis Toolkit pipeline for variant calling. Restriction fragment length Polymorphism with StuI and Sanger sequencing at a candidate variant locus were used to validate NGS data in N=21 non-diabetic mice across five generations.
Results
We identified a missense variant (nt 1407 CàA, Asn469Lys (N469K) in the Splicing Factor, Arginine/Serine-Rich 8 (Srsf8, Sfswap) gene. Analysis of this variant indicated a significant association of high plasma Esm-1 with presence of the homozygous variant compared to wild-type mice (4.54±0.45 vs. 1.25 ±0.26 ng/mL, p<0.0001). We found low (< 3 ng/mL) plasma Esm-1 in 10 out of 11 wild-type but only 2 out of 10 heterozygous or homozygous mutant mice. Heterozygote variant mice had an intermediate phenotype, indicating a gene dosage effect. Asn-469 is located in a binding domain indispensable for binding of Sfswap to similar splicing factors and is evolutionarily conserved. Further experiments to edit this variant in mouse endothelial cells and to quantify secreted Esm-1 are forthcoming.
Conclusion
Our findings reveal that the Sfswap mutant genotype is highly correlated with plasma Esm-1 levels in DN-susceptible mice. In addition to elucidating genetic drift within the DBA/2 mouse colony, these results may provide novel insights into the genetic regulation of Esm-1 and opposing roles of Sfswap and Esm-1 in protection from DN.
Funding
- NIDDK Support