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Abstract: FR-PO660

Mitochondrial DNA in Urinary Large Extracellular Vesicles as a Marker of Relapse in Children with Nephrotic Syndrome

Session Information

  • Pediatric Nephrology - II
    November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pediatric Nephrology

  • 1900 Pediatric Nephrology

Authors

  • Myette, Robert L., Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada
  • Xiao, Fengxia, Kidney Research Center, Ottawa, Ontario, Canada
  • Kennedy, Chris R., Kidney Research Center, Ottawa, Ontario, Canada
  • Burger, Dylan, Kidney Research Center, Ottawa, Ontario, Canada
Background

Nephrotic Syndrome (NS) is one of the most common causes of glomerulopathy in children. There has been an increased focus on reactive oxygen species (ROS) and mitochondrial injury in podocytes as drivers of proteinuric disease. We recently showed that podocyte-specific large extracellular vesicles (LEVs; 0.1-1.0 um) are increased in the urine of children with NS. The aim of this study was to characterize LEV mitochondrial DNA (mtDNA) content from those same children. We investigated this relationship further using cultured podocytes exposed to toxins in vitro.

Methods

We analyzed urine samples from a prospective cohort enrolling children 1-18y with NS. Podocyte specific LEVs were quantified using flow cytometry and nanoparticle tracking (NTA). Urinary LEV mtDNA was assessed using qPCR. Human immortalized podocytes (hPod) were used in cell culture experiments. Puromycin aminonucleoside (PAN; 25 ug/mL; 24 hours) and lipopolysaccharide (LPS; 25 ug/mL; 24 hours) were used as podocyte toxins.

Results

We analyzed 28 samples from 14 patients. Podocyte LEVs were significantly lower in remission vs. nephrosis (p<0.01). Urine protein to creatinine ratio correlated with elevated LEVs (p=0.0005). Patient urinary LEV mtDNA was higher in NS relapse compared to remission (p=0.04). In hPod cells, PAN treatment resulted in a 2.5-fold increase in hPod LEVs (p=0.03) while LPS caused a 3.5-fold increase (p=0.0004). The impact of PAN and LPS treatment on in vitro LEV production was abrogated by the antioxidant MITO-Tempol. Following treatment with PAN or LPS in vitro, we observed an ~25-fold increase in LEV mtDNA content (p<0.01).

Conclusion

In summary, LEVs may serve as a biomarker of podocyte injury in nephrotic syndrome in children and their mtDNA content can differentiate remission from relapse. hPods show similar characteristics when treated with common podocyte toxins, and ongoing studies aim to characterize this further.