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Abstract: SA-PO525

Deletion of Endothelial Nitric Oxide Synthase in BTBR ob/ob Mice Enhances Mesangiolysis and Worsens Diabetic Nephropathy

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Li, Xianwu, University of Washington, Seattle, Washington, United States
  • Hudkins, Kelly L., University of Washington, Seattle, Washington, United States
  • Holland, Alexander Lucas, University of Washington, Seattle, Washington, United States
  • Swaminathan, Shreya, University of Washington, Seattle, Washington, United States
  • Alpers, Charles E., University of Washington, Seattle, Washington, United States
Background

Leptin receptor deficient db/db mice with endothelial nitric oxide synthase (eNOS) deficiency and leptin deficient BTBR ob/ob mice have been useful models of moderately advanced diabetic nephropathy (DN). eNOS is essential for maintaining integrity and health of endothelial cells and podocytes. We postulated eNOS deficiency in BTBR ob/ob mice would exacerbate the severity of DN, allow testing of whether intact eNOS is essential or not to enable regression of DN with leptin replacement.

Methods


CRISPR/Cas9 was used to delete eNOS in BTBR ob +/- heterozygous mice. The resultant BTBR ob +/- /eNOS-/-mice were crossed to obtain BTBR ob/ob/eNOS-/- homozygous mice (eNOS ob/ob), and control BTBR wt eNOS-/- mice (eNOS BTBR). At 18 weeks, fasting glucose levels and timed (6 hour) urine samples were collected. The mice were then euthanized, and blood, urine and tissue samples collected.

Results

eNOS ob/ob mice exhibited robust hyperglycemia (517.5 mg/dl) and a marked increase in urine albumin-creatinine ratio (ACR) (791.9 vs 172.3 ug/mg in eNOS BTBR, p < 0.01). Compared to control mice, eNOS ob/ob mice had significantly higher mesangial matrix accumulation (29.5% vs 15.9%, p < 0.01, assessed as fraction of collagen IV staining of glomerular tuft area). Silver methenamine stain revealed striking mesangiolysis, affecting 31.1% of glomeruli in eNOS ob/ob vs. 9.5% in control mice (p < 0.01). Podocyte density decreased in eNOS ob/ob compared to control mice (90.5 vs 149.8 podos/x106 um3, p < 0.01). In addition, significantly higher amounts of urinary 8OHdG (a DNA/RNA marker of oxidative stress) were detected in eNOS ob/ob mice than in control mice (511.2 vs 35.9 ng/mg creatinine, p <0.05).

Conclusion



BTBR ob/ob mice with further knockout of eNOS exhibit dramatic albuminuria, reduced podocyte density, mesangial expansion and more extensive mesangiolysis than is featured in other models of murine DN including BTBR ob/ob mice of similar age. These characteristics more closely resemble advanced human DN. This mouse model could be useful for the development of drugs targeting mesangiolysis as well as for understanding the pathogenesis of DN.

Funding

  • NIDDK Support