Role of Acid Sphingomyelinase Knockout Mice in Protection Against Hyperhomocystenimia Induced Glomerular Injury
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1 Role of Acid Sphingomyelinase Knockout Mice in Protection Against Hyperhomocystenimia Induced Glomerular Injury Sophie S. Yuth Department of Pharmacology and Toxicology Medical College of Virginia Virginia Commonwealth University Summer Program for Undergraduate Research July 2009
2 Background Hyperhomocystenemia is known as a critical pathogenic factor in the progression of end stage renal disease (ESRD) and in the development of cardiovascular complications related to ESRD. Chronic elevations of plasma Hcys levels induce proteinuria, mesangial expansion and glomerulosclerosis. Elevated Hcys levels increase de novo ceramide synthesis in rat mesangial cells. This increased ceramide production enhances NADPH oxidase activity. Increased NADPH oxidase activity generates superoxide production and ultimately causes glomerular injury. However, nothing is known about hyperhomocysteinemia induced glomerular injury on mice lacking the acid sphingomyelinase gene.
3 Hypothesis To explore the role of acid sphingomyelinase and NAD(P)H oxidase in the development of hhcys-induced glomerular injury in ASM mice
4 Biosynthesis of Ceramide FF Diet hhcy ASM/Ceramide LR/platform NOX/O 2.- Glomerular Injury
5 Homocysteine HS O NH 2 OH S-Adenosyl - CH 3 methionine ATP S-Adenosyl - homocysteine SAHH Homocysteine H 2 O B 6 CBS Adenosine Cystathionine Methionine methionine synthase B 12 folate CH 3 B 6 GCS Cysteine Excreted in urine SO = 4 Taurine
6 Animal Model 8-weeks old ASM mice +/+ Control diet Control diet +/+ -/- -/- Folate free diet Folate free diet ASM mrna level, Plasma Hcy, Urinary protein excretion, Superoxide production and Glomerular injury markers
7 Plasma Hcys concentration in mice 45 * Plasma Hcys conc. (μm) # ASM +/+ ASM -/- * vs Control diet # vs WTFF diet 0 Control diet Folate free diet
8 ASM mrna expression in mice 3 ASM mrna level ASM +/+ ASM -/ Control diet Folate free diet
9 Morphological features of the glomeruli from different groups of mice WT ND KO ND WT FF KO FF Glomerular Damage Index ASM +/+ ASM -/- * vs Control diet # vs WTFF diet * # 0 Control Diet Folate free diet
10 Urinary total protein excretion in mice 0.42 U protein (mg/24h) ASM +/+ ASM -/- 0 Control diet Folate free Diet
11 Structure and function of podocytes
12 Podocin expression in mice treated with control or folate free diet 100x 400x 1000x WTND WTFF KOND KOFF
13 Nephrin expression in mice treated with control or folate free diet 400x 1000x Negative Control WTND WTFF KOND KOFF
14 Desmin expression in mice treated with control or folate free diet 100x 400x 1000x WTND WTFF KOND KOFF
15 Ceramide expression in mice treated with control or folate free diet 100x 400x 1000x WTND WTFF KOND KOFF
16 ASM expression in mice treated with control or folate free diet 400x 1000x WTND WTFF KOND KOFF
17 Superoxide production in ASM mice Fold change of superoxide production (nomalized to control) Control Diet * # Folate free diet ASM +/+ ASM -/- * vs Control diet # vs WTFF diet
18 Conclusions Mechanism: FF Diet- Hcy - ASM and Ceramide LR platform- Nox/O 2- - Glomerular injury Conclusions: ASM gene knockout mice attenuates folate free diet induced plasma homocysteine concentration, mrna level, urinary total protein excretion, and superoxide production compared to the wild type mice. ASM gene knockout protects against hyperhomocystenimia induced glomerular injury.
19 Acknowledgments Dr. Pin-Lan Li, MD, PhD Dr. Ningjun Li, MD Dr. Krishna Boini, PhD Dr. Qing Zhu, PhD (Carl) Dr. Chun Zhang, MD, PhD (Peter) Dr. Zhengchao Wang, PhD (Jackie) Dr. Fan Zhang, MD, PhD (Max) Dr. JunJun Hu, MD (June) Min Xia (Mark) Laura Laperle Christopher Brimson Jessica Cardwell Jennifer Doughman Shante Hinton VCU SPUR Program NIH - R01 DK ARRA Supplement Award
20 Thank you for everything!
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