williamsmadison
TRANSCRIPT
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Measuring Phenotypic Changes of Epithelial and Smooth Muscle Cells in Embryonic Ureters of a Mouse Model of Lethal Urinary Obstructions
Madison Williams, Kamehameha Schools MauiPrimary Mentor: Ben Fogelgren, PhD, John A. Burns School of MedicineSecondary Mentor: Noemi Polgar, PhD, John A. Burns School of Medicine
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Introduction • Congenital obstructions of the urinary tract in humans are the leading causes of kidney disease in children
• Most common cause of Congenital Obstructive Nephropathy (CON) is congenital Ureteropelvic Junction (UPJ) Obstruction.
• UPJ obstruction is a blockage in the ureters that causes hydronephrosis (water in the kidneys) and renal damage. Hydronephrosis is observed in prenatal ultrasounds in 1:100-200 pregnancies.
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Generation of Sec10 CKO mice to Study Urinary Tract Levels
Exocyst Complex
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Sec10 CKO pups most often die within hours of birth due to UPJ obstructions.
Lifespan <1 day Day 2 Days 3-7 Day 8+% CKO pups 95% 3% 1% <1%
Longer lived mice displayed the hypothesized phenotype:Shortened primary cilia, withcystic and fibrotic kidneys
Most neonates died from congenital obstructive nephropathy (CON)
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Normal Vs. Obstructed
Obstructed ureter of a newborn
mouse
Normal ureter of a newborn mouse
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F
Sec
10FL
/FL
Sec
10FL
/FL ; C
reK
sp
A
B
C
D
E
Sec10-CKO pups die at birth with severe bilateral hydronephrosis
E18.5
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Sec1
0FL/
FLSe
c10F
L/FL
;Ksp
-Cre
E-cadherin, SMANewborn Ureters
The UPJ obstruction in Sec10-CKO ureters is the result of overgrowth of surrounding mesenchymal cells
Question:
Why does this obstruction occur?
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Hypothesis of this Project • Sec10 Knockout in urothelial cells will weaken the epithelial barrier against urine
• Allows damage to the underlying Smooth Muscle cells
• Responds by over proliferating, causing UPJ obstruction
• We will measure cell proliferation rates in multiple stages of ureter development and compare between Sec10 CKO and normal controls
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Approach & Methods• Mouse husbandry and handling
• Dissection of kidney and urinary tracks at various stages of mouse embryonic development
• Fixed tissues, and sectioned using cryostat and microtome
• Stained sections with Hematoxylin and Eosin Y.
• Immunostaining of sections, followed by fluorescent microscopy
• Fluorescent microscopy using ImageJ, analysis software
• Statistical analysis using Excel
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• DAPI – (4',6-diamidino-2-phenylindole) binds and stains DNA
• Ki67 – protein present only in dividing cells
• SMA – (Smooth Muscle Actin) marker for smooth muscle cells
Results
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E 16
.5 fl
/fl
uret
erE
16.5
CKO
ur
eter
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E17.
5 fl/
flE1
7.5
CKO
uret
er
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Results
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Results
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Conclusion • Does not look like there was increased proliferation rate
• Small sample size
• Had difficulty with staining variability
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Acknowledgements Fogelgren Lab (UH - JABSOM)Ben FogelgrenNoemi Polgar, Ph.D. Vanessa Lui Amanda LeeFunding
NIH - NIDDK Hawaii Community Foundation March of Dimes Hepato/Renal Fibrocystic Disease Core Center (UAB) Core Support RCMI & BRIDGES Histopathology Core UAB Hepato/Renal Fibrocystic Disease Core Center Knock Out Mouse Project (KOMP) and Repository