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

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.

Generation of Sec10 CKO mice to Study Urinary Tract Levels

Exocyst Complex

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)

Normal Vs. Obstructed

Obstructed ureter of a newborn

mouse

Normal ureter of a newborn mouse

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

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?

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

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

• 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

E 16

.5 fl

/fl

uret

erE

16.5

CKO

ur

eter

E17.

5 fl/

flE1

7.5

CKO

uret

er

Results

Results

Conclusion • Does not look like there was increased proliferation rate

• Small sample size

• Had difficulty with staining variability

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