1. Abstract
Cystinuria is a rare autosomal recessive disorder characterized by
high concentrations of cystine in urine and repeated cystine stone
formation in the kidney, bladder, and/or ureter. We used
male Slc3a1 knockout mice of different ages to acquire
information about the production and development of the disorder.
Males are more severely affected than females, making our mice
an ideal model system. The exact pathology of how urinary stones
cause bladder obstruction is not fully understood, but we know
bladders of male knockout mice over 3 months of age exhibit
hypertrophy, decreased compliance, and decreased contractile
responses. We hypothesized that the urothelial cell surface,
possibly because of injury and/or increased proliferation, causes
gene expression changes. This brings about inflammation,
interstitial fibrosis, and smooth muscle hypertrophy. To determine
if the presence of cystine stones in the bladder leads to qualitative
and quantitative variations in gene expression in bladder cells, we
examined the expression of interleukin-1β (IL-1β), transforming
growth factor-β1 (TGF- β1), and insulin growth factor (IGF-1). IL-
1β is a proinflammatory cytokine that induces TGF- β1 and insulin
growth factor IGF-1, which contribute to tissue fibrosis and muscle
hypertrophy respectively. Starting with RNA extraction then
conversion to cDNA, we utilized RT-PCR and qPCR to test,
record, and analyze the expression levels of the genes. These
changes in expression with age underlie histopathological
changes associated with the dysfunctions found in mice with
Cystinuria, and studying them will provide further information
about the disorder and others of its kind.
Conclusion and Future Directions
Materials and Methods
Acknowledgments
Results
Figure 3: These are Standard Curve Plots of the genes PPIA, TGF-β1, and IGF-1 with relative R^2 values of 0.9962372, 0.9948246,
0.9991222, respectively
• Bladder tissue samples from four SLC(+/+) and three SLC (-/-
mice aged 8 months, and four SLC(+/+) and four SLC (-/-) aged
5 months were collected and underwent RNA extraction.
• The quality of mRNA was tested after proper purification and
extraction by RT-PCR, gel electrophoresis, and spectrometry.
• A ratio of RNA to distilled water was made for each sample,
then a 10x RT buffer, MgCl2, dNTP,R-Hex, Olgio-T, Inhibitor and
Reverse primers were added. The samples then underwent first-
strand synthesis to be quantified and converted to cDNA.
• The quality of cDNA and primers were tested after cDNA
synthesis by RT-PCR and gel electrophoresis.
• Quantitative real time PCR was carried out and the expression
of mRNA of TGF- β1 and IGF-1 were compared relatively to the
control gene PPIA.
We used the PCR primers described in Kanno et al., but the primers
for IL-1β did not work. BLAST search indicated that there was an error
in the primer sequence. We corrected this error and made a new
primer. Unfortunately, this primer amplified IL-1β poorly so we then
designed a new primer set, but I did not have time to test this primer
set as part of this poster presentation. With the primers that did work
the qPCR results indicate there is no statistically significant difference
between the genotypes or age groups in expression of TGF-β1 or
IGF-1. The T-test reveals the samples’ expression of each gene
respectively are very close in value, yielding high p-values. The
standards were created and diluted properly, the curve is a straight
line, and the unknowns fall within the standard range. This indicates
that the cDNA used was at a normal concentration and did not need to
be diluted further. Sample 7743, a 5 month old knockout mouse,
showed outlier expression levels for IGF-1, levels significantly larger
than that of similar samples of the same age and genotype. After two
different extractions of RNA to make new cDNA, sample 7743 showed
much higher levels each time a qPCR was run. This may be because
another gene could possibly affect the expression of IGF-1. Further
research needs to be conducted to verify or rule out this possibility.
With more time, IL-1β could be tested to see if there is any significant
differences in expression between age and/or genotype. After
acquiring accurate data about mRNA expression, expression of these
genes at the protein level may be further investigated.
Thank you to Dr. Amrik Sahota, Min Yang, and Dr. Jay A.
Tischfield for their help throughout the course of the summer on
this project. Thank you to the Human Genetics Institute of New
Jersey and the Aresty Undergraduate Research Center for
providing me with the opportunity and funding to conduct and
present my research.
References
• Kanno, Y., Mitsui, T., Kitta, T., Moriya, K., Tsukiyama, T., Hatakeyama, S. and
Nonomura, K. (2015), The inflammatory cytokine IL-1β is involved in bladder remodeling
after bladder outlet obstruction in mice. Neurourol. Urodyn.. doi: 10.1002/nau.22721
• Sahota, A., Parihar, J.S., Capaccione, K.M.,Yang, M.,Noll, K., Gordon, D., Reimer,D.,
Yang,III., Buckley, B.T., Polunas,M., Reuhl, K.R., Lewis,M.R., Ward,M.D., Goldfarb,D.S.,
Tischfield,J.A. "Novel Cystine Ester Mimics For The Treatment Of Cystinuria-Induced
Urolithiasis In A Knockout Mouse Model."Urology 84.5 (2014): 1249.e9-1249.e15. ISSN
0090-4295.
Tissue
Samples
mRNA cDNA
Gene
Expression
Levels
RNA
Extraction and
Purification
First-
Strand
Synthesis
Quantitative
Polymerase Chain
Reaction (qPCR)
Molecular Pathophysiology of Bladder Outlet Obstruction
Amanda Gallagher, Min Yang, Jay A. Tischfield, and Amrik Sahota
Department of Genetics, Rutgers University, New Brunswick, New Jersey
Figure 4: A T-test of 5 month and 8 month knockout TGF-β1 and IFG-1 expression
Figure 1: The expression of TGF-β1 in age 5 month and 8 month mice relative to PPIA was calculated utilizing the average of each
sample’s recorded quantity of mRNA.
T-Test Analysis
Figure 2: The expression of IGF-1 in age 5 month and 8 month mice relative to PPIA was calculated utilizing the average of each
sample’s recorded quantity of mRNA.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
SLC(+/+) SLC(+/+) SLC(+/+) SLC(+/+) SLC(-/-) SLC(-/-) SLC(-/-)
2024 2025 2026 2027 7610 7611 7600
0.46
0.77
0.15
1.48
0.53
0.06
1.55
RatioofmRNAExpression
Sample and Genotype
TGF-Beta 1 Relative mRNA Expression in 8 Month Old Mice
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
SLC(+/+) SLC(+/+) SLC(+/+) SLC(+/+) SLC(-/-) SLC(-/-) SLC(-/-)
2024 2025 2026 2027 7610 7611 7600
0.65
0.45
1.41
0.52
1.21
0.92
1.66
RatioofmRNAExpression
Sample and Genotype
IGF- 1 Relative mRNA Expression in 8 Month Old Mice
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
SLC(+/+) SLC(+/+) SLC(+/+) SLC(+/+) SLC(-/-) SLC(-/-) SLC(-/-) SLC(-/-)
2028 2029 7749 7650 7740 7741 7743 7660
0.40
0.75
1.18
1.06 1.01
0.65
1.75
1.39
RatioofmRNAExpression
Sample and Genotype
TGF-Beta 1 Relative mRNA Expression in 5 Month Old Mice
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
SLC(+/+) SLC(+/+) SLC(+/+) SLC(+/+) SLC(-/-) SLC(-/-) SLC(-/-) SLC(-/-)
2028 2029 7749 7650 7740 7741 7743 7660
0.30 0.35
1.61
0.58
0.72
0.41
3.35
0.61
RatioofmRNAExpression
Samples and Genotype
IGF-1 Relative mRNA Expression in 5 Month Old Mice