Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to
complications of diabetic kidney disease (DKD). TPr blockade appears to have
therapeutic potential. Several rodent models of DKD show attenuation of renal damage
and proteinuria upon administration of the TPr antagonist, S18886. However, the
cellular targets that underlie the injurious effects of TPr activation in DKD remain to be
elucidated.
A pilot study in our laboratory subjected a conditionally-immortalized mouse
podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical
stretch (an in vitro simulator of increased glomerular capillary pressure associated with
glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of
podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded
a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM
L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are
increased in podocytes during the onset of DKD resulting in maladaptive effects on this
key glomerular filtration barrier cell type.
We showed that enhanced TPr signaling threatens podocytes viablility. Cultured
podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more
vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) ,
TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001,
n=3). To further support these in vitro findings, we developed a transgenic mouse with
podocyte-specific overexpression of TPr. A construct consisting of a desensitization
resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into
pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR.
Our in vitro results suggest that increased TPr expression in podocytes of
diabetic mice may contribute to filtration barrier damage and have important implications
in the development and progression of DKD.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOU./en#10393/19759 |
| Date | 08 February 2011 |
| Creators | Shaji, Roya |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | thesis |
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