演題分類番 4 腎臓 3d 血管内皮 Key Words 314 endothelial cell 375 glomerulus 523 mechanoreceptor Title Cyclic stretch on glomerular endothelial cells, a new in vitro model for glomerular hypertension 糸球体高血圧の新しいin vitroモデルとしての糸球体内皮細胞に対する周期的伸展刺激の解析

1. Summary
Method
Cyclic stretch on glomerular endothelial cells, a new in vitro model for
glomerular hypertension
Yasuyuki Irie, Yukiko Kondo, Eiichi Taira
Department of Pharmacology, Iwate Medical School, Morioka 020-8505, Iwate, Japan
The prevalence of obesity and metabolic syndrome
(MetS) has increased over the past several decades and is
expected to continue to increase. Observational studies
have reported an association between metabolic
syndrome (MetS) and microalbuminuria or proteinuria
and chronic kidney disease (CKD). However, at this point,
the precise mechanism of this association remains to be
elucidated. Because this microalbuminuria occurs as a
result of the glomerular hypertension, we aimed to
analyze the molecular mechanism of glomerular
hypertension. In the present study, we developed an in
vitro model for glomerular hypertension, making use of
mechanical stress on cultured glomerular endothelial
cells. Using microarray analysis, we examined the effect
of cyclic stretch on GEN-T cells, which is derived from
glomerular endothelial cells. We found that several genes
were induced by the cyclic stretch and confirmed by
quantitative RT-PCR. Time course analysis as well as signal
transduction analysis using specific inhibitors was
performed.
When Ang II is increased, greater AT1 receptor-mediated constriction of efferent
than afferent arterioles increases single nephron glomerular filtration rate and
raises intraglomerular pressure, causing glomerular hypertension. Sustained or
severe increases in intraglomerular pressure can lead to glomerular basement
membrane damage, glomerular endothelial dysfunction, and ultimately,
extravasation of protein into Bowman’s capsule. In addition to hypertension,
conditions like diabetes that are associated with increased oxidative stress
(increased formation of reactive oxygen species) independent of hypertension
and glyco-oxidative modification of proteins (advanced glycation endproducts or
AGEs) comprising the glomerular basement membrane can lead to extravasation
of protein
http://www.hypertensiononline.org
It is now well-established in experimental models in rodents that increased
glomerular pressure results in the development of focal and segmental
glomerulosclerosis, proteinuria and progressive renal functional
deterioration. In humans, direct measurement of glomerular capillary
pressure is impossible. However, it is widely accepted that glomerular
hypertension is present in different clinical situations, like diabetic
nephropathy, chronic renal failure associated with glomerulonephritis, some
forms of essential hypertension and cadaveric kidney transplantation. Many
studies were performed on the effects of protein-restricted diet and/or
angiotensin converting enzyme inhibition on the rate of progression of renal
failure in these renal diseases. Although controversial, the overall results
suggest that these therapeutic strategies may reduce the rate of progression,
particularly in diabetic nephropathy.
1. We used GEN-T cells derived from the bovine glomerular endothelial cell.
2. We used a stretch culture device , model : ST-140 ( STREX ), for cyclic stretch. The cells were cultured under cyclic stretch with 106%
elongation and 1Hz cycle for 48 hours. We coated the stretch chamber with collagen followed by gelatin treatment.
3. The transcriptome of stretched and non stretched cells were analyzed by micro array.
4. In order to clarify the exact mechanism for the cell to detect the stretch stimulation, selectin P (SELP), mRNA [NM_174183] was examined
for relative quantity with or without inhibitors of cellular processes by real-time RT-PCR.
Method
Background
Stretch system : model SBT-140 ( STREX )
Micro array analysis for stretch-induced change of transcriptome in GEN-T cells
Relative quantitation of P-Selectin (SELP) mRNA expression in GEN-T cells; S: stretched, NS: non-stretched, 0; without inhibitors
Relative amount of transcripts was shown as folds of that from non-stretched and vehicle treated GEN-T cells.
Ca2+ blockers
M: 10uM miberandil, V:1uM verapamil
TRP channel blocker
G: 100uM Gd3+, L: 100uM La3+
purinoceptor antagonist
and ERK inhibitor
D: 10uM PD98059, P: 50uM PPADS
Conclusion
1. We developed a novel in vitro model for glomerular endothelial dysfunction in the glomerular
hypertension making use of mechanical cycle stretch.
2. After 48 hours, the expression of selectin P (SELP) was induced by cycle stretch. The magnitude of
induction was 3-14 folds compared to non-stretched control.
3. PKC pathway, T-type calcium channels, P2 purinoceptor were shown to be involved in the induction of
SELP expression in GEN-T cells by cycle stretch stimulation.
4. In parallel with the SELP mRNA induction, the adhesion of thrombocyte to GEN-T cells increased by cycle
stretch stimulation of GEN-T cells.
Because no in vitro models has been established for the glomerular endothelial
dysfunction so far and because there might be an important mechanisms for CKD
to develop systemic endothelial dysfunction in this process, here we present a
novel model for glomerular hypertension. This model is composed by a
combination of glomerular endothelial cell line and mechanical cycle stretch,
which mimics the expanded glomerular vessel in glomerular hypertension.
The aim of research
Fe a t u re Nu
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De s c rip t io n
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Se t 01 _PVa l
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Se t 01 _g Pro
Sig n a l
Se t 01 _g Sig
Ev a l
Se t 01 _rPro
Sig n a l
Se t 01 _rSig
Ev a l
KEGG Pa t h wa y
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 2.1 6 0.000 31.2 2 138.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .92 0.000 37.3 2 140.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .82 0.000 24.0 2 84.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .81 0.000 30.3 2 106.7 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .74 0.000 22.6 2 75.2 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .72 0.000 37.6 2 123.3 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .52 0.000 12.2 2 35.0 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .42 0.000 11.5 2 30.8 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .40 0.006 16.7 2 44.2 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .33 0.000 11.7 2 29.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .33 0.000 14.3 2 35.8 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .29 0.000 36.5 2 89.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .24 0.000 23.5 2 55.5 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .24 0.000 78.3 2 184.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .21 0.000 40.4 2 93.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 9 0.000 104.4 2 239.0 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 9 0.000 11.9 2 27.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 6 0.000 44.4 2 99.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 4 0.000 23.3 2 51.5 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 4 0.000 247.6 2 545.7 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 4 0.000 1924.9 2 4235.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 4 0.000 1852.1 2 4070.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 2 0.000 17.6 2 38.2 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .1 1 0.000 45.7 2 98.3 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
42687 Bos taurus s e le c tin P (SELP), mRNA [NM_174183] 1 .08 0.000 54.8 2 116.0 2 Ce ll adhe s ion mole c ule s (CAMs )
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .08 0.000 1412.0 2 2980.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .07 0.000 122.1 2 255.6 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .06 0.000 16.2 2 33.8 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .06 0.000 1467.3 2 3057.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .06 0.001 9.9 2 20.6 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .05 0.000 125.7 2 259.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .05 0.000 13.1 2 27.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .05 0.000 14.1 2 29.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .05 0.001 11.3 2 23.4 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .04 0.001 12.8 2 26.2 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .03 0.000 1659.4 2 3399.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .03 0.000 1543.3 2 3161.5 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .03 0.000 27.2 2 55.6 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .03 0.000 144.6 2 294.6 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .02 0.002 13.2 2 26.9 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .02 0.000 405.8 2 822.3 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .02 0.000 48.0 2 97.3 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .02 0.000 148.7 2 301.0 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .01 0.000 253.0 2 509.2 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .01 0.000 104.0 2 209.1 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
##### ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃ 1 .00 0.000 62.6 2 125.5 2 ＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃＃
Protein kinase blockers
H: 10uM H89, V:1uM Calphostin C
Thrombocyte adhesion to GEN-T cells
Freshly prepared concentrated platelet fruction was stained with DiI fluorescent dye and incubated with non-stretched or stretched GEN-T
cells. After substantial washing, the number of adhesive platelets were counted.
Typical visual fields of microscopy and the quantified results from 10 chambers of each treatment were shown.
Non-stretch Stretch