Osmotic stress regulates mineralocorticoid receptor expression in a novel aldosterone-sensitive cortical collecting duct cell line.

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Submitted on 23 Oct 2009

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expression in a novel aldosterone-sensitive cortical

collecting duct cell line.

Say Viengchareun, Peter Kamenicky, Marie Teixeira, Daniel Butlen, Géri

Meduri, Nicolas Blanchard-Gutton, Christine Kurschat, Aurelie Lanel,

Laetitia Martinerie, Soshana Sztal-Mazer, et al.

To cite this version:

Say Viengchareun, Peter Kamenicky, Marie Teixeira, Daniel Butlen, Géri Meduri, et al.. Osmotic

stress regulates mineralocorticoid receptor expression in a novel aldosterone-sensitive cortical collecting

duct cell line.. Mol Endocrinol, 2009, 23 (12), pp.1948-62. �10.1210/me.2009-0095�. �inserm-00413313�

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Figure 1

0.01 0.02 0.03 0.04 0.05 Bound/Unbound 0.4 0.6 Bound Aldosterone (nM) N = 30 fmol/mg of protein Kd = 1.30 +/- 0.33 nM 500 1000 1500 2000 2500

Total Aldo Spiro

0

*******

Dpm

C

D

E

F

G

H

Fractions [A] [B] 0 100 200 300 400 0 1 2 3 4 Hours + NAD + NADP Control dpm (X10 -2) dpm (X10 -2) 1 4 7 10 13 16 140 100 60 20 0 + NAD, 15 min 0.2

KC

3AC

1 M c

ells

R c

ells

WB: MR anti-MR 39N MR 150 kDa 100 kDa 0 α-Tubulin 50 kDa

A

B

-3 β = 1.1 x 10 +/- 0.06 x 10 -3

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Figure 2

A

B

MW +

-

+

-αENaC βENaC γENaC αENaC βENaC γENaC 0 5 10 15 20 25 30 0 10 20 30 40 50 60 Minutes 10-5 M Amiloride Isc ( μA/cm 2 ) 0 20 60 100 140 180 Control + Aldo

**

120 140 160 180

*

NS

*******

Na+

mmol/l 2 4 6 8 10

*

NS

*******

mmol/l 0

K+

0 20 40 60 80

**

Relative expression (Sgk1/ 18S) Sgk1 0 50 100 150 Relative expression (α ENaC/ 18S) αENaC KC3A WT Ki _KC3AC1 WT Ki dn

C

D

E

F

0

* ***

Isc (% of control) A B A B A B

Control + Aldo + Aldo + Spiro Control A B + Aldo + Aldo + Spiro A B A B

0 0.5 1 2 4 6 24 Hours

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Figure 3

C

A

B

MR α

-

Tubulin C Actino 0.64 0.35 100 56 % of control Ratio 0 3 6 9 24 Cycloheximide (hours) MR α - Tubulin % of control Ratio Hours

MR

Relative MR expression (MR/18S) 100 83 91 60 41 0.74 0.62 0.67 0.44 0.31 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 9 + Actino 150 kDa 100 kDa 50 kDa 150 kDa 100 kDa 50 kDa

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Figure 4

Hours

G

H

F

0 1 2 4 6 Relative MR expression (MR/18S) MR Iso Hyper % of control 100 31 73 28 0.84 0.31 0.61 0.24 Ratio α - Tubulin Cyclo Hyper + Cyclo Relative MR expression (MR/18S) MR

Iso Hyper Cyclo Hyper + Cyclo 0 20 40 60 80 100 MR

*

*******

0 20 60 100 140 180 + Raffinose 150 kDa 100 kDa 50 kDa

D

E

*******

Hyper Iso Control Raffinose 0.2 M Relative T onEBP expression (T onEBP/18S) TonEBP α-Tubulin 180 kDa 50 kDa Hyper Iso

+

-

Raffinose Hyper Iso Relative

AldoR expression (AldoR/18S)

Aldose Reductase TonEBP mRNA Control Raffinose 0.2 M 0 0.5 1.5 2.5 3.5 4.5 0 2 4 6 8 10 12 14

_***

TonEBP Protein

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Figure 5

5' 3'

A

Tis11b gene ATG 2 1

D

Tis11d

β- actin

Tis11b

B

Iso Hyper

C

3' 9 TGA hMR gene 0 1 2 3 4 5 0 1 2 4 6 Hours Relative T onEBP expression (T onEBP/18S) Relative T is1 1b expression (T is1 1b/18S)

**

*******

0 1 2 3 4 5 6 42 kDa 45 kDa 60 kDa

TGGAAANNYNY

TA TGGAAATCGGA

Human

TA TB TC

TB TGGAAAGTAGT

TC TGGAAAAAAAA

TD TGGAAACCGCG

TonE consensus

Mouse

TB TGGAAAGTGAC

Rat

TB CGGAAAGTGAC

TD

TC TGGGAAACAAC

TD TGGAAACCGCA

5' 8 A1 A2 A3 AURE consensus TonEBP + Raffinose A4 A5 A6

UU[AUUUA]A/UA/U

A1 UUAUUUAAU

Human

A2 AUUUA

A3 AUUUA

A4 AUUUA

A5 AUUUAAUUUA

A6 AUUUA

Tis11b + Raffinose

E

0 1 2 4 6 Hours 0 2 4 6 8 Relative T is1 1b expression (T is1 1b/18S)

*******

_**

Iso Hyper Actino Hyper + Actino

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Figure 6

*******

Hypo Iso Relative MR expression (MR/18S) MR Hypo Iso 0 1 2 3 4 5 6 7

E

1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 Hours Relative MR expression (MR/18S)

D

MR Hypo Hypo + Actino MR α - Tubulin % of control Ratio 0.86 153 0.84 148 0.56 0.29 100 52 Actino Hypo + Actino 150 kDa 100 kDa 50 kDa

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Figure 7

MR

α

- Tubulin

150 kDa 100 kDa 50 kDa Control Deprivation Water Intoxication Water

0 0.5 1.0 1.5 2.0 2.5 3.0 Relative MR expression (MR/ α -T ubulin) Control Water

Deprivation Intoxication Water