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Bertrand Gondouin (Author)Queue SummaryReviewer Area

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BLOOD/2011/394726

Role of indolic uremic solutes on tissue factor production via aryl hydrocarbon receptor pathway

Bertrand Gondouin, Claire Cerini, Laetitia Dou, Ariane Duval-Sabatier, Anneleen Pletinck, Raymond Calaf, Noemie Jourde,

Stephane Poitevin, Laurence Camoin-Jau, Laurent Arnaud, Raymond Vanholder, Philippe Brunet, Francoise Dignat-

George, and Stephane Burtey

Decision: Reject; Decision Date: 31 Dec 2011

Date Received: 9 Dec 2011

Editor: David Lillicrap

Article Type: Regular Article

Secondary Scientific Category: Thrombosis and Hemostasis

Primary Scientific Category: Vascular BiologyCorresponding Author: Stephane Burtey

Keywords: COAGULATION, Biochemistry and structural biology of hemostatic proteins, Coagulation co-factors;

VASCULAR BIOLOGY; VASCULAR BIOLOGY, Endothelial cells; Indole acetic acid; Indoxyl sulfate; Tissue factor; aryl

hydrocarbon receptor; chronic kidney disease

Supplemental Files: 0

Reviewer 1 Comments for the Author



Reviewer 1 Comments for the Author...

The manuscript describes a correlation between plasma TF levels and levels of

the uremic solutes IS and IAA in patients with chronic kidney disease and shows

the upregulation of cell surface TF in endothelial cells and PBMC in response

to IS and IAA. The authors also describe a new signalling mechanism leading to

TF expression involving activation of the aryl hydrocarbon receptor. However,there are some points that need addressing:

Major points:

1. The maximum concentrations of IS (1 mM or 250 !g/ml) and IAA (50 M or 9

!g/ml) used for the in vitro experiments are above the physiological

concentrations found in the serum of CKD patients as shown in Figure 1. In

fact, the highest concentration of IS found in vivo was


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these concentrations of uremic solutes affect cell viability?

2. Figure 6G shows TF activity of cells transfected with siRNA and treated with

IAA. Was TF mRNA and protein expression also reduced in these cells following

treatment with IAA? Figures 6B and 6C show the levels of AHR expression 48 h

after transfection of cells with the control and AHR siRNA. However, in the

methods section it is stated that the effects on TF expression were carried out

72 h after transfection. AHR mRNA and protein levels should therefore also be

shown at 72 h post-transfection with siRNA. Figure 7 shows only TF mRNA and TF

activity. Was TF protein expression also reduced following treatment of cells

with TCDD?

3. Figure 5 shows that the NFkB inhibitor (10 !M) wedelolactone only

partially inhibited TF expression in response to IS and IAA in HUVEC. Was this

concentration of wedelolactone shown to completely inhibit NFkB in these cells?

A dose-response curve is needed to prove that complete inhibition of NFkB only

partially blocks TF upregulation by IS and IAA.

4. In the introduction it is stated that uremic solutes have been shown to

induce the release of microparticles from endothelial cells. The effect of

uremic solutes on the release of TF-positive microparticles from endothelial

cells and PBMC would strengthen this study, and possibly explain increased

plasma TF levels in CKD patients.

5. The authors identify the AHR signalling pathway as a modulator of TF

expression in response to IS and IAA. However, since there are no known AHR

response elements in the promoter of the TF gene, how do the authors propose

that AHR activation upregulates the expression of TF?

6. Does sTF refer to full-length TF associated with microparticles, or

alternatively spliced TF in the plasma? Did sTF in plasma

have procoagulant activity?

Minor points:

1. Error in the x-axis of Figure 2A and in the y-axis of Figure 2F.

2. In the abstract the authors have stated that TF production is increased in

endothelial and PBMC via AHR activation. However, they have not shown the

involvement of AHR signalling in PBMC.


This study analyzes the regulation of tissue factor (TF) by indole uremic solutes.

Major comments

1/ A commercial ELISA is used to measure soluble TF levels in plasma.

Significant concerns have been raised about the specificity of this ELISA.

Therefore, other measures of circulating TF should be performed to confirm the

observed changes.

2/ There is considerable variation in the basal levels of TF protein (25-125


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pg/ml) and TF activity (10-50 ng/ml) and fold changes in the different

experiments. This is important because some of the changes are quite modest and

would be lost if there is a high basal expression. The SD in some experiments is

also very large (i.e. Figure 2E).

3/ The in vitro studies have focused on ECs but PBMCs generally express higher

levels of TF. More studies should be performed with PBMCs.

4/ The western blot shown in Figure 2C is not convincing because TF is

glycosylated and runs as a smear rather than a distinct band. How many

experiments were performed?

5/ More than one AhR siRNA should be used. Where is the control without siRNA?

6/ Do IS and IAA activate NF-KB?

7/ It is unclear why the AHR inhibitor abolishes TF induction whereas the NF-KB

only give partial inhibition. Why does the AHR inhibitor reduce basal TF

expression? Has toxicity been examined?

8/ Has LPS contamination been excluded?

9/ Has an AHR site been identified in the TF promoter?

10/ Please remove for the first time from the manuscript.

Minor comments

1/ Y axis of Figure 2F.

2/ Why is the control in Figure 4A not 1?

3/ Wededlolactone is not a typical NF-KB inhibitor. Provide a reference. Other

inhibitors should be used.

4/ Y axis of Figure 6 B should be decrease.

5/ Error bars need to be added to the controls in Figure 7A.

6/ When are the samples collected relative to dialysis?

7/ HUVECs are prepared not extracted.


Cardiovascular mortality is substantially increased in patients with chronic

kidney disease (CKD), however, the underlying mechanisms are still poorly

understood. Here, Gondouin and colleagues investigated in 73 hemodialysis (HD)

patients and 50 non-dialized CKD patients whether indolic uremic solutes induce

Tissue Factor (TF) production and thereby shifting the coagulation cascade in

CKD patients towards a pro-trombotic state. Since elevated levels of soluble


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Tissue Factor sTF is in e to increase morta ity in patients wit coronary

heart disease, an elevated production of sTF under uremic conditions may also

add to the increased cardiovascular mortality in CKD patients.

The authors convincingly demonstrated that sTF is elevated in HD and undialyzed

CKD patients compared to controls and that sTF is correlated with indoxyl

sulfate (IS) in CKD patients and indole-3-acetic acid (IAA) in HD patients. Both

indolic uremic solutes can induce TF expression and subsequent procoagulant

activity in endotheial vells under in vitro conditions. The expression of TF

through both indolic uremic solutes can be reduced but not completely inhibited

by wedelolactone, an inhibitor of the NF-kB signalling pathway. Microarray

experiments using HUVEC incubated with IS revealed an up-regulation of genes

regulated by the transcription factor aryl hydrocarbon receptor (AHR), a major

mediator of organism response to xenobiotics. Here the authors demonstrate for

the first time that indolic uremic solutes modulate TF production via the AHR

pathway.

Specific comments:

1) What genes are up- regulated after incubation of HUVECS with IAA for 4 hours?

How many of these genes match with the ones seen in the IS experiments.

2) The authors have nicely shown the effect of AHR on IS mediated TF production.But what role plays AHR on the IAA mediated TF production. The authors should

also measure the effect of AHR silencing on TF production.

3) Since PBMNC are easily available form HD and CKD patients, the authors should

repeat their AHR silencing experiments on TF expression and activity induced by

IS and IAA in PBMNCs.


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