Review of 'The anti-coagulants ASIS or APC do not protect against renal ischemia/ reperfusion injury'

The paper by Loubele and coworkers is a logical extension of published work on the effect of anticoagulant drugs on ischemia reperfusion injury. In a well characterized ischemia reperfusion model in mice, in which one kidney is clamped and subsequently reperfused while the contralateral kidney is removed, the authors show no effect of the anticoagulants ASIS and APC on ischemia reperfusion injury. Importantly, the dosages applied were previously shown to be effective in different ischemia reperfusion models. The analyses of ischemia reperfusion injury were thorough: kidney function markers, histology, inflammatory and hemostasis markers (protein and/or mRNA) and apoptosis were not appreciably different between vehicle and ASIS or APC treated animals. The study has been well executed and I agree with the main conclusions. I have a few minor comments aimed at further improvement of the manuscript. First, an analysis of kidney injury markers and stainings for inflammatory cells would have made the study complete. Second, the lack of effect of the anticoagulants (in particular of ASIS) on fibrin deposition in the kidney is unexpected given the observations that the drugs result is systemic anticoagulant activity, and the increased in kidney tissue factor after ischemia reperfusion. Are there indications that the fibrin deposition in the kidney originates from intrinsic (rather than TF-mediated) activation of coagulation? Third, the conclusion that the amount of tissue factor decreases after 2 hours of reperfusion seems unjustified. As the authors employ an activation assay, it is no surprise that TF activity in ASIS treated animals is decreased as the TF in the cell homogenate is likely (partially) occupied by ASIS. Forth, the remark in the materials and methods section ‘for ASIS the optimal dose was determined by a dose-finding study’ is unclear. What was the endpoint of such a dose-finding study? Finally, the argument that anticoagulants did have effects in previous studies perhaps because in those studies both kidneys were clamped is unclear. Why would this difference in experimental set-up explain differences between the published and present study?