Kidney Week

Abstract: FR-PO187

Adenosine Triphosphate Coincubation with Alkaline Phosphatase Reduces H2O2-Induced Endothelial Barrier Disruption

Session Information

• AKI: Mechanisms
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Steenvoorden, Thei Sybe, Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Thei Sybe Steenvoorden, MD, MSc

• Podieh, Fabienne, Amsterdam UMC Locatie VUmc, Amsterdam, Noord-Holland, Netherlands

Fabienne Podieh, MSc

• Tellez Garcia, Juan Miguel, Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Juan Miguel Tellez Garcia, MD

• Bemelman, Frederike J., Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Frederike J. Bemelman, MD, PhD

• Hordijk, Peter, Amsterdam UMC Locatie VUmc, Amsterdam, Noord-Holland, Netherlands

Peter Hordijk, PhD

• Vogt, Liffert, Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Liffert Vogt, MD, PhD

Background

Endothelial damage mediated by oxygen radicals is a key part of ischemia-reperfusion-induced acute kidney injury (IRI-AKI). Nucleotides e.g., adenosine triphosphate (ATP), are released from cells in response to IRI and can cause endothelial barrier disruption. ATP's base, adenosine (ADO), however, might improve barrier function. Alkaline phosphatase (ALP) can dephosphorylate ATP to form ADO. ALP treatment has been shown to protect mice against IRI-AKI. It is unknow if ALP can protect endothelium against IRI.

Methods

We exposed quiescent monolayers of human umbilical vein endothelial cells (HUVECs) to H₂O₂ (300µM and 400µM) and measured the trans-endothelial electrical resistance with Electric Cell-Substrate Impedance Sensing (ECIS). Confocal imaging was used to assess VE-cadherin. HUVECs were co-incubated with either ALP (10IU/ml bovine intestinal ALP), placebo, 100µM ATP, or 100µM ATP with ALP.

Results

Exposure of HUVECs to H₂O₂ led to strong reduction of elctrical resistance, indicating loss of barrier function. Co-treatment with 100 µM of ATP alleviated some of the induced damage. This effect was enhanced by the addition of ALP (Figure A). In the low H₂O₂ concentration, ALP addition resulted in partial rescue of endothelial barrier function (Figure B), however, there were large inter-experimental differences. Partial recovery of endothelial barrier function could be confirmed by immunofluorescent staining of VE-cadherin.

Conclusion

Co-incubation of ATP with ALP reduces H₂O₂ -induced barrier disruption. ALP may protect HUVECs against high levels of oxygen radicals, however, it is unlikely that HUVECs release enough substrate to guarantee stable protection during reperfusion. To determine if ADO generated from ATP dephosphorylation is responsible for the observed barrier protection, endothelial barrier function measurements will be repeated with ADO receptor inhibition.

Normalized resistance over endothelial monolayer. Red: untreated. Green: H2O2 only. Blue: AP treated with H2O2 with (A) or without (B) ATP. Gold: placebo and H2O2.

Funding

• Commercial Support – Health Holland B.V. and Alloksys Life Sciences B.V.