Deciphering the role of the blood-brain barrier in uremic toxins-induced neuropathies

Project Information

BBB-UT

Grant agreement ID: 101107439

DOI

10.3030/101107439

Project closed

EC signature date 14 March 2023

Start date 1 May 2023

End date 30 April 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 187 624,32

Coordinated by

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 1 - BBB-UT (Deciphering the role of the blood-brain barrier in uremic toxins-induced neuropathies)

Reporting period: 2023-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

Importance of the project: Chronic kidney disease (CKD) is a common and long-term pathology characterized by a progressive loss of kidney structure and function. Currently, CKD is underdiagnosed mainly because of a lack of symptoms until the advanced stages. Without effective patient care, the disease develops into End-Stage Kidney Disease (ESKD) when the kidneys are no longer able to carry out their cleansing function. Moreover, several CKD patients suffer from associated pathologies, including neurological disorders such as cognitive impairment or dementia. These CKD-related neuropathies worsen the quality of life of the patients and are still poorly understood. Over the past years, the substantial burden of CKD and the increased prevalence of ESKD have been highlighted. This trend is likely due to the ageing population, Western lifestyle evolution and the significant increase in related diseases such as diabetes and hypertension. In Europe, CKD affects nearly 100 million people, and it is projected to become the fifth leading cause of death globally by 2040. In addition, CKD is among the most expensive diseases for health care systems, with a cost estimated at EUR 140 billion annually in Europe. Face to these dismal projections, there is an urgent need to implement an EU action focused on CKD, including CKD-associated pathologies.

Research hypothesis, objectives and collaborative network: I hypothesize that the reduction in kidney tubular transporters’ functionality in CKD leads to (1) the systemic retention of protein-bound uremic toxins (PBUTs), (2) a disruption of the blood-brain barrier (BBB) integrity and (3) a reduction in the activity of BBB-endothelial cell (BBBec) membrane transporters. This phenomenon could play a part in the evolution of CKD-associated neurological pathologies (e.g. uremic encephalopathy and cognitive impairment) driven by the kidney-brain crosstalk impairment. This project aimed to develop an appropriate and innovative multi-organs-on-chip model to decipher underlying mechanisms of CKD-induced neurotoxicity. The project was achieved using a combined biofabrication and experimental approach, following research objectives:
(i) Development of an appropriate physiologically based BBB-on-chip
(ii) Characterization of the impact of PBUTs on BBB integrity and propriety
(iii) Elaboration of a multi-organ-on-chip system to recapitulate the kidney-brain crosstalk
(iv) Assessment of kidney tubular function on BBB uremic toxins-exposure and toxicity
This fellowship was carried out at the Division of Pharmacology of Utrecht Institute for Pharmaceutical Sciences (Utrecht University, NL) under the supervision of Prof. dr. Roos Masereeuw. This fellowship aimed at developing bioengineered, animal-free and advanced in vitro models to tackle a major healthcare problem through a collaborative and multi-scale approach (from polymer to human) including biofabrication and 3D printing and in vitro barrier function modelling.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the past years, I have focused on developing a robust organ-on-chip system supporting the BBB structure and function, and transposable to other organs. The model was adapted from an in-house system, designed with Autodesk Fusion 360 software, and printed using a resin printer. It supports a rocker-based perfusion as well as a vasculature-like scaffold named hollow fiber membrane (HFM). The presence of this semi-permeable tube-like membrane allows proper perfusion while considering the influence of curvature on cell physiology. I optimised the cell culture protocol-on-chip for endothelial cells and pericytes, as well as the hydrogel composition for 3D astrocyte culture. Building on this stand-alone system, I developed a multi-organ-on-chip platform that integrates multiple organs in series, with flow driven by a pump-based system. It is worth mentioning that we aimed to develop a cost-effective and easy-to-use system, potentially transferable to industrial applications.
In the meantime, I exposed the cells forming the BBB (i.e. endothelial cells, pericytes and astrocytes) in isolation to a PBUTs cocktail at different concentrations. I measured the cytokine release as an indicator of inflammatory processes and the protein expression of key membrane transporters involved in the brain-to-blood and blood-to-brain exchanges. Results showed a time- and concentration-dependent differential impact of the PBUTs on the different cell types that needs to be confirmed. Results obtained alongside the projects were presented at conferences and published in open-access international peer-reviewed journals. It is worth mentioning that the results obtained during the BBB-UT project have paved the way for future research. In the near future, I aim to explore the influence of the uremic environment on stroke severity and recovery in patients with chronic kidney disease. For this, my pre-application for a junior Kolff talent grant at the Dutch Kidney Foundation was selected for a full proposal, which is due 17 August 2025.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Currently, the understanding of CKD-induced neurological disorders is still challenging at both experimental and clinical levels, mainly due to the systemic scale and multifactorial parameters associated with these pathologies. Few studies have focused on the PBUTs-BBB relationship, and no barrier functional study has been conducted in this respect. The BBB-UT project provides new insights into differential effects of the PBUTs on the BBB and led to the development of an innovative advanced in vitro model encompassing a 3D perfusable scaffold and support rocker-based and pump-driven perfusion as well as multi-organ connection. I identified different time- and concentration-dependent effects of a PBUTs cocktail (combining nine PBUTs) on the cell types forming the BBB in isolation. PBUTs seem to promote cytokine release by the pericytes and astrocytes, while leading to a decrease in protein expression of the NaK-ATPase pump and the transporter GLUT1 in endothelial cells. Those findings show a direct effect of the toxins on BBB cell components that might contribute to further pathological mechanisms. Disruption of transporter function may disturb cellular and tissue homeostasis and alter local drug pharmacokinetics. Additionally, cytokine production by the BBB could trigger neuroinflammation, promoting barrier breakdown and subsequent PBUTs leakage. Ongoing experiments aim to validate these results and further explore the mechanisms involved, with particular emphasis on bioenergetic profiles and a broader analysis of transporter expression. In addition, we developed a bioengineered, 3D perfusable human brain microvascular model based on hollow fiber membranes (HFMs), incorporating endothelial cells and pericytes within a custom-designed, biocompatible, and cost-effective resin-printed chip. Efforts are made on astrocytes incorporation in our system to emulate tri-culture mixing and the BBB physiological structure. The adaptability and connection-based nature of the system support the study of dual-organ interaction and eventual addition of other organs in series. Further research is needed to facilitate the transition towards industrial applications.

Graphical abstract - BBB-UT project