Three-dimensional cell models for renal replacement therapy and drug screening

The kidneys are organs with a central role in homeostasis. Their principal function is the excretion of the organic and inorganic wastes of metabolism and the reabsorption of valuable nutrients. These functions are mediated by the proximal tubule epithelial cells (PTEC) containing segment, via the concerted action of different transporters (e.g. organic anion transporter 1, OAT1). Aging, diabetes and hypertension lead to kidney malfunction. This can progress to chronic kidney disease (CKD), in which the kidneys no longer function well enough to meet the daily needs and, as a consequence, uremic toxins (UTs) accumulate in the patients’ blood. The first aim of this thesis was to develop and characterize the functionality of an in vitro bioartificial kidney device (BAK) for the removal of uremic toxins. We developed the bioartificial kidney tubule as an in vitro platform that was validated by studying the protein-bound UTs, indoxyl sulfate (IS) and kynurenic acid (KA), removal. This novel tool, named bioengineered kidney tubules, is the combination of polyethersulfone hollow fiber membranes (PES-HFM) with conditionally immortalized proximal tubules epithelial cells (ciPTEC) overexpressing OAT1. Additionally, we characterized the effect of HSA on IS transepithelial transport using the bioengineered tubules, with attention given to the modified form of HSA as present in CKD patients. Also, we characterized the transport kinetics of IS in free and bound fractions to either healthy- or CKD-HSA. CKD-HSA, with its low binding and high affinity for IS, promoted a lower turnover of the free IS fraction, accompanied with less IS secretion. Furthermore, bioengineered kidney tubules demonstrated active secretion of IS from plasma of healthy donors and of CKD patients, demonstrating that, despite unfavorable binding kinetics, plasma can still be cleared from UTs in patients. We describe the first BAK upscaling attempt. In this respect, a three-PES-HFM bioreactor with a total surface area of 4 cm2 was built and characterized. Subsequently, ciPTEC were cultured in the upscaled devices and organic cation transporter 2 (OCT2) function was demonstrated. The second aim of this thesis was to use natural scaffolds instead of synthetic ones for the development of a new 3D kidney model. Decellularized organs constitute a promising field within regenerative medicine. These newly generated scaffolds can provide an alternative to synthetic ones, as the native extracellular matrix expected to guide tissue development in a physiologically relevant manner. Recellularizing these native scaffolds with human cell sources can better recapitulate the in vivo situation. The novel platform was applied in nephrotoxicity testing by culturing ciPTEC-OAT1 on decellularized native kidney scaffolds from rats and compared to standard 2D ciPTEC-OAT1 cultures. To validate the platform, three different nephrotoxicants were studied: cisplatin, tenofovir and cyclosporin A. The 3D model demonstrated increased sensitivity to cisplatin and tenofovir toxicity, related to improved physiology of the platform. Altogether, this work shows two ways of developing 3D renal cell models to study kidney physiology, pharmacology and pathology, and providing novel solutions within the regenerative nephrology field

Three-dimensional cell models for renal replacement therapy and drug screening

The kidneys are organs with a central role in homeostasis. Their principal function is the excretion of the organic and inorganic wastes of metabolism and the reabsorption of valuable nutrients. These functions are mediated by the proximal tubule epithelial cells (PTEC) containing segment, via the concerted action of different transporters (e.g. organic anion transporter 1, OAT1). Aging, diabetes and hypertension lead to kidney malfunction. This can progress to chronic kidney disease (CKD), in which the kidneys no longer function well enough to meet the daily needs and, as a consequence, uremic toxins (UTs) accumulate in the patients’ blood. The first aim of this thesis was to develop and characterize the functionality of an in vitro bioartificial kidney device (BAK) for the removal of uremic toxins. We developed the bioartificial kidney tubule as an in vitro platform that was validated by studying the protein-bound UTs, indoxyl sulfate (IS) and kynurenic acid (KA), removal. This novel tool, named bioengineered kidney tubules, is the combination of polyethersulfone hollow fiber membranes (PES-HFM) with conditionally immortalized proximal tubules epithelial cells (ciPTEC) overexpressing OAT1. Additionally, we characterized the effect of HSA on IS transepithelial transport using the bioengineered tubules, with attention given to the modified form of HSA as present in CKD patients. Also, we characterized the transport kinetics of IS in free and bound fractions to either healthy- or CKD-HSA. CKD-HSA, with its low binding and high affinity for IS, promoted a lower turnover of the free IS fraction, accompanied with less IS secretion. Furthermore, bioengineered kidney tubules demonstrated active secretion of IS from plasma of healthy donors and of CKD patients, demonstrating that, despite unfavorable binding kinetics, plasma can still be cleared from UTs in patients. We describe the first BAK upscaling attempt. In this respect, a three-PES-HFM bioreactor with a total surface area of 4 cm2 was built and characterized. Subsequently, ciPTEC were cultured in the upscaled devices and organic cation transporter 2 (OCT2) function was demonstrated. The second aim of this thesis was to use natural scaffolds instead of synthetic ones for the development of a new 3D kidney model. Decellularized organs constitute a promising field within regenerative medicine. These newly generated scaffolds can provide an alternative to synthetic ones, as the native extracellular matrix expected to guide tissue development in a physiologically relevant manner. Recellularizing these native scaffolds with human cell sources can better recapitulate the in vivo situation. The novel platform was applied in nephrotoxicity testing by culturing ciPTEC-OAT1 on decellularized native kidney scaffolds from rats and compared to standard 2D ciPTEC-OAT1 cultures. To validate the platform, three different nephrotoxicants were studied: cisplatin, tenofovir and cyclosporin A. The 3D model demonstrated increased sensitivity to cisplatin and tenofovir toxicity, related to improved physiology of the platform. Altogether, this work shows two ways of developing 3D renal cell models to study kidney physiology, pharmacology and pathology, and providing novel solutions within the regenerative nephrology field

Hypertensive heart disease and obesity: a complex interaction between hemodynamic and non hemodynamic factors.

The worldwide prevalence of obesity has nearly doubled, with an increase in obesity-related cardiovascular disease and mortality. Several factors are involved in the genesis of hypertension and hypertensive heart disease (HHD) in overweight/obesity. This review is focused on bridging factors between excessive adiposity and HHD, presenting a unifying hypothesis of vascular-metabolic syndrome, where an "handicap" of the natriuretic peptide system has a central role both in adipocyte dysmetabolism as well as in increased blood pressure and HHD

Upscaling of a living membrane for bioartificial kidney device

The limited removal of metabolic waste products in dialyzed kidney patients leads to high morbidity and mortality. One powerful solution for a more complete removal of those metabolites might be offered by a bioartificial kidney device (BAK), which contains a hybrid "living membrane" with functional proximal tubule epithelial cells (PTEC). These cells are supported by an artificial functionalized hollow fiber membrane (HFM) and are able to actively remove the waste products. In our earlier studies, conditionally immortalized human PTEC (ciPTEC) showed to express functional organic cationic transporter 2 (OCT2) when seeded on small size flat or hollow fiber polyethersulfone (PES) membranes. Here, an upscaled "living membrane" is presented. We developed and assessed the functionality of modules containing three commercially available MicroPES HFM supporting ciPTEC. The HFM were optimally coated with L-Dopa and collagen IV to support a uniform and tight monolayer formation of matured ciPTEC under static culturing conditions. Both abundant expression of zonula occludens-1 (ZO-1) protein and limited diffusion of FITC-inulin confirm a clear barrier function of the monolayer. Furthermore, the uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+), a fluorescent OCT2 substrate, was studied in absence and presence of known OCT inhibitors, such as cimetidine and a cationic uremic solutes mixture. The ASP+ uptake by the living upscaled membrane was decreased by 60% in the presence of either inhibitor, proving the active function of OCT2. In conclusion, this study presents a successful upscaling of a living membrane with active organic cation transport as a support for BAK device

Biotechnological challenges of bioartificial kidney engineering

Contains fulltext : 137070.pdf (publisher's version ) (Closed access)With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed

Associations Between Body Mass Index, Ambulatory Blood Pressure Findings, and Changes in Cardiac Structure: Relevance of Pulse and Nighttime Pressures

Ambulatory blood pressure monitoring (ABPM) is central in the management of hypertension. Factors related to BP, such as body mass index (BMI), may differently affect particular aspects of 24-hour ABPM profiles. However, the relevance of BMI, the most used index of adiposity, has been underappreciated in the determination of specific aspects of 24-hour ABPM profiles in hypertension. The authors evaluated the association between BMI and aspects of ABPM together with their associations with cardiac remodeling in 1841 patients. A positive association of BMI with 24-hour, daytime, and nighttime pulse pressure in untreated normal weight and overweight/obese hypertensive patients and a positive association of BMI with nocturnal BP parameters in treated overweight/obese hypertensive patients was observed. The clinical relevance of these findings was supported by the positive significant correlations of BMI-related BPs with left ventricular mass and atrial diameter

A bioartificial kidney device with polarized secretion of immune modulators

The accumulation of protein-bound toxins in dialyzed patients is strongly associated with their high morbidity and mortality. The bioartificial kidney device (BAK), containing proximal tubule epithelial cells (PTEC) seeded on functionalized synthetic hollow fiber membranes (HFM), may be a powerful solution for the active removal of those metabolites. In an earlier study, we developed an upscaled BAK containing conditionally immortalized human PTEC (ciPTEC) with functional organic cationic transporter 2 (OCT2). Here, we first extended this development to a BAK device having cells with the organic anionic transporter 1 (OAT1), capable of removing anionic uremic wastes. We confirmed the quality of the ciPTEC monolayer by confocal microscopy and paracellular inulin-FITC leakage, as well as, by the active transport of anionic toxin, indoxyl sulfate (IS). Furthermore, we assessed the immune-safety of our system by measuring the production of relevant cytokines by the cells after lipopolysaccharide (LPS) stimulation. Upon LPS treatment, we observed a polarized secretion of pro-inflammatory cytokines by the cells: 10-fold higher in the extraluminal space, corresponding to the urine compartment, as compared to the intraluminal space, corresponding to the blood compartment. To the best of our knowledge, our work is the first to show this favorable cell polarization in a BAK upscaled device

A modular test structure for CMOS mismatch characterization

In this work a new test structure for mismatch characterization of CMOS technologies is presented. The test structure is modular, with a reduced area and it can be inserted in the space between the dies (scribe lines) on the wafers. The test structure has been implemented in a standard 0.18-μm digital CMOS technology

Role of Vitamin D in Maintaining Renal Epithelial Barrier Function in Uremic Conditions

Contains fulltext : 182172.pdf (publisher's version ) (Open Access)As current kidney replacement therapies are not efficient enough for end-stage renal disease (ESRD) treatment, a bioartificial kidney (BAK) device, based on conditionally immortalized human proximal tubule epithelial cells (ciPTEC), could represent an attractive solution. The active transport activity of such a system was recently demonstrated. In addition, endocrine functions of the cells, such as vitamin D activation, are relevant. The organic anion transporter 1 (OAT-1) overexpressing ciPTEC line presented 1alpha-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1) and vitamin D receptor (VDR), responsible for vitamin D activation, degradation and function, respectively. The ability to produce and secrete 1alpha,25-dihydroxy-vitamin D(3), was shown after incubation with the precursor, 25-hydroxy-vitamin D(3). The beneficial effect of vitamin D on cell function and behavior in uremic conditions was studied in the presence of an anionic uremic toxins mixture. Vitamin D could restore cell viability, and inflammatory and oxidative status, as shown by cell metabolic activity, interleukin-6 (IL-6) levels and reactive oxygen species (ROS) production, respectively. Finally, vitamin D restored transepithelial barrier function, as evidenced by decreased inulin-FITC leakage in biofunctionalized hollow fiber membranes (HFM) carrying ciPTEC-OAT1. In conclusion, the protective effects of vitamin D in uremic conditions and proven ciPTEC-OAT1 endocrine function encourage the use of these cells for BAK application