Research Anja Verhulst

Abstract

Arterial media calcification (AMC) is defined as the accumulation of calcium-phosphate crystals in the medial layer of the arterial wall and is a major cardiovascular complication during aging and in patients with chronic kidney disease (CKD), diabetes and osteoporosis. AMC develops as a result of calcifying vascular smooth muscle cells (VSMC). Recently, however, the laboratory of Pathophysiology found strong evidence for the contribution of endothelial dysfunction (primarily characterized by reduced nitric oxide (NO) bioavailability) to AMC development. In this project, we will evaluate how endothelial cells (EC) can act as primary sensors of circulating triggers for AMC. We will use an in vitro EC/VSMC coculture model that will allow an in-depth investigation of the molecular effects of different calcification triggers on EC function, EC/VSMC interaction (with specific attention to NO signaling) and VSMC calcification. This detailed investigation of the endothelial phenotype during AMC could also potentially lead to the identification of new endothelial targets to treat AMC. Next, the therapeutic potential of restoring NO bioavailability in AMC will be preclinically evaluated in CKD and non-CKD induced AMC. In this way, we aim to contribute to the search for save, efficient arterial media calcification treatments, independent of patient population specific risk factors and not affecting bone health, as often seen with compounds that directly inhibit VSMC calcification.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Opdebeeck Britt
• Fellow: Adriaensen Saar

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Arterial media calcification or the deposition of hydroxy-apatite (calcium-phosphate) crystals in the medial layer of the vessel wall occurs as a result of ageing and is accelerated in chronic kidney disease (CKD), diabetes and osteoporosis. Arterial media calcification has severe cardiovascular consequences including arterial stiffening, hypertension, impaired coronary perfusion, cardiac stroke and left ventricular hypertrophy, ultimately leading to heart failure. Consequently, this pathology has a high impact on patients' quality of live, economic activity and survival. Currently available therapies lack efficacy since they do not directly target the calcification process itself, however, act indirectly by controlling its risk factors. Furthermore, studies performed in the Laboratory of Pathophysiology repeatedly showed that compounds efficiently targeting arterial media calcification compromise physiological bone mineralization, which can be attributed to the fact that arterial media calcification resembles highly to physiological bone mineralization. This project aims to contribute to the search for save, efficient, arterial media calcification treatments, independent of patient population specific risk factors and not affecting bone health.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Opdebeeck Britt
• Fellow: Adriaensen Saar

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

The Vevo LAZR-X is an imaging platform for preclinical applications capable of acquiring in vivo anatomical, functional and molecular data. It combines ultra high frequency ultrasound with photoacoustic imaging (a new biomedical imaging modality based on the use of lasergenerated ultrasound) for high resolution images as well as software for analysis and quantification. This equipment will be used in the context of the study of (cardio)vascular diseases, genetics of the heart, heart valves and aortic dissection, kidney diseases and their effects on the heart and blood vessels, and for cancer research.

Researcher(s)

• Promoter: De Meyer Guido
• Co-promoter: De Keulenaer Gilles
• Co-promoter: Heidbuchel Hein
• Co-promoter: Loeys Bart
• Co-promoter: Smits Evelien
• Co-promoter: Van Craenenbroeck Emeline
• Co-promoter: Verhulst Anja
• Co-promoter: Verstraeten Aline

Research team(s)

• Physiopharmacology (PHYSPHAR)

Project type(s)

• Research Project

Abstract

Arterial media calcification (AMC) is the deposition of calcium-phosphate crystals in the medial layer of the arterial wall and is an independent risk factor for cardiovascular morbidity/mortality. Efficient treatment for this lethal, highly prevalent pathology is lacking. Vascular smooth muscle cells, the key cell type in AMC, under oxidative stress transdifferentiate into cells with bone-forming capacity or die. It is not clear, however, which cell death type is most important. Ferroptosis, a recently discovered regulated type of cell death, is the result of iron-catalyzed, oxidative stress-induced membranous lipid peroxidation. Several arguments from literature and preliminary results from our lab put forward a role for iron accumulation, lipid peroxidation/ferroptosis in the process of AMC. This project aims to further substantiate this role by (i) exploring the AMC-aggravating effect of iron and (ii) the genetic induction of vascular smooth muscle cell-lipid peroxidation, in order to put forward lipid peroxidation as a novel target to treat AMC. Lipid peroxidation can be prevented by membranous radical trapping. Since potent membranous radical traps were developed in-house, we will have the unique opportunity to test these patented molecules for their ability to halt AMC. AMC-therapy development often stumbled over osseous side-effects (crystal deposition was inhibited in bone next to vessels). Membranous radical trapping therapy should overcome this problem.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Vanden Berghe Tom
• Fellow: Van den Branden Astrid

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Arterial media calcification is a severe cardiovascular complication in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. This disease is a master of camouflage by disguising itself as physiological bone mineralization, which makes it extremely difficult to find efficient and safe therapies against it, i.e. without affecting bone metabolism. The more as CKD and osteoporosis patients already suffer from bone mineralization defects. The candidate's FWO pre-doc project showed for the first time that the ATP analogue ?,?-meATP completely prevented all calcification in vascular smooth muscle cell cultures and efficiently prevented the development of arterial media calcification in rats, however also provoked deleterious effects on bone mineralization. Also other studies performed in the Laboratory of Pathophysiology showed repeatedly that compounds targeting arterial media calcification compromises physiological bone mineralization. Therefore, this FWO Post-doctoral junior project proposal focusses on developing a Poly(lactic-co-glycolic acid) (PGLA) nanoparticle conjugated to anti-elastin antibody drug delivery system to target ?,?-meATP selectively to the blood vessels, bypassing the bone compartment, and increase its bioavailability. PGLA nanoparticles have a high biocompatibility and biodegradability and are FDA approved for human therapy. The nanoparticles will be tested on in vitro and in vivo arterial calcification models.

Researcher(s)

• Promoter: Verhulst Anja
• Fellow: Opdebeeck Britt

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

The proposed Scientific Research Network aims to stimulate the investigation of save/efficient treatments for arterial media calcifications in Flanders. The specific aims of are to further investigate: (1) the role of extracellular nucleotides in arterial media calcification prevention/treatment, (2) the bone-vascular axis and (3) beneficial impact of arterial media calcification treatment on arterial stiffness.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Guns Pieter-Jan

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Disorders of mineral metabolism, specifically calcium and phosphorus homeostasis, are common in patients with chronic kidney disease (CKD), diabetes and osteoporosis. CKD is a world-wide recognized public health problem affecting 8-16% of the world population. Also the prevalence of diabetes and osteoporosis is high (12.3% for diabetes and 30% for osteoporosis of all postmenopausal women) and still increasing. Essentially, these three disorders of mineral metabolism typically share interconnected features including renal failure, vascular calcification and aberrant bone metabolism. CKD represents a progressive loss of renal function over a period of months or years ultimately leading to end-stage renal disease, which inevitably requires renal replacement therapy, i.e. dialysis and kidney transplantation. Vascular calcification in the medial layer of blood vessels is a major clinical problem and the most important cause of death in CKD patients. Structures similar to bone and cartilage are detected in the calcified arterial wall thereby mimicking bone formation. In addition, the disturbed mineral metabolism in CKD patients leads to the development of renal osteodystrophy which ultimately result in a reduced bone strength and increased incidence of bone fractures. The concomitant occurrence of a disturbed bone metabolism with a pathological calcification of the vessel wall in CDK patients is referred to as "the calcification paradox", which is also observed in diabetes and osteoporosis patients. The strong increase in the number of elderly boosts the prevalence of aging-related disorders such as CKD, diabetes and osteoporosis, and herewith the interest of pharmaceutical companies in clinical as well as preclinical research on these disorders. During the last decade, the Laboratory of Pathophysiology has developed unique animal models for the in vivo investigatation of different aspects of this interconnected triad (kidney, vessels and bone) in mineral metabolism disorders. These animal models besides investigation of fundamental mechanisms underlying these pathophysiological processes also allow to intervene in these processes by candidate therapeutics. This unique combination of animal models, substantiated by fundamental pathological knowledge, resulted in a cutting edge platform for preclinical (animal model) drug testing, which successfully attracted contract research with industrial partners resulting in an income of 500k€/year during the last decade. From our unique position at the interface between academics and industry, we note that industrial interest is shifting from symptomatic treatment (hypertension or hyperphosphatemia) towards a direct interference with CKD and vessel wall calcification. To anticipate this trend and level-up the valorization potential of our current platform of animal models, this IOF-service platforms project aims to expands its portfolio and setup novel innovative animal models to guarantee a further increase in industrial revenue.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Vanden Berghe Tom
• Co-promoter: Vervaet Benjamin

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Arterial stiffening is a hallmark of vascular aging and is associated with a high risk of cardiovascular disease and end-organ failure in kidney and brain (dementia). No effective therapies to combat this life threatening pathology currently exist, which is largely due to the lack of knowledge of the underlying molecular mechanisms. Therefore, this project aims to identify novel druggable targets which opens up new perspectives for discovery and evaluation of inventive therapies for this growing epidemic. Endothelial dysfunction and vascular calcification are two well known pathological processes leading to arterial stiffness. Established mouse models of both will be used to identify the molecular signaling pathways responsible for arterial stiffness by use of quantitative proteomics coupled with bioinformatic analysis. We will also explore the reciprocal relationship between different pathological processes leading to arterial stiffness. As impaired autophagy has been suggested to play a role in vascular aging, this project will also focus on its role in vascular calcification and stiffness. To explore whether autophagy is a candidate target for future intervention studies, complementary in vivo studies will be performed that investigate the effect of both autophagy induction and deficiency on vascular calcification and stiffness. Ultimately, this project aims to prevent or possibly reverse stiffening of large arteries to reduce long-term risk for end-organ damage.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Fransen Paul
• Co-promoter: Maudsley Stuart

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Cardiovascular calcification significantly contributes to cardiovascular disease, which is the leading cause of mortality and a major cause of morbidity in Europe. Cardiovascular calcification occurs in both rare monogenic (e.g. pseudoxanthoma elasticum) and in common acquired diseases (e.g. atherosclerosis and chronic kidney disease). Importantly, cardiovascular calcification is an active but incompletely understood process regulated by a variety of (epi)genetic and environmental factors, acting both systemically and locally. Despite its major clinical impact, no specific therapeutic strategies targeting cardiovascular calcification are applied in current clinical practice. In 2019, the Physiopharmacology and Pathophysiology research groups of the University of Antwerp were both part of the "eRaDiCal" consortium (H2020-MSCA-ITN call) aimed to investigate risk factors and underlying mechanisms across rare and common ectopic calcification disorders for the development of adequate diagnostic, preventive and therapeutic solutions to target cardiovascular calcification. eRaDiCal received an excellent reviewer score (94.8%, reserve list), but was not funded. As part of its strategic research plan, the University of Antwerp provides funding for excellent H2020 proposals to facilitate and encourage a successful resubmission. The teams of Physiopharmacology and Pathophysiology will use the budget to substantiate the evidence base on the role of autophagy in arterial calcification and stiffness. The intention is to recruit a (part-time) postdoc and/or PhD student to obtain preliminary data during the next year.

Researcher(s)

• Promoter: Guns Pieter-Jan
• Co-promoter: Verhulst Anja

Research team(s)

• Physiopharmacology (PHYSPHAR)

Project type(s)

• Research Project

Abstract

The calcification paradox is seen in chronic kidney disease (CKD) and osteoporosis patients and refers to the occurrence of a bone pathology characterized by a disturbed bone turnover/mineralisation in combination with calcification of the medial layer of the vessel wall; i.e. arteriosclerosis. This process of vascular calcification, which shares many similarities to bone development, is a rapidly progressive aspect of cardiovascular disease and is linked to increased morbidity and mortality particularly in CKD patients. As safe therapies to treat these mineralisation defects are urgently needed, this topic raised great interest during the last years. A pathway that might clarify the co-occurrence of mineralisation defects at the level of bone and vasculature is the Wnt/beta-catenin signalling pathway. It is known that this pathway regulates bone formation, however, its role in vascular calcification is less clear. The most obvious strategy in elucidating the potential (patho-)physiological roles of this cascade is by studying the functions of sclerostin and Dickkopf-related protein 1 (DKK1), both well-known inhibitors of the Wnt/beta-catenin signalling. Additionally, concerns are raised against the cardiovascular safety of anti-Scl and anti-DKK1 antibodies which are currently tested in clinical trials as a treatment for osteoporosis. Therefore, clarifying the mechanisms underlying this paradox is essential for the development of safe preventive and therapeutic treatments.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Verhulst Anja
• Fellow: De Maré Annelies

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Vascular stiffening is a growing epidemic associated with a high risk of cardiovascular disease, and end organ-damage in kidney and brain. No effective therapies to combat this life threatening vascular pathology currently exist, which is largely due to the lack of knowledge on the underlying molecular mechanisms. Therefore, this project aims to identify novel druggable targets which opens up new perspectives for discovery and evaluation of inventive therapies. Endothelial dysfunction and vascular calcification are well known pathological processes leading to vascular stiffness. Established mouse models of endothelial dysfunction and vascular calcification will therefore be used to identify the molecular signaling pathways responsible for vascular stiffness by means of a proteomic approach of arterial tissue. Additionally, we will explore the reciprocal relationship between endothelial dysfunction, vascular calcification and stiffness. As impaired autophagy has been suggested to play a role in vascular aging, this project will also focus on its role in vascular calcification and stiffness. To explore whether autophagy is a candidate target for future intervention studies, complementary in vivo studies will be performed that investigate the effect of both autophagy induction and defective autophagy on vascular calcification and stiffness. Broader ambitions of this project are to reduce the impact and lower the economic burden of vascular stiffness on society.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Neven Ellen
• Co-promoter: Verhulst Anja
• Fellow: Van den Bergh Geoffrey

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Over the past decades metformin is the optimal first-line drug for type 2 diabetes mellitus (T2DM) with 70 million prescriptions in the United States in 2013. Only in the last few years, it has become clear that metformin exerts benign pleiotropic actions beyond its prescribed use and ongoing investigations focus on its anti-ageing and anti-cancer properties. Recent data from preclinical and clinical studies are additionally pointing towards a putative beneficial impact of metformin on the kidney and cardiovascular system. Chronic kidney disease (CKD) is a world-wide recognized public health problem affecting 8–16% of the world population and represents a progressive loss of renal function over a period of months or years ultimately leading to end stage renal disease (CKD stage 5) which inevitably requires renal replacement therapy, i.e. dialysis or kidney transplantation. CKD also confers a markedly increased risk of cardiovascular disease (mainly vascular calcification) which accounts for 50% of all deaths in this population. Current treatment strategies for bot CKD and vascular calcification mainly focus on controlling important risk factors, however to date, effective treatment directly targeting the kidney and/or the vessels is lacking. Therefore the current project aims to investigate the possibility to use metformin in the treatment/prevention of CKD and its most important complication vascular calcification. Hereto the following objectives are put forward: (1) to investigate whether metformin is able to slow down or arrest the progression of CKD in rats with mild to moderate CKD and (2) to investigate whether metformin can prevent calcification in the arteries in an established rat model of vascular calcification. For the whole project, we will study the effect of metformin at the functional, structural (histopathological) and mechanistic level of the kidney and the aorta. Therefore, we will use both conventional and novel, more challenging techniques. If this project manages to demonstrate that this agent is able to retard or effectively halt the progression of CKD and to prevent, slow down or even arrest the development of vascular calcification, one of the major comorbidities of CKD, a major step forward will be taken in the treatment of this expanding population in our ageing society. Undoubtedly, such a finding will have far-reaching clinical and social benefits for the patients as well as a significant financial relieve for health insurance systems, taken into account the low cost of this generic drug (few euros/month) versus the high financial impact of dialysis and transplantation.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: Neven Ellen
• Co-promoter: Vervaet Benjamin
• Fellow: Corremans Raphaëlle

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Vascular calcification or the deposition of calcium-phosphate crystals in the arteries has a severe impact on morbidity/mortality in elderly and patients with chronic kidney disease (CKD) and diabetes mellitus (DM) by inducing severe cardiovascular events. In our ageing society (CKD and DM are ageing diseases), treatment for vascular calcification is warranted. However, current therapies only consist of controlling risk factors and therefore effective therapies to prevent/cure vascular calcification are lacking. For this reason we aim to investigate new treatment strategies that directly inhibit arterial calcification in two ways (i) directly interfering with crystal formation in the vessel wall and (ii) directly targeting the predominant cells in arterial calcification; by targeting respectively cell receptor independent and dependent effects of extracellular nucleotides on the vascular calcification process. Treatments will not only be tested for their ability to prevent vascular calcifications but also to attenuate the progression of pre-existing calcifications as most patients present themselves with a given degree of vascular calcification. Since vascular calcification shares many features with the bone formation process it is furthermore important to test new therapies inhibiting vascular calcifications for negative effects on the bone. In conclusion, the aim of this project is to develop effective therapies for vascular calcification without side-effects on the bone

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Neven Ellen
• Co-promoter: Verhulst Anja
• Fellow: Opdebeeck Britt

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

The calcification paradox is seen in chronic kidney disease (CKD) and osteoporosis patients and refers to the occurrence of a bone pathology characterized by a disturbed bone turnover/mineralisation in combination with calcification of the medial layer of the vessel wall; i.e. arteriosclerosis. This process of vascular calcification, which shares many similarities to bone development, is a rapidly progressive aspect of cardiovascular disease and is linked to increased morbidity and mortality particularly in CKD patients. As safe therapies to treat these mineralisation defects are urgently needed, this topic raised great interest during the last years. A pathway that might clarify the co-occurrence of mineralisation defects at the level of bone and vasculature is the Wnt/beta-catenin signalling pathway. It is known that this pathway regulates bone formation, however, its role in vascular calcification is less clear. The most obvious strategy in elucidating the potential (patho-)physiological roles of this cascade is by studying the functions of sclerostin and Dickkopf-related protein 1 (DKK1), both well-known inhibitors of the Wnt/beta-catenin signalling. Additionally, concerns are raised against the cardiovascular safety of anti-Scl and anti-DKK1 antibodies which are currently tested in clinical trials as a treatment for osteoporosis. Therefore, clarifying the mechanisms underlying this paradox is essential for the development of safe preventive and therapeutic treatments.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Verhulst Anja
• Fellow: De Maré Annelies

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

In the frame of this project analyses of heavy metals (lead, zinc, copper, chrome and cadmium) by using flame- and electrothermal atomic absorption spectrometry in urine and serum samples were performed.

Researcher(s)

• Promoter: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Cardiovascular disease is responsible for a substantial part of mortality in patients with chronic kidney disease (CKD) or diabetes (also a cause of CKD). Vascular calcification (VC) is a rapidly progressive, prominent aspect of cardiovascular disease in these patients as well as in those with osteoporosis. Remarkably, pathological VC in these patient groups goes along with disturbed bone metabolism. This association is called the calcification paradox. Development of new, safe therapies to treat the mineralization defects in bone and vessels is urgently needed. Sclerostin (Scl) is a protein produced by bone cells (osteocytes), inhibiting bone formation and mineralization. VC is regulated similarly to that of developing bone: vascular smooth muscle cells (VSMC) transdifferentiate to cells with a bone-like phenotype and proteins involved in bone formation also regulate the development of VC. Scl expression has been found in VSMC during calcification and CKD patients with VC were observed to have higher serum Scl. The Laboratory of Pathophysiology was the first to find an association between increased serum Scl and decreased mortality in hemodialysis patients. Hence, it is not surprising that during the last years the potential role of Scl in vascular pathophysiology and the calcification paradox has received increasing interest. This project aims to elucidate the complex role of Scl in mineralistation defects of bone and vessels and their reciprocal effects onto each other -

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Verhulst Anja
• Fellow: De Maré Annelies

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

The current project aims to elucidate the role of sclerostin in vascular calcification and its complex relationship with disturbed physiological bone mineralization, in particular in patients with chronic kidney disease. This research is necassary in order to develop safe therapies for these pathologies.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Behets Geert
• Co-promoter: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

The proposed project aims at getting profound molecular insight in cell cycle control of the proximal tubular epithelial cell in injured kidneys, an event recently recognized to play a critical role in the progression of acute kidney injury to chronic kidney disease.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Verhulst Anja
• Co-promoter: Vervaet Benjamin

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

In the present project we will evaluate the usefulness of lanthanum carbonate in the prevention of hyperoxaluria/renal calcifications by investigating its capability to limit intestinal oxalate absorption and/or promote oxalate secretion.

Researcher(s)

• Promoter: D'Haese Patrick
• Fellow: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Goals of the project: 1. To investigate the bio-physicochemical mechanism of crystal clearance and the role of pH and inflammation therein. 2. To study the fundamental effects of acute hyperphosphatemia induced by therapeutic oral sodium phosphateintake on (i) the systemic regulators of the phosphate balance, (ii) the tubular epithelial phenotype and (iii) renal function, and investigate if these effects can be eliminated by limiting or inhibiting phosphate absorption in the intestine.

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: De Broe Marc
• Co-promoter: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Aim: Investigate renal and intestinal oxalate handling as new targets for preventive therapies in the field of renal calcifications. Objectives: 1. Investigate the transepithelial transport of oxalate in human renal cells. 2. Investigate the effects of intestinal oxalate binding on oxalate excretion and incidence of renal calcifications.

Researcher(s)

• Promoter: D'Haese Patrick
• Fellow: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Erythropoietin (EPO) has recently been shown to protect the kidney both functionally and morphologically against acute renal failure induced by ischemia/reperfusion injury, as seen for example after kidney transplantation. The mechanisms underlying this renoprotective effect are not known. This project aims to elucidate the mechanisms of the EPO-induced renoprotection both in vivo (ischemic rat model) and in vitro (human renal tubular cells culture), this in order to justify further clinical studies.

Researcher(s)

• Promoter: Verhulst Anja
• Co-promoter: D'Haese Patrick
• Co-promoter: Ysebaert Dirk

Research team(s)

• Pathophysiology
• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

This project deals with the study of primary, tubular human kidney cell cultures as a possible in vitro model of renal molecule transport. At first instance we are interested in the transport of molecules that are possibly nephrotoxic (for example drugs which are in most cases orgnanic anions or kations) because of their transport by the kidney.

Researcher(s)

• Promoter: Verhulst Anja

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

In order to get a better insight in causes and prevention of nephrocalcinosis and -lithiasis, this project aims to answer the following questions: -is a non-differentiated distal tubular phenotype that is associated with damage/regeneration, the causal factor of crystal retention? -what is the role of osteopontin (OPN), CD44 and hyaluronic acid (HA) in the process of crystal retention? -can crystal retention be prevented by anti-inflammatory therapies?

Researcher(s)

• Promoter: D'Haese Patrick
• Co-promoter: Verhulst Anja

Research team(s)

• Pathophysiology

Project type(s)

• Research Project

Abstract

Osteopontin (OPN) is a protein present in different tissues and organs including the kidney. In the kidney, the constitutive expression of this glycosylated phosphoprotein is confined to the apical cell surface in the lis of Henle and more distal parts of the nephron. After acute renal damage OPN is also present in perinuclear vesicles in proximal tubular cells. OPN function is unknown, but a role in tissue remodelling is suggested both by its molecular structure and upregulated expression during increased cell turnover in a variety of tissues. However, knowledge concerning its expression, metabolism and activity is insufficient to define the function of OPN in a precise and well-founded manner. Its renal biology has never been studied in cultures of human kidney cells. The general aim of this project is to gain a better insight in the function of renal OPN. Primary kidney cell cultures will allow the study of the heterogenic nephron cell population in both a mixed and separated manner. First, expression and metabolism of OPN will be studied, either or not after induction of cell damage, which can be considered as an in vitro analogue of acute renal failure. Next, blocking of OPN expression during further experiments may reveal some of its activities related to tissue remodelling. In a first part of this project primary kidney cell cultures will be established. Human mixed and separated (for example distal versus proximal) cultures have already been developed in our laboratory. Immunocytochemical identification and separation of cells is based on specific cell surface markers. Mixed cultures of rat and mice kidney cells will also be developed in view of the availability of OPN knockout mice and to allow the comparison with in vivo rat models of acute renal failure, respectively. The second part of this study will entail the development of detection methods for OPN expression and phosphorylation in cell culture. Both the mRNA level (Northern blotting, in situ hybridisation) and the protein level (immunocytochemistry, immunoblotting and ELISA) will be studied. During part three of this project OPN binding and possible internalisation by tubular cells will be studied by the addition of labeled OPN, either or not phosphorylated, to primary kidney cell cultures. In a fourth part it will be investigated whether induction of cell damage by hypoxia, lipopolysaccharide or HgCl2 results in a different expression and metabolization pattern of OPN. A number of parameters in relation to tissue remodelling will be examined (before and after cell damage) in kidney cell cultures from OPN knockout versus wild-type mice during the fifth part of this study. Parameters that will be examined are viability, growth, adhesion, differentiation, vimentin expression and inhibition of NO synthase (iNOS). Observed differences may be abrogated by adding recombinant or native bovine OPN to the cultures. In order to confirm the observations in knockout mice, OPN function will be studied during the sixth and last part of this study by the addition of oligodeoxynucleotides to the cell cultures before and after induction of cell damage. A dose-dependent effect may be observed. Moreover it will enable to perform measurements in the same system before and after blocking of OPN synthesis. The results of this research project may contribute to the development of diagnostic applications and the implementation of new therapeutic approaches of acute renal failure.

Researcher(s)

• Promoter: De Broe Marc
• Fellow: Verhulst Anja

Research team(s)

Project type(s)

• Research Project

Abstract

Osteopontin (OPN) is a protein present in different tissues and organs including the kidney. In the kidney, the constitutive expression of this glycosylated phosphoprotein is confined to the apical cell surface in the lis of Henle and more distal parts of the nephron. After acute renal damage OPN is also present in perinuclear vesicles in proximal tubular cells. OPN function is unknown, but a role in tissue remodelling is suggested both by its molecular structure and upregulated expression during increased cell turnover in a variety of tissues. However, knowledge concerning its expression, metabolism and activity is insufficient to define the function of OPN in a precise and well-founded manner. Its renal biology has never been studied in cultures of human kidney cells. The general aim of this project is to gain a better insight in the function of renal OPN. Primary kidney cell cultures will allow the study of the heterogenic nephron cell population in both a mixed and separated manner. First, expression and metabolism of OPN will be studied, either or not after induction of cell damage, which can be considered as an in vitro analogue of acute renal failure. Next, blocking of OPN expression during further experiments may reveal some of its activities related to tissue remodelling. In a first part of this project primary kidney cell cultures will be established. Human mixed and separated (for example distal versus proximal) cultures have already been developed in our laboratory. Immunocytochemical identification and separation of cells is based on specific cell surface markers. Mixed cultures of rat and mice kidney cells will also be developed in view of the availability of OPN knockout mice and to allow the comparison with in vivo rat models of acute renal failure, respectively. The second part of this study will entail the development of detection methods for OPN expression and phosphorylation in cell culture. Both the mRNA level (Northern blotting, in situ hybridisation) and the protein level (immunocytochemistry, immunoblotting and ELISA) will be studied. During part three of this project OPN binding and possible internalisation by tubular cells will be studied by the addition of labeled OPN, either or not phosphorylated, to primary kidney cell cultures. In a fourth part it will be investigated whether induction of cell damage by hypoxia, lipopolysaccharide or HgCl2 results in a different expression and metabolization pattern of OPN. A number of parameters in relation to tissue remodelling will be examined (before and after cell damage) in kidney cell cultures from OPN knockout versus wild-type mice during the fifth part of this study. Parameters that will be examined are viability, growth, adhesion, differentiation, vimentin expression and inhibition of NO synthase (iNOS). Observed differences may be abrogated by adding recombinant or native bovine OPN to the cultures. In order to confirm the observations in knockout mice, OPN function will be studied during the sixth and last part of this study by the addition of oligodeoxynucleotides to the cell cultures before and after induction of cell damage. A dose-dependent effect may be observed. Moreover it will enable to perform measurements in the same system before and after blocking of OPN synthesis. The results of this research project may contribute to the development of diagnostic applications and the implementation of new therapeutic approaches of acute renal failure.

Researcher(s)

• Promoter: De Broe Marc
• Fellow: Verhulst Anja

Research team(s)

Project type(s)

• Research Project