La hyalinose segmentaire et focale (HSF) est une maladie rénale sévère dont la physiopathologie est complexe. La récidive de la maladie après transplantation rénale et l’obtention de sa rémission après un traitement par immunoadsorption (IA) illustre l’implication d’un facteur circulant dans sa physiopathologie, capable de se fixer à la protéine A. Récemment, suPAR a été rapporté comme agent causal et marqueur de la HSF. Le premier objectif de notre travail a été de vérifier si suPAR se fixe à la protéine A. Le deuxième objectif a été d’identifier le facteur circulant responsable de la récidive de la HSF après transplantation rénale, à partir de l’analyse par spectrométrie de masse des protéines liées à la colonne de protéine A après (IA). Premièrement, nous avons mesuré la concentration de suPAR par un test ELISA parmi les protéines fixées à la colonne de protéine A après IA chez 7 patients atteints de HSF récidivantes et dans le sérum de 13 patients atteints de HSF récidivantes et de 11 contrôles sains. Le sérum des patients a été immunoadsorbé in vitro sur bille de protéine A sépharose. Nous avons quantifié suPAR avant et après la procédure et dans l’éluat des protéines fixées à la protéine A. La concentration de suPAR est plus élevée chez les patients atteints de HSF récidivantes par rapport aux groupes contrôles. La concentration de suPAR est très faible dans les proteines éluées à partir de la colonne de protéine A, indiquant que suPAR ne se lie pas à la protéine A et n’est pas le facteur circulant élué par les colonnes de protéines A. Deuxièmement, nous avons identifié le FC à partir des protéines fixées à la colonne de protéine A par une caractérisation des protéines par spectrométrie de masse chez des patients traités pour récidive de HSF et chez un patient contrôle. Nous avons recherché le FC dans le sérum de patient atteint de HSF, de patient ayant une néphropathie diabétique et chez des contrôles sains. L’effet de la protéine recombinante du FC a été testé in vitro sur une culture de podocytes et in vivo chez la souris. Nous avons identifié une forme sérique de CASK (calcium calmoduline sérine thréonine kinase), à partir des protéines fixées à la colonne de protéine A après IA. CASK est présente uniquement dans le sérum de patients atteints de HSF et non dans les groupes contrôles. In vitro, la protéine recombinante de CASK (CASKr) induit une redistribution de l’actine du cytosquelette des podocytes en culture par une interaction avec CD98. CASKr altére la perméabilité des podocytes à l’abumine et induit in vivo une protéinurie chez la souris associé à un effacement des pédicelles.En conclusion, suPAR ne se fixe pas à la protéine A ni in vivo ni in vitro. Une forme sérique de CASK est impliqué dans la récidive de la HSF avec comme cible potentiel CD98 sur le podocyte. / Focal and segmental glomerulosclerosis (FSGS) is a serious disease, the pathogenesis of which is unknown. Its recurrence after transplantation (Tx) and its partial remission after treatment with immunoadsorption (IA) on a protein A column indicate the existence of a circulating factor (CF) responsible for the disease that is able to bind to a protein A column. Recently, the soluble receptor of urokinase (suPAR) was described as the factor responsible for FSGS. The first aim of my work was to test the capacity of suPAR to bind to protein A and to be eliminated by IA. The second aim was to identify the CF responsible of the recurrence of the disease after renal transplantation from the analysis of proteins eluted from protein-A columns from patients with rFSGS who had undergone therapeutic (IA). First, we measured suPAR in eluates of protein A columns from 7 patients with recurrent FSGS after Tx (rFSGS) treated with IA, and in the serum of 13 patients with rFSGS and 11 healthy donors (HD). Additionally, the plasma of these patients was immunoadsorbed in vitro on a protein A Sepharose column and we quantified suPAR in the eluates and in pre- and post-column samples. The concentration of suPAR was higher in the plasma of patients with rFSGS than in the plasma of HD patients. However, the concentration of suPAR was similar before and after IA on protein A for the rFSGS and HD samples. The suPAR concentration was very low in the eluates from protein A columns incubated with plasma from HD or rFSGS patients. However, 85% of rFSGS patients showed a decrease in immunoglobulin G and proteinuria. Secondly, we analyzed proteins eluted from protein-A columns from patients with rFSGS who had undergone therapeutic immunoadsorption. Compared to control a differential band was identified by mass spectrometry. The expression of this protein was tested by immunochemical methods in sera from healthy controls, from patients with proteinuria caused by diabetic nephropathy, and from rFSGS patients. The effect of the recombinant protein was evaluated in vitro (podocytes) and in vivo experiments (mice). A soluble form of calcium/calmodulin-dependent serine/threonine kinase (CASK) eluted from protein-A columns was identified by mass spectrometry. CASK was immunoprecipitated only in the sera from patients with rFSGS. Recombinant CASK induced reorganization of the actin cytoskeleton of cultured podocytes through an interaction with CD98 at the cell surface. In vitro, CASK increased the permeability of podocyte monolayers, and induced proteinuria and foot-process effacement in miceIn conclusion, suPAR does not significantly bind to protein A in vitro or in vivo. Soluble CASK acts as a permeability factor in patients with rFSGS bindinding CD98 on podocytes.

Motivation Soluble urokinase plasminogen activator receptor (suPAR) is a known inflammatory marker, which is found in various body fluids. SuPAR reflects the immune and pro–inflammatory status of patients caused by HIV and tuberculosis, amongst others. However, recent studies have shown that suPAR is related to cardiovascular function. The cardiovascular health of the black South African population is a major health concern as this group suffers mostly from hypertension and stroke, leading to an alarming increase in cardiovascular morbidity and mortality. SuPAR may be able to contribute to early detection and prevention of cardiovascular diseases. No studies regarding the associations of suPAR with cardiovascular function have been investigated on black South Africans. Objectives To investigate suPAR as a possible marker of cardiovascular function in African and Caucasian men and women, by determining possible gender and ethnic–specific associations of suPAR with cardiovascular function. Methodology There were 207 African and 314 Caucasian men and women (aged 20–79 yrs.) included in this study. High–sensitivity C–reactive protein, glucose, lipids and creatinine were determined in fasting serum and suPAR was analyzed in plasma samples. Blood pressure was measured using the OMRON apparatus (HEM–747), with a 5–min rest interval between measurements. The Finometer device was used to determine the Windkessel compliance and the carotid dorsalis–pedis pulse wave velocity (PWV) was measured with the Complior (SP acquisition system) on the left side of each subject in the supine position. The means, adjusted means and proportions were compared between the groups by using independent t–tests, analysis of co–variance and the chi–square test, respectively. Associations were investigated between cardiovascular variables and suPAR using single and multiple regression analyses with either pulse wave velocity, systolic blood pressure, diastolic blood pressure or Windkessel compliance as dependent variable. Covariates included were age, body mass index, smoking, alcohol use, physical activity, glucose and high–density lipoprotein cholesterol. Results and conclusion SuPAR levels were significantly higher in Africans (P<0.001) compared to Caucasians. After adjusting for body mass index, suPAR increased significantly with age in all groups, except for African women. Moreover, the suPAR levels of African men and women were significantly higher than the Caucasians within each age quartile. While adjusting for age and body mass index, the cardiovascular profiles of the African and Caucasian men were less favourable compared to women, but suPAR levels were significantly higher in Caucasian women compared to men. In single regression, various measures of cardiovascular function correlated with suPAR in African men and Caucasian men and women. After adjusting for confounders the associations disappeared in Caucasian women, and remained nonsignificant in the African women. However, the association between PWV and suPAR remained significant in African men (B=0.19; P=0.030), while the association of systolic blood pressure (B=0.20; P=0.017), diastolic blood pressure (B=0.17; P=0.020) and Windkessel compliance (B=–0.14; P=0.004) with suPAR remained significant in Caucasian men. In conclusion, Africans presented higher suPAR levels compared to Caucasians, even when stratified by age. Gender specific associations indicated that suPAR was associated with arterial stiffness in African and Caucasian men only, therefore, indicating that suPAR could be a possible biomarker for predicting cardiovascular dysfunction. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Motivation Soluble urokinase plasminogen activator receptor (suPAR) is a known inflammatory marker, which is found in various body fluids. SuPAR reflects the immune and pro–inflammatory status of patients caused by HIV and tuberculosis, amongst others. However, recent studies have shown that suPAR is related to cardiovascular function. The cardiovascular health of the black South African population is a major health concern as this group suffers mostly from hypertension and stroke, leading to an alarming increase in cardiovascular morbidity and mortality. SuPAR may be able to contribute to early detection and prevention of cardiovascular diseases. No studies regarding the associations of suPAR with cardiovascular function have been investigated on black South Africans. Objectives To investigate suPAR as a possible marker of cardiovascular function in African and Caucasian men and women, by determining possible gender and ethnic–specific associations of suPAR with cardiovascular function. Methodology There were 207 African and 314 Caucasian men and women (aged 20–79 yrs.) included in this study. High–sensitivity C–reactive protein, glucose, lipids and creatinine were determined in fasting serum and suPAR was analyzed in plasma samples. Blood pressure was measured using the OMRON apparatus (HEM–747), with a 5–min rest interval between measurements. The Finometer device was used to determine the Windkessel compliance and the carotid dorsalis–pedis pulse wave velocity (PWV) was measured with the Complior (SP acquisition system) on the left side of each subject in the supine position. The means, adjusted means and proportions were compared between the groups by using independent t–tests, analysis of co–variance and the chi–square test, respectively. Associations were investigated between cardiovascular variables and suPAR using single and multiple regression analyses with either pulse wave velocity, systolic blood pressure, diastolic blood pressure or Windkessel compliance as dependent variable. Covariates included were age, body mass index, smoking, alcohol use, physical activity, glucose and high–density lipoprotein cholesterol. Results and conclusion SuPAR levels were significantly higher in Africans (P<0.001) compared to Caucasians. After adjusting for body mass index, suPAR increased significantly with age in all groups, except for African women. Moreover, the suPAR levels of African men and women were significantly higher than the Caucasians within each age quartile. While adjusting for age and body mass index, the cardiovascular profiles of the African and Caucasian men were less favourable compared to women, but suPAR levels were significantly higher in Caucasian women compared to men. In single regression, various measures of cardiovascular function correlated with suPAR in African men and Caucasian men and women. After adjusting for confounders the associations disappeared in Caucasian women, and remained nonsignificant in the African women. However, the association between PWV and suPAR remained significant in African men (B=0.19; P=0.030), while the association of systolic blood pressure (B=0.20; P=0.017), diastolic blood pressure (B=0.17; P=0.020) and Windkessel compliance (B=–0.14; P=0.004) with suPAR remained significant in Caucasian men. In conclusion, Africans presented higher suPAR levels compared to Caucasians, even when stratified by age. Gender specific associations indicated that suPAR was associated with arterial stiffness in African and Caucasian men only, therefore, indicating that suPAR could be a possible biomarker for predicting cardiovascular dysfunction. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Background: Fibulin-1, an extracellular matrix component and mediator in cardiac fibrosis, is expressed in cardiac valves, heart muscles and blood vessels and may contribute to different cardiovascular pathological conditions such as hypertension, aortic valve stenosis, atrial fibrillation and coronary artery disease. The most conspicuous functions of fibulin-1 include cell adhesion and cell migration within the extracellular matrix (ECM). This was found to reflect vascular dysfunction contributing to the development of fibrosis in the myocardium by means of changes in the ECM, possibly as a result of inflammation. Inflammatory mediators such as C-reactive protein (CRP) and albumin have been investigated over the years for the role they play in the inflammatory processes. However, one inflammatory mediator, soluble urokinase-type plasminogen activator receptor (suPAR), only emerged as a potential biomarker in the development of sclerotic disease. SuPAR is a soluble bioactive form of the urokinase-type plasminogen activator receptor (uPAR) secreted by inflammatory cells such as macrophages, endothelial cells and monocytes. The most profound functions of suPAR such as cell migration and cell adhesion contribute to the development of diseases such as infection, autoimmune diseases, cancer and atherosclerosis. Motivation and aim: This study was motivated by an awareness of the limited data on the potential link between fibulin-1 and suPAR, along with other markers of inflammation (CRP and albumin). We aimed to compare the levels of a marker of cardiac fibrosis (fibulin-1) and inflammatory mediators (suPAR, CRP and albumin) in African and Caucasian men and women. A second aim was to explore fibulin-1 and its potential association with these inflammatory markers independent of haemodynamic and metabolic risk factors in a bi-ethnic cohort from South Africa. Methodology: Data from the cross-sectional SAfrEIC study (South African study regarding the role of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function) were used, which initially included 756 participants. Our study population comprised 290 Africans (men: n=130; women: n=160) and 343 Caucasians (men: n=160; women: n=183). We excluded HIV-infected participants (n=115) as well as those with missing data (n=8). Traditional cardiovascular measurements together with the relevant biochemical analyses were done. T-tests and Chi-square tests were used to compare means and proportions between groups, respectively. Single and partial correlations were performed to determine the relationship of fibulin-1 with suPAR, CRP and albumin, with adjustments for age. SuPAR, CRP and albumin were divided into tertiles to explore the association with fibulin-1 levels, while adjusting for age, body mass index (BMI) and diastolic blood pressure (DBP) by using analysis of covariance (ANCOVA). Multiple regression analysis was performed to explore independent associations. Results: Participants were divided into African and Caucasian men and women due to significant interactions of the main effects of ethnicity and gender on the association of fibulin-1 with suPAR (ethnicity: F(633)=7.29; p<0.001 and gender: F(633)=7.99; p<0.001). Fibulin-1 levels were higher in African men (p=0.010), whereas CRP was higher in African women (p<0.001) compared to their Caucasian counterparts. In both gender groups suPAR levels were higher and albumin lower in Africans compared to Caucasians (p<0.006). In single regression analyses, a positive correlation existed between fibulin-1 and suPAR in African (r=0.19; p=0.028) and Caucasian men (r=0.37; p<0.001), also in African (r=0.193; p=0.028) and Caucasian women (r=0.14; p=0.036). After adjustments were applied for age, this correlation remained in African (r=0.23; p=0.010) and Caucasian men (r=0.22; p=0.005) only. An inverse correlation was found between fibulin-1 and albumin in African men (r=-0.28; p=0.002), but not in Caucasian men (r=-0.09; p=0.245). No significant correlation was found between fibulin-1 and CRP in any group. Forward stepwise regression analysis was performed in men and the previous associations between fibulin-1 and suPAR were confirmed in African and Caucasian men; along with the inverse relationship of fibulin-1 with albumin (Adj. R2=0.217; β=–0.210; p=0.013) in African men only. Conclusion: Fibulin-1 was positively associated with suPAR in African and Caucasian men, but not in women. We also found fibulin-1 to be negatively associated with albumin in African men only. These results are indicative of the presence of potential subclinical low-grade inflammation as depicted by suPAR within the extracellular matrix. This low-grade inflammation may contribute to the potential onset of cardiac fibrosis or vascular sclerosis among these South African men with lower albumin levels. / MSc (Physiology), North-West University, Potchefstroom Campus, 2014

Background: Fibulin-1, an extracellular matrix component and mediator in cardiac fibrosis, is expressed in cardiac valves, heart muscles and blood vessels and may contribute to different cardiovascular pathological conditions such as hypertension, aortic valve stenosis, atrial fibrillation and coronary artery disease. The most conspicuous functions of fibulin-1 include cell adhesion and cell migration within the extracellular matrix (ECM). This was found to reflect vascular dysfunction contributing to the development of fibrosis in the myocardium by means of changes in the ECM, possibly as a result of inflammation. Inflammatory mediators such as C-reactive protein (CRP) and albumin have been investigated over the years for the role they play in the inflammatory processes. However, one inflammatory mediator, soluble urokinase-type plasminogen activator receptor (suPAR), only emerged as a potential biomarker in the development of sclerotic disease. SuPAR is a soluble bioactive form of the urokinase-type plasminogen activator receptor (uPAR) secreted by inflammatory cells such as macrophages, endothelial cells and monocytes. The most profound functions of suPAR such as cell migration and cell adhesion contribute to the development of diseases such as infection, autoimmune diseases, cancer and atherosclerosis. Motivation and aim: This study was motivated by an awareness of the limited data on the potential link between fibulin-1 and suPAR, along with other markers of inflammation (CRP and albumin). We aimed to compare the levels of a marker of cardiac fibrosis (fibulin-1) and inflammatory mediators (suPAR, CRP and albumin) in African and Caucasian men and women. A second aim was to explore fibulin-1 and its potential association with these inflammatory markers independent of haemodynamic and metabolic risk factors in a bi-ethnic cohort from South Africa. Methodology: Data from the cross-sectional SAfrEIC study (South African study regarding the role of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function) were used, which initially included 756 participants. Our study population comprised 290 Africans (men: n=130; women: n=160) and 343 Caucasians (men: n=160; women: n=183). We excluded HIV-infected participants (n=115) as well as those with missing data (n=8). Traditional cardiovascular measurements together with the relevant biochemical analyses were done. T-tests and Chi-square tests were used to compare means and proportions between groups, respectively. Single and partial correlations were performed to determine the relationship of fibulin-1 with suPAR, CRP and albumin, with adjustments for age. SuPAR, CRP and albumin were divided into tertiles to explore the association with fibulin-1 levels, while adjusting for age, body mass index (BMI) and diastolic blood pressure (DBP) by using analysis of covariance (ANCOVA). Multiple regression analysis was performed to explore independent associations. Results: Participants were divided into African and Caucasian men and women due to significant interactions of the main effects of ethnicity and gender on the association of fibulin-1 with suPAR (ethnicity: F(633)=7.29; p<0.001 and gender: F(633)=7.99; p<0.001). Fibulin-1 levels were higher in African men (p=0.010), whereas CRP was higher in African women (p<0.001) compared to their Caucasian counterparts. In both gender groups suPAR levels were higher and albumin lower in Africans compared to Caucasians (p<0.006). In single regression analyses, a positive correlation existed between fibulin-1 and suPAR in African (r=0.19; p=0.028) and Caucasian men (r=0.37; p<0.001), also in African (r=0.193; p=0.028) and Caucasian women (r=0.14; p=0.036). After adjustments were applied for age, this correlation remained in African (r=0.23; p=0.010) and Caucasian men (r=0.22; p=0.005) only. An inverse correlation was found between fibulin-1 and albumin in African men (r=-0.28; p=0.002), but not in Caucasian men (r=-0.09; p=0.245). No significant correlation was found between fibulin-1 and CRP in any group. Forward stepwise regression analysis was performed in men and the previous associations between fibulin-1 and suPAR were confirmed in African and Caucasian men; along with the inverse relationship of fibulin-1 with albumin (Adj. R2=0.217; β=–0.210; p=0.013) in African men only. Conclusion: Fibulin-1 was positively associated with suPAR in African and Caucasian men, but not in women. We also found fibulin-1 to be negatively associated with albumin in African men only. These results are indicative of the presence of potential subclinical low-grade inflammation as depicted by suPAR within the extracellular matrix. This low-grade inflammation may contribute to the potential onset of cardiac fibrosis or vascular sclerosis among these South African men with lower albumin levels. / MSc (Physiology), North-West University, Potchefstroom Campus, 2014

Thesis ((Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2010.

Thesis ((Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2010.