Cardiovascular disease (CVD) is highly prevalent in chronic kidney disease (CKD) patients. Whilst this can in part be explained by the high incidence of traditional CVD risk factors such as hypertension and diabetes evident in CKD patients, recent focus has been on non-traditional risk factors and their role in CVD progression. These include endothelial dysfunction, arterial stiffness, inflammation and oxidative stress. The potent vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of CKD and the CVD associated with it. Further understanding of the mechanisms by which it contributes to CKD and CVD pathogenesis, specifically its interactions with non-traditional risk factors are still required. Additionally, the potential applications of ET antagonists in renal disease have not been fully explored. This thesis aims to investigate the role of ET-1 in the development of renal disease and the associated inflammation, hypertension and vascular dysfunction through a series of in vitro, in vivo and clinical studies. I have demonstrated using in vitro techniques that murine macrophages (Mϕ) express both endothelin A (ETA) and endothelin B (ETB) receptors but that ET-1 does not elicit either a classical pro-inflammatory or alternative anti-inflammatory phenotype in Mϕ. I was however, able to show that M display chemokinesis towards ET-1 and M ETB receptors provide a novel clearance mechanism for ET-1 through receptor mediated dynamin-dependent endocytosis In an in vivo study I investigated whether ET-1 mediates the progressive renal injury after renal ischaemia reperfusion injury (IRI) that leads to the development of CKD. I demonstrated that endothelin A receptor antagonism provided long term beneficial effects reducing blood pressure and preventing progressive kidney injury, inflammation, and the development of fibrosis resulting from an episode of acute kidney injury (AKI). Similar benefits were observed with calcium channel blockade, suggesting hypertension may mediate some of the long term effects of renal IRI and anti-hypertensive treatments could prevent the development of CKD after AKI. Finally, in a clinical study I showed for the first time that CKD patients lack the diurnal variation in arterial stiffness that is seen in matched subjects without CKD. Alteration in the circadian variation of the ET-1 system may contribute to this. In summary, my studies have furthered our understanding of the role of ET-1 in CKD progression and the cardiovascular risk associated with it. Mϕ were shown to express both ET receptors and a novel mechanism of ET-1 clearance was observed in Mϕ. Using an in vivo model of AKI I was able to identify ETA receptor antagonism as a novel therapeutic agent in preventing the development of CKD caused by AKI where data are limited. Finally, alterations in the circadian rhythm of the cardiovascular system is emerging as an important factor in disease pathogenesis. Here the diurnal variation in arterial stiffness was described for the first time in a group of CKD patients and matched controls.

Tight control of blood glucose and blood pressure (BP) reduces cardiovascular risk in early Type 1 diabetes mellitus (T1DM). Increased BP normally increases renal medullary perfusion and sodium excretion. This is called acute pressure natriuresis. Inadequate acute pressure natriuresis disrupts circadian regulation of BP, which predicts hypertension. The peptide, endothelin-1 (ET-1), regulates BP via ETA and ETB receptors. ETA receptor antagonists reduce BP and restore its circadian rhythm. Two hypotheses were investigated. First, that acute pressure natriuresis is impaired in early T1DM, prior to established nephropathy, and this is associated with elevated BP. Second, that the mechanism is an ETA receptor-mediated blunting of medullary perfusion which can be reversed with insulin and ETA receptor antagonism. Experimental acute pressure natriuresis was induced in young, early T1DM (2-3 weeks post streptozotocin) Sprague Dawley rats and healthy controls. Despite maintaining glomerular filtration rate, early T1DM suppressed urinary flow (UV, 22.9±2.9 v. 93.7±11.1μl/min/gkw) and sodium excretion (UNaV, 3.2±0.7 v. 22.7±3.3μmol/min/gkw) rates by >80%, and reduced gradients of pressure diuresis (linear, 1.9 to 0.3) and natriuresis (non-linear k, 0.05 to 0.01) curves. Insulin treatment lowered blood glucose (16.8±1.8 to 9.3±0.6mmol/l) and restored gradients of the responses. Tissue and urine analyses did not suggest structural nephropathy. In early T1DM rats, changes in BP on radiotelemetry were consistent with impaired circadian regulation of BP and precursors of hypertension: 24-hour diastolic BP rose (92.3±0.4 to 97.1±0.5mmHg), and the circadian dip in diastolic BP fell (6±1 to 2±1%). Atrasentan (ETA receptor antagonist, 5mg/kg/day orally) reduced diastolic dipping in early T1DM (3±1 to 1±1%) while additional ETB receptor antagonism (A-192621, 10mg/kg/day orally) reversed this, suggesting that ETA, and not ETB receptors, mediate impairment of acute pressure natriuresis. To address this, renal blood flow was measured during experimental acute pressure natriuresis and ET receptor antagonism. Early T1DM suppressed the normal rise in medullary perfusion (flux, 227.2±26.7 v. 115.4±10.3%) by ~90%. Suppressed medullary flux was unaffected by insulin (112.2±6.8%), despite restoration of UV and UNaV. In controls, atrasentan reduced UV (15.7±4.9 v. 38.6±6.2μl/min/gkw), UNaV (1.7±0.5 v. 16.7±1.4μmol/min/gkw), FENa (3.4±1.4 v. 15.0±2.4%) and medullary flux (122.2±26.7%) by 60 to 90% of control values, while A-192621 increased UNaV (26.6±6.9μmol/min/gkw) and FENa (21.6±3.4%), but not medullary flux, by ~50%. ET receptor antagonism did not modify early T1DM+/-insulin effects. Diabetic status had no effect on renal ET-1 and ET receptor expression. These results support the first hypothesis but disprove the second. Early T1DM blunts medullary perfusion and acute pressure natriuresis, and increases diastolic BP. Insulin restores natriuresis but not medullary flow. Therefore, targeting medullary perfusion may reduce cardiovascular risk in early T1DM, but this is not achievable with selective ETA receptor antagonists. Novel natriuretic (ETA) and anti-natriuretic (ETB) roles for ET receptors, which are not apparent in early T1DM during severe, experimental rises in BP, appear to contribute to daily regulation of BP, and may preclude the use of selective ETA receptor antagonists in T1DM prior to nephropathy.

- / Background: Endothelin-1 (ET-1), a strong vasoconstrictive substance acting via stimulation of specific receptors (ET-A and ET-B), has been implicated in the development of renal scarring. Activation of endothelin system was observed in experimental models of glomerular diseases and this was attributed to the toxic action of proteinuria to the tubular epithelial cells. However, we have not enough information about the role of endothelin system in human glomerular diseases and in renal diseases without proteinuria like obstructive nephropathy. The aim of this study was to examine the endothelin system in patients with primary glomerular diseases and in experimental animals with unilateral ureteric obstruction. Patients and Methods: Thirty-seven patients with different types of primary glomerulonephritides and 14 controls were included in the study. Patients presented by either nephrotic syndrome (n=25) or mild proteinuria (<1g/24h, n=12). The expression of ET-A and ET-B receptors in the renal tissue was examined immunohistochemically. At the time of biopsy, urinary ET-1 was determined by RIA. Experimental animals and Methods: Twenty –day old opossum pups (n=6) underwent surgical ligation of the left ureter. Sham operated animals, non-operated controls and normal human kidneys were also used. Animals were sacrificed at 2 (n=2), 3 (n=1), 4 (n=1), 5(n=1) and 8 (n=1) weeks post surgery and their kidneys were examined. Sham operation was performed at equivalent times in pups that served as control. The expression of ET-A and ET-B receptors in the renal tissue was examined immunohistochemically. Results: The expression of both receptors was mainly localized within tubular epithelial cells and was significantly higher in patients with glomerulonephritides compared to controls. The expression of ET-B receptors was higher in nephrotic compared to non-nephrotic patients while no difference was observed in the expression of ET-A receptors. Urinary excretion of ET-1 was increased in patients compared to healthy subjects (579±146 ng/24h vs. 410±78 ng/24h, p<0.01) and it was higher in nephrotic compared to non-nephrotic patients (617±167 ng/24h vs. 485±71 ng/24h, p<0.05). A significant positive correlation of the excreted ET-1 with the degree of proteinuria (r= 0.338, p<0.05) and the extent of immunostaining for ET-B receptors (r=0.427, p<0.05) was observed. The expression of ET-B receptors and the excretion of ET-1 were significantly decreased in patients who present remission of the nephrotic syndrome under immunosuppressive therapy. In tubular epithelial cells of the experimental animals there was a temporal increase in the expression of ET-A receptors with duration of obstruction while there was no significant difference between the expression of ET-B receptors in obstructed kidneys and controls. Conclusions: this study provides evidence that the endothelin system is activated in renal diseases and proteinuria seems to be related only in part to this activation. Further investigation is needed to ascertain if the activation of endothelin system has a causative role in the progression of renal diseases.

Motivation: The prevalence of obesity is the highest among African women in South Africa. Since obesity is a major cardiovascular risk factor, African women in South Africa could be regarded as a high risk group. However, investigations on obesity-related hypertension are limited in this population group. The associations of body fat distribution and hormones such as leptin and endothelin-1 with cardiovascular function have not yet been determined in these women. It has been determined that endothelin-1 is a role player in the development and/or maintenance of hypertension in various population groups, especially African Americans. Endothelin-1 has also been found to be involved in obesity-related hypertension in non-African population groups. It has been indicated that the obesity-related hormone, leptin, also plays a role in obesity-related hypertension, especially in African Americans. Leptin levels have been found to be higher in obese hypertensive African American women compared to an obese normotensive control group. Since the above-mentioned two hormones playa prominent role in obesity and hypertension in African American and non-African population groups, the lack of data on African women in South Africa serves as motivation to conduct this investigation. Aim: To investigate obesity-related hypertension in African women through the determination of associations between various anthropometric and endocrinological variables with cardiovascular, especially vascular function. Methodology: Manuscripts presented in Chapters 2, 3 and 4 made use of data from the POWIRS (Profiles of Obese Women suffering from the Insulin Resistance Syndrome) I project where African women were selected from a government institution in the North West Province. A group of 98 women were divided into lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. Anthropometric and cardiovascular measurements were taken and the lipid profile, leptin and endothelin-1 levels determined. The analysis of covariance (ANCOVA) was used to show significant differences between groups while adjusting for age. Partial correlation coefficients were used to show associations between various variables while adjusting for age. Stepwise linear regression analysis was also used to show associations between variables. The study presented in Chapter 5 made use of both POWIRS I and II, which are studies including Africans and Caucasians, respectively. The methodology of the two studies was the same. All subjects gave informed consent in writing and the Ethics Committee of the North-West University approved the study. The reader is referred to the "Materials and Methods" section of Chapters 2-5 for a more elaborate description of the subjects, study design and analytical methods used in each article. vii Results and conclusions of the individual manuscripts > Results from Chapter 2 showed that the volume loading effect associated with obesity was present in both overweight/obese normotensive and overweight/obese hypertensive groups, however, the accommodating effect observed in the overweight/obese normotensive group was absent in the overweight/obese hypertensive group due to decreased vascular function. This was confirmed by a high pulse pressure. Decreased vascular functioning was associated with the abdominal skin fold. This suggests that abdominal subcutaneous fat may either be a marker of visceral fat, or may in itself contribute to increased cardiovascular risk in Africans. > Results from Chapter 3 showed a negative result. Plasma endothelin-1 levels were similar for the lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. After re-dividing the groups into normotensive and hypertensive, and then into lean and overweight/obese, still no differences could be obtained. Additionally, no correlations could be obtained between endothelin-1 and cardiovascular function in any of the groups. These findings suggest that endothelin-1 is not implicated in obesity-related hypertension in African women. > In Chapter 4, leptin levels were elevated in both overweight/obese normotensive and hypertensive groups compared to the lean normotensive group. However, leptin levels did not differ between the two overweight/obese groups. Even though leptin levels were the same, leptin was directly and positively associated with systolic blood pressure and pulse pressure and negatively with arterial compliance only in the overweight/obese hypertensive group, independent of obesity, insulin resistance, hyperinsulinemia and age. > In Chapter 5 the volume loading, as well as the accommodating effect, that is, decreased total peripheral resistance and increased arterial compliance, was present in both African and Caucasian obese groups compared to their lean controls. Even though leptin levels, body mass index and age were similar for both African and Caucasian obese groups, the accommodating effect seemed to be more prominent in the obese Caucasian group, explaining a lower diastolic blood pressure compared to the obese African group. Leptin showed a favourable negative association with diastolic blood pressure and total peripheral resistance in the obese Caucasian group, but not in the obese African group. This may indicate that leptin predominantly exerts pathological influences on obese African women, as determined previously in Chapter 4. / Thesis (Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over-expression of endothelin 1 (ET 1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET 1 mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, and phosphorylation, Nav1.5 mRNA and protein, Na+ conductance, K+ channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET 1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model.

Binary transgenic (BT) mice with doxycycline (DOX)-suppressible cardiac-specific over-expression of endothelin 1 (ET 1) exhibit progressive heart failure, QRS prolongation, and death following DOX withdrawal. However, the molecular basis and reversibility of the electrophysiological abnormalities in this model were not known. Here we assess the mechanisms underlying ET 1 mediated electrical remodelling, and its role in heart failure. Prior attempts to prevent this model of ET-1 induced cardiomyopathy with ET receptor antagonism were not beneficial. We now propose to evaluate the effectiveness of blocking the synthesis of ET-1 with CGS 26303, a dual inhibitor of endothelin converting enzyme (ECE) and neutral endopeptidase. BT vs. littermate control mice were withdrawn from DOX and serially studied with ultrasound biomicroscopy, octapolar catheters, multi-electrode epicardial mapping, histopathology, Western blot, immunohistochemistry and qRT-PCR. Prolonged ventricular activation and depressed rate of ventricular activation were detected as early as 4 wks after transgene activation, when structure and function of the heart remained unaffected. By 8 wks of ET-1 over-expression, biventricular systolic and diastolic dysfunction, myocardial fibrosis, cardiomyocyte hypertrophy, prolonged ventricular activation and repolarization, depressed rate of ventricular activation, and abnormal atrioventricular nodal function were observed. Within 4 wks of ET-1 induction, reduction were observed in connexin-43 mRNA, protein, and phosphorylation, Nav1.5 mRNA and protein, Na+ conductance, K+ channel interacting protein-2 mRNA and Kv4.2 mRNA. Chromatin immunoprecipitation revealed that nuclear factor κB preferentially binds to Cx43 and Nav1.5 promoters. Importantly, the associated electrophysiological abnormalities at this time point were reversible upon suppression of ET 1 over-expression and completely prevented the development of structural and functional remodelling. Treatment with CGS-26303 (5 mg/kg/day) failed to improve survival, or hemodynamic and contractile decline. ET-1-mediated ventricular conduction delays correlates with gap junction and ion channel remodelling, and precedes heart failure. The sequence and reversibility of this phenotype suggest that a primary abnormality in electrical remodelling may contribute to the pathogenesis of heart failure. CGS 26303 failed to prevent this cardiomyopathic phenotype. These data suggest that chronically high levels of bigET-1, as seen in heart failure, may induce increased ECE activity and/or non-ECE ET-1 synthesis, thus circumventing the efficacy of ECE blockade in this model.

Motivation: The prevalence of obesity is the highest among African women in South Africa. Since obesity is a major cardiovascular risk factor, African women in South Africa could be regarded as a high risk group. However, investigations on obesity-related hypertension are limited in this population group. The associations of body fat distribution and hormones such as leptin and endothelin-1 with cardiovascular function have not yet been determined in these women. It has been determined that endothelin-1 is a role player in the development and/or maintenance of hypertension in various population groups, especially African Americans. Endothelin-1 has also been found to be involved in obesity-related hypertension in non-African population groups. It has been indicated that the obesity-related hormone, leptin, also plays a role in obesity-related hypertension, especially in African Americans. Leptin levels have been found to be higher in obese hypertensive African American women compared to an obese normotensive control group. Since the above-mentioned two hormones playa prominent role in obesity and hypertension in African American and non-African population groups, the lack of data on African women in South Africa serves as motivation to conduct this investigation. Aim: To investigate obesity-related hypertension in African women through the determination of associations between various anthropometric and endocrinological variables with cardiovascular, especially vascular function. Methodology: Manuscripts presented in Chapters 2, 3 and 4 made use of data from the POWIRS (Profiles of Obese Women suffering from the Insulin Resistance Syndrome) I project where African women were selected from a government institution in the North West Province. A group of 98 women were divided into lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. Anthropometric and cardiovascular measurements were taken and the lipid profile, leptin and endothelin-1 levels determined. The analysis of covariance (ANCOVA) was used to show significant differences between groups while adjusting for age. Partial correlation coefficients were used to show associations between various variables while adjusting for age. Stepwise linear regression analysis was also used to show associations between variables. The study presented in Chapter 5 made use of both POWIRS I and II, which are studies including Africans and Caucasians, respectively. The methodology of the two studies was the same. All subjects gave informed consent in writing and the Ethics Committee of the North-West University approved the study. The reader is referred to the "Materials and Methods" section of Chapters 2-5 for a more elaborate description of the subjects, study design and analytical methods used in each article. vii Results and conclusions of the individual manuscripts > Results from Chapter 2 showed that the volume loading effect associated with obesity was present in both overweight/obese normotensive and overweight/obese hypertensive groups, however, the accommodating effect observed in the overweight/obese normotensive group was absent in the overweight/obese hypertensive group due to decreased vascular function. This was confirmed by a high pulse pressure. Decreased vascular functioning was associated with the abdominal skin fold. This suggests that abdominal subcutaneous fat may either be a marker of visceral fat, or may in itself contribute to increased cardiovascular risk in Africans. > Results from Chapter 3 showed a negative result. Plasma endothelin-1 levels were similar for the lean normotensive, overweight/obese normotensive and overweight/obese hypertensive groups. After re-dividing the groups into normotensive and hypertensive, and then into lean and overweight/obese, still no differences could be obtained. Additionally, no correlations could be obtained between endothelin-1 and cardiovascular function in any of the groups. These findings suggest that endothelin-1 is not implicated in obesity-related hypertension in African women. > In Chapter 4, leptin levels were elevated in both overweight/obese normotensive and hypertensive groups compared to the lean normotensive group. However, leptin levels did not differ between the two overweight/obese groups. Even though leptin levels were the same, leptin was directly and positively associated with systolic blood pressure and pulse pressure and negatively with arterial compliance only in the overweight/obese hypertensive group, independent of obesity, insulin resistance, hyperinsulinemia and age. > In Chapter 5 the volume loading, as well as the accommodating effect, that is, decreased total peripheral resistance and increased arterial compliance, was present in both African and Caucasian obese groups compared to their lean controls. Even though leptin levels, body mass index and age were similar for both African and Caucasian obese groups, the accommodating effect seemed to be more prominent in the obese Caucasian group, explaining a lower diastolic blood pressure compared to the obese African group. Leptin showed a favourable negative association with diastolic blood pressure and total peripheral resistance in the obese Caucasian group, but not in the obese African group. This may indicate that leptin predominantly exerts pathological influences on obese African women, as determined previously in Chapter 4. / Thesis (Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Diss. (sammanfattning) Göteborg : Göteborgs universitet , 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Abstract The heart responds to increased hemodynamic stress by increased cardiac myocyte size, enhanced protein synthesis and altered gene expression. Regulation of hypertrophic adaptation involves a number of neural and hormonal factors, which act on the cardiovascular system. The aim of the present study was to elucidate the regulation of gene expression of natriuretic peptides and adrenomedullin (AM) in cardiac overload in vivo. Furthermore, the interactions of AM and angiotensin II (Ang II) in cardiac function and development of left ventricular hypertrophy were studied both in vivo and in vitro. The effects of cardiac hypertrophy on the regulation of natriuretic peptides (atrial natriuretic peptide, ANP and B-type natriuretic peptide, BNP) and AM gene expression were studied during pressure overload in the hearts of two hypertensive rat strains, angiotensinogen-renin transgenic rats and spontaneously hypertensive rats as well as their normotensive control strains. Increased workload resulted in rapid upregulation of both BNP and AM gene expression in all rat strains; the response of AM was, however, augmented in hypertensive rats. Direct left ventricular wall stretch induced AM gene expression in isolated, perfused rat hearts, whereas stretching of cultured cardiac myocytes downregulated AM mRNA levels. In cultured cardiac cells exposed to Ang II, endothelin-1 or the α-agonist phenylephrine, Ang II-induced myocyte hypertrophy was selectively inhibited by AM. In vivo, AM interacted with Ang II in circulation by attenuating the hypertensive effects of Ang II, and in the heart by augmenting the Ang II-induced improvement in cardiac systolic function. However, AM had no direct modulatory effects on Ang II-induced left ventricular hypertrophy. These results show that cardiac wall stretch is a major stimulus for the early induction of AM gene expression in both normal and hypertrophied ventricle, and the response in hypertrophied myocardium is augmented. Furthermore, cardiac non-muscle cells may be involved in mediating effects of direct stretch. In vitro, AM acts as a selective inhibitor of Ang II-induced myocyte hypertrophy, suggesting a cardioprotective role for AM to counteract the local renin-angiotensin system and Ang II in cardiac hypertrophy and heart failure. Circulating AM appears to act mainly as a regulator of vascular tone and cardiac function.