Anna Wickman

Cardiac Insulin-like Growth Factor I and Growth Hormone Receptor Expression in Renal Hypertension

The aim of the present study was to investigate the role of insulin-like growth factor I in the development of cardiac hypertrophy in two-kidney, one clip hypertension by relating growth hormone receptor and insulin-like growth factor I receptor mRNA levels to insulin-like growth factor I gene transcription using a solution hybridization/RNase protection assay. Two-kidney, one clip hypertension was induced in male Wistar rats, and experiments were performed 2, 4, 7, and 12 days after surgery. Systolic blood pressure was elevated 2, 7, and 12 days after clipping (P < .001). Left ventricular weights were increased 2, 4, 7, and 12 days after surgery (P < .01). Associated with the rise in blood pressure, left ventricular insulin-like growth factor I mRNA was increased 2, 7, and 12 days after surgery (P < .01). Furthermore, growth hormone receptor and insulin-like growth factor I receptor gene expression increased specifically in the left ventricle of renal hypertensive rats (P < .05 and P < .001, respectively). Left ventricular growth hormone receptor mRNA peaked 7 days after induction of renal artery stenosis. These results show that insulin-like growth factor I, growth hormone receptor, and insulin-like growth factor I receptor mRNA increase in the pressure-overloaded left ventricle of two-kidney, one clip rats, suggesting a role for insulin-like growth factor I and the growth hormone/insulin-like growth factor I axis in the development of cardiac hypertrophy.

Angiotensin II, type 2 receptor is not involved in the angiotensin II-mediated pro-atherogenic process in ApoE-/- mice.

Objective Angiotensin II (Ang II) accelerates atherogenesis in ApoE−/− mice via the angiotensin II, type 1 receptor (AT1) while the type 2 receptor (AT2) is suggested to counteract atherogenesis. To confirm and further explore this possibility, we studied the effect of AT2 receptor antagonism on Ang II-accelerated atherosclerosis. Methods ApoE−/− mice were fed a standard or high cholesterol diet (1.25%) for 4 weeks. Mice on each diet were treated with either Ang II (0.5 μg/kg per min) or Ang II in combination with PD123319 (3 mg/kg per day). Plaque distribution was assessed by en face quantification of the thoracic aorta and in cross-sections of the aortic root. Mean arterial pressure (MAP) was measured. AT1 and AT2 receptor expression were analysed using real-time polymerase chain reaction (PCR) and the localization of the AT2 receptor protein confirmed with immunohistochemistry. Results Ang II infusion increased MAP only in mice on a standard diet (P < 0.001). Regardless of diet, Ang II-infused mice had 22–30 times increased plaque area in the thoracic aorta (P < 0.001 for both). Ang II had no effect on plaque in the aortic root. Plaque area was not affected by PD123319. AT2 receptor was heavily expressed in the plaques and increased six- to ninefold by a high cholesterol diet and Ang II infusion (P < 0.01). Conclusion Ang II increases the extent of atherosclerosis in ApoE−/− mice. Despite up-regulation of the AT2 receptor, we found no support for an effect of the AT2 receptor on atherogenesis in this model.

Brief losartan treatment in young spontaneously hypertensive rats abates long-term blood pressure elevation by effects on renal vascular structure.

Objective We studied the importance of regional vascular structural changes for the long-term antihypertensive effect of brief angiotensin II receptor blockade with losartan in young spontaneously hypertensive rats (SHRs). Design/methods SHRs were treated from 3 to 8 weeks of age with losartan (SHRLos, 30 mg/kg per day in drinking water) or vehicle (SHRCon). Mean arterial blood pressure (MAP) was measured using a telemetric technique from 12 to 25 weeks of age. Indices of vascular structure in the renal and hindquarter limb (HQ) were assessed using a haemodynamic perfusion technique at 13–15 weeks of age. Results MAP in SHRLos was reduced by 20–30 mmHg throughout the study (P < 0.001) and left ventricular weights were reduced (P < 0.05). The slope of the pressure/flow relationship was significantly changed (P < 0.001) in both kidneys and HQ vascular beds, suggesting greater average lumen dimensions in SHRLos. Pressure–glomerular filtration rate (GFR) curves of SHRLos kidneys were shifted to the left (P < 0.001), suggesting that the reduction in renal vascular resistance was predominantly preglomerular. The changes in structural indices of the heart and HQ closely followed the reduction in MAP. However, resistance at maximal dilatation in SHRLos kidneys was changed out of proportion to the lowering in MAP (P < 0.01). Conclusions Brief losartan treatment in young SHRs reduces long-term MAP. The reduced MAP is associated with higher average renal and skeletal muscle vascular dimensions at maximal dilatation, predominantly in the pre-capillary vasculature. The reduction in vascular resistance of the kidney appears to be out of proportion to the reduction in MAP and it may be speculated that this is of primary importance in the long-term hypotensive effect of brief angiotensin II antagonism in SHRs.

Endothelial nitric oxide synthase protein is reduced in the renal medulla of two-kidney, one-clip hypertensive rats

Objective We studied endothelial nitric oxide synthase (eNOS) expression in the kidneys of two-kidney, one-clip renal hypertensive rats (2K1C) before and after removal of the clip (unclipping, UC). We hypothesised that the haemodynamic changes induced by 2K1C and UC would change eNOS expression in the two kidneys. Methods Six weeks after inducing 2K1C, mean arterial pressure (MAP) was measured in conscious rats and hypertension reversed by UC. Left and right kidney eNOS protein in cortex and outer medulla was semi-quantified using immunoblotting. Groups were; normotensive (n = 10), 2K1C (n = 10), 3 h (n = 10), 48 h (n = 7) and 4 weeks (n = 7) after UC. The effect of 7 days of aldosterone or angiotensin II (Ang II) infusion on medullary eNOS protein was tested as well as the effect of l-NAME (nitric oxide (NO) synthase inhibitor) on medullary blood flow (MBF) in anaesthetized 2K1C. Results UC reduced MAP from 178 ± 5 to 134 ± 3 mmHg after 3 h and normalized MAP at 48 h and 4 weeks. The medulla from 2K1C kidneys contained about 33% less eNOS protein compared with normotensive kidneys (P < 0.05). This difference was still evident at 3 h (P < 0.05), but completely reversed at 48 h and 4 weeks after UC. Similar levels of eNOS expression were seen in the left and right kidney at all time points. Cortical eNOS was increased in kidneys from 2K1C. Neither Ang II nor aldosterone affected eNOS expression in the medulla. MBF was under similar influence of NO in 2K1C compared with normotensive kidneys. Conclusions 2K1C is associated with reduced levels of eNOS protein in the renal medulla of both clipped and contralateral kidney. eNOS expression in right and left kidney was not changed despite expected large changes in haemodynamics of the two kidneys. The reduced level of eNOS may be associated with a reduction in MBF and thus be of patho-physiological importance in renovascular hypertension.

ANGIOTENSIN-CONVERTING ENZYME INHIBITION IN PIGLETS INDUCES PERSISTENT RENAL ABNORMALITIES

1. We have previously shown that neonatal angiotensin‐converting enzyme (ACE) inhibition or angiotensin II type I (AT1) receptor antagonism during the first three postnatal weeks in the rat produces persistent abnormalities in renal function and histology, indicating an essential role for the reninangiotensin system (RAS) in normal renal development.

Induction of Growth Hormone Receptor and Insulin-Like Growth Factor-I mRNA in Aorta and Caval Vein During Hemodynamic Challenge

Induction of two-kidney, one clip hypertension (renal hypertension) is characterized by a slow increase in left ventricular tension and aortic wall stress, as opposed to aortocaval fistula or shunt volume overload, which induces a marked and rapid onset of wall stress in the caval vein and right ventricle. In the present study, we applied hemodynamic challenge to study the growth response involving gene expression of insulin-like growth factor-I (IGF-I) and growth hormone receptor (GH-R) mRNA in aorta and caval vein. Volume overload and pressure overload were induced in Wistar rats by means of shunt and renal hypertension, respectively. Systolic pressure was measured before excision of the great vessels, which was performed between 2 and 12 days postoperatively. Aortic and caval vein IGF-I and GH-R mRNA expressions were measured by means of a solution hybridization assay, and the caval vein was analyzed for IGF-I protein by immunohistochemistry. In the volume-distended but not pressurized caval vein in shunt rats, verified by telemetry recordings, there was an eightfold increase in IGF-I and 3.5-fold increase in GH-R mRNA at day 4 versus control. The IGF-I protein appeared to be localized in smooth muscle cells. In the aorta of the renal hypertension group, changes were of a slower onset. At day 7, there was a fourfold increase in IGF-I and five-fold increase of GH-R mRNA expressions versus sham-operated rats. Both the shunt caval vein and renal hypertension aorta showed evidence of a structural adaptation of the growth response. The present study suggests that acute elevation in vascular wall stress is an important triggering factor for overexpression of IGF-I and GH-R mRNA in great vessels. The growth hormone/insulin-like growth factor axis may be an important link in mediating structurally adaptive growth responses in the blood vessel wall.

Endothelial dysfunction in growth hormone transgenic mice

Acromegaly [overproduction of GH (growth hormone)] is associated with cardiovascular disease. Transgenic mice overexpressing bGH (bovine GH) develop hypertension and hypercholesterolaemia and could be a model for cardiovascular disease in acromegaly. The aims of the present study were to investigate the effects of excess GH on vascular function and to test whether oxidative stress affects endothelial function in bGH transgenic mice. We studied the ACh (acetylcholine)-induced relaxation response in aortic and carotid rings of young (9-11 weeks) and aged (22-24 weeks) female bGH transgenic mice and littermate control mice, without and with the addition of a free radical scavenger {MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride]}. We also measured mRNA levels of eNOS (endothelial nitric oxide synthase) and EC-SOD (extracellular superoxide dismutase). Intracellular superoxide anion production in the vascular wall was estimated using a dihydroethidium probe. Carotid arteries from bGH transgenic mice had an impaired ACh-induced relaxation response (young, 46 +/- 7% compared with 69 +/- 8%; aged, 52 +/- 5% compared with 80 +/- 3%; P < 0.05), whereas endothelial function in aorta was intact in young but impaired in aged bGH transgenic mice. Endothelial dysfunction was corrected by addition of MnTBAP in carotid arteries from young mice and in aortas from aged mice; however, MnTBAP did not correct endothelial dysfunction in carotid arteries from aged bGH transgenic mice. There was no difference in intracellular superoxide anion production between bGH transgenic mice and control mice, whereas mRNA expression of EC-SOD and eNOS was increased in aortas from young bGH transgenic mice compared with control mice (P < 0.05). We interpret these data to suggest that bGH overexpression is associated with a time- and vessel-specific deterioration in endothelial function, initially caused by increased oxidative stress and later by other alterations in vascular function.

Cardiovascular and renal phenotyping of genetically modified mice: A challenge for traditional physiology

1. The advent of techniques to genetically modify experimental animals and produce directed mutations in both a conditional and tissue‐specific manner has dramatically opened up new fields for physiologists in cardiovascular and renal research.

Haemodynamically significant plaque formation and regional endothelial dysfunction in cholesterol-fed ApoE-/- mice.

Flow-mediated vasodilation is suggested as one of the mechanisms involved in arterial expansive remodelling, which is thought to be a defence mechanism in atherogenesis. In the present study, we tested the hypothesis that lumen obstructive plaque formation is associated with failure of NO (nitric oxide)-dependent vasodilation in conduit vessels. Cardiac function and aortic root flow velocities were assessed using high-resolution echocardiography and two-dimensional-guided pulsed Doppler in ApoE(-/-) (apolipoprotein E-deficient) mice fed a standard or high-cholesterol diet. Endothelial function in the proximal and mid-descending aortic regions was studied using a myograph technique. Flow velocity at the aortic root of cholesterol-fed ApoE(-/-) mice was significantly increased as a result of lumen narrowing, detected via histological analysis. NO-dependent vasodilatory responses were selectively impaired in the atherosclerosis-prone vascular regions in cholesterol-fed ApoE(-/-) mice. In conclusion, consumption of a high-cholesterol diet results in lumen obstructive plaque formation in ApoE(-/-) mice, which significantly alters aortic root haemodynamics. This phenomenon is associated with impaired NO-dependent vasodilation in vessel segments known to be prone to atherosclerosis.

Genetic Disruption of Protein Kinase STK25 Ameliorates Metabolic Defects in a Diet-Induced Type 2 Diabetes Model

Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type littermates with a high-fat diet and showed that STK25 deficiency suppressed development of hyperglycemia and hyperinsulinemia, improved systemic glucose tolerance, reduced hepatic gluconeogenesis, and increased insulin sensitivity. Stk25−/− mice were protected from diet-induced liver steatosis accompanied by decreased protein levels of acetyl-CoA carboxylase, a key regulator of both lipid oxidation and synthesis. Lipid accumulation in Stk25−/− skeletal muscle was reduced, and expression of enzymes controlling the muscle oxidative capacity (Cpt1, Acox1, Cs, Cycs, Ucp3) and glucose metabolism (Glut1, Glut4, Hk2) was increased. These data are consistent with our previous study of STK25 knockdown in myoblasts and reciprocal to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes.