Regulation of angiogenesis involves tight cell-to-cell and cell-to-extracellular-matrix interactions. Various reports demonstrate that the Wnt signaling pathways participate in this regulation. Using a three-dimensional aortic ring culture combined with an ex vivo retroviral infection approach, we evaluated the effects of two Wnt growth factors, Wnt-1 and Wnt-4, on the formation and growth of new capillaries. Our results show that Wnt-1 had no effect, whereas Wnt-4 was a potent inhibitor of capillary outgrowth in vitro.

Renin-producing cells have been the object of intense research efforts for the past fifty years within the field of hypertension. Two decades ago, research focused on the concept and characterization of the intrarenal renin-angiotensin system. Early morphological studies led to the concept of the juxtaglomerular apparatus, a minute organ that links tubulovascular structures and function at the single nephron level. The kidney, thus, appears as a highly “topological organ” in which anatomy and function are intimately linked. This point is reflected by a concurrent and constant development of functional and structural approaches. After summarizing our current knowledge about renin cells and their distribution along the renal vascular tree, particularly along glomerular afferent arterioles, we reviewed a variety of imaging techniques that permit a fine characterization of renin synthesis, storage, and release at the single-arteriolar, -cell, or -granule level. Powerful tools such as multiphoton microscopy and transgenesis bear the promises of future developments of the field.

Clinical studies suggest a strong link between tissue calcification and pressure hyperpulsatility in end stage renal disease patients. Using a Wistar rat model of arterial elastocalcinosis and hyperpulsatility [vitamin D and nicotine (VDN) treatment], we evaluated the relative importance of tissue calcification and hyperpulsatility in the etiology of renal failure. VDN rats showed significant increases in aortic wall calcium content (50 times; 992+/-171 vs. control 19+/-1 micromol/g dry wt) and pulse pressure (1.5 times; 61+/-4 vs. control 40+/-2 mmHg). Significant renal calcification (16 times; 124+/-27 vs. control 8.1+/-0.7 micromol/g dry wt) occurred mainly within the media of the preglomerular vasculature and in the areas of interstitial fibrosis in VDN. Extensive renal damages (5 times; 26+/-5% of collapsed-atrophic or sclerotic glomeruli, or glomerular cysts vs. control 5.2+/-0.3%; 28 times; 61+/-12% areas of focal, cortical areas exhibiting interstitial fibrosis per section vs. control 2.2+/-0.6%) were observed histologically. The glomerular filtration rate significantly decreased (880+/-40 vs. control 1,058+/-44 microl.min(-1).g kidney wt(-1)). Albuminuria increased six times (1.6+/-0.4 vs. control 0.27+/-0.04 mg/24 h). There were significant linear relationships between albuminuria and pulse pressure (r2=0.408; n=24) or renal calcium content (r2=0.328; n=24; P<0.05) and between glomerular filtration rate and pulse pressure (r2=0.168; n=27). To our knowledge, this study provides the first evidence of links between both 1) hyperpulsatility and renal dysfunction, and 2) renal calcification and renal dysfunction. Given the increasing frequency of end-stage renal disease, this model could prove useful for preclinical evaluation of drugs that prevent or attenuate hyperpulsatility and/or tissue calcification.

Cardiac vessel density (beta-actin immunolabeling) and angiogenic capacity of coronary artery explants (culture in collagen gel) was determined in hypertrophied heart obtained by exercise training (10 wk) or ANG II infusion for 10 days. A group of rats received ANG II the last 10 days of training. The heart weight index was similarly elevated after exercise, and ANG II-hypertension compared with controls (3.16 +/- 0.09 and 3.11 +/- 0.11 vs. 2.68 +/- 0.08 mg/g, respectively), whereas tail cuff pressure (TCP) increased only in sedentary rats infused with ANG II. Vessel density was increased by 36% in trained rats and reduced by 30% in ANG II-infused rats. The number of sprouts generated by coronary rings was reduced by 50% in ANG II-infused rats and increased by 50% in exercise trained rats compared with controls (35 +/- 4 and 113 +/- 5 vs. 71 +/- 1 sprouts per ring, respectively). Exercise-training partly prevented the hypertensive effect of ANG II (TCP of 141 +/- 5 mmHg), whereas heart weight index (3.66 +/- 0.06 mg/g body wt) was not lowered. Myocardial vessel density was normalized, and sprouting from coronary rings increased by 50% in trained rats infused with ANG II compared with sedentary normotensive rats. Cardiac VEGF (Western blot analysis) was higher in hypertensive rats and not affected by exercise. Facing a similar increase in cardiac mass, intense training, but not ANG II hypertension, is accompanied by an increase in vascular density of the heart. The effect of training is unlikely related to changes in resting VEGF and may represent enhanced angiogenic capacity of the coronary vascular bed.

Schwann cells (Sc), associated axons, and nearby vascular endothelium constitute a functional trilogy of major importance during the development and regrowth of peripheral vascular nerves. The goal of the present study is to provide a technique of triple fluorescence confocal imaging of these cell types along renal preglomerular vessels. We took advantage of a protein S100B/EGFP transgenic mouse to visualize Sc. The endothelium was labeled with an intravenous injection of fluorescently tagged lectin, and after tissue processing, adrenergic nerves were revealed with an antibody against the marker protein synaptophysin. As a validation step, we found that EGFP-positive perivascular cells with prominent cell bodies and extensive, multidirectional cell processes were protein S100B positive. They were identified as Sc and indirectly assumed to be unmyelinated Sc. By contrast, we found strong EGFP expression in proximal epithelial cells and in the epithelium lining thin limbs of Henle. This epithelial fluorescence was not associated with immunoreactive protein S100B and thus corresponded to ectopic EGFP expressions in this mouse strain. Sc were organized in bundles or as a meshwork surrounding the preglomerular vasculature from arcuate arteries to afferent arterioles. No Sc were detected in the medulla. Although most Sc were closely apposed to adrenergic varicosities, many varicosities were not associated with detectable Sc processes. The present technique, and the capacity of confocal microscopy to yield three-dimensional imaging, allow the study of the microtopology of Sc and related sympathetic axons in the renal perivascular interstitium.

Recent clinical studies have suggested that the kidney is susceptible to increased central pulse pressure. We evaluated renal function in a rat model of increased central pulse pressure. The later was induced by arterial elastocalcinosis following one day's treatment with vitamin D and nicotine (VDN, Wistar rats). Rats were studied for up to 90 days following VDN treatment.Central arterial pulse pressure (PP) and renal function - estimated from glomerular filtration rate (GFR) and urine albumin excretion (Ualb) - were measured in anesthetized rats.[GRAPHICS]Central PP increased then stabilized following VDN treatment (x1.5 at Day16 to x1.8 at Day45 and Day90). Urine albumin excretion was significantly higher in VDN than in CTRL rats at Day2 and Dayl 6, and then normalized. GFR decreased at Day 16 and remained lower in VDN than in CTRL rats at Day9O (- 13%).In conclusion, the increase in central PP is associated with renal dysfunction in the VDN rat model, as 3 month's exposure to high PP levels decreased GFR. However, other confounding factors could be involved during the initial phase.

Des observations cliniques recentes suggerent que le rein est vulnerable face a une augmentation de la pression pulsee centrale. L'objectif de cette etude est de produire un modele animal de ce phenomene en evaluant la fonction renale dans un modele murin normotendu d'hyperpulsatilite arterielle due a une elastocalcinose arterielle (rat VDN : une administration de vitamine D + nicotine au Jour 0). Pendant trois mois suite au traitement VDN, la pression pulsee (PP) et la fonction renale, evaluee par le taux de filtration glomerulaire (TFG) et l'excretion urinaire d'albumine (Ualb), sont mesurees sous anesthesie. Jour 2 Jour 16 Jour 45 Jour 90 PP (mmHg) C 27 ± 2 48±5* 54±9* 34 ± 2 V 29±1 33±2 30±3 56±5* log10Ualb C 1,7 ±0,1 2,3 ±0,1 2,1 ±0,1 2,3±0,1 V 2,4±0,2* 2,9±+0,1* 2,3±0,1 2,5±0,1 TFG C nd 966±105 nd 1026±52 (mL. min-1.g rein -1 ) V 685 ± 63* 888± 39* (m ± esm, C = controle, V = VDN, *: P < 0,05 versus controle, nd = non determine) Suite au traitement VDN, la PP augmente puis atteint un plateau. L'albuminurie est fortement augmentee dans la phase precoce suivant le traitement VDN puis se restaure. Le TFG, tres diminue au Jour 16, reste plus faible au Jour 90 (- 13 %) chez les rats VDN. En conclusion, l'hyperpulsatilite semble etre responsable d'une dysfonction renale chez le rat VDN. Trois mois d'exposition a une forte hyperpulsatilite diminuent le TFG. Cependant, dans la phase precoce, d'autres facteurs confondant pourraient etre impliques.

Reversal by the mineralocorticoid receptor antagonist spironolactone on cardiac and renal abnormalities, associated with long-term (since weaning) administration of a high (2 and 8% NaCl chow, HS2 and HS8) sodium diet, was assessed in Sprague-Dawley rats. At the age of 5 mo, spironolactone (20 or 100 mg/kg, gavage) or placebo were given for 14 days to HS2 and HS8 rats. A group fed a regular diet (0.8% NaCl, NS) remained untreated. High sodium intake had no detectable effect on blood pressure; however, cardiac mass index and cross-sectional area of the carotid artery, as well as albuminuria, were increased only in the HS8 group compared with the control group on NS diet. In addition, a marked reduction in glomerular filtration rate (by 40%), associated with a nonproportional fall in renal plasma flow (thus resulting in a decrease in filtration fraction), was observed only in the HS8 group. No change in cardiac and renal fibrosis was detected. Production of the reactive oxygen species (ROS) by aortic tissue was increased in HS8 rats, whereas ROS production by the heart was unaffected. Only the high dose of spironolactone was effective, as it markedly reversed the cardiac hypertrophy and renal hypofiltration associated with the HS8 feeding. The changes were observed in the absence of any effect on systemic blood pressure and production of ROS. These observations favor aldosterone's role in the deleterious effects of marked and prolonged increases in sodium intake.

Specific treatment of age-related aortic wall arteriosclerosis and stiffening is lacking. Because ligands for peroxisome proliferator-activated receptor γ have beneficial effects on the arterial wall in atherosclerosis, via an antiinflammatory mechanism, we investigated whether long-term pioglitazone (Pio) treatment protects against another form of vascular wall disease, arteriosclerosis. We evaluated, in a rat model of elastocalcinotic arteriosclerosis (hypervitaminosis D and nicotine [VDN]), whether Pio (3 mg · kg −1 per day for 1.5 month PO) attenuated arteriosclerosis and its consequences: aortic wall rigidity, increased aortic pulse pressure, and left ventricular hypertrophy. In VDN rats, medial calcification was associated with monocyte/macrophage infiltration and induction of tumor necrosis factor α and interleukin 1β. Pio increased nuclear peroxisome proliferator-activated receptor γ immunostaining in the aortic wall, decreased tumor necrosis factor α ( P <0.05 versus VDN Pio − ), tended to decrease interleukin 1β mRNA expression ( P =0.08 versus VDN Pio − ), blunted aortic wall calcification (271±69, P <0.05 versus VDN Pio − 562±87 μmol · g −1 dry weight) and prevented fragmentation of elastic fibers (segments per 10 000 μm 2 : 8.4±0.3; P <0.05 versus VDN Pio − 10.5±0.6). Pio reduced aortic wall stiffness (elastic modulus/wall stress: 4.8±0.6; P <0.05 versus VDN Pio − 10.0±1.6), aortic pulse pressure (30±2 mm Hg; P <0.05 versus VDN Pio − 39±4) and left ventricular hypertrophy (1.58±0.05 g · kg −1 ; P <0.05 versus VDN Pio − 1.76±0.06). In conclusion, long-term Pio treatment attenuates aortic wall elastocalcinosis and, thus, lowers aortic wall stiffness, aortic pulse pressure, and left ventricular hypertrophy.

BACKGROUND:Nitric oxide is an important vasodilator released from endothelial cells by the calcium-dependent endothelial nitric oxide synthase (NOS). We considered it important to investigate how shear stress/perfusion pressure influenced endothelial cell calcium concentration, nitric oxide release, and autoregulation of the afferent arteriole, since this arteriole controls glomerular filtration rate (GFR) and renin release.METHODS:We used an isolated perfused juxtamedullary nephron preparation and measured calcium with Fura 2, nitric oxide with 4-amino-5 methylamino-2', 7'-difluorescein (DAF-FM) and diameter with an imaging system. A mathematical model was applied to calculate changes in nitric oxide concentration and shear stress/wall tension during perfusion with and without erythrocytes at perfusion pressures varying from 50 to 150 mm Hg.RESULT:Cell-free perfusion increased nitric oxide concentration and abolished autoregulation; addition of erythrocytes or l-arginine analog N-nitro-l-arginine methyl ester (L-NAME) decreased nitric oxide concentration and reinstated autoregulation. Elevated perfusion pressure/elevated shear stress increased nitric oxide release and surprisingly decreased the endothelial cell calcium concentration, with perfusion pressure increase from 50 to 150 mm Hg, using blood perfusion endothelial calcium concentration decreased from 186 +/- 39 to 76 +/- 25 nmol/L and with cell-free perfusion from 116 +/- 33 to 56 +/- 21 nmol/L.CONCLUSION:Nitric oxide scavenging by erythrocytes has a high impact on arteriolar nitric oxide concentration and autoregulatory response. Nitric oxide measurements in endothelial cells of the afferent arteriole showed that increased perfusion pressure/shear stress increased nitric oxide release, while simultaneously endothelial cell calcium concentration decreased, possibly indicating a feedback control of this calcium by nitric oxide release.

High sodium intake is associated with the development of cardiac hypertrophy in man and rats independently of the rise of blood pressure. In addition, the reduced capillary density observed in striated muscle of rats fed a high salt diet suggests that angiogenesis is altered. The aim of the present experiments was to investigate the angiogenic capacities of vascular rings isolated from the aorta and the coronary artery of rats. Vascular rings (external diameter of 1 669 +/- 9 and 323 +/- 26 microm for aorta and coronary artery. respectively) are cultured in a three-dimensional collagen type I lattice and a standard medium (DMEM+HAMF12) containing 152 mM of sodium. Sodium ion associated with chloride or citrate is added to the standard medium to achieve a final concentration of sodium of 160 and 176 mM. The role of sodium-proton exchanger is evaluated through the addition of amiloride to the culture medium. Sprouts formed from vascular explants are counted every second day until days 8 to 10. Kinetics of new vessels formation and the number of sprouts were similar in aortic and coronary rings (83 +/- 5 and 95 +/- 5 sprouts, respectively). Elevation of sodium chloride concentration inhibits by 50 to 80% the neo-vessels formation in both the aorta and the coronary artery. Anti-angiogenic effect of the high sodium medium was not affected when citrate was substituted to chloride. Amiloride (3.10(-5) M) reduced the number of sprouts formed in the standard medium; however, it counteracted the anti-angiogenic effect of elevated sodium concentration. These results indicate that high extracellular concentration of sodium and not chloride anion is accompanied by a deleterious effect on angiogenic capacities of cultured aortic and coronary artery rings through modifications of trans-membrane sodium exchanges.

Major aspects of renal function such as glomerular filtration, blood flow autoregulation, tubular reabsorption, and medullary concentrating ability rely on an exquisite paracrine-autocrine control of renal microvascular resistances, and on the coordinated network behavior of the vasculature (,). However, vascular and microvascular networks of mammalian kidneys are mostly inaccessible to microscopic observation, which greatly limits the direct study of renal microcirculation. There have been ongoing efforts to overcome this technical difficulty. In the seventies, glomerular microcirculation was observed in adult renal tissue grafted into a chamber in the rabbit ear (), and in neonatal renal tissue grafted into a chamber in the hamster cheek pouch (,). At the same time, micropuncture techniques were applied to the Munich-Wistar rat, which is endowed with surface glomeruli. This greatly increased our knowledge of single-nephron dynamics and filtration (,). By the mid-eighties, three additional techniques were implemented for real-time microscopic evaluation of single renal arteriolar dynamics (). Steinhausen et al. () took advantage of the tubular destruction provoked by hydronephrosis to visualize all segments of the renal microvasculature. Using an adaptation of the tubular microperfusion techniques (), Edwards () and Osgood et al. () perfused single glomerular arterioles of rabbits and dogs in vitro. Peti-Peterdi ( Chapter 8) and Pallone ( Chapter 30) present further refinements of this elegant technique elsewhere in this book.

Intrarenal distribution and function(s) of the peripheral benzodiazepine receptor (PBR) remain uncertain. The goals of this study were to (1) develop a specific anti-rat PBR antibody and (2) map intrarenal immunoreactive PBR (irPBR) in untreated rats and in rats that received chronic angiotensin II infusion (200 ng/kg per min, subcutaneously, 17 d). A polyclonal rabbit antibody was raised against the C-terminal end of rat PBR (aa 159 to 169). The antibody specifically recognized a single 18-kD protein in whole kidney extracts, and confocal microscopy showed exclusive mitochondrial localization of irPBR in cultured rat glial C6 cells. In control rats, irPBR was found along thick ascending limbs of Henle's loops, including the macula densa area, along distal tubules, and along collecting ducts. Vascular smooth-muscle cells were PBR-positive. General irPBR distribution was unaffected by angiotensin II treatment (systolic BP, 205 +/- 9 mmHg). However, irPBR appeared in parietal glomerular epithelial cells, atrophic proximal tubules, and infiltrating mononuclear cells. In conclusion, the results suggest previously unsuspected roles of PBR in the control of glomerular dynamics and in proximal tubular injury/repair processes.