Urinary mitochondrial DNA copy number identifies renal mitochondrial injury in renovascular hypertensive patients undergoing renal revascularization: A Pilot Study.

AIMS: Patients with renovascular hypertension (RVH) exhibit elevated urinary mtDNA copy numbers, considered to constitute surrogate markers of renal mitochondrial injury. The modest success of percutaneous transluminal renal angioplasty (PTRA) in restoring renal function in RVH has been postulated to be partly attributable to acute reperfusion injury. We hypothesized that mitoprotection during revascularization would ameliorate PTRA-induced renal mitochondrial injury, reflected in elevated urinary mtDNA copy numbers and improve blood pressure and functional outcomes 3 months later. METHODS: We prospectively measured urinary copy number of the mtDNA genes COX3 and ND1 using qPCR in RVH patients before and 24 hrs after PTRA, performed during IV infusion of vehicle (n = 8) or the mitoprotective drug elamipretide (ELAM, 0.05 mg/kg/h, n = 6). Five healthy volunteers (HV) served as controls. Urinary mtDNA levels were also assessed in RVH and normal pigs (n = 7 each), in which renal mitochondrial structure and density were studied ex-vivo. RESULTS: Baseline urinary mtDNA levels were elevated in all RVH patients vs HV and directly correlated with serum creatinine levels. An increase in urinary mtDNA 24 hours after PTRA was blunted in PTRA+ELAM vs PTRA+Placebo. Furthermore, 3-months after PTRA, systolic blood pressure decreased and estimated glomerular filtration rate increased only in ELAM-treated subjects. In RVH pigs, mitochondrial damage was observed using electron microscopy in tubular cells and elevated urinary mtDNA levels correlated inversely with renal mitochondrial density. CONCLUSIONS: PTRA leads to an acute rise in urinary mtDNA, reflecting renal mitochondrial injury that in turn inhibits renal recovery. Mitoprotection might minimize PTRA-associated mitochondrial injury and improve renal outcomes after revascularization.

Micro-RNAS Regulate Metabolic Syndrome-induced Senescence in Porcine Adipose Tissue-derived Mesenchymal Stem Cells through the P16/MAPK Pathway.

Mesenchymal stem cells (MSCs) constitute an important repair system, but may be impaired by exposure to cardiovascular risk factors. Consequently, adipose tissue-derived MSCs from pigs with the metabolic syndrome (MetS) show decreased vitality. A growing number of microRNAs (miRNAs) are recognized as key modulators of senescence, but their role in regulating senescence in MSC in MetS is unclear. We tested the hypothesis that MetS upregulates in MSC expression of miRNAs that can serve as post-transcriptional regulators of senescence-associated (SA) genes. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet ( n = 6 each). Next-generation miRNA sequencing (miRNA-seq) was performed to identify miRNAs up-or down-regulated in MetS-MSCs compared with Lean-MSCs. Functional pathways of SA genes targeted by miRNAs were analyzed using gene ontology. MSC senescence was evaluated by p16 and p21 immunoreactivity, H2AX protein expression, and SA-ß-Galactosidase activity. In addition, gene expression of p16, p21, MAPK3 (ERK1) and MAPK14, and MSC migration were studied after inhibition of SA-miR-27b. Senescence biomarkers were significantly elevated in MetS-MSCs. We found seven upregulated miRNAs, including miR-27b, and three downregulated miRNAs in MetS-MSCs, which regulate 35 SA genes, particularly MAPK signaling. Inhibition of miR-27b in cultured MSCs downregulated p16 and MARP3 genes, and increased MSC migration. MetS modulates MSC expression of SA-miRNAs that may regulate their senescence, and the p16 pathway seems to play an important role in MetS-induced MSC senescence.

Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis.

PURPOSE: Diets rich in plant-derived polyphenols such as olive oil (OO) and/or catechins such as epigallocatechin 3-gallate (EGCG) have been shown to reduce the incidence of cardiovascular diseases, potentially by improving endothelial function, an important surrogate for atherosclerosis. The possible augmentation of endothelial function with the combined efforts of OO and EGCG is intriguing, yet unknown. METHODS: Eighty-two patients with early atherosclerosis (presence of endothelial dysfunction) were enrolled in this double-blind, randomized trial with 52 completing the study. The aim of the study was to compare the effect of a daily intake of 30 ml simple OO, with 30 ml of EGCG-supplemented OO, on endothelial function as well as on inflammation and oxidative stress after a period of 4 months. Endothelial function was assessed noninvasively via peripheral arterial tonometry (Endo-PAT®). RESULTS: After 4 months, when OO and EGCG-supplemented OO groups were combined, OO significantly improved endothelial function (RHI, 1.59 ± 0.25-1.75 ± 0.45; p < 0.05). However, there were no significant differences in results between the two olive oil groups. Interestingly, with OO supplementation there was a significant reduction in inflammatory parameters: sICAM (196 to 183 ng/mL, p = < 0.001); white blood cells (WBCs) (6.0 × 109/L-5.8 × 109/L, p < 0.05); monocytes (0.48 × 109/L to 0.44 × 109/L, p = 0.05); lymphocytes (1.85 × 109/L to 1.6 × 109/L, p = 0.01); and platelets (242-229 × 109/L, p = 0.047). CONCLUSIONS: Improvement in endothelial dysfunction in patients with early atherosclerosis in association with significant reduction in leukocytes may suggest an important role of early cellular inflammatory mediators on endothelial function. The current study supports one potential mechanism for the role of olive oil, independent of EGCG, modestly supplemented to a healthy cardiovascular diet.

Circulating humanin levels are associated with preserved coronary endothelial function.

Humanin is a small endogenous antiapoptotic peptide, originally identified as protective against Alzheimer's disease, but subsequently also found on human endothelium as well as carotid artery plaques. Endothelial dysfunction is a precursor to the development of atherosclerotic plaques, which are characterized by a highly proinflammatory, reactive oxygen species, and apoptotic milieu. Previous animal studies demonstrated that humanin administration may improve endothelial function. Thus the aim of this study was to test the hypothesis that patients with coronary endothelial dysfunction have reduced systemic levels of humanin. Forty patients undergoing coronary angiography and endothelial function testing were included and subsequently divided into two groups based on coronary blood flow (CBF) response to intracoronary acetylcholine (normal ≥ 50% increase from baseline, n = 20 each). Aortic plasma samples were obtained at the time of catheterization for the analysis of humanin levels and traditional biomarkers of atherosclerosis including C-reactive protein, Lp-Pla(2), and homocysteine. Baseline characteristics were similar in both groups. Patients with coronary endothelial dysfunction (change in CBF = -33 ± 25%) had significantly lower humanin levels (1.3 ± 1.1 vs. 2.2 ± 1.5 ng/ml, P = 0.03) compared with those with normal coronary endothelial function (change in CBF = 194 ± 157%). There was a significant and positive correlation between improved CBF and humanin levels (P = 0.0091) not seen with changes in coronary flow reserve (P = 0.76). C-reactive protein, Lp-Pla(2), and homocysteine were not associated with humanin levels. Thus we observed that preserved human coronary endothelial function is uniquely associated with higher systemic humanin levels, introducing a potential diagnostic and/or therapeutic target for patients with coronary endothelial function.

Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model.

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

Low vasa vasorum densities correlate with inflammation and subintimal thickening: potential role in location--determination of atherogenesis.

OBJECTIVES: To assess the role of coronary vasa vasorum (VV) spatial distribution in determining the location of early atherosclerotic lesion development. METHODS AND RESULTS: Six, 3-month-old, female, crossbred swine were fed 2% high-cholesterol (HC) diet for 3 months prior to euthanasia. Six other pigs were fed normal diet (N) for the entire 6 months. Right coronary arteries were harvested and scanned intact with micro-CT (20mum cubic-voxel-size). After scanning, randomly selected cross-sectional histological sections were stained for nuclear-factor kappaB (NF-kappaB), hypoxia-inducible factor-1alpha (HIF-1alpha), macrophages, von-Willebrand-factor, dihydroethidium (DHE), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The number of positive stained cells, as well as intima-to-media ratio, were compared with VV density (#/mm(2)) obtained from micro-CT images (which closely matched the location of the histological sections) in each of four equal quadrants of the coronary vessel wall. In normal, as well as HC pigs, the number of NF-kappaB (r=0.73 and 0.70), HIF-1alpha (r=0.74 and 0.77), TNF-alpha (r=0.58 and 0.72) and IL-6 (r=0.70 and 0.72) positive cells as well as the expression of DHE (Kendall tau coefficient -0.64 and -0.63) inversely correlated with VV density. In HC the VV density also inversely correlated with intima/media ratios (r=0.65). CONCLUSIONS: Our data suggest that low VV density territories within the coronary vessel wall are susceptible to hypoxia, oxidative stress and microinflammation and may therefore be starting points of early atherogenesis.

Effect of gestational hypercholesterolaemia on omental vasoreactivity, placental enzyme activity and transplacental passage of normal and oxidised fatty acids.

OBJECTIVE: Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring. DESIGN: Comparison between normo- and hypercholesterolaemic mothers (n = 36 each) and their children. SETTING: Obstetric wards, hospitals of the University of Naples and Regione Campania. POPULATION: Healthy primiparas delivering by caesarean section. METHODS: Biochemical measurements of oxidative stress and serum leptin in cord plasma and placenta, immunochemistry of placenta microvessels, and vasoreactivity studies were performed. MAIN OUTCOME MEASURES: Oxidative status (i.e. lipid composition and content of oxidised fatty acids, activity of pro- and antioxidant enzymes, immunohistochemical presence of oxidation-specific epitopes) in maternal and cord blood and in placental tissue, as well as vascular reactivity in omental arteries. RESULTS: Hypercholesterolaemia during pregnancy was associated with extensive changes in fatty acid composition of both maternal and cord blood lipids, sufficient to alter vasoreactivity of omental vessels. Results also indicated that the placenta is not only subject to substantial oxidative stress, but that it may further increase fetal oxidative stress through changes of pro- and antioxidant enzyme activities. CONCLUSIONS: The placenta plays an important role in both transmitting and enhancing pathogenic effects of gestational hypercholesterolaemia.

The importance of reendothelialization after arterial injury.

Atherosclerosis is still the principal cause of morbidity and mortality in Western countries and although a significant progress has been made in the understanding of its pathophysiology, the determinants of atherosclerotic plaque instability are still poorly understood. The endothelium plays a pivotal role for the development, progression, and complication of atherosclerosis. Endothelial dysfunction is widely recognized as one of the early alteration in the vessel wall preceding the development of the plaque. However, considering the plethora of vascular functions which are regulated by endothelium, it plays a pivotal role throughout the atherosclerotic process and indeed the loss of endothelial cells, leading to plaque denudation, is one of the main causes of plaque complication. It is therefore conceivable that the maintenance of the endothelial layer physical continuity and function is crucial for the prevention of atherosclerosis. In the presence of cardiovascular risk factors, endothelial cells are continuously injured and repaired by the proliferation of resident cells and circulating endothelial progenitor cells. Indeed the number of circulating endothelial progenitor cells has been identified as an predictor of cardiovascular events. The increase in bone marrow release of endogenous progenitor cells or the enhancement of their homing in arterial denuded sites or in intravascular stent surface, are currently pursued to reduce atherosclerosis development/complication and intrastent restenosis, respectively. However, some challenges may arise from procedures enhancing endothelialization, including unwanted angiogenesis which may favor neoplasia progression and paradoxically atherosclerotic plaque expansion and complication.

In vivo veritas: thrombosis mechanisms in animal models.

Experimental models have enhanced our understanding of atherothrombosis pathophysiology and have played a major role in the search for adequate therapeutic interventions. Various animal models have been developed to simulate thrombosis and to study in vivo parameters related to hemodynamics and rheology that lead to thrombogenesis. Although no model completely mimics the human condition, much can be learned from existing models about specific biologic processes in disease causation and therapeutic intervention. In general, large animals such as pigs and monkeys have been better suited to study atherosclerosis and arterial and venous thrombosis than smaller species such as rats, rabbits, and dogs. On the other hand, mouse models of arterial and venous thrombosis have attracted increasing interest over the past two decades, owing to direct availability of a growing number of genetically modified mice, improved technical feasibility, standardization of new models of local thrombosis, and low maintenance costs. To simulate rupture of an atherosclerotic plaque, models of arterial thrombosis often involve vascular injury, which can be achieved by several means. There is no animal model that is sufficiently tall, that can mimic the ability of humans to walk upright, and that possesses the calf muscle pump that plays an important role in human venous hemodynamics. A number of spontaneous or genetically engineered animals with overexpression or deletion of various elements in the coagulation, platelet, and fibrinolysis pathways are now available. These animal models can replicate important aspects of thrombosis in humans, and provide a valuable resource in the development of novel concepts of disease mechanisms in human patients.

Vascular wall elasticity measurement by magnetic resonance imaging.

The goal of this current study was to determine whether an MRI-based elastography (MRE) method can visualize and assess propagating mechanical waves within fluid-filled vessels and to investigate the feasibility of measuring the elastic properties of vessel walls and quantitatively assessing stenotic lesions by using MRE. The ability to measure the Young's modulus-wall thickness product was tested using a thin-walled latex vessel model. Also tested in vessel models was the ability to quantitate the degree of stenosis by measuring transmitted and reflected mechanical waves. This method was then applied to ex vivo porcine models and in vivo human arteries to further test its feasibility. The results provide preliminary evidence that MRE can be used to quantitatively assess the stiffness of blood vessels, and provide a non-morphologic method to measure stenosis. With further development, it is possible that the method can be implemented in vivo.

Mild renal insufficiency is associated with reduced coronary flow in patients with non-obstructive coronary artery disease.

Patients with chronic kidney disease (CKD) have increased risk for cardiovascular events. However, the association between these pathophysiological processes is unclear. Therefore, this study was designed to determine the association between early CKD and coronary microvascular disease in patients with normal or mildly diseased coronary arteries. A total of 605 patients with normal or mildly diseased coronary arteries based on angiography underwent coronary flow reserve (CFR) evaluation using intracoronary adenosine. Patients were divided based on glomerular filtration rate (GFR). CKD was defined as calculated GFR<60 ml/min/1.73 m(2). Patients with normal GFR (> or =60 ml/min/1.73 m(2), n=481) had higher CFR compared to those with CKD (n=124, CFR=3.0+/-0.8 vs 2.6+/-0.6, P<0.001, respectively). Patients with abnormal GFR were more likely to be older and of female gender, with greater prevalence of hypertension. Multiple logistic regression analysis adjusted for the aforementioned risk factors further supported the observed relationship. The current study shows that reduced renal function is associated with attenuated coronary vasodilator capacity in patients without obstructive coronary artery disease. The correlation between low GFR and reduced CFR may suggest parallel alterations in the renal and coronary microcirculation at the early stage of disease. Impairment in both microcirculatory beds may reflect an unmeasured risk factor induced by blunted renal function and add a burden to the increased propensity for cardiovascular events in CKD.

Microarray analysis: a novel research tool for cardiovascular scientists and physicians.

The massive increase in information on the human DNA sequence and the development of new technologies will have a profound impact on the diagnosis and treatment of cardiovascular diseases. The microarray is a micro-hybridisation based assay. The filter, called microchip or chip, is a special kind of membrane in which are spotted several thousands of oligonucleotides of cDNA fragments coding for known genes or expressed sequence tags. The resulting hybridisation signal on the chip is analysed by a fluorescent scanner and processed with a software package utilising the information on the oligonucleotide or cDNA map of the chip to generate a list of relative gene expression. Microarray technology can be used for many different purposes, most prominently to measure differential gene expression, variations in gene sequence (by analysing the genome of mutant phenotypes), or more recently, the entire binding site for transcription factors. Measurements of gene expression have the advantage of providing all available sequence information for any given experimental design and data interpretation in pursuit of biological understanding. This research tool will contribute to radically changing our understanding of cardiovascular diseases.

Renal blood flow in hypercholesterolemic pigs is increased by chronic antioxidant treatment.

Oxygen radical species can influence vascular tone, and antioxidants may have hemodynamic and vascular effects. To date, the vascular effects of chronic intervention with a combination of antioxidant vitamins E and C on renal blood flow (RBF) in hypercholesterolemia (which increases oxidative stress) have not been fully defined. The aim of this intervention study was to explore the involvement of increased oxidative stress in pig RBF disturbance by using chronic dietary antioxidant vitamin intervention. Responses of RBF to the acetylcholine (Ach) were measured in vivo using electron beam computed tomography (EBCT). Acetylcholine significantly increased RBF in normal and hypercholesterolemic + vitamins (P < 0.05 for both), but not in hypercholesterolemic pigs (P=0.1). In normocholesterolemic + vitamins pigs, Ach infusion did not induce any further increase in RBF, but RBF was similar to that observed in normal and hypercholesterolemic + vitamins under the same conditions, and tended to be higher than in hypercholesterolemic pigs (P=0.06). Thus, antioxidants improve RBF in hypercholesterolemic pigs and this effect may help to prevent renal diseases and hypertension in animals.

Electron beam computerized tomography assessment of in vivo single kidney glomerular filtration rate and tubular dynamics during chronic partial unilateral ureteral obstruction in the pig.

PURPOSE: The assessment of hydronephrosis due to chronic partial ureteral obstruction is controversial. We determined whether a new radiographic technique for assessing kidney function, electron beam computerized tomography (CT), can detect altered renal physiology due to chronic partial ureteral obstruction. We also compared and contrasted electron beam CT with standard well tempered diuretic mercaptoacetyltriglycine (MAG-3) urography. MATERIALS ANDS METHODS: Six pigs underwent creation of unilateral partial ureteral occlusion or sham operation. Three weeks after surgery diuretic enhanced MAG-3 renal scan was done and 48 hours later contrast enhanced electron beam CT was performed. RESULTS: Mean differential function plus or minus standard error of mean of the obstructed kidney was 5.6% +/- 2.4% on MAG-3 renography. In contrast, electron beam CT revealed significantly preserved mean renal function at 24.5% +/- 2.7% (p <0.01). Electron beam CT analysis of tubular function revealed persistent glomerular filtration and filtrate flow through the proximal tubules and loop of Henle with a selective decrease in distal tubular flow, which were findings suggestive of proximal tubular sparing that were not demonstrated by nuclear renography. CONCLUSIONS: Renal function on MAG-3 renography is primarily determined by measuring kidney perfusion and tubular secretion of the isotope. In contrast, electron beam CT determines renal function via quantifying the in vivo single kidney glomerular filtration rate and by assessing renal tubular function. This study documents that electron beam CT of differential renal function is significantly different from that of MAG-3 renography. To our knowledge which of these 2 radiographic studies is most clinically applicable is unknown to date.

Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia.

OBJECTIVE: Experimental hypercholesterolemia is associated with vasa vasorum neovascularization, unknown to occur before or after initial lesion formation. Thus, this study was performed to determine the temporal course of neovascularization of coronary vasa vasorum in relation to endothelial dysfunction, a hallmark of early atherosclerosis. METHODS: Female domestic pigs were fed a normal diet (Group 1), a hypercholesterolemic diet for 2 and 4 weeks (Group 2), or a hypercholesterolemic diet for 6 and 12 weeks (Group 3). In vitro analysis of relaxation response to bradykinin served as an index for epicardial endothelial function. Spatial pattern and density of coronary vasa vasorum were assessed by three-dimensional microscopic computed tomography. RESULTS: Relaxation response of coronary arteries to bradykinin was normal in both Group 1 (93+/-6%) and Group 2 (89+/-7%) but impaired in Group 3 (71+/-11%; P<0.05 vs. Group 1 and 2). In contrast, density of coronary vasa vasorum was significantly higher in both Group 2 (4.88+/-2.45 per-mm(2)) and Group 3 (4.50+/-1.37 per-mm(2)) compared to Group 1 (2.97+/-1.37 per-mm(2); P<0.05 vs. Group 2 and 3). CONCLUSION: This study demonstrates that coronary vasa vasorum neovascularization occurs within the first weeks of experimental hypercholesterolemia and prior to the development of endothelial dysfunction of the host vessel, suggesting a role for vasa vasorum neovascularization in the initial stage of atherosclerotic vascular disease.

Coronary microvascular functional reserve: quantification of long-term changes with electron-beam CT preliminary results in a porcine model.

PURPOSE: To evaluate the ability of electron-beam computed tomography (CT) to help quantify long-term changes in coronary microvascular functional reserve in a porcine model. MATERIALS AND METHODS: Electron-beam CT-based intramyocardial blood volume and perfusion and Doppler ultrasonography (US)-based intracoronary blood flow were obtained in 13 pigs at baseline and again 3 months later. Measurements were obtained at rest and after the administration of adenosine. The short-term variation during 30 minutes of electron-beam CT measurements was assessed in nine additional pigs. RESULTS: Short-term variation of blood volume and perfusion averaged 8% and 9%, respectively, and was similar for both weight groups at rest and after adenosine administration. At rest, intracoronary blood flow, blood volume, and perfusion remained unchanged from baseline to follow-up. Long-term increases (percentage change with adenosine relative to that at rest) in blood volume and perfusion reserves were consistent with increasing intracoronary blood flow reserves. Despite these long-term changes in intracoronary blood flow, blood volume, and perfusion, the blood volume-to-perfusion relationship suggests a similar blood volume distribution among different microvascular functional components in normal porcine myocardium at both weight groups. CONCLUSION: Electron-beam CT may be of value for quantifying long-term changes in intramyocardial microvascular function.

Functional assessment of the circulation of the single kidney.

Functional alterations in the renal circulation that can contribute to abnormal renal perfusion have been demonstrated in various models of renal injury. To detect impairments in renal vascular function, renal flow reserve can be determined by repeated measurements of renal blood flow (RBF) during pharmacological challenge with short-acting vasodilators that should increase RBF in kidneys that are not severely damaged structurally. Among the invasive techniques for such measurements, the most readily available is probably the intravascular Doppler, which can be employed during renal angiography for rapid evaluation of changes in RBF during intrarenal injections of vasoactive substances. High-resolution tomographic imaging techniques, like electron-beam x-ray computed tomography, further offer the potential for noninvasive measurements of renal parenchymal perfusion and function, in association with either intrarenal or systemic injections of vasoactive substances. Acetylcholine is a potent short-acting renal vasodilator that can be useful to assess the response of the renal microcirculation, define renal flow reserve, and examine the endothelium-dependent responses of RBF. Such assessments of the function of the renal circulation can assist in evaluation of patients with systemic or renal disease for early detection and monitoring of renovascular injury.

Noninvasive measurement of concurrent single-kidney perfusion, glomerular filtration, and tubular function.

To assess the reliability of electron beam computed tomography (EBCT), measurements of single-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and intratubular contrast medium concentration (ITC) of radiographic contrast media were quantified in anesthetized pigs before and after acetylcholine-induced vasodilation and diuresis. EBCT measurements were compared with those obtained with intravascular Doppler and inulin clearance. The capability of EBCT to detect chronic changes in single-kidney function was evaluated in pigs with unilateral renal artery stenosis, and their long-term reproducibility in normal pigs was studied repeatedly at 1-mo intervals. EBCT-RBF (ml/min) correlated with Doppler-RBF as RBF(EBCT) = 45 + 1.07 * RBF(Doppler), r = 0.81. EBCT-GFR (ml/min) correlated with inulin clearance as GFR(EBCT) = 11.7 + 1.02 * GFR(inulin), r = 0.80. During vasodilation, RBF and GFR increased, whereas ITC decreased along the nephron. In renal artery stenosis, single-kidney GFR decreased linearly with the degree of stenosis, and ITC increased along the nephron, indicating increased fluid reabsorption. EBCT-RBF, GFR, and ITC were similar among repeated measurements. This approach might be invaluable for simultaneous quantification of regional hemodynamics and function in the intact kidneys, in a manner potentially applicable to humans.