Monitoring kidney size to interpret MRI-based assessment of renal oxygenation in acute pathophysiological scenarios.

AIM: Tissue hypoxia is an early key feature of acute kidney injury. Assessment of renal oxygenation using magnetic resonance imaging (MRI) markers T2 and T2 * enables insights into renal pathophysiology. This assessment can be confounded by changes in the blood and tubular volume fractions, occurring upon pathological insults. These changes are mirrored by changes in kidney size (KS). Here, we used dynamic MRI to monitor KS for physiological interpretation of T2 * and T2 changes in acute pathophysiological scenarios. METHODS: KS was determined from T2 *, T2 mapping in rats. Six interventions that acutely alter renal tissue oxygenation were performed directly within the scanner, including interventions that change the blood and/or tubular volume. A biophysical model was used to estimate changes in O2 saturation of hemoglobin from changes in T2 * and KS. RESULTS: Upon aortic occlusion KS decreased; this correlated with a decrease in T2 *, T2 . Upon renal vein occlusion KS increased; this negatively correlated with a decrease in T2 *, T2 . Upon simultaneous occlusion of both vessels KS remained unchanged; there was no correlation with decreased T2 *, T2 . Hypoxemia induced mild reductions in KS and T2 *, T2 . Administration of an X-ray contrast medium induced sustained KS increase, with an initial increase in T2 *, T2 followed by a decrease. Furosemide caused T2 *, T2 elevation and a minor increase in KS. Model calculations yielded physiologically plausible calibration ratios for T2 *. CONCLUSION: Monitoring KS allows physiological interpretation of acute renal oxygenation changes obtained by T2 *, T2 . KS monitoring should accompany MRI-oximetry, for new insights into renal pathophysiology and swift translation into human studies.

Pharmacodynamic assessment of diuretic efficacy and braking in a furosemide continuous infusion model.

INTRODUCTION: Diuretic failure is a potential life-ending event but is unpredictable and poorly understood. The objectives of this study were to evaluate pharmacodynamic markers of furosemide-induced diuresis and to investigate mechanisms of diuretic braking in dogs receiving constant rate infusion (CRI) of furosemide. ANIMALS: Six healthy male dogs. METHODS: Raw data and stored samples from one arm of a previously published study were further analyzed to mechanistically investigate causes of diuretic braking in these dogs. Urine volume was recorded hourly during a 5-h furosemide CRI. Urine and blood samples were collected hourly to measure serum and urine electrolytes, urine aldosterone, and plasma and urine furosemide. Serum electrolyte fractional excretion was calculated. Urine sodium concentration was indexed to urine potassium (uNa:uK) and urine furosemide (uNa:uFur) concentrations, plasma furosemide concentration was indexed to urine furosemide concentration (pFur:uFur), and urine aldosterone was indexed to urine creatinine (UAldo:C). Temporal change and the relationship to urine volume were evaluated for these measured and calculated variables. RESULTS: Urine volume was significantly correlated with urine electrolyte amounts and with uNa:uK. The ratio of pFur:uFur decreased during the infusion, whereas furosemide excretion was unchanged. CONCLUSIONS: There was a strong relationship between urine volume and absolute urine electrolyte excretion. Urine volume was strongly correlated to uNa:uK, giving it potential as a spot indicator of urine production during diuresis. The decrease in uNa:uK over time during the infusion is consistent with mineralocorticoid modification of urinary electrolyte excretion, supporting renin-angiotensin-aldosterone activation as a cause of diuretic braking in this model.

Assessment of temperature-induced hERG channel blockade variation by drugs.

Drug-induced QT prolongation has been reported in humans and animals. This potentially lethal effect can be induced by drugs interacting with a cardiac potassium channel, namely hERG (human ether-a go-go-related gene) leading to arrhythmia or torsade de pointes (TdP). Hence, in vitro evaluation of therapeutics for their effects on the rapid delayed rectifier current (IKr) mediated by the K(+) ion channel encoded by hERG is a valuable tool for identifying potential arrhythmic side effects during drug safety testing. Our objective was to evaluate the temperature-induced hERG channel blockade variation by human and veterinary drugs using the IonFlux 16 system. A panel of eight drugs was tested for IKr inhibition at both ambient (23 °C) and physiological (37 °C) temperatures at various concentrations using IonFlux 16, an automated patch clamp system. Our results established that both amiodarone (IC(50) = 0.56 µM at 23 °C and 0.30 µM at 37 °C) and ß-estradiol (IC(50) = 24.72 µM at 23 °C and 8.17 µM at 37 °C) showed a dose-dependent IKr blockade with a higher blockade at 37 °C. Whereas, blockade of IKr by both ivermectin (IC(50) = 12.52 µM at 23 °C and 24.41 µM at 37 °C) and frusemide (IC(50) = 12.58 µM at 23 °C and 25.55 µM at 37 °C) showed a dose-dependent IKr blockade with a lower blockade at 37 °C. Gentamicin, enrofloxacin, xylazine and albendazole did not block IKr at both the assessed temperatures. Collectively, these results demonstrate that the effect of temperature variation should be taken into consideration during the evaluation of test drugs for their hERG channel blockade potential.

Measurement of renal tissue oxygenation with blood oxygen level-dependent MRI and oxygen transit modeling.

Blood oxygen level-dependent (BOLD) MRI data of kidney, while indicative of tissue oxygenation level (Po2), is in fact influenced by multiple confounding factors, such as R2, perfusion, oxygen permeability, and hematocrit. We aim to explore the feasibility of extracting tissue Po2 from renal BOLD data. A method of two steps was proposed: first, a Monte Carlo simulation to estimate blood oxygen saturation (SHb) from BOLD signals, and second, an oxygen transit model to convert SHb to tissue Po2. The proposed method was calibrated and validated with 20 pigs (12 before and after furosemide injection) in which BOLD-derived tissue Po2 was compared with microprobe-measured values. The method was then applied to nine healthy human subjects (age: 25.7 ± 3.0 yr) in whom BOLD was performed before and after furosemide. For the 12 pigs before furosemide injection, the proposed model estimated renal tissue Po2 with errors of 2.3 ± 5.2 mmHg (5.8 ± 13.4%) in cortex and -0.1 ± 4.5 mmHg (1.7 ± 18.1%) in medulla, compared with microprobe measurements. After injection of furosemide, the estimation errors were 6.9 ± 3.9 mmHg (14.2 ± 8.4%) for cortex and 2.6 ± 4.0 mmHg (7.7 ± 11.5%) for medulla. In the human subjects, BOLD-derived medullary Po2 increased from 16.0 ± 4.9 mmHg (SHb: 31 ± 11%) at baseline to 26.2 ± 3.1 mmHg (SHb: 53 ± 6%) at 5 min after furosemide injection, while cortical Po2 did not change significantly at ∼58 mmHg (SHb: 92 ± 1%). Our proposed method, validated with a porcine model, appears promising for estimating tissue Po2 from renal BOLD MRI data in human subjects.

Echocardiographic assessment of hemodynamic changes produced by two methods of inducing fluid deficit in dogs.

BACKGROUND: Hydration status is important to the cardiovascular system because of its effects on preload. Decreased preload can alter echocardiographic measurements of systolic and diastolic function, potentially confounding interpretation of results. HYPOTHESIS/OBJECTIVES: Mild fluid deficits are associated with measurable echocardiographic changes that are validated by physical and biochemical markers of decreased intravascular volume. ANIMALS: Twenty-five healthy staff/student-owned dogs with no evidence of cardiac or renal disease. METHODS: Prospective, interventional laboratory study. Dogs were randomly assigned to water deprivation (WD) alone for 8 hours (n = 13) or to furosemide treatment (FTx, 2.5mg/kg IV) followed by WD for 8 hours (n = 12). Echocardiograms, biochemical sampling, and physical parameters were measured at baseline, and after 4 and 8 hours. RESULTS: Both protocols induced fluid deficit as indicated by significant (P < .00001) decreases in weight at 4 hours (WD, 1.1%; FTx, 3.7%) and 8 hours (WD, 2.7%; FTx, 4.5%). Furosemide significantly decreased left ventricular end-diastolic volume (54.3 +/- 19.3-42.1 +/- 17.3 mL, P < .0001), cardiac index (4.2 +/- 1.1-2.9 +/- 0.9 L/min/M2, P < .0001), and mitral valve E wave velocity (0.79 +/- 0.2-0.66 +/- 0.2 m/s, P = .0004). These changes were accompanied by significant increases in blood urea nitrogen concentration (13.8 +/- 2.6-14.8 +/- 2.7 mg/dL, P = .04), vasopressin concentration (1.4 +/- 1.2-3.3 +/- 1.9 pg/mL, P = .045), and PCV (49.8 +/- 4.5-53.2 +/- 6.5%, P = .006). Effects of water deprivation alone were similar, but less pronounced. CONCLUSIONS AND CLINICAL IMPORTANCE: Mild fluid deficits have measurable hemodynamic effects in dogs. Hydration status should be considered when evaluating cardiac function by echocardiogram.

Assessment of diuretic effects and changes in plasma aldosterone concentration following oral administration of a single dose of furosemide or azosemide in healthy dogs.

OBJECTIVE: To determine the diuretic effects and changes in plasma aldosterone concentration (PAC) following oral administration of a single dose of furosemide or azosemide in healthy dogs. ANIMALS: 8 mixed-breed dogs. PROCEDURES: A single dose of furosemide (2 mg/kg), azosemide (1, 5, or 10 mg/kg), or placebo (bifidobacterium [1 mg/kg]) was administered orally (in random order at 7-day intervals) to each dog (5 treatments/dog). Urine and blood samples were collected before (2 hours after evacuation of the urinary bladder; baseline) and at intervals for 24 hours after drug treatment to assess urine volume and plasma and urine biochemical variables. RESULTS: Compared with baseline values, treatment with furosemide and azosemide (5 and 10 mg/kg) increased urine output for 1 to 2 hours and 2 to 4 hours, respectively. The 24-hour urine volume and urinary sodium excretion were significantly increased following furosemide and azosemide (5 and 10 mg/kg) treatments, compared with effects of placebo; these increases were dose dependent for azosemide, and increases were similar for furosemide and the 5 mg/kg dose of azosemide. Compared with other treatments, 24-hour urinary potassium excretion was significantly increased with azosemide at 10 mg/kg. Azosemide (5 and 10 mg/kg) significantly increased plasma total protein concentration and decreased plasma potassium concentration, compared with baseline values. Compared with the effect of placebo, PAC was significantly increased by furosemide and the 10 mg/kg dose of azosemide. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy dogs, a moderate dose of azosemide caused sufficient diuretic action and increased PAC to a lesser extent than furosemide.

Application of red laser video-rate scanning confocal microscopy to in vivo assessment of tubular function in the rat: selective action of diuretics on tubular diameter.

This study examined the use of a red laser illuminated, video-rate scanning confocal reflection microscopy (VRSCM) system, with improved structural and functional imaging at high temporal resolution, to visualize physiological changes in the kidney in response to pharmacological stimuli. We applied VRSCM to superficial nephrons in vivo and measured temporal changes in the diameter of proximal and/or distal tubular segments in response to the administration of three major classes of diuretics with known selective actions at specific nephron sites. Mannitol caused measurable increases in both proximal and distal tubular diameter, whereas frusemide and hydrochlorothiazide caused dilation of the distal tubules only. The findings indicate that VRSCM is capable of detecting and quantifying predicted dynamic changes in renal tubular diameter.

Electrophysiological assessment of sensations arising from the bladder: are there objective criteria for subjective perceptions?

PURPOSE: Initial bladder filling sensation, first and strong desire to void are subjective perceptions that occur periodically during the urine storage mode of bladder function, representing sensory input from the lower urinary tract. To our knowledge methods for evaluating sensory bladder function are not available. We studied a simple electrophysiological procedure for the objective assessment of bladder sensations using sympathetic skin responses and surface pelvic floor electromyography. MATERIALS AND METHODS: Informed consent was provided by 8 healthy male subjects, who were administered 20 mg. furosemide and 1 l. fluid to drink. Palmar and plantar sympathetic skin responses, and surface pelvic floor electromyogram were continuously recorded during bladder filling, voluntary pelvic floor contraction and voiding. RESULTS: First desire to void evoked simultaneous sympathetic skin responses and pelvic floor contractions. This pattern appeared periodically with the desire to void sensation as well as with strong desire to void at maximum bladder capacity and it correlated well with the subjective sensation of the subjects. Voluntary pelvic floor contraction decreased the subjective intensity of the desire to void sensation as well as sympathetic skin response activity for the same short period. During voiding sympathetic skin responses almost complete absence of sympathetic skin responses was observed. CONCLUSIONS: Sensations arising from the bladder induce combined activation of sympathetic skin responses and pelvic floor activity. This coherence indicates synchronized activation and inactivation of the autonomic and somatic pathways necessary for appropriate urine storage and coordinated voiding. Our observations may introduce a new approach for objectively assessing subjective sensations arising from the urinary tract.

Assessment of echocardiographic left ventricular mass before and after acute volume depletion.

Left ventricular mass calculations are often performed to assess the need or effectiveness of antihypertensive drug therapy. However, there are multiple potential errors that may affect the accuracy of these calculations, which can possibly include acute changes in preload. Therefore, to assess the hypothesis that acute volume depletion might alter calculated left ventricular mass, 15 normotensive healthy male volunteers underwent standard M-mode echocardiographic evaluations (at the level of the chordae tendineae guided by two-dimensional echocardiography) before and 2 h after 40 mg of intravenous furosemide. One patient was eliminated due to hypotension prior to the final echocardiogram. The echocardiograms were blinded to patient identity and the time sequence and read separately by two investigators. Four to five cycles were read per echocardiogram by each investigator. All values measured were the mean of the two investigators. Echocardiographic measurements were derived by both the American Society of Echocardiography and Penn conventions. An average urine volume of 1728 mL was collected, and the mean weight change 2 h after furosemide administration was 1.78 kg (P = .001). Penn left ventricular diastolic diameter (1.8 mm, P = .015) and left ventricular mass index (10 g/m2, P = .04) were significantly decreased; however, there was no significant change in septal, posterior, or relative wall thicknesses. As it is unreasonable to believe that acute remodeling of the left ventricle resulted in a decline in left ventricular mass in 2 hours, it is concluded that acute volume changes resulted in a decrease in left ventricular mass measurement due to the influence of diastolic diameter on the calculation of cardiac mass.

A new visual analogue scale for assessment of dyspnoea in congestive heart failure.

A new (quality of life) visual analogue scale of dyspnoea has been designed to evaluate the severity of dyspnoea in patients with congestive heart failure (CHF). The index has 10 numerical values, rated on a scale from 0-10, each value corresponding to an intensity of breathlessness. Since dyspnoea is a primary symptom that restricts the quality of life in patients with congestive heart failure, the new scale provided a simple, easily comprehensible and inexpensive instrument to assess the severity of breathlessness. We also report the sensitivity of the visual scale of dyspnoea before and after the administration of intravenous injection of a diuretic (frusemide) in CHF patients.

Sodium transport in hypertension: assessment of membrane-associated defects in South African black and white hypertensives.

Sodium transport was studied in red cells taken from four groups of patients, whites or blacks, and normotensive or hypertensive, with approximately ten patients in each group. There were no differences between normotensive and hypertensive whites. Normotensive blacks had higher rates of sodium-proton exchange than did the other groups, and hypertensive blacks had higher rates of efflux via the sodium-potassium ATPase than did other groups. These data support proposals that hypertension in black subjects is linked to abnormalities of control of intracellular sodium.

Assessment of high-energy phosphorus compounds in the rat kidney by in situ 31P nuclear magnetic resonance spectroscopy: effect of ischemia and furosemide.

31P nuclear magnetic resonance (NMR) spectroscopy of the in situ rat kidney was performed by a surface coil method, and the effects of ischemia and furosemide infusion were assessed. 31P NMR spectra of the kidney subjected to 30 min of ischemia returned completely to the pre-ischemic level after 60 min of reperfusion. But the 31P NMR spectra after 60 min of ischemia did not recover, even after 120 min of reperfusion. Levels of beta-ATP and inorganic phosphate (Pi) decreased and the chemical shift of Pi increased after intravenous infusion of furosemide. This increase in chemical shift might signal an alkalotic change in intracellular pH. Furosemide infusion prior to ischemia is thought to protect the kidney from injury induced by 60 min of warm ischemia. The chemical shift of Pi returned to the pre-ischemic level earlier than beta-ATP and Pi. In conclusion, according to the findings of 31P NMR spectroscopy, furosemide infusion prior to ischemia may be effective in protecting the kidney against ischemic injury. But the change in Pi peak and the causes of the dissociation of Pi and beta-ATP should be examined further.

An isolated perfused rat kidney preparation designed for assessment of glomerular permeability characteristics.

The aim of the present investigation was to modify the widely used isolated perfused rat kidney preparation to make it more suitable for studies of glomerular permeability to macromolecules. Both kidneys were perfused in situ using separate pumps in two of each other independent systems with Tyrode-solution containing human serum albumin (18.2 g l-1). Sodium nitroprusside was administered to induce dilatation and to maintain constant vascular resistance (PRU100) during the experiments. The addition of sodium nitroprusside decreased vascular resistance from 0.17 +/- 0.05 to 0.09 +/- 0.02 mmHg min-1 100 g-1 ml-1 and increased urine flow and glomerular filtration rate. The temperature of the perfusate was reduced from 37 degrees C to 8 degrees C to inhibit tubular reabsorption of protein and fluid, resulting in a urine to plasma concentration ratio of [51Cr]EDTA of 1.26 +/- 0.07. Furosemide reduced the urine to plasma concentration ratio for [51Cr]EDTA further to 1.15 +/- 0.02 and increased glomerular filtration rate. Moreover, by performing the studies at low temperatures (8 degrees C) in the presence of sodium nitroprusside and furosemide it was possible to achieve low and stable albumin fractional clearance values close to those prevailing in vivo. Thus, the described technique, allowing simultaneous perfusions of both kidneys with different solutions, pressures and flows, seem to be well suited for studies of macromolecular transport across glomerular capillaries.

A comparison of simultaneously collected arterial, mixed venous, jugular venous and cephalic venous blood samples in the assessment of blood-gas and acid-base status in the dog.

Blood samples were collected simultaneously from the pulmonary artery, jugular vein, cephalic vein, and carotid artery in awake dogs. Blood-gas and acid-base values were measured from these blood samples in normal dogs and in dogs after production of metabolic acidosis and metabolic alkalosis. The values obtained from each of the venous sites were compared with those obtained from arterial blood to determine if venous blood from various sites accurately reflected acid-base balance and could therefore be used in the clinical patient. The results of this study demonstrated significant differences between the blood from various venous sites and the arterial site for PCO2 and pH in all acid-base states. Significant differences for standard bicarbonate (SHCO3) were found only when jugular and cephalic venous blood were compared with arterial blood in dogs with a metabolic acidosis. No significant differences were found for BE when blood from the venous sites was compared with arterial blood. The values for pH, HCO3, TCO2, BE, and SHCO3 measured on blood collected at the various venous sites were found to correlate well with those obtained from arterial blood, with a correlation coefficient of 0.99 for HCO3, TCO2, BE, and SHCO3. These correlation coefficients, together with similar values in BE at all collection sites, indicate that, in the dog with normal circulatory status, blood from any venous site will accurately reflect the acid-base status of the patient.

Osmotic challenges in the assessment of bronchial hyperresponsiveness.

It is now well recognized that a change in osmolarity of the periciliary fluid is a potent stimulus to airway narrowing and may be a common cause for provoking an attack of asthma. This has led to the use of nonisotonic aerosols to document bronchial hyperresponsiveness in patients with clinical asthma. These aerosols are generated by ultrasonic nebulizers, and the most commonly used are water, hyperosmolar sodium or potassium chloride, and dextrose. An increase in specific airway resistance of 100% or a reduction in FEV1 of 20% is considered as an abnormal response to these aerosols. There are a number of factors that affect the airway response to these aerosols, and these include the osmolarity of the solution and its ionic content and pH. In addition responses may be affected by a previous challenge or medication. It is not known precisely how these aerosols act to induce airway narrowing, but it is not by a direct action on smooth muscle. It is thought that a change in osmolarity of the airways causes the release of inflammatory mediators, which then act to narrow the airways. For this reason challenge by nonisotonic aerosols is different from challenge by histamine and methacholine and may be preferable for the diagnosis of asthma and assessment of its treatment.

The effect of loop diuretics on the vestibular system. Assessment by recording the vestibular evoked response.

Whether or to what extent loop diuretics, e.g., ethacrynic acid and furosemide, affect the vestibular system is controversial. We studied this problem by recording in cats the short-latency vestibular evoked response (VsER) to acceleration stimuli by skin electrodes before and after local or systemic administration of loop diuretics. The effect on the VsER was minimal in contrast to the major changes that appeared in the auditory evoked response, in which, among the waves known to originate from the brain stem, the most affected was N1. These findings suggest that the vestibular end-organ function is minimally affected by loop diuretics and that the changes in the vestibulo-ocular reflex reported by several authors might be due to the effect of the drugs on the central nervous system. Thus, recording of the VsER in experimental animal models might serve as a useful tool for direct evaluation of the effect of certain drugs and conditions on the vestibular system.

Methodological assessment of assays for intracellular concentration and transmembrane fluxes of sodium and potassium in erythrocytes of man.

The reliability of the assay of intracellular Na+ and K+ concentration; Na+,K+-ATPase activity; Na+-K+ cotransport and Na+-Li+ countertransport in erythrocytes was investigated. These assays were also applied in normal male subjects with and without family history of hypertension. The various assays are reproducible as indicated by the intra-assay variation and are stable over time as shown by the inter-assay variation. A delay between blood sampling and analysis, however, result in an increase in intracellular Na+ concentration and decreases in Na+,K+-ATPase activity; Na+-K+ cotransport and Na+-Li+ countertransport. Compared to the white normal males without family history of hypertension (n = 43) red blood cells of white normal males with such a history (n = 17) have a higher (p less than 0.01) intracellular Na+ concentration. This can be at least partly due to their lower (p less than 0.001) furosemide-sensitive Na+ efflux rate. Also, their ouabain-resistant 86Rb-uptake was lower (p less than 0.05). The potassium concentration in the red blood cells was similar in both groups.