Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition.

Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

Differential impedance spectroscopy for monitoring protein immobilization and antibody-antigen reactions.

This work describes the theoretical and experimental approaches for monitoring the interfacial biomolecular reaction between immobilized antibody and the antigen binding partner using novel differential impedance spectroscopy. The prerequisite of any biosensor is the immobilization of macromolecules onto the surface of a transducer. It is clear that the function of most macromolecules changes from what is observed in solution once immobilization has occurred. In the worst case, molecules entirely lose their binding activity almost immediately after immobilization. Certain conditions (e.g., denaturation, interfacial effects based on ionic strength, surface charge, dielectric constants, etc.) at interfaces are responsible for alterations of binding activity; it is not clear whether a combination of such processes is understood. However, these processes in combination must be reliably modeled in order to predict the outcome for most macromolecules. This work presents the theoretical and practical means for elucidating the surface reactivity of biomolecular reagents using ion displacement model with antibody-antigen (Ab-Ag) reaction as the test case. The Ab-Ag reaction was directly monitored using a dual-channeled, impedance analyzer capable of 1 measurement/s using covalent immobilization chemistry and polymer-modified electrodes in the absence of a redox probe. The evidence of Ab-Ag binding was revealed through the evolution of differential admittance. The surface loading obtained using the covalent immobilization chemistry was 9.0 x 10(16)/cm2, whereas with polymer-modified electrodes, the surface loading was 9.0 x 10(15)/cm2, representing a 10 times increase in surface reactivity. The proposed approach may be applicable to monitoring other surface interfacial reactions such as DNA-DNA interactions, DNA-protein interactions, and DNA-small molecule interactions.

Role of sodium in contrast medium-induced polymorphic ventricular tachycardia: results in a rabbit model of lengthened QT interval.

RATIONALE AND OBJECTIVES: The authors (a) compared the proarrhythmic effects of ioxaglate (152 mmol/L sodium) and iohexol (no sodium) in a rabbit model and (b) assessed the effect of adding 150 mmol/L sodium to isotonic iohexol. MATERIALS AND METHODS: Either ioxaglate (320 mg of iodine per milliliter) or iohexol (350 mg of iodine per milliliter) was selectively injected into the right coronary artery (1.5 mL over 30 seconds) of 10 rabbits, some of which also received the alpha 1-adrenergic receptor agonist methoxamine. To validate the model, the class III antiarrhythmic agent clofilium was injected intravenously during methoxamine infusion. Frontal electrocardiography was performed continuously to detect polymorphic ventricular tachycardia (PVT). In a second study, the authors assessed the frequency of arrhythmias after injection of isotonic iohexol solution (145 mg of iodine per milliliter), either alone or with 150 mmol/L sodium. RESULTS: Methoxamine significantly lengthened the QT, QTc, and RR intervals (P < .05). The use of clofilium alone induced no PVT, whereas five of eight methoxamine-infused rabbits developed PVT after clofilium injection (P = .03). Both contrast media prolonged the repolarization period. Iohexol alone induced a higher frequency of PVT than did ioxaglate alone (P = .0006). Methoxamine infusion did not potentiate the frequency of PVT in the ioxaglate-injected rabbits. The addition of sodium to isotonic iohexol prevented the occurrence of PVT (P = .0006). CONCLUSION: Although ioxaglate prolonged the repolarization period, it did not cause a higher frequency of arrhythmia when injected in association with methoxamine. Iohexol, which contains no sodium, induced a high frequency of arrhythmia. The addition of a physiologic concentration of sodium to isotonic iohexol can prevent ventricular arrhythmias.

Comparative effects of low- and high-osmolar contrast media on the renal function during early degenerative gentamicin-induced nephropathy in rats.

The nephrotoxic potentials of a high-osmolar contrast medium, diatrizoate, and of a low-osmolar contrast medium, ioxaglate, were compared during early degenerative gentamicin-induced nephropathy in the rat. Male rats (13-22/group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery, and either diatrizoate or ioxaglate was administered (1 ml/min for 3 min) via an aortic puncture into the remaining kidney. Some of the rats received chronic treatment with gentamicin (50 mg/kg/day i.m., 4 days), starting 2 days before and ending 1 day after contrast medium administration. Two control groups, only one of which received gentamicin, were subjected to a 3-min renal ischemia. The creatinine clearance (CrCl) per 100 g body weight was determined before and 24 and 48 h after contrast medium injection. A second study (6 rats/group) evaluated urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and the histologic appearance of the kidneys (blinded analysis) in the same experimental groups. Gentamicin induced a significant decrease in CrCl at baseline (0.35 +/- 0.19 vs. 0.41 +/- 0.19 ml/min; p < 0.01) and an increase in urinary NAG (128 +/- 92 vs. 39 +/- 57 mumol/h/mmol creatinine; p < 0.01). Taking into account these differences at baseline, univariate repeated-measures analysis showed that on day 1 diatrizoate caused a more marked decrease in CrCl than ioxaglate (p < 0.05), whether or not gentamicin was also administered. On day 2, the depressant effect of diatrizoate associated with gentamicin persisted (CrCl vs. day 0 = -0.19 +/- 0.10 ml/min), while that of diatrizoate alone returned to baseline (-0.05 +/- 0.24 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Iobitridol, a new nonionic low-osmolality contrast agent, and iohexol. Impact on renal histology in the rat.

RATIONALE AND OBJECTIVES: To compare the histologic effects on rat tubular cells of two nonionic contrast media with equivalent osmolalities and viscosities. METHODS: Histologic, functional (creatinine clearance), and biochemical (proteinuria and enzymuria) profiles of iohexol and iobitridol (both at 350 mg I/mL) were compared in the uninephrectomized rat. A control group (n = 14) received compared isotonic saline solution. Test substances (3 mL) were injected into the kidney at a rate of 1 mL/minute while transitory ischemia was induced by clamping the aorta above the renal artery. RESULTS: In terms of their (moderate) effects on creatinine clearance, proteinuria, and urinary N-acetyl-beta-D-glucosaminidase activity, no statistically significant difference was detected between the two low-osmolar contrast agents either 24 or 48 hours after injection. However, blinded histologic analysis of the kidneys showed significantly greater epithelial cell vacuolization in the proximal convoluted tubules of the outer cortex with iohexol (14 of 14 rats versus 3 of 14 rats for iobitridol; P < .001). The same degree of vacuolization in the inner cortex was observed for all three substances. Iobitridol also induced fewer congestive lesions in the glomerular capillaries than iohexol (4 of 14 versus 10 of 14, respectively; P < .05) and saline (5 of 6; P < .05). It is difficult to explain the lesser degree of cytoplasmic vacuolization using standard physicochemical parameters. CONCLUSION: Although iobitridol and iohexol showed similar functional and biochemical profiles when selectively injected into the single remaining kidney of rats, iobitridol induced significantly less tubular vacuolization and capillary congestion than iohexol.

Modulation of the renal effects of contrast media by endothelium-derived nitric oxide in the rat.

RATIONALE AND OBJECTIVES: A possible involvement of endothelium derived relaxing nitric oxide (NO) in the pathogenesis of iodinated contrast media (CM)-induced nephrotoxicity was investigated in the rat. METHODS: Male rats (6 to 12 per group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery and a low-osmolar contrast medium (CM), ioxaglate, was injected (1 mL/min; 3 minutes) via an aortic puncture in the single remaining kidney. Contrast medium was injected with or without the NO-synthase inhibitor L-NAME (100 mg/kg intravenously [i.v.] 5 minutes before CM). One group received L-Arginine, the physiological precursor of NO (100 mg/kg i.v.), 5 minutes before L-NAME. Phenylephrine (300 micrograms/kg; 30 min) was used as a vasoconstrictive NO-independent control. The effects of iohexol, another low-osmolar CM, on creatinine clearance (CrCl) were also studied with and without pretreatment with L-NAME. A control group was subjected to a 3-minute renal ischemia only. Creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined before, and 24 and 48 hours after CM administration. Blinded histologic analysis was carried out after completion of the study. RESULTS: When administered alone, neither L-NAME nor L-arginine modified CrCl. Ioxaglate mildly but significantly decreased CrCl at 24 hours (-26.5% of preinjection value). This was similar to the effect observed in the control group subjected to ischemia only. When associated with L-NAME, ioxaglate markedly decreased CrCl (-58 + 11% at 24 hours, P < .05 vs. ioxaglate alone). A similar interaction was noted in the case of iohexol. L-NAME also markedly increased ioxaglate-induced urinary NAG excretion. Phenylephrine had a similar impact on renal function. L-arginine pretreatment reduced the increase in serum creatinine induced by L-NAME+ioxaglate (68 + 17 mumol/L vs. 175 + 59 mumol/L for L-NAME+ioxaglate; P < .05) and urinary NAG excretion. Ioxaglate alone induced only tubular epithelial vacuolization. When associated with L-NAME, this CM induced tubular and vascular lesions, as well as necrosis in the outer medulla. Such histologic effects were clearly inhibited by L-arginine. CONCLUSION: These data indicate that L-NAME, a specific inhibitor of NO-synthase, and phenylephrine, accentuate the nephrotoxicity of CM in the rat. This is consistent with results from the literature showing that CM-toxicity is enhanced by renal ischemia.