Simultaneous measurement of kidney function by dynamic contrast enhanced MRI and FITC-sinistrin clearance in rats at 3 tesla: initial results.

Glomerular filtration rate (GFR) is an essential parameter of kidney function which can be measured by dynamic contrast enhanced magnetic resonance imaging (MRI-GFR) and transcutaneous approaches based on fluorescent tracer molecules (optical-GFR). In an initial study comparing both techniques in separate measurements on the same animal, the correlation of the obtained GFR was poor. The goal of this study was to investigate if a simultaneous measurement was feasible and if thereby, the discrepancies in MRI-GFR and optical-GFR could be reduced. For the experiments healthy and unilateral nephrectomised (UNX) Sprague Dawley (SD) rats were used. The miniaturized fluorescent sensor was fixed on the depilated back of an anesthetized rat. A bolus of 5 mg/100 g b.w. of FITC-sinistrin was intravenously injected. For dynamic contrast enhanced perfusion imaging (DCE-MRI) a 3D time-resolved angiography with stochastic trajectories (TWIST) sequence was used. By means of a one compartment model the excretion half-life (t1/2) of FITC-sinistrin was calculated and converted into GFR. GFR from DCE-MRI was calculated by fitting pixel-wise a two compartment renal filtration model. Mean cortical GFR and GFR by FITC-sinistrin were compared by Bland-Altman plots and pair-wise t-test. Results show that a simultaneous GFR measurement using both techniques is feasible. Mean optical-GFR was 4.34 ± 2.22 ml/min (healthy SD rats) and 2.34 ± 0.90 ml/min (UNX rats) whereas MRI-GFR was 2.10 ± 0.64 ml/min (SD rats) and 1.17 ± 0.38 ml/min (UNX rats). Differences between healthy and UNX rats were significant (p<0.05) and almost equal percentage difference (46.1% and 44.3%) in mean GFR were assessed with both techniques. Overall mean optical-GFR values were approximately twice as high compared to MRI-GFR values. However, compared to a previous study, our results showed a higher agreement. In conclusion, the possibility to use the transcutaneous method in MRI may have a huge impact in improving and validating MRI methods for GFR assessment in animal models

Conformational Sampling of the Intrinsically Disordered C-Terminal Tail of DERA Is Important for Enzyme Catalysis

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (ecDERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the ecDERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, ecDERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the ecDERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis

Reliability of transcutaneous measurement of renal function in various strains of conscious mice.

Measuring renal function in laboratory animals using blood and/or urine sampling is not only labor-intensive but puts also a strain on the animal. Several approaches for fluorescence based transcutaneous measurement of the glomerular filtration rate (GFR) in laboratory animals have been developed. They allow the measurement of GFR based on the elimination kinetics of fluorescent exogenous markers. None of the studies dealt with the reproducibility of the measurements in the same animals. Therefore, the reproducibility of a transcutaneous GFR assessment method was investigated using the fluorescent renal marker FITC-Sinistrin in conscious mice in the present study. We performed two transcutaneous GFR measurements within three days in five groups of mice (Balb/c, C57BL/6, SV129, NMRI at 3-4 months of age, and a group of 24 months old C57BL/6). Data were evaluated regarding day-to-day reproducibility as well as intra- and inter-strain variability of GFR and the impact of age on these parameters. No significant differences between the two subsequent GFR measurements were detected. Fastest elimination for FITC-Sinistrin was detected in Balb/c with significant differences to C57BL/6 and SV129 mice. GFR decreased significantly with age in C57BL/6 mice. Evaluation of GFR in cohorts of young and old C57BL/6 mice from the same supplier showed high consistency of GFR values between groups. Our study shows that the investigated technique is a highly reproducible and reliable method for repeated GFR measurements in conscious mice. This gentle method is easily used even in old mice and can be used to monitor the age-related decline in GFR

Plot of concentrations (mmol/ml) versus signal intensities (arbitrary units) obtained from a phantom consisting of tubes filled with water and different contrast agent concentrations (0, 0.01, 0.025, 0.05, 0.1 0.25, 0.5, 1.0, 2.5 mmol/ml).

<p>The error bars depict the standard deviation within a ROI of size 0.3² placed within the tubes.</p

Image examples obtained by the MR measurements.

<p>A) depicts one slice of a T2 weighted morphological MR image used for cortex delineation, B) shows a parametric map of the tubular flow (filtration) calculated from the DCE- MRI perfusion data in units of ml/min/100 ml tissue. The map is superimposed onto the corresponding slice of the T2 weighted image.</p

Optical devices for FITC-sinistrin clearance measurement.

<p>A: original devices with LEDs and photodiode for signal excitation and reception (bottom side) and electronics (upper side). B: the devices are shielded by copper foil, only the LEDs, the photodiode and the battery plug are spared. The device is powered by a small battery that was covered in copper foil, too.</p

Example of FITC-sinistrin clearance curve from one animal.

<p>The dashed line depicts the background signal, which is subtracted from the signal for quantification of the GFR.</p

Bland-Altman plot of GFR values estimated by both methods.

<p>On the x-axis, the average of both methods is plotted whereas the y-axis shows the differences between the methods.</p

Conformational Sampling of the Intrinsically Disordered C‑Terminal Tail of DERA Is Important for Enzyme Catalysis

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (<i>ec</i>DERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the <i>ec</i>DERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, <i>ec</i>DERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the <i>ec</i>DERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis