Daniel Schock-Kusch

Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves

Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here, we describe a technique allowing determination of renal function based on transcutaneously measured elimination kinetics of fluorescein isothiocyanate (FITC)-sinistrin, the FITC-labeled active pharmaceutical ingredient of a commercially available marker of GFR. A low cost device transcutaneously excites FITC-sinistrin at 480  nm and detects the emitted light through the skin at 520  nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of renal function. Due to miniaturization, the whole device fits on the back of freely moving rats, and requires neither blood sampling nor laboratory assays. As proof of principle, comparative measurements of transcutaneous and plasma elimination kinetics of FITC-sinistrin were compared in freely moving healthy rats, rats showing reduced kidney function due to unilateral nephrectomy and PKD/Mhm rats with cystic kidney disease. Results show highly comparable elimination half-lives and GFR values in all animal groups. Bland-Altman analysis of enzymatically compared with transcutaneously measured GFR found a mean difference (bias) of 0.01 and a -0.30 to 0.33 ml/min per 100 g body weight with 95% limit of agreement. Thus, with this device, renal function can be reliably measured in freely moving rats eliminating the need for and influence of anesthesia on renal function.

Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats

BACKGROUND Inulin/sinistrin (I/S) clearance is a gold standard for an accurate assessment of glomerular filtration rate (GFR). Here we describe and validate an approach for a transcutaneous determination of GFR by using fluorescein-isothiocyanate-labelled sinistrin (FITC-S) in rats. METHODS Using a small animal imager, fluorescence is measured over the depilated ear of a rat after the injection of FITC-S. The decay curve of fluorescence is used for the calculation of half-life and GFR. The thus obtained transcutaneous data were validated by simultaneously performed enzymatic and fluorometric measurements in plasma of both FITC-S and sinistrin. RESULTS The results of enzymatic sinistrin determination versus transcutaneous half-life of FITC-S or plasma fluorescence correlated well with each other (R(2) > 0.90). Furthermore, Bland-Altman analyses proved a good degree of agreement of the three methods used. The measurements performed in healthy animals as well as different models of renal failure demonstrate its appropriateness in a wide range of renal function. CONCLUSIONS The transcutaneous method described offers a precise assessment of GFR in small animals. As neither blood and/or urine sampling nor time-consuming lab work is required, GFR can be determined immediately after the clearance procedure is finished. This method, therefore, simplifies and fastens GFR determinations in small lab animals compared to conventional bolus clearance techniques based on blood sampling. A low-cost device for the measurement of transcutaneous fluorescence intensity over time is under construction.

Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging

BACKGROUND Glomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report a novel technique to count and size every glomerulus in the rat kidney using magnetic resonance imaging (MRI). METHODS The ferromagnetic nature of cationized ferritin allowed visualization of single glomeruli in high-resolution susceptibility-weighted MRI. A segmentation algorithm was used to identify and count all glomeruli within the whole kidney. To prove our concept, we estimated total glomerular number and mean glomerular volume of each kidney using design-based stereology. RESULTS The glomerular counts obtained with MRI agreed well with estimates obtained using traditional methods [MRI, 32 785 (3117); stereology, 35 132 (3123)]. For the first time, the glomerular volume distribution for the entire kidney is shown. Additionally, the method is substantially faster than the current methods. CONCLUSIONS MRI provides a new method for measuring these important microanatomical markers of disease risk and leads the way to in vivo analysis of these parameters, including longitudinal studies of animal models of CKD.

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.

Progression of Glomerular Filtration Rate Reduction Determined in Conscious Dahl Salt-Sensitive Hypertensive Rats

Sequential changes in glomerular filtration rate (GFR) during development of hypertension in the conscious Dahl salt-sensitive (SS) rat were determined using a new method for measurement. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocyanate (FITC)-sinistrin were measured by transcutaneous excitation and real time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure (MAP) by telemetry) were studied while maintained on a 0.4% NaCl diet and on days 2,5,7,14 and 21 after switching to 4.0% (HS) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. MAP rose progressively from the last day of 0.4% (130±2 mmHg) reaching significance by day 5 of HS and averaged 162±7 mmHg by day 21. Urine albumin excretion was significantly elevated (3×) by day 7 of HS in SS rats. GFR became reduced on day 14 of HS falling from 1.53±0.06 ml/min/100g bwgt to 1.27±0.04. By day 21, GFR had fallen 28% to 1.1±0.04 ml/min/100g bwgt (t1/2 28.4±1.1 min.) No significant reductions of creatinine clearance (Ccre) were observed throughout the study in response to HS demonstrating the insensitivity of Ccre measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of GFR was a consequence and not a cause of the hypertension and that this non-invasive approach could be used in these conscious SS rats for a longitudinal assessment of renal function.Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate–sinistrin were measured by transcutaneous excitation and real-time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure by telemetry) were studied, while maintained on a 0.4% NaCl diet and on days 2, 5, 7, 14, and 21 after switching to 4.0% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t1/2 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.

Progression of Glomerular Filtration Rate Reduction Determined in Conscious Dahl Salt-Sensitive Hypertensive RatsNovelty and Significance

Sequential changes in glomerular filtration rate (GFR) during development of hypertension in the conscious Dahl salt-sensitive (SS) rat were determined using a new method for measurement. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocyanate (FITC)-sinistrin were measured by transcutaneous excitation and real time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure (MAP) by telemetry) were studied while maintained on a 0.4% NaCl diet and on days 2,5,7,14 and 21 after switching to 4.0% (HS) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. MAP rose progressively from the last day of 0.4% (130±2 mmHg) reaching significance by day 5 of HS and averaged 162±7 mmHg by day 21. Urine albumin excretion was significantly elevated (3×) by day 7 of HS in SS rats. GFR became reduced on day 14 of HS falling from 1.53±0.06 ml/min/100g bwgt to 1.27±0.04. By day 21, GFR had fallen 28% to 1.1±0.04 ml/min/100g bwgt (t1/2 28.4±1.1 min.) No significant reductions of creatinine clearance (Ccre) were observed throughout the study in response to HS demonstrating the insensitivity of Ccre measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of GFR was a consequence and not a cause of the hypertension and that this non-invasive approach could be used in these conscious SS rats for a longitudinal assessment of renal function.Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate–sinistrin were measured by transcutaneous excitation and real-time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure by telemetry) were studied, while maintained on a 0.4% NaCl diet and on days 2, 5, 7, 14, and 21 after switching to 4.0% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t1/2 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.

Online feedback-controlled renal constant infusion clearances in rats

Constant infusion clearance techniques using exogenous renal markers are considered the gold standard for assessing the glomerular filtration rate. Here we describe a constant infusion clearance method in rats allowing the real-time monitoring of steady-state conditions using an automated closed-loop approach based on the transcutaneous measurement of the renal marker FITC-sinistrin. In order to optimize parameters to reach steady-state conditions as fast as possible, a Matlab-based simulation tool was established. Based on this, a real-time feedback-regulated approach for constant infusion clearance monitoring was developed. This was validated by determining hourly FITC-sinistrin plasma concentrations and the glomerular filtration rate in healthy and unilaterally nephrectomized rats. The transcutaneously assessed FITC-sinistrin fluorescence signal was found to reflect the plasma concentration. Our method allows the precise determination of the onset of steady-state marker concentration. Moreover, the steady state can be monitored and controlled in real time for several hours. This procedure is simple to perform since no urine samples and only one blood sample are required. Thus, we developed a real-time feedback-based system for optimal regulation and monitoring of a constant infusion clearance technique.

Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging

BACKGROUND The aim of this study was the assessment of kidney morphology and glomerular filtration rate (GFR) in rat models of polycystic kidney disease and a healthy control group of Sprague-Dawley rats (SD rats). The performance of two non-invasive GFR estimation methods-3.0 Tesla magnetic resonance imaging (MRI) and optical imaging were investigated. Data of GFR assessment was compared to surrogate markers of kidney function and renal histology. METHODS Optical imaging of GFR was performed transcutaneously in a small animal imaging system with the fluorescent renal marker fluorescein-isothiocyanate-labelled-sinistrin. Morphologic and dynamic renal imaging was done on a clinical 3.0T MR scanner. Renal perfusion analysis was performed with a two-compartment filtration model. RESULTS The healthy SD rats showed physiological levels of creatinine and urea, indicating normal kidney function. These parameters were elevated in the small animal groups of polycystic kidney disease. For the calculation of perfusion and filtration parameters of kidney function in MRI, a 2D turbo FLASH sequence was performed and allowed to distinguish between normal GFR of healthy rats and reduced GFR of rats with polycystic kidney disease. Also, MRI GFR varied among two different rat strains of polycystic kidney disease, according to their status of renal function impairment. Optical imaging GFR confirmed higher GFR values in healthy rats compared to ill rats but did not show different results among the two rat strains of polycystic kidney disease. For this reason, MRI and optical imaging GFR estimation presented an intra-method bias. CONCLUSIONS Both non-invasive estimation methods of GFR, MRI and optical imaging, can differentiate between healthy rats and animals with limited kidney function. Furthermore, optical imaging, unlike MRI, seems to consider that disease progression with increase of renal polycystic deterioration does not correlate with decrease of GFR in the initial stage of compensatory hyperfiltration.

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.

A Novel Analysis Technique for Transcutaneous Measurement of Glomerular Filtration Rate With Ultralow Dose Marker Concentrations

Objective: A novel high-precision approach [lifetime-decomposition measurement (LTDM)] for the assessment of the glomerular filtration rate (GFR) based on clearance measurements of exogenous filtration marker. Methods: The time-correlated single photon counting (TCSPC) acquisition in combination with a new decomposition method allows the separation of signal and background from transcutaneous measurements of GFR. Results: The performance of LTDM is compared versus the commercially available NIC-kidney patch-based system for transcutaneous GFR measurement. Measurements are performed in awake Sprague Dawley (SD) rats. Using the standard concentration required for the NIC-kidney system [7-mg/100-g body weight (b.w.) FITC-Sinistrin] as reference, the mean difference (bias) of the elimination curves GFR between LTDM and NIC-kidney was 4.8%. On the same animal and same day, the capability of LTDM to measure GFR with a FITC-Sinistrin dose reduced by a factor of 200 (35-μg/100-g b.w.) was tested as well. The mean differences (half lives with low dose using LTDM compared with those using first, the NIC-Kidney system and its standard concentration, and second, LTDM with the same concentration as for the NIC-Kidney system) were 3.4% and 4.5%, respectively. Conclusion: We demonstrate that with the LTDM strategy substantial reductions in marker concentrations are possible at the same level of accuracy. Significance: LTDM aims to resolve the issue of the currently necessary large doses of fluorescence tracer required for transcutaneous GFR measurement. Due to substantially less influences from autofluorescence and artifacts, the proposed method outperforms other existing techniques for accurate percutaneous organ function measurement.