RESUMEN
Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.
Asunto(s)
Tasa de Filtración Glomerular/inmunología , Administración Cutánea , Animales , Masculino , RatonesRESUMEN
Transcutaneous measurement of the glomerular filtration rate (tGFR) is now frequently used in animal studies. tGFR allows consecutive measurements on the same animal, including multiple measurements on a daily basis, because no blood sampling is required. Here we derive and validate a novel kinetic model for the description of transcutaneously measured FITC-Sinistrin excretion kinetics. In contrast to standard 1- to 3-compartment models, our model covers the complete kinetic, including injection and distribution of the tracer in the plasma compartment. Because the model describes the complete progression of the measurement, it allows further refinement by correcting for baseline shifts observed occasionally during measurement. Possible reasons for shifts in the background signal include photo bleaching of the skin, autofluorescence, changes of physiological state of the animals during the measurements, or effects arising from the attachment of the measurement device. Using the new 3-compartment kinetic model with modulated baseline (tGFR3cp.b.m), tGFR measurements in rats can reach comparable precision as those from GFR measurements assessed using a gold standard technique based on constant infusion of a tracer. Moreover, the variability of simultaneous (parallel) measurements, as well as repeated tGFR measurements in the same animals, showed higher precision when tGFR3cp.b.m was compared with the 1-compartment tGFR1cp model.
Asunto(s)
Tasa de Filtración Glomerular , Modelos Animales , Modelos Teóricos , Animales , Biometría , Cinética , Masculino , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Tasa de Filtración Glomerular/fisiología , Pruebas de Función Renal/métodos , Riñón/fisiología , Algoritmos , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Fluoresceínas/análisis , Fluoresceínas/metabolismo , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Masculino , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Glomerular filtration rate (GFR) is considered the best parameter for the assessment of renal function, being usually determined on the basis of urine or plasma clearance of exogenous renal markers. The common methodology is invasive, time consuming and cumbersome, with multiple blood and/or urine sampling and following laboratory assays required. The method detailed here allows to transcutaneously determine the renal function in awake animals, in a non-invasive and efficient manner by using an electronic device which detects the fluorescence emitted through the skin from the renal marker FITC-Sinistrin. A crucial target has been to improve the fixation of the device, which is dependent on the skin structure. For validation, the technique has been compared with the classical clearance method, and its robustness has been demonstrated in healthy and diseased murine models. Moreover, the method allows sequential measurements in the same individual. Thus progression and recovery of renal failure can be followed. Therefore, its future application in humans would allow an accurate and appropriate prediction and monitoring of patients with established kidney disease over time. Furthermore, it will be possible to observe those patients under other pathological conditions with associated risk of developing renal problems.
Asunto(s)
Biomarcadores/análisis , Tasa de Filtración Glomerular/fisiología , Oligosacáridos/análisis , Insuficiencia Renal/diagnóstico , Absorción Cutánea , Animales , Ratas , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Estado de Conciencia/fisiología , Pruebas de Función Renal , Piel/metabolismo , Envejecimiento/fisiología , Animales , Fluoresceínas/metabolismo , Tasa de Filtración Glomerular/fisiología , Semivida , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligosacáridos/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.
Asunto(s)
Fluoresceínas , Tasa de Filtración Glomerular , Riñón/fisiología , Oligosacáridos , Animales , Estado de Conciencia , Colorantes Fluorescentes , Ratones , Miniaturización , Oligosacáridos/orina , Fenómenos Fisiológicos del Sistema UrinarioRESUMEN
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.
Asunto(s)
Tasa de Filtración Glomerular , Pruebas de Función Renal/métodos , Riñón/fisiología , Animales , Simulación por Computador , Retroalimentación Fisiológica , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/farmacocinética , Infusiones Parenterales , Masculino , Modelos Biológicos , Nefrectomía , Oligosacáridos/administración & dosificación , Oligosacáridos/sangre , Sistemas en Línea , Ratas , Ratas Sprague-DawleyRESUMEN
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.