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Transcutaneous measurement of the glomerular filtration rate ( t GFR) is now frequently used in animal studies. t GFR 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 ( t GFR 3cp.b.m ), t GFR 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 t GFR measurements in the same animals, showed higher precision when t GFR 3cp.b.m was compared with the 1-compartment t GFR 1cp model.

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.

Recombinant human erythropoietin (rhEPO) is used to treat anemia in chronic renal insufficiency. Erythropoietin (EPO) immunogenicity can lead to EPO-resistant anemia. Conjugating proteins with polyethylene glycol (PEG) can prolong elimination half-life and diminish protein immunogenicity. We investigated the efficacy of new erythropoietic agents, synthesized by single (Ro 50-3821) and multiple (MIX) integrations of PEG and succinimidyl butanoic acid with rhEPO, in rats with chronic renal insufficiency. Sprague-Dawley rats with surgically induced renal insufficiency received Ro 50-3821 or MIX subcutaneously (s.c.) over 4-12 weeks compared to rhEPO and NaCl. Hemoglobin and antibody levels served as primary efficacy and safety variables. Dosing intervals and dose-response characteristics were investigated. Ro 50-3821 (2.5 microg/kg once weekly) increased hemoglobin levels by 7 g/dl after 4 weeks compared to 1 g/dl in NaCl controls (P0.05). MIX (2.5 microg/kg once weekly) and rhEPO (0.25 microg/kg three times weekly) increased hemoglobin levels by 3 g/dl. Ro 50-3821 administered for 12 weeks (0.75 microg/kg once weekly) increased hemoglobin levels (from 13 to 19 g/dl) more effectively than rhEPO (0.75 microg/kg once weekly, decline from 13 to 11 g/dl, P0.05). No antibodies against Ro 50-3821 were detected after 12 weeks of treatment. Antibodies against rhEPO were seen in 69% of animals (P0.00001). Ro 50-3821 increased hemoglobin levels with once weekly s.c. dosing. Multiple pegylated EPO is less effective. In rats, rhEPO failed to increase hemoglobin levels with once weekly long-term dosing. Antibody formation following rhEPO may explain this finding. Therefore, Ro 50-3821 may provide important clinical advantages compared to unpegylated EPO. It can be administered in longer dosing intervals and has a lower risk of unfavorable immunological responses.

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.

The use of the colorimetric Jaffé method for the measurement of creatinine in mouse and rat plasma has been criticized as prior studies have shown a dramatic overestimation. We compared a colorimetric picric acid, an enzymatic, and a high-performance liquid chromatography (HPLC) method to assess their appropriateness for routine measurements of creatinine in plasma of healthy and diseased mice (n=61) and rats (n=56). For the colorimetric Jaffé method a pronounced overestimation is confirmed. Additionally the method showed interference with hemoglobin already in a very low, non-visible concentration range in rat plasma. The enzymatic measurement demonstrated a hemoglobin interference in mice, only when hemolysis was visible. The comparison between HPLC and the enzymatic measurement gave a good agreement between both methods in both species. Therefore the enzymatic method fulfills the requirements for a routine screening test for plasma creatinine in healthy as well as diseased mice and rats Kiover a broad concentration range.