Can Ferumoxytol be Used as a Contrast Agent to Differentiate Between Acute and Chronic Inflammatory Kidney Disease?: Feasibility Study in a Rat Model.

OBJECTIVES: Ferumoxytol, an intravenous iron supplement, can be used in off-label mode as a contrast agent in magnetic resonance imaging. The aim of this study was to assess whether ferumoxytol can be used as a marker of inflammation in animal models of acute and chronic inflammatory kidney diseases. MATERIAL AND METHODS: The institutional animal care committee approved this study. A total of 18 rats were examined: 6 healthy Sprague Dawley rats as a control group; 6 rats with polycystic kidney disease (PKD) as a model for chronic inflammatory disease; Thy-1, an antibody triggering glomerulonephritis, was injected in 6 rats as a model for acute inflammation. Each rat was examined directly before and 24 hours after intravenous administration of ferumoxytol at a dose of 30 mg Fe/kg body weight. T2* times of renal tissue were approximated using a multiecho sequence. Changes in relative T2* times and T2 signal intensity after ferumoxytol injection were calculated. RESULTS: Statistically significant differences between the 3 groups were found: the T2* times of both, Thy-1 and PKD rats were statistically significant different compared with the control group (T2* time ratio after/before: Thy-1, 0.21; PKD, 0.19, control, 0.28; P = 0.002). The highest T2 signal loss in the renal cortex was observed in the Thy-1 rats (T2 signal intensity ratio after/before: Thy-1, 0.49; PKD, 0.79; control, 0.78; P = 0.0005). CONCLUSIONS: Ferumoxytol-enhanced magnetic resonance imaging allows detection and differentiation of acute and chronic inflammatory kidney disease based on different patterns of parenchymal ferumoxytol depositions. Ferumoxytol thus might help to differentiate between different types of inflammation in various kidney diseases.

Evaluation of the Metabolic Response to Cyclopamine Therapy in Pancreatic Cancer Xenografts Using a Clinical PET-CT System.

OBJECTIVES: We analyzed the effects of anti-hedgehog signaling on the (18)F-FDG uptake of pancreatic cancer xenografts (PCXs) using a clinically implemented positron emission tomography (PET)-computer tomography (CT) scanner with high-resolution reconstruction. METHODS: PCXs from two pancreatic cancer cell lines were developed subcutaneously in nude mice and injected intraperitoneally with a low dose of cyclopamine for 1 week. (18)F-FDG PET-CT was performed using a new-generation clinical PET-CT scanner with minor modifications of the scanning protocol to adapt for small-animal imaging. The data set was reconstructed and quantified using a three-dimensional workstation. RESULTS: MiaPaCa-2 cells, which respond to cyclopamine, showed decreased (18)F-FDG uptake without a change in tumor size. For hip tumors, the maximum standardized uptake value (SUV(max)) was reduced by -24.5 ± 9.2%, the average SUV (SUV(avg)) by -33.5 ± 7.0%, and the minimum SUV (SUV(min)) by -54.4 ± 11.5% (P < .05). For shoulder tumors, SUV(max) was reduced by -14.7 ± 7.5%, SUV(avg) by -12.6 ± 6.3, and SUV(min) by -30.3 ± 16.7% (P < .05). Capan-1 cells, which do not respond to cyclopamine, did not show significant SUV changes. CONCLUSIONS: The new generations of clinically implemented PET-CT scanners with high-resolution reconstruction detect a minimal response of PCX to low-dose short-term cyclopamine therapy without changes in tumor size and offer potential for preclinical translational imaging.

The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568. CONCLUSIONS: PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis. These data suggest that effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts.

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.

First multimodal embolization particles visible on x-ray/computed tomography and magnetic resonance imaging.

OBJECTIVES: Embolization therapy is gaining importance in the treatment of malignant lesions, and even more in benign lesions. Current embolization materials are not visible in imaging modalities. However, it is assumed that directly visible embolization material may provide several advantages over current embolization agents, ranging from particle shunt and reflux prevention to improved therapy control and follow-up assessment. X-ray- as well as magnetic resonance imaging (MRI)-visible embolization materials have been demonstrated in experiments. In this study, we present an embolization material with the property of being visible in more than one imaging modality, namely MRI and x-ray/computed tomography (CT). Characterization and testing of the substance in animal models was performed. MATERIALS AND METHODS: To reduce the chance of adverse reactions and to facilitate clinical approval, materials have been applied that are similar to those that are approved and being used on a routine basis in diagnostic imaging. Therefore, x-ray-visible Iodine was combined with MRI-visible Iron (Fe3O4) in a macroparticle (diameter, 40-200 µm). Its core, consisting of a copolymerized monomer MAOETIB (2-methacryloyloxyethyl [2,3,5-triiodobenzoate]), was coated with ultra-small paramagnetic iron oxide nanoparticles (150 nm). After in vitro testing, including signal to noise measurements in CT and MRI (n = 5), its ability to embolize tissue was tested in an established tumor embolization model in rabbits (n = 6). Digital subtraction angiography (DSA) (Integris, Philips), CT (Definition, Siemens Healthcare Section, Forchheim, Germany), and MRI (3 Tesla Magnetom Tim Trio MRI, Siemens Healthcare Section, Forchheim, Germany) were performed before, during, and after embolization. Imaging signal changes that could be attributed to embolization particles were assessed by visual inspection and rated on an ordinal scale by 3 radiologists, from 1 to 3. Histologic analysis of organs was performed. RESULTS: Particles provided a sufficient image contrast on DSA, CT (signal to noise [SNR], 13 ± 2.5), and MRI (SNR, 35 ± 1) in in vitro scans. Successful embolization of renal tissue was confirmed by catheter angiography, revealing at least partial perfusion stop in all kidneys. Signal changes that were attributed to particles residing within the kidney were found in all cases in all the 3 imaging modalities. Localization distribution of particles corresponded well in all imaging modalities. Dynamic imaging during embolization provided real-time monitoring of the inflow of embolization particles within DSA, CT, and MRI. Histologic visualization of the residing particles as well as associated thrombosis in renal arteries could be performed. Visual assessment of the likelihood of embolization particle presence received full rating scores (153/153) after embolization. CONCLUSIONS: Multimodal-visible embolization particles have been developed, characterized, and tested in vivo in an animal model. Their implementation in clinical radiology may provide optimization of embolization procedures with regard to prevention of particle misplacement and direct intraprocedural visualization, at the same time improving follow-up examinations by utilizing the complementary characteristics of CT and MRI. Radiation dose savings can also be considered. All these advantages could contribute to future refinements and improvements in embolization therapy. Additionally, new approaches in embolization research may open up.

Paclitaxel encapsulated in cationic liposomes: a new option for neovascular targeting for the treatment of prostate cancer.

Neovascular targeting is an established approach for the therapy of prostate cancer (PCa). Cationic liposomes have been shown to be absorbed by immature vascular endothelial cells due to negative electric charge of their outer cell membrane. We aimed to evaluate the antitumoural efficacy of paclitaxel encapsulated in cationic liposomes for the treatment of PCa. Tumours were generated by subcutaneous injection of 10(6) MatLu tumour cells into the right hind leg of 21 male Copenhagen rats. After tumour growth, the animals were treated by an i.v. infusion with either 5% glucose (Gl), paclitaxel (Pax), cationic liposomes (CL) or paclitaxel encapsulated in cationic liposomes (EndoTAG-1) on days 12, 14, 16 and 19. Treatment was initiated on day 12 after tumour inoculation at mean tumour volumes of 0.31+/-0.13 mm(3). On the last day of treatment, animals treated with EndoTAG-1 had the significantly lowest tumour volumes with 2.49+/-0.84 cm(3) vs. Pax (5.59+/-0.45 cm(3)) vs. CL (3.87+/-1.25 cm(3)) vs. GL (5.17+/-1.70 cm(3)). The quantification of MVD showed the lowest count for EndoTAG-1-treated tumours (11.78+/-2.68 vessels/mm(2)) followed by Gl (15.64+/-6.68 vessels/mm(3)), Pax (18.22+/-9.50 vessels/mm(3)) and CL (40.9+/-32.8 vessels/mm(3)). The data confirm that neovascular targeting with EndoTAG-1 is a promising new method for the treatment of PCa by reducing the primary tumour mass and demonstrating benefits in the suppression of angiogenesis in comparison with the conventional treatment.

Morphologic and dynamic renal imaging with assessment of glomerular filtration rate in a pcy-mouse model using a clinical 3.0 Tesla scanner.

UNLABELLED: OBJECTIVE Morphologic and dynamic renal imaging is necessary for characterization of kidney function in renal insufficiency. Assessment of renal perfusion and the glomerular filtration rate (GFR) are essential, as the serum creatinine level and urea are not sensitive at an early stage of kidney damage. Currently available GFR estimation methods are time consuming, expensive, and lead to radiation exposure for the patient. Therefore, the aim was to determine the feasibility of morphologic and contrast-enhanced dynamic magnetic resonance imaging for GFR assessment in pcy (polycystic kidneys and fibrosis) mice, using a clinical 3.0 Tesla scanner. MATERIALS AND METHODS: Fourteen pcy-mice were anesthetized and an internal jugular vein catheter was implanted to which a dedicated extension tube with a 0.28 mm inner diameter was connected, filled with 1:100 microL diluted gadobutrol (Gadovist Bayer Schering Pharma, Berlin, Germany). Imaging of the mice was performed with a dedicated 8-element mouse coil (Rapid Biomedical, Rimpar, Germany) plugged into a clinical 32-channel 3.0 Tesla magnetic resonance-scanner (Magnetom Verio, Siemens Medical Solution, Erlangen, Germany). In this study, different morphologic sequences comprising a T1-w 3D volume-interpolated breathhold examination, T2-w 2D half-Fourier acquired turbo spin echo (HASTE), T2-w 2D BLADE-TSE with fat saturation, and a T2-w 3D SPACE were acquired. The dynamic sequence performed for assessment of GFR, was a 2D SR-Turbo FLASH sequence. Image analysis and data assessment was performed by 2 radiologists who were experienced in assessment of human kidney disease. A 3-point scale for visual assessment of renal cystic changes in the pcy-mice was applied. The appearance of cysts, considering a detailed demarcation of the cyst with an enhancing rim and a hypointense core, were assessed as detailed: (1) faint (2) and not to be differentiated, (3) findings in the morphologic sequences. Quantitative parameters of renal function (cortex plasma flow mL/100 mL/min, cortex plasma volume mL/100 mL, and PT sec) were fitted to a 2-compartment model and compared with blood samples of creatinine and urea. Histologic progression of cysts and fibrosis in the pcy-mice was analyzed. RESULTS: The T2-w 3D SPACE and T1-w 3D volume-interpolated breathhold examination sequence post contrast with thinnest slice thickness of 1 to 1.2 mm were well suited for delineation of the kidneys with detailed demarcation of the cysts (image quality score: 1.14 +/- 0.37 and 1.2 +/- 0.70, respectively). The T2-w 2D BLADE-TSE proved feasible, too (image quality score: 1.28 +/- 0.59). The T2-w 2D HASTE sequence with minimally achievable slice thickness of 2 mm was not suitable for morphologic assessment (image quality score: 2.9 +/- 0.37). The HASTE sequence suffered from blurring artifacts which further decreased the conspicuity of small cystic changes. The 2D SR-Turbo FLASH sequence showed the renal first pass of the contrast agent and enabled assessment of GFR. The data after time resolved 2D SR-Turbo FLASH perfusion analysis was used for GFR evaluation and demonstrated better GFR values in the young pcy-mice (0.558 mL/min) compared with the older pcy-mice (0.066 mL/min). CONCLUSION: Application of dedicated coils with a clinical 3.0 Tesla magnetic resonance-scanner have proved feasible for morphologic and dynamic renal imaging with assessment of GFR in pcy-mice.

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.

Faster rates of post-puberty kidney deterioration in males is correlated with elevated oxidative stress in males vs females at early puberty.

BACKGROUND: Post-puberty deterioration of kidneys is more rapid in males than in females. To reveal the underlying molecular mechanisms for this difference, we analyzed gender-dependent gene expression in kidneys of three groups of 36 day-old rats. RESULTS: The number of genes exhibiting gender-dependent expression was highly influenced by the genetic background of the rat group examined. 373, 288 and 79 genes showed differential gene expression between males and females (p = 0.001) in US, Mhm and Mhm*BN rats, respectively. Of all gender dependently expressed genes, only 39 genes were differentially expressed in all tested groups and the direction of expression change was the same for those genes for all groups. The gene expression profile suggests higher metabolic and transport activities, enhanced cell proliferation, elevated oxidative stress, and altered vascular biology in males. Furthermore, elevated levels of superoxide anion (two- to three-fold) in males compared to females were detected at early puberty, but neither at pre-puberty nor at late puberty/early adulthood. CONCLUSION: Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.

Enhanced iNOS gene expression in the steatotic rat liver after normothermic ischemia.

BACKGROUND: Impaired hepatic microcirculation in the steatotic liver has been identified as a considerable factor for increased vulnerability after ischemia/reperfusion (I/R). Changes in regulation and synthesis of vasoactive mediators, such as nitric oxide (NO) and endothelin (ET-1), may result in functional impairment of postischemic sinusoidal perfusion. The aim of the current study was to assess the impact of I/R injury on postischemic gene expression of NO and ET-1 in steatotic livers. MATERIALS AND METHODS: Male Sprague-Dawley rats with or without hepatic steatosis (induced by carbon tetrachloride treatment) were subjected to normothermic I/R injury. Steady-state mRNA levels were assessed using RT-PCR to study the expression of genes encoding ET-1, NO synthase (endothelial cell NO synthase and inducible NO synthase, iNOS). Immunohistochemistry was performed for detection of iNOS. RESULTS: I/R injury was followed by increased iNOS gene expression (RT-PCR/immunohistochemistry) in animals with hepatic steatosis, predominately in hepatocytes with fatty degeneration. A mild increase in mRNA levels for ET-1 was found in steatotic rat livers. I/R induced a further increase in ET-1 gene expression in some but not all reperfused steatotic livers. CONCLUSIONS: We show an enhanced gene expression of iNOS in postischemic steatotic rat livers. Hepatocytes with fatty degeneration appear to be the major source for NO generation. Furthermore, I/R may also induce ET-1 gene expression. Dysregulation of sinusoidal perfusion by NO and ET-1 is therefore likely to contribute to I/R injury of the steatotic liver.

Tissue response to subcutaneous implantation of glucose-oxidase-based glucose sensors in rats.

Considerable progress in improved control of disturbed glucose metabolism can be expected by continuous glucose monitoring. The aim of the study was to evaluate in male Sprague-Dawley rats tissue response to implantation of a new amperometric glucose-oxidase-based glucose sensor (NTS) compared to a commercially available sensor system CGMS of MiniMed. Both sensors were tested under working conditions over a period of 3 days. Using NTS, glucose in interstitial fluid reflected glucose in arterial blood even in rapidly changing hyper- and hypoglycaemia whereas the CGMS did not detect the experimentally induced glucose changes adequately. Gene expression profiling was performed using Affymetrix chips. Acute phase response to injury by sensor application for a short time is indicated by down regulation of the increase in mRNA of proteases e.g. metallothionein-1alpha and matrix metalloprotease-3 at day 3. Improvement of anabolic situation is suggested by decrease in mRNA of insulin-like growth factor binding protein whereas increase of heme oxygenase and hypoxia-inducible factor may be related to defense mechanisms. Changes of mRNA together with slight fibrous capsule formation suggest good histocompatibility. Comparability of the patterns of changed mRNA in tissue surrounding SCGM with and without operating voltage as shown in dendrogram indicates no contribution of hydrogen peroxide to worsening biocompatibility. Beside established histological investigations of foreign body reaction weeks or months after implantation, gene expression profiling provides additional information to biocompatibility already early after implantation.

Continuous glucose monitoring in interstitial fluid using glucose oxidase-based sensor compared to established blood glucose measurement in rats.

Glucose monitoring is of importance for success of complex therapeutic interventions in diabetic patients. Its impact on treatment and glycemic control is demonstrated in large clinical trials. Up to eight blood glucose measurements per day are recommended. Notwithstanding, a substantial number of diabetic patients cannot or will not monitor their blood glucose appropriately. Considerable progress in control of disturbed metabolism in diabetic patients can be expected by continuous glucose monitoring. The aim of the study was to evaluate the performance of a new amperometric glucose oxidase-based glucose sensor in vitro and in vivo after subcutaneous implantation into rats. For in vitro testing current output of sensors was measured by exposure to increasing and decreasing glucose concentrations up to 472 mg dL(-1) over a time period of 7 days. After subcutaneous implantation of sensors into interscapular region of male rats glucose in interstitial fluid was evaluated and compared to glucose in arterial blood up to 7 days. Hyper- and hypoglycaemia were induced by intravenous application of glucose and insulin, respectively. Current of each implanted sensor was converted into glucose concentration using the first blood glucose measurement only. A change of current with glucose of 0.35 nA mg(-1)dL(-1) indicates high sensitivity of the sensor in vitro. The response time (90% of steady state) was calculated by approximately 60s. Test strips for blood glucose measurement as reference for sensor readings was found as an appropriate and rapidly available method in rats by comparison with established hexokinase method in an automated lab analyzer with limits of agreement of +32.8 and -25.7 mg dL(-1) in Bland-Altman analysis. In normo- and hypoglycaemic range sensor readings in interstitial fluid correlated well with blood glucose measurements whereas hyperglycaemia was not reflected by the sensor completely when blood glucose was changing rapidly. The data given characterize a sensor with high sensitivity, long term stability and short response time. A single calibration of the sensor is required only in measurement periods up to 7 days. The findings demonstrate that the sensor is a highly promising candidate for assessment in humans.

Direct fluorometric analysis of a newly synthesised fluorescein-labelled marker for glomerular filtration rate.

There is an obvious and growing medical need for an accurate determination of kidney function in the diagnosis and management of renal diseases. The glomerular filtration rate (GFR) is the accepted gold standard measurement of kidney function. Several approaches to estimate the GFR are available, but most of them are inconvenient and, therefore, of limited acceptance. A new method of quantification with fluorescein-isothiocyanate (FITC) sinistrin (FS), a novel GFR marker, has been evaluated. The method is based on the fluorescence label of FS and can be performed with a standard fluorometer. To control the interference of protein with the fluorescence signal, a calibration function was developed. The accuracy of the fluorometric method established is comparable to the so-called "gold standard" of enzymatic determination of polyfructosan. Moreover, FS is easy to handle and requires low-cost instruments. Our results demonstrate the potential of the direct fluorometric analysis of the new FITC-labelled marker of being a precise, simple, rapid and cost-effective method for diagnosing disturbed kidney function and monitoring its treatment efficacy. The dramatic decrease in analytical effort will result in a significantly higher acceptability of GFR determination.

Fluorescein-labeled sinistrin as marker of glomerular filtration rate.

There is an obvious and growing medical need for an accurate and easy to handle determination of glomerular filtration rate (GFR) for a broad spectrum of indications. Newly synthesized fluorescein-isothiocyanate (FITC)-sinistrin (FS) with various degrees of labeling was selected by its physicochemical properties and good tolerability out of a number of dye-labeled compounds intended for use as GFR markers for characterization of its pharmacological profile. With respect to solubility FS is more convenient in handling compared to FITC-inulin (FI). Up to 100 mg ml(-1) of FS can be dissolved in aqueous solvents at room temperature, whereas FI can only be solubilized after warming up to 55 degrees C. This reveals a considerable advantage of FS over FI in preparation of galenical formulations for intended i.v. application. A fluorometric method was established to determine FS concentration in blood serum with a comparable accuracy to the established enzymatic method for polyfructosanes. Similar concentration time curves in blood serum of FS measured fluorometrically and enzymatically suggest no relevant change of pharmacokinetic behavior by dye labeling. This notion is supported by the rapid renal and missing of biliary excretion. On the basis of these results, FS is superior in handling to the available GFR markers and makes it highly interesting as a novel diagnostic drug.