Detection of glomerular complement C3 fragments by magnetic resonance imaging in murine lupus nephritis.

One of the challenges of treating patients with glomerulonephritis is to accurately assess disease activity. As renal biopsies are routinely stained for deposits of C3 activation fragments and glomerular C3 deposits are found in most forms of glomerulonephritis, we sought to determine whether a relatively noninvasive measure of C3 fragment deposition in the kidney can serve as a good biomarker of disease onset and severity. We recently developed a magnetic resonance imaging (MRI)-based method of detecting glomerular C3 and used this to track the progression of renal disease in the MRL/lpr mouse model of lupus nephritis using superparamagnetic iron oxide nanoparticles conjugated to complement receptor type 2 as a targeting agent. Quantitative immunofluorescence showed that glomerular C3b/iC3b/C3d deposition progressively increased with disease activity, a finding replicated by the T2-weighted MRI. The T2 relaxation times decreased with disease activity in the cortex and medulla of the MRL/lpr but not in MRL/Mpj control mice. Thus, MRI contrast agents targeted to glomerular C3 fragments can be used to noninvasively monitor disease activity in glomerulonephritis. As therapeutic complement inhibitors are used in patients with renal disease, this method, should it become feasible in humans, may identify those likely to benefit from complement inhibition.

Silibinin prevents lung tumorigenesis in wild-type but not in iNOS-/- mice: potential of real-time micro-CT in lung cancer chemoprevention studies.

PURPOSE: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS(-/-) mice. EXPERIMENTAL DESIGN: Male B6/129-Nos2(tm1Lau) (iNOS(-/-)) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks. RESULTS: Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS(-/-) mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors. CONCLUSIONS: The lack of effect of silibinin in iNOS(-/-) mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents.

Distinct patterns of kidney and liver cyst growth in pkd2(WS25/-) mice.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that results in the development of cystic kidneys and liver. Pkd2(WS25/-) mice are a key genetic mouse model of human ADPKD that recapitulate the 'molecular recessive' nature of human ADPKD. Providing the foundation for future long-term studies, the present work documents distinct patterns of long-term cyst growth in the kidneys and liver of male and female pkd2(WS25/-) mice. METHODS: Gravimetric measurements documented the progression of kidney and liver growth in male and female pkd2(WS25/-) mice over 12 months. A fast imaging with steady-state precision-magnetic resonance imaging (FISP-MRI) technique to measure kidney and liver organ and cyst volumes was optimized and validated. Longitudinal FISP-MRI analyses of changes in cyst volumes were performed in pkd2(WS25/-) mice over 15 months. RESULTS: Male and female pkd2(WS25/-) mice had significant increases in kidney weights after 4 months of age. The progression of kidney growth was minimal after 4 months of age. Liver cyst growth in male pkd2(WS25/-) mice was minimal after 4 months of age but showed an accelerated rate of growth after 8 months of age. Female pkd2(WS25/-) mice also showed accelerated growth but this was delayed in time when compared with male pkd2(WS25/-) mice. CONCLUSIONS: Pkd2(WS25/-) mice are a genetic mouse model that recapitulates the early phenotypic characteristics of human ADPKD kidney cystogenesis. Male pkd2(WS25/-) mice consistently display a late progression in liver growth that is seen in clinically impacted livers of human ADPKD patients.

Assessment of the in vivo antitumor effects of ENMD-2076, a novel multitargeted kinase inhibitor, against primary and cell line-derived human colorectal cancer xenograft models.

PURPOSE: This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC). EXPERIMENTAL DESIGN: HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and (18)FDG-positron emission tomography were conducted to assess the antiproliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition. RESULTS: In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in (18)FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition. CONCLUSIONS: ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC.

Renal inflammation: targeted iron oxide nanoparticles for molecular MR imaging in mice.

PURPOSE: To determine the feasibility of T2-weighted magnetic resonance (MR) imaging in the noninvasive quantification of renal inflammation by using superparamagnetic iron oxide (SPIO) nanoparticles targeted to tissue-bound C3 activation fragments in a mouse model of lupus nephritis. MATERIALS AND METHODS: All animal procedures were approved by the University of Colorado-Denver animal care and use committee. SPIO nanoparticles were encapsulated by using amine-functionalized phospholipids. A recombinant protein containing the C3d-binding region of complement receptor type 2 (CR2) was then conjugated to the surface of the SPIO nanoparticle. Five MRL/lpr mice (a model of lupus nephritis) and six C57BL/6 wild-type mice were assessed with T2-weighted MR imaging at baseline and after SPIO injection. The same five MRL/lpr mice and three C57BL/6 mice also underwent MR imaging after injection of CR2-targeted SPIO. A series of T2-weighted pulses with 16 echo times was used to enable precise T2 mapping and calculation of T2 relaxation times in the cortex and outer and inner medulla of the kidneys, as well as in the spleen, muscle, and fat. The effects of treatment and animal genotype on T2 relaxation times were analyzed with repeated-measures analysis of variance. RESULTS: At baseline, the T2-weighted signal intensity in the kidneys of MRL/lpr mice was higher than that in the kidneys of wild-type mice. Injection of untargeted SPIO did not alter the T2-weighted signal in the kidneys in either strain of mice. Injection of CR2-targeted SPIO in MRL/lpr mice, however, caused a significant accumulation of targeted iron oxide with a subsequent decrease in T2 relaxation times in the cortex and outer and inner medulla of the kidneys. No changes in T2 relaxation time were observed in the wild-type mice after injection of targeted SPIO. CONCLUSION: Injection of CR2-conjugated SPIO caused a significant reduction in T2-weighted MR imaging signal and T2 relaxation time in nephritic kidneys.

Toxicity of MRI and CT contrast agents.

Anatomical and physiological imaging using CT and MRI are playing a critical role in patients' diagnosis, disease characterization and treatment planning. CT- and MRI-based protocols increasingly require an injection of iodinated CT and gadolinium (Gd)-based MRI contrast media. Although routinely used in clinical practice, iodinated and to a less extent Gd-based contrast media possess side effects: life-threatening contrast-induced nephropathy (CIN) is associated with CT and nephrogenic systemic fibrosis (NSF) with MRI contrast agents. CIN is defined as an acute decline in renal functions (serum creatinine increase > 0.5 mg/dl) after administration of iodinated contrast media. Patients with moderate-to-severe chronic kidney disease are considered the highest risk group for development of CIN. CIN is more common with ionic high-osmolar contrast CT media. NSF is a rare condition characterized by the formation of connective tissue in the skin and systemically in the lung, liver, heart and kidney. Patients with end stage kidney disease, acute kidney injury and stage 4-5 chronic kidney disease are at a high risk for NSF. The nonionic linear Gd-chelates are associated with the highest risk of NSF. This review summarizes the incidence, symptoms, safety profile of various CT and MRI contrast agents based on their physiochemical properties.

Magnetic resonance spectroscopy of living tissues.

The comprehensive work of both clinical and basic science colleagues has demonstrated a clear proof of concept for "in vitro discovered- in vivo validated" biomarkers in translational metabolic profiling research using magnetic resonance techniques. Major tissue metabolites (initially discovered by high-resolution in vitro techniques on cancer specimens) can be translated into in vivo protocols based on noninvasive magnetic resonance spectroscopy (MRS). Using (1)H- and (31)P-MRS on living animals or patients, a decrease in citrate and polyamines in prostate cancer, an increase of cholines in breast cancer, as well as a decreased NAA and an increased lactate in gliomas during cancer progression can be assessed noninvasively. MRS can be used to follow up conventional cytotoxic as well as targeted anticancer therapies, which has been extensively done in animal models of cancer. This review focuses on applications and protocol development for in vivo (1)H- and (31)P-MRS on small animal models as well as on larger animals in cancer research, diagnosis, and treatment.