Magnetic Resonance Imaging Biomarkers of Renal Structure and Function

The kidney's major role in filtration depends on its high blood flow, concentrating mechanisms, and biochemical activation. The kidney's greatest strengths also lead to vulnerability for drug-induced nephrotoxicity and other renal injuries. The current standard to diagnose renal injuries is with a percutaneous renal biopsy, which can be biased and insufficient. In one particular case, biopsy of a kidney with renal cell carcinoma can actually initiate metastasis. Tools that are sensitive and specific to detect renal disease early are essential, especially noninvasive diagnostic imaging. While other imaging modalities (ultrasound and x-ray/CT) have their unique advantages and disadvantages, MRI has superb soft tissue contrast without ionizing radiation. More importantly, there is a richness of contrast mechanisms in MRI that has yet to be explored and applied to study renal disease.

The focus of this work is to advance preclinical imaging tools to study the structure and function of the renal system. Studies were conducted in normal and disease models to understand general renal physiology as well as pathophysiology. This dissertation is separated into two parts--the first is the identification of renal architecture with ex vivo MRI; the second is the characterization of renal dynamics and function with in vivo MRI. High resolution ex vivo imaging provided several opportunities including: 1) identification of fine renal structures, 2) implementation of different contrast mechanisms with several pulse sequences and reconstruction methods, 3) development of image-processing tools to extract regions and structures, and 4) understanding of the nephron structures that create MR contrast and that are important for renal physiology. The ex vivo studies allowed for understanding and translation to in vivo studies. While the structure of this dissertation is organized by individual projects, the goal is singular: to develop magnetic resonance imaging biomarkers for renal system.

The work presented here includes three ex vivo studies and two in vivo studies:

1) Magnetic resonance histology of age-related nephropathy in sprague dawley.

2) Quantitative susceptibility mapping of kidney inflammation and fibrosis in type 1 angiotensin receptor-deficient mice.

3) Susceptibility tensor imaging of the kidney and its microstructural underpinnings.

4) 4D MRI of renal function in the developing mouse.

5) 4D MRI of polycystic kidneys in rapamycin treated Glis3-deficient mice.

In vivo visualization of abdominal malignancies with acoustic radiation force elastography.

The utility of acoustic radiation force impulse (ARFI) imaging for real-time visualization of abdominal malignancies was investigated. Nine patients presenting with suspicious masses in the liver (n = 7) or kidney (n = 2) underwent combined sonography/ARFI imaging. Images were acquired of a total of 12 tumors in the nine patients. In all cases, boundary definition in ARFI images was improved or equivalent to boundary definition in B-mode images. Displacement contrast in ARFI images was superior to echo contrast in B-mode images for each tumor. The mean contrast for suspected hepatocellular carcinomas (HCCs) in B-mode images was 2.9 dB (range: 1.5-4.2) versus 7.5 dB (range: 3.1-11.9) in ARFI images, with all HCCs appearing more compliant than regional cirrhotic liver parenchyma. The mean contrast for metastases in B-mode images was 3.1 dB (range: 1.2-5.2) versus 9.3 dB (range: 5.7-13.9) in ARFI images, with all masses appearing less compliant than regional non-cirrhotic liver parenchyma. ARFI image contrast (10.4 dB) was superior to B-mode contrast (0.9 dB) for a renal mass. To our knowledge, we present the first in vivo images of abdominal malignancies in humans acquired with the ARFI method or any other technique of imaging tissue elasticity.

In vivo small animal micro-CT using nanoparticle contrast agents.

Computed tomography (CT) is one of the most valuable modalities for in vivo imaging because it is fast, high-resolution, cost-effective, and non-invasive. Moreover, CT is heavily used not only in the clinic (for both diagnostics and treatment planning) but also in preclinical research as micro-CT. Although CT is inherently effective for lung and bone imaging, soft tissue imaging requires the use of contrast agents. For small animal micro-CT, nanoparticle contrast agents are used in order to avoid rapid renal clearance. A variety of nanoparticles have been used for micro-CT imaging, but the majority of research has focused on the use of iodine-containing nanoparticles and gold nanoparticles. Both nanoparticle types can act as highly effective blood pool contrast agents or can be targeted using a wide variety of targeting mechanisms. CT imaging can be further enhanced by adding spectral capabilities to separate multiple co-injected nanoparticles in vivo. Spectral CT, using both energy-integrating and energy-resolving detectors, has been used with multiple contrast agents to enable functional and molecular imaging. This review focuses on new developments for in vivo small animal micro-CT using novel nanoparticle probes applied in preclinical research.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Optimization of treatment strategy used during shockwave lithotripsy to maximize stone fragmentation efficiency.

BACKGROUND AND PURPOSE: Previous studies have demonstrated that treatment strategy plays a critical role in ensuring maximum stone fragmentation during shockwave lithotripsy (SWL). We aimed to develop an optimal treatment strategy in SWL to produce maximum stone fragmentation. MATERIALS AND METHODS: Four treatment strategies were evaluated using an in-vitro experimental setup that mimics stone fragmentation in the renal pelvis. Spherical stone phantoms were exposed to 2100 shocks using the Siemens Modularis (electromagnetic) lithotripter. The treatment strategies included increasing output voltage with 100 shocks at 12.3 kV, 400 shocks at 14.8 kV, and 1600 shocks at 15.8 kV, and decreasing output voltage with 1600 shocks at 15.8 kV, 400 shocks at 14.8 kV, and 100 shocks at 12.3 kV. Both increasing and decreasing voltages models were run at a pulse repetition frequency (PRF) of 1 and 2 Hz. Fragmentation efficiency was determined using a sequential sieving method to isolate fragments less than 2 mm. A fiberoptic probe hydrophone was used to characterize the pressure waveforms at different output voltage and frequency settings. In addition, a high-speed camera was used to assess cavitation activity in the lithotripter field that was produced by different treatment strategies. RESULTS: The increasing output voltage strategy at 1 Hz PRF produced the best stone fragmentation efficiency. This result was significantly better than the decreasing voltage strategy at 1 Hz PFR (85.8% vs 80.8%, P=0.017) and over the same strategy at 2 Hz PRF (85.8% vs 79.59%, P=0.0078). CONCLUSIONS: A pretreatment dose of 100 low-voltage output shockwaves (SWs) at 60 SWs/min before increasing to a higher voltage output produces the best overall stone fragmentation in vitro. These findings could lead to increased fragmentation efficiency in vivo and higher success rates clinically.

Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by beta-adrenergic receptor stimulation and blockade.

BACKGROUND: Impaired myocardial beta-adrenergic receptor (betaAR) signaling, including desensitization and functional uncoupling, is a characteristic of congestive heart failure. A contributing mechanism for this impairment may involve enhanced myocardial beta-adrenergic receptor kinase (betaARK1) activity because levels of this betaAR-desensitizing G protein-coupled receptor kinase (GRK) are increased in heart failure. An hypothesis has emerged that increased sympathetic nervous system activity associated with heart failure might be the initial stimulus for betaAR signaling alterations, including desensitization. We have chronically treated mice with drugs that either activate or antagonize betaARs to study the dynamic relationship between betaAR activation and myocardial levels of betaARK1. METHODS AND RESULTS: Long-term in vivo stimulation of betaARs results in the impairment of cardiac +betaAR signaling and increases the level of expression (mRNA and protein) and activity of +betaARK1 but not that of GRK5, a second GRK abundantly expressed in the myocardium. Long-term beta-blocker treatment, including the use of carvedilol, improves myocardial betaAR signaling and reduces betaARK1 levels in a specific and dose-dependent manner. Identical results were obtained in vitro in cultured cells, demonstrating that the regulation of GRK expression is directly linked to betaAR signaling. CONCLUSIONS: This report demonstrates, for the first time, that betaAR stimulation can significantly increase the expression of betaARK1 , whereas beta-blockade decreases expression. This reciprocal regulation of betaARK1 documents a novel mechanism of ligand-induced betaAR regulation and provides important insights into the potential mechanisms responsible for the effectiveness of beta-blockers, such as carvedilol, in the treatment of heart failure.

In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.

BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary beta2-adrenergic receptor gene delivery.

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Development of a decision aid to inform patients' and families' renal replacement therapy selection decisions.

BACKGROUND: Few educational resources have been developed to inform patients' renal replacement therapy (RRT) selection decisions. Patients progressing toward end stage renal disease (ESRD) must decide among multiple treatment options with varying characteristics. Complex information about treatments must be adequately conveyed to patients with different educational backgrounds and informational needs. Decisions about treatment options also require family input, as families often participate in patients' treatment and support patients' decisions. We describe the development, design, and preliminary evaluation of an informational, evidence-based, and patient-and family-centered decision aid for patients with ESRD and varying levels of health literacy, health numeracy, and cognitive function. METHODS: We designed a decision aid comprising a complementary video and informational handbook. We based our development process on data previously obtained from qualitative focus groups and systematic literature reviews. We simultaneously developed the video and handbook in "stages." For the video, stages included (1) directed interviews with culturally appropriate patients and families and preliminary script development, (2) video production, and (3) screening the video with patients and their families. For the handbook, stages comprised (1) preliminary content design, (2) a mixed-methods pilot study among diverse patients to assess comprehension of handbook material, and (3) screening the handbook with patients and their families. RESULTS: The video and handbook both addressed potential benefits and trade-offs of treatment selections. The 50-minute video consisted of demographically diverse patients and their families describing their positive and negative experiences with selecting a treatment option. The video also incorporated health professionals' testimonials regarding various considerations that might influence patients' and families' treatment selections. The handbook was comprised of written words, pictures of patients and health care providers, and diagrams describing the findings and quality of scientific studies comparing treatments. The handbook text was written at a 4th to 6th grade reading level. Pilot study results demonstrated that a majority of patients could understand information presented in the handbook. Patient and families screening the nearly completed video and handbook reviewed the materials favorably. CONCLUSIONS: This rigorously designed decision aid may help patients and families make informed decisions about their treatment options for RRT that are well aligned with their values.

Selecting renal replacement therapies: what do African American and non-African American patients and their families think others should know? A mixed methods study.

BACKGROUND: Little is known regarding the types of information African American and non-African American patients with chronic kidney disease (CKD) and their families need to inform renal replacement therapy (RRT) decisions. METHODS: In 20 structured group interviews, we elicited views of African American and non-African American patients with CKD and their families about factors that should be addressed in educational materials informing patients' RRT selection decisions. We asked participants to select factors from a list and obtained their open-ended feedback. RESULTS: Ten groups of patients (5 African American, 5 non-African American; total 68 individuals) and ten groups of family members (5 African American, 5 non-African American; total 62 individuals) participated. Patients and families had a range (none to extensive) of experiences with various RRTs. Patients identified morbidity or mortality, autonomy, treatment delivery, and symptoms as important factors to address. Family members identified similar factors but also cited the effects of RRT decisions on patients' psychological well-being and finances. Views of African American and non-African American participants were largely similar. CONCLUSIONS: Educational resources addressing the influence of RRT selection on patients' morbidity and mortality, autonomy, treatment delivery, and symptoms could help patients and their families select RRT options closely aligned with their values. Including information about the influence of RRT selection on patients' personal relationships and finances could enhance resources' cultural relevance for African Americans.