Children s Hospital of Philadelphia. Annual Progress Report: 2010 Formula Grant. Reporting Period. July 1, 2011 June 30, Formula Grant Overview

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1 Children s Hospital of Philadelphia Annual Progress Report: 2010 Formula Grant Reporting Period July 1, 2011 June 30, 2012 Formula Grant Overview The Children s Hospital of Philadelphia received $3,548,977 for grant award period January 1, 2011 to December 31, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose Magnetic Resonance Imaging and Neurocognitive Assessment in Chronic Kidney Disease - Utilizing state of the art neuroimaging technology, including standard magnetic resonance imaging (MRI), arterial spin labeling (ASL), resting state functional MRI (fmri), and a concurrent matched control group in a cross-sectional study of 180 individuals, aged 8-25 years, we will test the hypothesis that subclinical vascular disease affects cognitive function in chronic kidney disease (CKD). Using novel pattern recognition methods, we will integrate these measures to develop a multi-parametric imaging phenotype in CKD that can identify high-risk groups. We hypothesize that level of kidney function, hypertension (HTN) and anemia affect resting cerebral blood flow (CBF) and functional performance. Achievement of our aims will enable targeted interventions to prevent and treat the cognitive deficits associated with CKD. Anticipated Duration of Project 1/1/ /31/2014 Project Overview To identify the causal mechanisms underlying the cognitive decline seen in CKD, we propose to use state of the art multimodal MRI and cognitive testing in a cross-sectional study of 90 subjects with Stage III, IV and V CKD (estimated GFR <90 ml/min/1.73m2), aged 8 25 years, and 90 controls matched on age and socioeconomic status, using insurance status as a proxy. Our specific aims are: 1. To demonstrate differences in brain imaging between the group of individuals with CKD and age-matched controls, manifested by a higher prevalence of subcortical hyperintensities on T2- weighted (FLAIR) MRI and reduction in ASL signal in subjects with CKD. We hypothesize that HTN and anemia in CKD cause alterations in CBF measured using ASL perfusion MRI, as well as hypoxic-ischemic injury in the brain, which can be measured via multi-parametric MRI (combination of FLAIR, T2, Proton density and T1-weighted images). Children s Hospital of Philadelphia 2010 Formula Grant Page 1

2 2. To explore the impact of CKD on structural brain integrity as measured by anatomic MRI with quantitative volumetric measurements with a particular focus on prefrontal cortex and frontotemporal regions. We hypothesize that evidence of frontotemporal brain injury will be correlated with deficits in executive function in subjects with kidney disease. 3. To characterize the impact of longer duration and increased severity of CKD, HTN and anemia on neurocognitive abilities, and correlate these with fmri findings. We hypothesize that cognitive deficits will be correlated with changes in regional CBF and/or connectivity within specific brain networks supporting these neurocognitive functions. 4. To develop individual-patient biomarkers by combining structural, ASL and fmri data in multi-parametric classification. Our goal is to ultimately find imaging patterns that in future studies can predict high risk of neurocognitive decline and thereby identify subjects for targeted interventions. Principal Investigator Susan L. Furth, MD, PhD Professor, Pediatrics Children's Hospital of Philadelphia 3615 Civic Center Boulevard Philadelphia, PA Other Participating Researchers Abbas Jawad, PhD; Divya Moodalbail, MD; Kevin Meyers, MD; Madhura Pradhan, MD; Jerilynn Radcliffe, PhD; Rebecca Ruebner, MD; Robert Schultz, PhD; Karuna Shekdar, MD - employed by The Children s Hospital of Philadelphia Christos Davatzikos, PhD; John Detre, PhD; Ruben Gur, PhD - employed by University of Pennsylvania Stephen Hooper, PhD employed by University of North Carolina Expected Research Outcomes and Benefits We will identify differences in brain structure and functional performance between CKD and control group children and young adults. Structural MRI (smri) will assess the regional distribution of grey matter, white matter and cerebrospinal fluid (CSF), which will determine whether there are correlations between CKD related clinical variables, brain atrophy and cognitive performance, and whether or not these relationships are spatially heterogeneous. Structural MRI will provide measurements of the regional distribution of cerebrovascular disease (CVD), as HTN is exceedingly common in CKD and is one of the highest risk factors for CVD. Additionally, ASL MRI will provide measurements of regional blood perfusion, which will provide mechanistic insights into the pathogenesis of observed structural changes based on correlations between CKD-related clinical variables, brain atrophy and cognitive performance, and whether or not these relationships are spatially heterogeneous. Using both ASL MRI and resting state fmri we will examine brain activity in the absence of external stimuli and this will Children s Hospital of Philadelphia 2010 Formula Grant Page 2

3 allow us to evaluate functional changes in these patients that correlate with clinical and cognitive variables. State of the art pattern recognition methods will allow us to integrate all these types of measurements and develop a multi-parametric imaging phenotype in CKD. This phenotype can be used to predict subsequent cognitive decline in future longitudinal studies so that interventions can be implemented to improve functional performance in this population. We are studying children and young adults with CKD, as this will allow us to identify neurocognitive and neuroanatomic changes due to kidney disease in the absence of underlying CVD, which is ubiquitous in adults with CKD. The long term benefit of this project will be improvement in the health status of the 1 in 9 Americans with CKD, as cognitive impairment may affect adherence to the complex medical regimens which these patients are routinely prescribed, and may contribute substantially to decreasing the adverse outcomes and substantial cost associated with end stage renal disease (>$36 billion in 2007). Summary of Research Completed Literature Review: The study team conducted an extensive and thorough literature review of neuroimaging studies in adult and pediatric CKD populations. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method was used to identify a comprehensive body of literature including 43 scholarly articles. These studies were summarized and results were compared across adult and pediatric populations to isolate gaps in the existing literature. Intermittent dialysis treatments such as hemodialysis (HD), when compared to stable CKD and continuous dialysis modalities are associated with recurrent osmotic changes in the brain, which increases risk for brain atrophy and brain density changes. Neuroimaging studies have revealed associations between moderate to severe CKD and white matter hyperintensities, highlighting the growing importance of kidney disease as a possible determinant of cerebrovascular disease and/or as a marker of microangiopathy. Brain MRI studies in adults have shown that CKD maintained by HD increases the prevalence of silent cerebral infarction (SCI). Methods: Informed consent was obtained before conduct of study procedures and unique study IDs were assigned to protect confidentiality. Case report forms were created and are kept in a locked office to keep identifying information separate. Fasting blood draw, urinalysis, and urine pregnancy test (for girls greater than 11 years of age) were performed in the Clinical and Translational Research Center (CTRC). The traditional neurocognitive battery was administered by the CTRC Neurobehavioral Core and a computerized battery was administered by the University of Pennsylvania (UPenn), Department of Psychiatry. The MRI scans were conducted in CHOP s Radiology department. The ABPMs were worn for 24 hours following study visit. By ABPM, BP loads were calculated as the percentage of readings > 95 th %ile for age, gender and height during waking and sleeping periods. The ambulatory BP index was calculated as the mean ambulatory BP, by wake or sleep state, divided by the corresponding 95 th percentile (an index of 1 indicates an ambulatory BP equal to the threshold value for a clinical diagnosis of HTN, and an index of 1.1 is 10% above the threshold). Questionnaires were used to collect demographics, medical, family, and developmental history. A secure REDCap database was created to compile data. Structural MRI are pre-processed by bias correction, removal of extra-cranial material on T1- Children s Hospital of Philadelphia 2010 Formula Grant Page 3

4 weighted image (skull-stripping), and tissue segmentation into gray matter, white matter and CSF. Fig. 1 illustrates the processing pipeline. Voxel Based Morphometry analysis is applied on Regional Analysis of Volumes Examined in Normalized Space (RAVENS) maps to determine regional patterns of brain changes between CKD and control subjects. Functional MRI data are analyzed by both complex network analysis and spatiotemporal independent component analysis (ICA). The researchers focus on correlation matrices between regions of interest (ROI) associated with cognitive functions of interest in this study derived from a priori structural templates as well as segmentations of smri data. Arterial Spin Labeling was achieved using pseudo continuous ASL at 3 Tesla. All quantitative CBF maps were calculated from the ASL series using the single-blood-compartment model and normalized to Montreal Neurological Institute space with Statistical Parametric Mapping 8 (SPM8). Standard anatomical regions of gray matter, white matter and global brain areas based on the template were applied to the normalized images in SPM8 to extract the CBF values for comparison. Secure databases were developed to compile smri, fmri, and ASL MRI data and a secure File Transfer Protocol (FTP) server was created to facilitate the transfer of information between CHOP and UPenn. A comprehensive neuropsychological test battery (WASI Vocabulary, WASI Similarities, WASI Block Design, WASI Matrix Reasoning, Wechsler Memory Scales-III, WISC-IV Digit Span, Continuous Performance Test-II, and Delis-Kaplan Executive Function Scales) was administered during the study visit. These tests of language, memory, visuospatial functioning, and executive and attention are scored relative to age-based norms. Mean z-score and standard deviation (SD) were derived for each domain. The researchers report initial comparisons across functional domains between CKD subjects and controls in Table 3. These preliminary results illustrate poorer functioning overall for the CKD group as compared with the controls, although visuospatial skills, executive functioning and attention were relatively weaker for the CKD group. A survey measure of executive function, Behavior Rating Inventory for Executive Functioning (BRIEF), which was completed by either the parent (ages 8 to 18 yrs.) or the older subject (ages 19 to 25 yrs.) was analyzed. Scores for the two versions of BRIEF were combined for comparison purposes and results were reported in terms of t-scores and SD in Table 3.The CKD group shows slightly higher scores (indicating greater impairment) relative to the controls. A computerized neurocognitive test developed by Dr. Ruben Gur was also administered to study participants (Gur, R.C. et al, J Neurosci Methods 187(2): ; 2010). Results from this computerized battery will be analyzed similarly to those for the neuropsychological test battery. The steering committee met via bimonthly conference call and quarterly in-person meetings to review study progress. No serious adverse events were reported during this time period. Institutional review board (IRB) approval was obtained and maintained for all study procedures. Results: 21 CKD subjects and 7 controls were enrolled. Demographic data is available in Table 1. Control subject recruitment has intentionally lagged CKD cohort recruitment to allow for matching based on SES using insurance provider as a proxy. Overall, there were no differences in chemistry panels between the two groups, except CKD subjects when compared to controls had lower mean carbon-di-oxide (25.35 vs. 27.7meq/L), hemoglobin (Hb) (12.57 vs g/dl) Children s Hospital of Philadelphia 2010 Formula Grant Page 4

5 and higher mean blood urea nitrogen (37.20 vs mg/dl) and creatinine (2.92 vs. 0.69mg/dl). These findings are consistent with clinical patterns seen in CKD. Ambulatory blood pressure monitoring from 25 out of 28 subjects yielded 22 successful studies. Preliminary data suggest higher waking systolic blood pressure (BP) load and sleeping diastolic BP load in CKD subjects (Table 2). This data is also consistent with clinical patterns in CKD. As recruitment continues, we will assess CBF changes and smri changes in CKD with high BP and low Hb. Neurocognitive data: Results from the neuropsychological test battery has been preliminarily examined and is presented in Table 3. Neuropsychological findings indicate relative weaknesses in attention, verbal memory, visuospatial skills and set shifting, as evidenced by the more negative z-scores in comparison to language and inhibitory control, which had near-normal performance. However, wide variability in functioning was found within each domain. Results of BRIEF indicate the presence of mild executive dysfunction, particularly in areas of planning, organization, and working memory as evidenced by higher t-scores in the Metacognitive Index (MI) and Global executive Composite (GEC), as shown in Table 3. Neuroimaging data: All 28 subjects had neuroimaging studies. Regional patterns of brain changes between CKD and control subjects in preliminary analysis are illustrated in Figs. 2 and 3. A sample smri result demonstrating ischemic lesions segmented using multimodal segmentation technique trained on lesions manually delineated by an expert radiologist is presented in Fig. 4. The left panel of Fig. 5 displays a preliminary fmri group connectivity matrix for CKD participants using ROIs derived from Shirer et al., This panel color-codes pearson correlations (over time) between sets of brain areas (which are organized into known functional subdivisions). As the researchers collect additional control participants, they will be able to directly compare CKD and control network matrices (and their distributional properties: Lynall et al., 2010). The right panel of Fig. 5 illustrates a fronto-parietal independent component from an ICA on a pooled fmri dataset of 17 CKD and 6 control participants. This component reflects coordinated, distributed brain activity over time. ICA analyses yield global maps as well as individual subject weightings single numbers reflecting each subject s contribution to each independent component. With more participants, CKD and control participant weightings can be compared via t-tests to isolate independent components (connectivity clusters) that differ between groups. Current ASL MRI data analysis includes 20 CKD and 7 control subjects, with good quality CBF maps. Preliminary results demonstrated that CKD subjects when compared to the controls showed higher CBF in gray matter (80.83±12.66 vs ±11.02 ml/100 g/min), white matter (65.35±10.61 vs ±5.02 ml/100 g/min), and global brain areas (76.18±12.40 vs ±9.82 ml/100 g/min). The researchers used linear regression to evaluate the relationship between hematocrit (HCT) and CBF (in combined CKD and control groups) in these brain areas, and observed inverse correlation between HCT and CBF (Table 4). Although the sample scanned to date is small, the findings suggest that CBF is increased in CKD as compared to control subjects. Only some of this difference can be explained by known modulators of CBF such as age, gender and HCT. Children s Hospital of Philadelphia 2010 Formula Grant Page 5

6 Milestones : During this reporting period, 5 of 5 milestones were met: 1. Ongoing screening for eligible subjects - All new and follow-up patients in nephrology and dialysis clinics at CHOP were screened. All screening data were entered into a secure log according to IRB-approved pre-screening procedures. 2. Identify 50 eligible subjects Of the 79 eligible subjects, the researchers were able to approach 66 (84%) and enroll 21 (32%). Reasons for not enrolling include waiting for removal of orthodontic braces (prohibitive of MRI)/ unable to miss school/ refusal/ transfer to adult care center far from CHOP. 3. Perform first study visits including neurocognitive testing and neuroimaging Study visits and neuroimaging were completed for 28 subjects. Neurocognitive testing has been completed for Assess number of eligible CKD and control subjects identified through nephrology clinic, if not achieving recruitment goals, assess alternative methods to identify eligible subjects (consider increasing upper age limit, recruit from neighboring nephrology centers, and primary care centers for controls) and submit updated recruitment plan to IRB - The number of eligible subjects at CHOP nephrology clinic may lead to challenges in meeting original recruitment goals. After discussion with the steering committee, it was decided that broadening the eligibility criteria to include subjects with less severe kidney disease would not undermine the goals of the study. Therefore, the following were approved by the IRB on May 25, 2012: 1) Expand inclusion kidney function criteria to egfr<90 ml/min/1.73m 2 2) Expand recruitment at Jefferson and Dupont pediatric nephrology clinics using existing CHOP-IRB-approved recruitment materials. 3) Expand recruitment strategies for control subjects to include CHOP s outpatient adolescent clinic and primary care satellite clinics. A letter of support has been written by the Chief of the Division of Adolescent Medicine, and the researchers have received IRB approval to send recruitment letters to matched controls identified by the Pediatric Research Consortium (PeRC). 5. Ongoing steering committee meetings to assess progress in recruitment, and quality of data collection - The steering committee met quarterly in person and bi-monthly via conference call. Each meeting followed a data-driven agenda and all members individually met with PI Susan Furth to review data transfer and analysis practices. Children s Hospital of Philadelphia 2010 Formula Grant Page 6

7 Table 1: Demographics of CKD and Control Subjects Demographic Number (%) of CKD Cohort (n=21) Number (%) of Control Cohort (n=7) Gender Male 16 (76%) 3 (43%) Race White Black Other 16 (76%) 4 (19%) 0 (0%) 5 (71%) 1 (14%) 1 (14%) Ethnicity Non-Hispanic 20 (95%) 4 (57%) Age 8-16 yrs yrs 14 (67%) 7(23%) 5 (71%) 2 (29%) Maternal Education High school or greater 19 (90%) 7 (100%) Annual Household Income <$36,000 3 (14%) 1 (14%) Table 2: ABPM data CKD Cohort (n=21) Control Cohort (n=7) Systolic Diastolic Systolic Diastolic Load* Index + Load Index Load Index Load Index (%) (%) (%) (%) Waking 23 ± ± ± ± ± ± ± ±0.07 Sleeping 19 ± ± ± ± ± ± ± ±0.04 Total 22 ± ± ± ± ± ± ± ±0.04 Dipping 11% ± 6% 19% ± 7% 13% ± 5% 17% ± 5% *Load: Percentage of readings > 95 th %ile for age, gender and height Reference values for BP Load: Normal <25%; Intermediate 25 50%; Abnormal >50% + Index: Mean ambulatory BP, by wake or sleep state/ corresponding 95 th percentile Reference values for Dipping: Normal > 10% Children s Hospital of Philadelphia 2010 Formula Grant Page 7

8 Table 3: Comparison of neuropsychological test battery scores in CKD and control subjects CKD subjects (n=18) Control subjects (n=7) Domains Language Attention Inhibitory Control Planned Problem solving Set Shifting Visuo-spatial skills Verbal Memory Visual Work Memory BRIEF Summary Scales Behavioral Regulation Index (BRI) Metacognitive Index (MI) Global Executive Composite (GEC) Domain Mean z-scores BRIEF t-score Domain Mean z-scores BRIEF t-score Note. Scores for the Neuropsychology Domains are reported in z-scores with a Mean = 0 and a Standard Deviation of 1, with higher scores reflecting a better performance. Scores for the BRIEF summary scales are reported in T-Scores with a Mean = 50 and a Standard Deviation of 10, with higher scores reflecting more impaired performance. Table 4: Relationship between Hematocrit and Cerebral Blood Flow for combined CKD and Control subjects Hematocrit and CBF Correlation Co-efficient (R) Not Controlled for Age and Gender Correlation Co-efficient (R) Controlled for Age and Gender Grey Matter -0.44, P= , P=0.038 between White Matter , P= , P=0.087 Global , P= , P=0.043 Children s Hospital of Philadelphia 2010 Formula Grant Page 8

9 Figure1: Structural MRI processing pipeline. Figure 2. Gray matter volumetric differences between CKD and Control groups. Blue indicates higher volume in Controls as compared to CKD group and vice versa for red. P<0.01 Figure 3: White matter volumetric differences between CKD and Control groups. Blue indicates higher volume in Controls as compared to CKD group and vice versa for red. P<0.01 Children s Hospital of Philadelphia 2010 Formula Grant Page 9

10 Figure 4: Segmented white matter lesions on a sample subject. Right: Functional scan, Left: Lesion mask overlaid in red. Figure 5: Resting State Connectivity Analyses. Left panel: average connectivity for 15 CKD participants.the figure color-codes positive correlations (pearson r-values) between brain areas (over time). Right panel: connectivity between parietal and frontal cognitive control regions in 17 CKD and 6 control participants, calculated using ICA. Children s Hospital of Philadelphia 2010 Formula Grant Page 10