Paul Brakeman

MR-Based Correction of Brain PET Measurements for Heterogeneous Gray Matter Radioactivity Distribution

Partial volume and mixed tissue sampling errors can cause significant inaccuracy in quantitative positron emission tomographic (PET) measurements. We previously described a method of correcting PET data for the effects of partial volume averaging on gray matter (GM) quantitation; however, this method may incompletely correct GM structures when local tissue concentrations are highly heterogeneous. We have extended this three-compartment algorithm to include a fourth compartment: a GM volume of interest (VOI) that can be delineated on magnetic resonance (MR) imaging. Computer simulations of PET images created from human MR data demonstrated errors of up to 120% in assigned activity values in small brain structures in uncorrected data. Four-compartment correction achieved full recovery of a wide range of coded activity in GM VOIs such as the amygdala, caudate, and thalamus. Further validation was performed in an agarose brain phantom in actual PET acquisitions. Implementation of this partial volume correction approach in [18F]fluorodeoxyglucose and [11C]-carfentanil PET data acquired in a healthy elderly human subject was also performed. This newly developed MR-based partial volume correction algorithm permits the accurate determination of the true radioactivity concentration in specific structures that can be defined by MR by accounting for the influence of heterogeneity of GM radioactivity.

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin–Darby canine kidney cells in three‐dimensional collagen gel culture, blockade of β1‐integrin by the AIIB2 antibody or expression of dominant‐negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA–ROCK I–myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Renal repair and recovery.

Objective:To review the cellular and molecular mechanisms of renal repair and recovery after acute kidney injury (AKI). Data Source:The data were summarized from published research articles. Results:In AKI, there is an acute inflammatory response, epithelial cell necrosis and apoptosis, and shedding of epithelial cells into the tubular lumen. Recent work demonstrates that repopulation of damaged renal tubules occurs primarily from proliferation of tubular epithelial cells and resident renal-specific stem cells, with some contribution of paracrine factors from bone marrow–derived mesenchymal stem cells. In addition, growth factors seem to play a critical role in the repair process in animal models of renal injury. However, attempts to use growth factors in the clinical setting to attenuate human AKI or accelerate renal repair have not yet been successful. The endothelium also plays a critical role in the pathogenesis of AKI. Lastly, in human studies, the effect of dialysis on renal recovery remains poorly understood. Conclusions:Experimental animal models of AKI demonstrate that renal recovery and repair involves proliferation of tubular epithelial cells and stem cell populations and the coordinated contribution of multiple growth factors. Future efforts to improve recovery from AKI and improve patient outcomes may include novel therapies based on manipulation of populations of stem cells and augmenting repopulation of renal tubules.

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells

The formation of epithelial tissues requires both the generation of apical–basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

Being Overweight Modifies the Association Between Cardiovascular Risk Factors and Microalbuminuria in Adolescents

OBJECTIVE. The goal was to determine the association between cardiovascular risk factors and microalbuminuria in a nationally representative sample of adolescents and to determine whether being overweight modifies this association. METHODS. We analyzed cross-sectional data from the National Health and Nutrition Examination Survey(1999–2004) for 2515 adolescents 12 to 19 years of age. Cardiovascular risk factors included abdominal obesity, impaired fasting glucose, diabetes mellitus, insulin resistance, high triglyceride levels, low high-density lipoprotein cholesterol levels, hypertension, smoking, and the metabolic syndrome. Microalbuminuria was defined as a urinary albumin/creatinine ratio of 30 to 299 mg/g in a random morning sample. Overweight was defined as BMI of ≥95th percentile, according to the Centers for Disease Control and Prevention 2000 growth charts. RESULTS. Microalbuminuria was present in 8.9% of adolescents. The prevalence of microalbuminuria was higher among nonoverweight adolescents than among overweight adolescents. The median albumin/creatinine ratio decreased with increasing BMI z scores. The association of microalbuminuria with cardiovascular risk factors differed according to BMI category. Among nonoverweight adolescents, microalbuminuria was not associated with any cardiovascular disease risk factor except for overt diabetes mellitus. Among overweight adolescents, however, microalbuminuria was associated with impaired fasting glucose, insulin resistance, hypertension, and smoking, as well as diabetes mellitus. CONCLUSION. For the majority of adolescents, microalbuminuria is not associated with cardiovascular risk factors. Among overweight adolescents, however, microalbuminuria is associated with cardiovascular risk factors. The prognostic importance of microalbuminuria in overweight and nonoverweight adolescents with regard to future cardiovascular and renal disease needs to be defined in prospective studies conducted specifically in children.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein–protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein–protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

Transient Hyperglycemia Affects the Extent of Ischemia-Reperfusion-induced Renal Injury in Rats

Background:Chronic hyperglycemia is known to increase renal injury, particularly during ischemia-reperfusion episodes. The goal of this study was to examine whether transient hyperglycemia during or after renal ischemia-reperfusion increased renal dysfunction. Methods:Male Lewis rats underwent sham operations or unilateral nephrectomies followed by contralateral renal ischemia-reperfusion. Hyperglycemic rats were given 25% dextrose to induce transient hyperglycemia lasting throughout the duration of ischemia (PI rats) or beginning 2 h after initiation of reperfusion (PR rats). Additional vehicle control rats received saline and underwent ischemia-reperfusion surgery as with PI and PR rats. Twenty-five minutes of mild renal ischemia followed by 24 h of reperfusion was induced by occluding the renal artery and vein. Results:Terminal serum creatinine concentrations were significantly higher in the PI rats when compared with the PR or vehicle control rats. Histology demonstrated significantly increased necrosis in the PI rats relative to PR and control animals. Tissue analyses demonstrated significantly higher heat shock protein 70, heat shock protein 32, and cleaved caspase-3 protein levels in the PI rats. Oxidative stress generated through the xanthine pathway in the PI group was significantly increased compared with the oxidative stress in the PR and vehicle control rats. In contrast, vascular endothelial growth factor and erythropoietin were significantly decreased in the PI rats compared with the PR rats and controls. Conclusions:Hyperglycemia that occurred during renal ischemia-reperfusion resulted in severe functional injury compared with normoglycemia or with hyperglycemia that occurred after reperfusion. Investigated molecular pathways are more profoundly affected by hyperglycemia that occurs before renal ischemia-reperfusion.

Preso1 dynamically regulates group I metabotropic glutamate receptors

Group I metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, are G protein–coupled receptors (GPCRs) that are expressed at excitatory synapses in brain and spinal cord. GPCRs are often negatively regulated by specific G protein–coupled receptor kinases and subsequent binding of arrestin-like molecules. Here we demonstrate an alternative mechanism in which group I mGluRs are negatively regulated by proline-directed kinases that phosphorylate the binding site for the adaptor protein Homer, and thereby enhance mGluR–Homer binding to reduce signaling. This mechanism is dependent on a multidomain scaffolding protein, Preso1, that binds mGluR, Homer and proline-directed kinases and that is required for their phosphorylation of mGluR at the Homer binding site. Genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, and Preso1−/− mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. Preso1 creates a microdomain for proline-directed kinases with broad substrate specificity to phosphorylate mGluR and to mediate negative regulation.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity

A fundamental process in biology is the de novo formation and morphogenesis of polarized tubules. Although these processes are essential for the formation of multiple metazoan organ systems, little is known about the molecular mechanisms that regulate them. In this study, we have characterized several steps in tubule formation and morphogenesis using the mouse kidney as a model system. We report that kidney mesenchymal cells contain discrete Par3-expressing membrane microdomains that become restricted to an apical domain, coinciding with lumen formation. Once lumen formation has been initiated, elongation occurs by simultaneous extension and additional de novo lumen generation. We demonstrate that lumen formation and elongation require afadin, a nectin adaptor protein implicated in adherens junction formation. Mice that lack afadin in nephron precursors show evidence of Par3-expressing membrane microdomains, but fail to develop normal apical-basal polarity and generate a continuous lumen. Absence of afadin led to delayed and diminished integration of nectin complexes and failure to recruit R-cadherin. Furthermore, we demonstrate that afadin is required for Par complex formation. Together, these results suggest that afadin acts upstream of the Par complex to regulate the integration and/or coalescence of membrane microdomains, thereby establishing apical-basal polarity and lumen formation/elongation during kidney tubulogenesis.