Phenotypic Transition of the Collecting Duct Epithelium in Congenital Urinary Tract Obstruction

Epithelial-mesenchymal transition (EMT) has emerged in recent years as an important process in the development of organ fibrosis in many human diseases. Our previous experience in a nonhuman primate model of obstructive nephropathy suggested that EMT of collecting duct epithelium contributes to the development of interstitial fibrosis. In this study we demonstrate for the first time in humans that obstructed fetal collecting duct epithelium undergoes transition to mesenchymal phenotype, characterized by decreased expression of epithelial markers, de novo expression of mesenchymal markers with subsequent loss of cell-cell interaction, disruption of the basement membrane, and increased deposition of extracellular matrix into the expanded interstitium of the obstructed kidney. The results of this study therefore support the previous findings from animal studies and suggest that EMT of the collecting duct epithelium might contribute to the development of interstitial fibrosis in human fetal obstructive nephropathy

Investigating the nutritional advice and support given to colorectal cancer survivors in the UK: is it fit for purpose and does it address their needs?

Background The present study assessed the quantity and quality of nutritional advice and support given to colorectal cancer survivors in the UK. Methods A descriptive cross‐sectional survey was completed by 75 colorectal cancer survivors recruited through social media and bowel cancer support groups in the UK. The survey consisted of open‐ended and closed questions that aimed to explore the nutritional needs, nutritional advice given and other sources of information accessed by colorectal cancer survivors. Results Sixty‐nine percent of respondents reported that they did not receive any nutritional advice or support from their healthcare team throughout diagnosis, treatment and post‐treatment. Colorectal cancer survivors accessed nutritional advice from a variety of sources, mainly cancer charity websites. Respondents expressed their desire for individualised advice relating to their nutritional problems. Conclusions The results obtained in the present study indicate that a high proportion of colorectal cancer patients are not receiving the nutritional support that they need to overcome nutritional difficulties. There is an urgent need to improve clinical practice to ensure colorectal patients receive nutritional advice that is both consistent between healthcare professionals and personalised throughout each stage of diagnosis, treatment and post‐treatment

Failure to ubiquitinate c-Met Leads to Hyperactivation of mTOR Signaling in a Mouse Model of Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated. in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cb1), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with alpha(3)beta(1) integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD

Comparative Immunohistochemical Study of Multicystic Dysplastic Kidneys With and Without Obstruction

Etiology of multicystic dysplastic kidney (MCDK) remains unknown. Not all cases are associated with obstruction. We compared by immunohistochemistry 17 cases of MCDK (10 cases with and seven without obstruction) to 17 controls and 20 fetal kidneys. TGF-β was negative in obstructive MCDKs and positive in nonobstructive MCDK. IGF2 was overexpressed in obstructive and underex-pressed in nonobstructive MCDKs. PAX2, BCL-2, and β-catenin were expressed equally in obstructive and nonobstructive dysplasia. TGF-β and IGF2 work by different mechanisms in obstructive and nonobstructive MCDKs, but there are no differences among PAX 2, BCL-2, and β-catenin in obstructive versus nonobstructive dysplasia

Characterizing the function of ATP8A2 and its role in neurodegenerative disease

ATP8A2 is a P4-ATPase that actively flips phosphatidylserine and to a lesser extent phosphatidylethanolamine across cell membranes to generate and maintain transmembrane phospholipid asymmetry. This transporter is expressed primarily in neuronal tissues, such as the brain, retina and spinal cord. The importance of this flippase is underscored by the findings that loss-of-function mutations in ATP8A2 are known to cause the neurodevelopmental disease known as CAMRQ4 in humans and related neurodegenerative disorders in mice. This thesis focuses on characterizing ATP8A2, investigating its regulatory mechanisms, the impact of disease-associated variants, and its involvement in specific cellular pathways. Little is known about the structural and functional properties of the cytosolic N- and C-terminal segments of this flippase. In addition, there has been uncertainty regarding the methionine start site of ATP8A2 and accordingly the size of the N-terminal segment. Here, we have used mass spectrometry to show that bovine ATP8A2, like its human counterpart has an extended N-terminal segment not apparent in the mouse ortholog. This segment greatly enhances the expression of ATP8A2 without affecting its cellular localization or PS-activated ATPase activity. Using a cleavable C-terminal protein and site-directed mutagenesis, we further show that the conserved GYAFS motif in the C-terminal segment plays a role in autoinhibition as well as efficient folding of ATP8A2 into a functional protein. With regards to understanding the impact of clinical mutations, five variants of ATP8A2 were studied. The expression, localization and ATPase activity of these variants were characterized using a variety of biochemical assays. Using various software programs predicting protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments. These findings suggest that such analysis is useful in identifying protein misfolding disease-associated variants. Finally a cellular model was developed to study the role of P4-ATPases in the process of protein trafficking. A P4-ATPase deficient cell line was developed to study the impact on the trafficking of the plasma membrane receptor VLDLR, also associated with CAMRQ. These investigations will help improve our understanding of the function of ATP8A2, and its role in neurodegenerative disease.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Association of PFO, Inflammatory Cytokines and White Blood Cells with Hemoglobin Mass in Men

59 pagesA patent foramen ovale (PFO) is a source of intracardiac shunt, and preliminary data suggests that those with a PFO have higher concentrations of inflammatory cytokines. Cytokines and white blood cells (WBCs) negatively impact red blood cell (RBC) regulation, but no studies have examined the effect of the PFO on this relationship. The purpose of this study was to investigate the relationships between the presence of a PFO, inflammatory cytokine concentrations, and WBCs on RBC mass. We hypothesized that those with a PFO would have both a higher WBC count and cytokine concentrations, negatively impacting hemoglobin (Hb) mass. Twenty healthy, male participants completed the study (10 with and 10 without a PFO). Participants underwent a comprehensive ultrasound screening with saline contrast echocardiography to determine the presence or absence of a PFO. Hb mass was measured twice on the same day via the 10-minute CO-rebreathe method, and venous blood samples were drawn for measurements of WBC counts and inflammatory cytokines. WBC counts were analyzed by QUEST diagnostics via flow cytometry. Cytokine analysis was done using the BioLegend 13-plex human inflammation panel and analyzed via flow cytometry. No differences were found for absolute (g) (p = 0.1096) and relative (g/kg) (p = 0.1382) Hb mass between PFO+ and PFO- participants. No differences were found for WBC count (p = 0.8680) between PFO+ and PFO- participants and for each of the respective immune cells; neutrophils (p = 0.5418), lymphocytes (p = 0.6721), monocytes (p = 0.5388), eosinophils (p = 0.5603), basophils (p = 0.2539). There was no differences in cytokine concentrations between PFO+ and PFO- participants. There was no significant relationship between WBC count and absolute or relative Hb mass. There was a relationship between IFN-α2 and the whole group data for absolute Hb mass (p = 0.0390, R2 = 0.2158). There was a relationship between MCP-1 levels and absolute Hb mass in PFO+ participants, but not in PFO- participants (p = 0.0258, R2 = 0.4823). Similarly, there was a relationship between IL-23 levels and absolute Hb mass in PFO+ participants, but not in PFO- participants (p = 0.0497, R2 = 0.5003). Conversely, there was a relationship between IL-10 levels and absolute Hb mass in PFO- participants, but not in PFO+ participants (p = 0.0392, R2 = 0.5350). Although there were no PFO differences in Hb mass, WBC counts or cytokine concentrations, the presence of a PFO may alter the relationship between Hb mass and the cytokines MCP-1, IL-23 and IL-10. In addition, we found a significant relationship with IFN-2 and absolute Hb mass irrespective of the presence or absence of a PFO, indicating a potential role for IFN-2 on Hb mass regulation