Characterization of Epithelial Sodium Channel Alpha Subunit Transcripts and Their Corresponding Mrna Expression Levels in Dahl S versus R Rats’ Kidney Cortex

S POSTER PRESENTATIONS, JUNE 20-21, 2007 Fifth Northern Lights Summer Conference University of Waterloo Coordinated by: Canadian Federation of Biological Societies 37 Waterloo, Ontario 51 Scientific Conference OB1 CHARACTERIZATION OF EPITHELIAL SODIUM CHANNEL ALPHA SUBUNIT TRANSCRIPTS AND THEIR CORRESPONDING MRNA EXPRESSION LEVELS IN DAHL S VERSUS R RATS’ KIDNEY CORTEX. MF Shehata, FHH Leenen, F Tesson, University of Ottawa Heart Institute. Source of Research Funds: CIHR, University of Ottawa, OGSST. AIMS/HYPOTHESIS: The á-subunit of the amiloridesensitive epithelial sodium channel (ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of á ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to investigate the mRNA expression levels of alternatively spliced forms of á ENaC in kidney cortex of Dahl salt resistant (R) versus salt sensitive (S) on normal and four-week high salt diet. METHODS: Using quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported á ENaC a and b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to the wildtype á ENaC mRNA levels. Using polymerase chain reaction, we identified, cloned and sequenced 2 novel non coding C-terminus spliced forms and examined their mRNA expression in Dahl S versus R rats. We also tested the presence of five previously reported lungspecific á ENaC spliced forms in Dahl rats’ kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980). RESULTS: Previously reported ENaC a and b variants were significantly higher in Dahl R versus S rats (P<0.05). Four-week high salt diet significantly increased á ENaC b (P<0.05), but not á ENaC a transcript abundance in Dahl R, but not S rats. The expression levels for the two newly identified non coding ENaC c and d variants were comparable in Dahl S versus R rats. Compared to á ENaC wt, á ENaC a, c and d were low abundance transcripts, in contrast to á ENaC b transcript abundance that was 32 +/3 folds higher than á ENaC wt. We could not identify any of the five previously reported lung-specific á ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rats’ kidney cortex. CONCLUSIONS/INTERPRETATION: á ENaC spliced forms, particularly, á ENaC b, might likely act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. OB2 Other Biological Fields OVEREXPRESSION OF HUMAN PROEGF CYTOPLASMIC DOMAIN CAUSES REDUCTION IN BODY AND ORGAN WEIGHT IN TRANSGENIC MICE. Aleksandra Glogowska1, Ekkehard Weber2*, Cuong Hoang-Vu3*, Ana A. Gratao4*, Eckhard Wolf4*, Marlon R. Schneider4*, Thomas Klonisch1, 1Dept. Human Anatomy & Cell Science, U of Manitoba, Winnipeg, R3E 0W3, Canada; Depts. of 2Physiological Chemistry & 3Surgery, MLU Halle, Halle, D-06097, Germany; 4Institute of Molecular Animal Breeding and Biotechnology, Gene Center, U of Munich, Munich, Germany. Source of Research Funds: Research found by German Cancer Research Council & Manitoba Health Research Council (MHRC). The cytoplasmic domains of proTGF-á-cyt and proHBEGF-cyt precursors were shown to encode protein binding motifs for Naked2 (NKD2) and promyelocytic leukemia zinc finger protein (PLFZ) nuclear transcriptional repressor, respectively. The cytoplasmic domains of proTGF-á and proAR were demonstrated to contain basolateral sorting information. The soluble cytoplasmic domain of neuregulin1 (NRG1-cyt) was shown to be a nuclear transcriptional suppressor for regulators of apoptosis and to associate with LIM-kinase implicating NRG1-cyt as a regulator of cytoskeletal processes. Here we show that transgenic mice overexpressing human proEGFcyt, but not a proEGFcyt splice form proEGFdel23, display significantly reduced body weight. The differences in body and organ weights were significant and more pronounced in female than in male transgenic animals. Detailed analysis revealed significantly reduced weights for kidney and brain tissues in proEGFcyt mice. A new polyclonal antiserum specific for proEGFcyt demonstrated the basolateral presence of proEGFcyt protein in distal tubules and collecting ducts in transgenic kidneys. Growth retardation had previously been shown in transgenic mice overexpressing total human proEGF. Here we show that over-expression of human cytoplasmic proEGF domain alone is sufficient to cause reduction in body weight. Further histological and molecular studies are in progress to determine the effects of proEGFcyt on specific organ functions in these proEGFcyt mice. ABSTRACTS POSTER PRESENTATIONS, JUNE 20-21, 2007S POSTER PRESENTATIONS, JUNE 20-21, 2007 Fifth Northern Lights Summer Conference University of Waterloo Coordinated by: Canadian Federation of Biological Societies 38 Waterloo, Ontario 51 Scientific Conference OB3 ENVIRONMENTAL TOXINS INDUCE METABOLIC AND STRUCTURAL CHANGES IN IMMORTALIZED HUMAN ENDOMETRIAL CELLS. Sabine Hombach-Klonisch1, Marco Peich2, Aleksandra Glogowska1, Anja Seifert2,Thomas Klonisch1 1Dept. of Human Anatomy and Cell Science, University of Manitoba, Faculty of Medicine, 130 Basic Medical Science Building, Winnipeg, MB, R3E 0W3, Canada; 2Dept. of Anatomy and Cell Biology, University of Halle, Halle, D-06097, Germany. Source of Research Funds: Research funding by Wilhelm Roux Research Fund, Medical Faculty MLU Halle. Environmental pollutants and Arylhydrocarbon/Dioxin Receptor (AhR-) agonists are xenobiotics and have been implicated in reduced female fertility and in the pathogenesis of endometriosis. The molecular mechanisms elicited by dioxin-type toxins in the human endometrium are not understood. We established a telomerase-immortalized human endometrial epithelial cell line (hTERT-EEC) as a novel invitro tool to investigate molecular actions of these AhR agonists. We show that hTERT-EEC express functional arylhydrocarbon-receptor (AhR). Exposure to TCDD (2,3,7,8tetrachlorodibenzo-p-dioxin) and coplanar polychlorinated biphenyls (PCB) results in a timeand dose-dependent transcriptional activation of AhR as indicated by xenobiotic response element (XRE-)luciferase assay and the induction of AhR target genes of p450 microsomal enzymes CYP1A1 and CYP1B1. TCDD did not change estrogen receptor(ER-) alpha protein levels suggesting that dioxin does not induce proteasomal degradation of ER in hTERT-EEC. However, TCDD induced marked alterations in membrane surface morphology as determined by scanning electron microscopy (SEM) suggesting a novel role of TCDD in the organization of sub-membranous cytoskeletal structures.