טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHermoni Adva
SubjectTranscriptional Regulation of the Renal Magnesium Channel
(Paracellin-1) Gene
DepartmentDepartment of Medicine
Supervisor Professor Israel Zelikovic


Abstract

    The kidney plays a major role in magnesium balance. Tubular reabsorption of Mg+2 is mediated by the Mg+2 channel protein, paracellin-1, which is encoded by the gene

PCLN-1 and exclusively expressed in the kidney. Hypermagnesemia and hypercalcemia as well as cisplatin decrease, whereas  hypomagnesemia and insulin stimulate magnesium reabsorption. The molecular mechanisms of the modulation of renal Mg+2 transport are unknown.

    The aim of this study was to examine whether changes in ambient Mg+2 and  Ca+2 concentrations, insulin and cisplatin affect the activity of the paracellin-1 gene promoter in renal cell lines.

    A 2.5kb human PCLN-1 (hPCLN-1) 5' flanking DNA sequence, as well as a 210bp segment (derived from this sequence) containing a peroxisome proliferator response element (PPRE) were cloned in luciferase reporter vectors and termed pJ2M and pJ210, respectively. The DNA constructs were transiently transfected into OK and HEK293 cells, that were exposed to media containing 1) 0mM (low), 0.7mM (normal) or 1.5mM (high) Mg+2 concentration  2) 1.0mM (normal) or 2.7mM (high) Ca+2 concentration

3) insulin(10-6M) 4) cisplatin (50µg/ml). Following 24 hours exposure, cells were assayed for luciferase activity. Mg+2 depletion significantly increased pJ2M-driven luciferase activity, whereas Mg+2 load decreased this activity when compared with conditions of normal Mg+2. Ca+2 load reduced reporter gene activity compared with cells exposed to normal Ca+2. Insulin treatment and cisplatin exposure caused a marked reduction in pJ2M-driven luciferase activity. Stimulation of the PPRE-harboring pJ210 construct with cisplatin decreased reporter gene activity, a decrease that was abolished in the absence of PPRE.

    Our findings provide the first direct evidence that paracellular, paracellin-1-mediated Mg+2 transport may be regulated at the transcriptional level by ambient Mg+2 or  Ca+2, insulin and cisplatin therapy. The cisplatin-induced effect is likely achieved via PPRE.