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Identification of the Claudin-14 Promoter and Calcium Sensing Receptor (CaSR) Responsive Elements Open Access


Other title
Calcium Sensing Receptor
Type of item
Degree grantor
University of Alberta
Author or creator
Alzamil, Jawad F
Supervisor and department
Todd Alexander, Department of Pediatrics
Examining committee member and department
Emmanuelle Cordat, Department of Physiology
Eytan Wine, Department of Pediatrics
Mike Walter, Department of Genetics
Maury Pinsk , Department of Pediatrics
Medical Sciences-Paediatrics

Date accepted
Graduation date
Master of Science
Degree level
One in ten Canadians will have a kidney stone during their life. Kidney stones cause significant pain and are expensive to treat due to recurrent emergency room visits and surgeries. The majority of kidney stones are composed of calcium and the greatest risk factor for kidney stones is hypercalciuria (i.e. urine with excess calcium), for which the etiology is unknown. A recent GWAS linked hypercalciuria to claudin-14, a gene we have shown increases expression in response to a calcium load, thereby inducing calciuria. Given this relationship we set out to 1) identify the claudin-14 promoter and 2) identify calcium sensing receptor (CaSR) sensitive signaling elements. To this end, we performed quantitative RT-PCR on the 5’ untranslated regions of the 3 mouse claudin-14 variants and found that the expression of the first variant increases after CaSR activation. In silico studies comparing 5’ of the 5’ UTR of multiple species identified a highly homologous 25 bp region approximately 400 bp 5’ of transcript variant-1, predicted to have promoter activity. We therefore cloned between 500-1500 bp 5’ of the 5’ UTR of the mouse variant 1 into the pGL3 Basic and Enhancer luciferase reporter constructs. As a positive control we cloned the SV40 promoter. We then expressed these constructs in both a HEK293 cell line and another renal tubular cell line over expressing the CaSR (OK-CaSR). Constructs containing 500, 700, 1000 or 1200 bp 5’ of the 5’ UTR of the mcldn-14 V1 didn’t show any promoter activity when transfected into OK-CaSR cell lines. In contrast, the construct containing the 1500 bp fragment 5’ of the 5’ UTR of mcldn-14 V1 demonstrates promoter activity when transfected into both HEK293 and OK-CaSR cell lines and responds to CaSR activation in the OK-CaSR cell line. We therefore conclude that the claudin-14 promoter is located between 1200 (exact length = 1243 bp) – 1500 (exact length = 1340 bp) 5’ to the 5’UTR of variant 1 and responds to CaSR activation. Future experiments will delineate the mechanism by which claudin-14 is regulated via this promoter.
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