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

• Author / Creator

  Alzamil, Jawad F

• 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.

• Subjects / Keywords

  • Cldn14
  • Claudin-14
  • Calcium Sensing Receptor
  • Hypercalciuria
  • CaSR

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3SB3X65V

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.