Investigating Vitamin D Insufficiency in Claudin-2 and Claudin-12 Knockout Mice

• Author / Creator

  Young, Kennedi

• Infants and children require a net positive calcium (Ca2+) balance to achieve optimal bone mineral density by early adulthood. Vitamin D is a hormone that increases blood Ca2+ levels by increasing intestinal absorption and renal reabsorption of Ca2+ as well as bone remodelling. In both the kidney and intestine, Ca2+ is absorbed both paracellularly, through claudin proteins that make up the tight junction, and transcellularly, through Ca2+ specific transporters and channels. Claudins-2 and -12 are tight junction proteins that mediate Ca2+ permeability across renal and intestinal cell culture epithelial layers. In studying a claudin-2 and claudin-12 double knockout (DKO) mouse model, we observed a decreased Ca2+ balance that resulted in hypocalcemia, hypercalciuria and decreased bone mineralization. Interestingly, DKO mice did not have an increase in serum calcitriol levels or upregulation of the genes CYP27B1 and CYP24A1 regulating serum calcitriol, despite a significant increase in serum parathyroid hormone (PTH) levels and decreased serum Ca2+ levels compared to WT. We hypothesize that claudin-2 and/or claudin-12 are necessary for efficient signalling of CYP24A1 and CYP27B1, and thus vitamin D production in the proximal tubule (PT). To address this, we have examined cell culture models in the PT, including HEK-293 and primary mouse proximal tubule cells. We have found that in HEK cells, PTH treatment results in increased expression of the CYP27B1 promoter, and that claudin-2 expression alone increases promoter expression to the same extent as PTH treated groups. Further, claudin-2 overexpression enhanced CYP24A1 mRNA transcription with calcitriol treatment. In primary mouse proximal tubule cells, the treatment with PTH results in significantly increased Cyp27b1 mRNA expression compared to controls, and notably, DKO cells have enhanced Cyp27b1 mRNA expression with PTH treatment relative to WT cells. CaSR transfection into HEK-293 cells exposed to high extracellular calcium levels displayed an increase in CYP24A1 mRNA expression which was suppressed in claudin-12 transfected cells relative to EV and claudin-2 transfected cells, consistent with the CaSR having a role in the transcriptional regulation of CYP27B1 and CYP24A1 which is dependent on extracellular calcium levels but not on claudin expression. The results of this study are consistent with claudin-2 and/or claudin-12 playing a role in vitamin D metabolism in the proximal tubule via regulation of CYP27B1 and CYP24A1 transcription. Further work is needed to determine the mechanism behind this effect and what, if any, interactions exist between the claudins and the CaSR. This work will tease out the role of the proximal tubule tight junction proteins claudin-2 and -12 in regulating vitamin D levels.

• Subjects / Keywords

  • Claudin
  • Vitamin D
  • Calcium
  • Proximal tubule

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-qcbg-5161

• 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.