LAUSR

Infants and children require a net positive calcium (Ca2+) balance to achieve optimal bone mineral density by early adulthood. Active vitamin D (calcitriol) 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. Claudin-2 and claudin-12 double knockout (DKO) mice have a perturbed Ca2+ balance resulting in hypocalcemia, hypercalciuria and decreased bone mineralization. Interestingly, DKO mice did not have increased calcitriol levels or upregulation of Cyp27b1 (the key enzyme regulating serum calcitriol levels), despite a significant increase in serum parathyroid hormone (PTH) levels and decreased serum Ca2+ compared to wild-type mice. We hypothesize therefore that claudin-2 and/or claudin-12 are necessary for optimal PTHR1 signalling to increase Cyp27b1 expression, and thus calcitriol production in the proximal tubule (PT). To address this, we first administered wild-type and double knockout mice calcitriol and found that they could increase their blood calcium levels, though not to the same extent as WT, consistent with the DKO animals not having vitamin D resistance. Next we put wild-type and DKO animals on a low calcium diet. The DKO mice were able to increase their serum calcitriol levels and Cyp27b1 expression under these conditions, consistent with attenuated regulation and not abolition of PTHR1 signalling. Next we examined PTHR1 signaling in HEK-293 cells and a CYP27B1 reporter construct. We found that in HEK cells, PTH treatment results in increased expression of the CYP27B1 promoter construct, and that claudin-2 expression alone, increased promoter expression to the same extent as PTH. Additionally, calcitriol suppressed CYP27B1 promoter expression, however this suppression was abolished when claudin-2, but not claudin-12, was expressed. In primary mouse proximal tubule cells, PTH significantly increased Cyp27b1 mRNA expression compared to controls. In future experiments we will investigate the effects of claudin-2 and/or claudin-12 on PTH-induced Cyp27b1 mRNA expression using primary proximal tubule cells from claudin-2, claudin-12 or claudin-2/12 DKO mice. This work will tease out the role of the proximal tubule tight junction proteins claudin-2 and -12 in regulating calcitriol levels. Funding: Alberta Innovates, NSERC and CIHR This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.