|Ph.D Student||Kladnitsky Orly|
|Subject||Molecular Mechanisms Responsible for the Regulation of|
the Renal Magnesium Channel (Paracellin-1) Gene
|Department||Department of Medicine||Supervisors||Professor Israel Zelikovic|
|Dr. Julia Rozenfeld|
|Full Thesis text|
The kidney is the principal organ responsible for Mg2 balance. Tubular reabsorption of Mg2 is mediated predominantly by the tight junction channel protein, claudin-16 (paracellin-1), encoded by the gene CLDN16. Tubular Mg2 reclamation is modulated by various factors including Mg2 availability, the hormone 1,25(OH)2VitD , and the immunomodulatory drug cyclosporine A (CsA) . Very little is known about the exact molecular mechanisms of this modulation.
The aim of this study was to investigate the involvement of claudin-16 in the renal response to changes in Mg2 availability, 1,25(OH)2VitD administration, and CsA treatment.
The results of the study include:
1. Effect of Mg2 availability: Mg2 deprivation increases and Mg2 load decreases claudin-16 protein and mRNA expression in the mouse kidney. Extracellular Mg2 concentration influences endogenous CLDN16 mRNA levels in HEK293 cells, an effect that is particularly pronounced in HEK293 cells expressing the calcium sensing receptor (CaSR). Mg2 level modulates human CLDN16 (hCLDN16) promoter activity by acting on the 5'-flanking 2.5kb hCLDN16 promoter region previously characterized by us.
2. Effect of 1,25(OH)2VitD: 1,25(OH)2VitD decreases claudin-16 protein abundance in mouse kidney and CLDN16 mRNA level in both mouse kidney and HEK293 cells. 1,25(OH)2VitD inhibits hCLDN16 promoter activity in renal cells, an inhibition that is CaSR-dependent as evidenced by experiments in CaSR-transfected HEK293 cells and OK cells transfected with dominant negative CaSR construct. 1,25(OH)2VitD reverses the expected increase in claudin-16 protein abundance and CLDN16 mRNA level in kidneys of Mg2 deprived mice, and reverses the expected increase in endogenous CLDN16 mRNA level and hCLDN16 promoter activity in Mg2 depleted HEK293 cells.
3. Effect of cyclosporine A: CsA decreases claudin-16 protein expression in mouse kidney, decreases CLDN16 mRNA levels in mouse kidney and in HEK293 cells, and inhibits hCLDN16 promoter activity in transfected renal cells. A 572-bp region between positions -2554 and -1982 may play a key role in the inhibition of hCLDN16 promoter activity by CsA.
We conclude that changes in Mg2 availability, administration of 1,25(OH)2VitD and treatment with cyclosporine A influence Mg2 transport in the renal tubule by regulating transcription of the claudin-16 gene, likely via distinct response elements to these modulators on the promoter of this gene.
The modulation of CLDN16 transcription by Mg2 availability and by 1,25(OH)2VitD involves the Ca2/Mg2 sensing receptor pathway. 1,25(OH)2VitD dampens the Mg2 depletion-induced stimulation of CLDN16 transcription. The 1,25(OH)2VitD-induced repression of renal Mg2 transport may serve, in part, as an adaptive mechanism to the 1,25(OH)2VitD-induced increase in intestinal Mg2 absorption.