Epigenetic regulation of microRNAs controlling CLDN14 expression as a mechanism for renal calcium handling

The kidney has a major role in extracellular calcium homeostasis. Multiple genetic linkage and association studies identified three tight junction genes from the kidney - claudin-14, -16, and -19 - as critical for calcium imbalance diseases. Despite the compelling biologic evidence that the claudin-14/16/19 proteins form a regulated paracellular pathway for calcium reabsorption, approaches to regulate this transport pathway are largely unavailable, hindering the development of therapies to correct calcium transport abnormalities. Here, we report that treatment with histone deacetylase (HDAC) inhibitors downregulates renal CLDN14 mRNA and dramatically reduces urinary calcium excretion in mice. Furthermore, treatment of mice with HDAC inhibitors stimulated the transcription of renal microRNA-9 (miR-9) and miR-374 genes, which have been shown to repress the expression of claudin-14, the negative regulator of the paracellular pathway. With renal clearance and tubule perfusion techniques, we showed that HDAC inhibitors transiently increase the paracellular cation conductance in the thick ascending limb. Genetic ablation of claudin-14 or the use of a loop diuretic in mice abrogated HDAC inhibitor-induced hypocalciuria. The genetic mutations in the calcium-sensing receptor from patients with autosomal dominant hypocalcemia (ADH) repressed the transcription of miR-9 and miR-374 genes, and treatment with an HDAC inhibitor rescued the phenotypes of cell and animal models of ADH. Furthermore, systemic treatment of mice with antagomiRs against these miRs relieved claudin-14 gene silencing and caused an ADH-like phenotype. Together, our findings provide proof of concept for a novel therapeutic principle on the basis of epigenetic regulation of renalmiRs to treat hypercalciuric diseases.