PROJECT SUMMARY/ABSTRACT Kidney stones affect 9% of the population and idiopathic hypercalciuria is the major pathogenic risk factor. Our long-term goal is to elucidate the pathogenesis of idiopathic hypercalciuria and develop novel therapies. The renal proximal tubule reabsorbs 70% of filtered calcium, which is believed to occur via the paracellular pathway. Claudin-2 is a paracellular cation channel that is expressed in the proximal renal tubule and in intestinal epithelium. In preliminary studies, we show that claudin-2 knockout mice have renal and absorptive hypercalciuria, leading to severe papillary nephrocalcinosis. Moreover, we find that claudin-2 gene variants are associated with kidney stone disease in humans. We hypothesize that claudin-2 mediates calcium reabsorption in the proximal tubule and calcium secretion in the colon. Loss of claudin-2 increases calcium delivery to the loop of Henle, and predisposes to inner medullary calcium deposition and hence kidney stone disease. The specific aims to test this hypothesis are: (1) Test whether claudin-2 mediates paracellular calcium reabsorption in the renal proximal tubule. We will use tubule micropuncture of claudin-2 global knockout mice, determine the contribution of the proximal tubule with kidney-specific conditional knockout mice, test the role of claudin-2 in the hypocalciuric effect of thiazides, and explore the effect of sex. (2) Test whether claudin-2 mediates intestinal secretion of calcium. We will generate intestine-specific claudin-2 knockout mice and perform tracer flux assays in everted intestinal sacs and Ussing chamber preparations. (3) Test whether human claudin-2 gene variants and protein expression are associated with urine calcium and kidney stones. We will conduct a gene association study in 3 U.S. population-based cohorts to test the association of claudin-2 gene polymorphisms with a history of kidney stones and calcium excretion. In a separate cohort of kidney stone formers and matched normal volunteers, we will test the association of claudin-2 protein abundance in urinary exosomes with hypercalciuric kidney stone disease.