Project Summary TRPV5-mediated renal Ca2+ reabsorption is critical for maintaining Ca2+ homeostasis. Klotho is a type-1 transmembrane protein predominantly produced in the kidney. Klotho exists either in the membranous klotho form or the soluble ectodomain that is shedded into urine or systemic circulation to function as a paracrine or endocrine factor. Previously, we have found that soluble klotho increases surface abundance of TRPV5 by removing terminal sialic acids from the N-linked glycan chains of the channel. Removal of sialic acids exposes underlying disaccharide N-acetyl-lactosamine, a ligand for galectin-1 that is ubiquitously present on the external surface of cells. Binding to galectin-1 at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. To support this novel hypothesis for mechanism of action for klotho, we propose the following studies to extend our in vitro findings into in vivo and to investigate pathophysiological relevance of our findings. Aim-1 will test the hypothesis that soluble klotho regulates renal Ca2+ reabsorption via TRPV5 in vivo and that it does so through the putative sialidase activity of klotho. To examine the role of soluble klotho in renal Ca2+ reabsorption in vivo, we will generate transgenic mice that express soluble klotho in the background of klotho-deficient mice (rescued from death by dietary phosphate and vitamin D restriction) and examine urinary calcium excretion, TRPV5 expression, several gene expression parameters related to TRPV5-mediated Ca2+ reabsorption, and patch-clamp recording of TRPV5 channel activity in the native renal tubules. We will also define domains of soluble klotho involved in klotho's putative sialidase activity in vitro and test the effect of mutant soluble Klotho carrying sialidase activity-inactivating mutations in vivo. Chronic kidney disease (CKD) is a klotho-deficient state. Aim 2 will test the hypothesis that defect in TRPV5-mediated renal Ca2+ reabsorption from soluble klotho deficiency contributes to secondary hyperparathyroidism in chronic kidney disease. Elevation of parathyroid hormone (PTH) occurs in patients with CKD as a response to combat hyperphosphatemia and hypocalcemia. Hyperparathyroidism yet causes multiple adverse consequences in CKD. We will test the hypothesis that decrease in soluble klotho and downregulation of TRPV5 plays a role in hyperparathyroidism of CKD by using two mouse models of CKD. The effect of transgenic delivery of wild-type or inactive mutant soluble klotho on serum PTH and Ca2+ levels, urinary Ca2+ excretion, and other mineral metabolites in CKD will be examined. These studies on the mechanism by which klotho stimulates renal calcium reabsorption and its role in mineral metabolism of CKD will be important for our understanding the pathophysiology of mineral disorder of CKD and may help management or deign future therapy of mineral disorder in CKD patients.