Putative anion transporter 1 (PAT1, SLC26A6), an intestinal epithelial Cl-/HCO3- exchanger, also mediates oxalate secretion critical for maintaining oxalate homeostasis via governing net intestinal oxalate transport. Intestinal PAT1 has recently emerged as a novel therapeutic target for nephrolithiasis, a kidney disease linked to defective oxalate secretion. In fact, Slc26a6-null mice exhibit defective oxalate secretion, hyperoxalemia, hyperoxaluria and high incidence of kidney stones. Also, patients with kidney stones display mutations in slc26a6 gene. In view of this critical role of PAT1 in normal physiology and pathophysiology of nephrolithiasis, an in-depth understanding of the mechanisms of its regulation in health and disease is warranted. There has been limited progress in understanding regulation of PAT1 due to technical challenges in dissecting its role from that of SLC26A3 (Down-regulated in Adenoma, DRA, another key intestinal epithelial anion exchanger), as both proteins are localized to IEC luminal membranes and mediate identical anion exchange activities. We have now uncovered a novel in vitro model utilizing intestinal epithelial SK-CO15 cell line that shows abundant expression of PAT1 with negligible DRA expression. Our preliminary data in SK-CO15 cells provide strong evidence for distinct mechanisms for modulation of PAT1 expression involving transcriptional activation by CDX1 and HNF4α, posttranscriptional repression by microRNAs, and posttranslational regulation by autophagy. Further, PAT1 was markedly reduced in response to infection by Cryptosporidium parvum, a diarrheal parasite that specifically infects the small intestine and is a common opportunistic pathogen in HIV patients known to be at high risk for nephrolithiasis. Our preliminary data envisage the critical need for detailed investigations of the regulatory mechanisms of PAT1 expression to unravel its underexplored role in health and disease. We, therefore, hypothesize that expression of intestinal epithelial PAT1 is regulated at transcriptional, posttranscriptional and posttranslational levels via novel mechanisms that are also altered in gut infection causing impaired PAT1 expression and function. Our hypothesis will be tested utilizing SK-CO15 cells, intestinal organoids, and mouse models. The Specific Aims are: 1. Elucidate transcriptional/posttranscriptional mechanisms of regulation of PAT1 expression and function in SK-CO15 cells and mouse enteroids; 2. Determine the role of autophagy in basal or C. parvum-induced modulation of PAT1 expression; 3. Elucidate the mechanisms of regulation of PAT1 expression in vivo in normal mice or in response to C. parvum infection. Overall, in addition to establishing a novel in vitro model to study PAT1 regulation, our studies should yield critical insights into the molecular mechanisms of intestinal PAT1 regulation and unravel potentially novel therapeutic targets for diseases associated with impaired intestinal PAT1 functio...