Abstract Nephron salt reabsorption is essential to salt and water homeostasis and its dysregulation is involved in the progression of kidney and cardiovascular diseases, including chronic kidney disease, hypertension and those started by diabetes and obesity. There are only a few methods to measure renal tubular ion transport at the single cell in live animals that were developed over 40 years ago. Using multi-photon imaging of the live mouse kidney we recently developed a method to monitor Thick Ascending Limb (TAL)-mediated urine concentration at the single tubule level. We propose to develop 2 transgenic mice lines expressing a Chloride-sensing genetically encoded probe that will allow measurement of cortical TAL and Distal Convoluted tubule (DCT) NaCl transport using 2-photon microcopy. In Specific Aim 1 we will generate inducible Umod-Cre, Slc12a3-Cre-SuperClomelen expressing transgenic mice that will allow the methodological development of NaCl transport measurements in vivo. In specific Aim 2, we will develop a method to measure NKCC2 and NCC activity in live mice by monitoring the initial rates of Cl entry using SuperClomeleon mice or ClopHensor expression in the distal tubule in vivo. Our preliminary data demonstrate the use of a ratiometric Cl-sensitive probe to measure intracellular chloride in epithelial cells and measurement of urinary concentrating capacity and luminal Na via intravital MP microscopy in mice. Our studies will demonstrate the use of genetically encoded Cl indicators for measurement of nephron NaCl transport and spur the development of new sensors (K, Na, Mg, cAMP, cGMP, etc) to be used in live mice.