Assaying and controlling the kidney cell function using a genetically encoded pH-sensor

点击翻译按钮获取中文摘要

Abstract: Assaying and controlling the proximal tubule using the nbce1A promoter and ion-sensors Bicarbonate absorption of filtered blood by the kidney is critical to not just blood pH homeostasis but also for life. The proximal tubule (PT) absorbs isotonic NaHCO3. Basal metabolism generates 70 mM H+, making it crucial to buffer this acid (CO2/HCO3-) or removed (low pH urine with titratable acids). Proximal tubule HCO3- absorption is mediated by basolateral NBCe1A (SLC4A4-A), the electrogenic Na+ bicarbonate cotransporter. The final phase of kidney acid excretion is accomplished through apical H+ extrusion by the α-intercalated cells (αIC). In the past several years, we have focused cellular and Drosophila experiments using genetically encoded ion-sensors (pH, Cl-, Ca2+). We developed a new but bright, red fluorescent protein (pHire), a pH-sensor that we have used in cells and to make transgenic flies. Here, we will test the lox-stop-lox-pHire (LSL-pHire) mouse we have made (Rosa26 targeted) as a tool for general kidney pH-regulation experiments. We will direct pHire to the PT using the SGLT2-Cre and to the αIC using KitcreERT2/+. From our mouse crosses, we will microdissect PT and collecting ducts (for αIC) and test intracellular pH-regulation. We will further test PT functional differences by SGLT2:pHire ± NBCe1A- knockout (Nbce1A-KO mouse). After tissue specific-validation, we will monitor mice to make sure that marker proteins as well as basic renal function and morphology are unchanged from wild-type and the LSL-pHire parent strain. We will follow nephron-segment specific antibodies morphology (cryosections) for comparison. Prior to sacrifice, we will longitudinally track animals: GFR measurements; total kidney volume (TKV) and cardiac function by ultrasound. The red-fluorescent pHire will allow us and others in the kidney and KUH community to quickly focus on specific-cell types for either pH experiments ± other gene knockdowns or sort cells for high enrichment of this cell population for RNAseq or protein analysis. Thus, pursuing these Aims creates a new set of acid/base- specific assessment tools for the entire KUH community.