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

摘要：利用nbce1a启动子和离子传感器检测和控制近端小管 肾脏对过滤后血液的碳酸氢盐吸收不仅对血液pH动态平衡至关重要，而且对 生活。近端小管(PT)吸收等张碳酸氢钠。基础代谢产生70 mM H，使其 对于缓冲这种酸(CO2/HCO3-)或去除(含可滴定酸的低pH尿液)至关重要。近端小管 HCO3-的吸收是由碱侧NBCe1A(SLC4A4-A)介导的，它是一种生电的小苏打 辅助传送器。肾酸排泄的最后阶段是通过顶端H排泄完成的。 α-插层细胞(αIC)。 在过去的几年里，我们利用基因编码进行了细胞和果蝇的实验 离子传感器(pH、Cl-、Ca2+)。我们开发了一种新的明亮的红色荧光蛋白(Phire)，这是一种pH传感器，可以 我们已经在细胞中使用，并制造出转基因果蝇。在这里，我们将测试lox-top-lox-phire(LSL-Phire)小鼠 我们已经将(ROSA26靶向)作为一般肾脏pH调节实验的工具。我们将执导菲尔 使用SGLT2-CRE的PT和使用KitcreERT2/的αIC。从我们的老鼠十字架上，我们将显微解剖 PT和集合管(用于αIC)，并检测细胞内pH调节。我们将进一步测试PT功能 SGLT2差异：Phire±NBCe1A-基因敲除(Nbce1A-KO小鼠)。 在组织特异性验证之后，我们将监测小鼠以确保标记蛋白以及基本的 肾功能和形态与野生型和LSL-Phire亲本株没有变化。我们会跟随 肾单位段特异性抗体形态(冰冻切片)作比较。在牺牲之前，我们将 纵向追踪动物：GFR测量；总肾脏体积(TKV)和心功能 超音波。 红色荧光菲尔将允许我们和肾脏和KUH社区的其他人迅速专注于 用于pH实验±其他基因敲除的特定细胞类型或用于高度浓缩该基因的分类细胞 用于RNAseq或蛋白质分析的细胞群。因此，追求这些目标会产生一套新的酸/碱- 为整个KUH社区提供了专门的评估工具。

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.