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

使用基因编码的 pH 传感器测定和控制肾细胞功能

基本信息

批准号：
10682466
负责人：
MICHAEL F. ROMERO
金额：
$ 19.88万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10682466
关键词：
Acids Animals Antibodies Apical Assessment tool Basal metabolic rate Bicarbonates Biological Assay Blood Buffers Carbon Dioxide Cardiac Output Cell Line Cell Separation Cell physiology Cell secretion Cells Communities Complementary DNA Cryoultramicrotomy Cultured Cells Cytomegalovirus Data Disease Doctor of Philosophy Dose Drosophila genus Duct (organ) structure EPHA3 gene Enterobacteria phage P1 Cre recombinase Enzymes Epithelium Excretory function Exons Fluorescent Dyes Genes Genetic Human Intercalated Cell Introns Ions Isotonic Exercise Kidney Knock-out Knockout Mice Knowledge Letters Life Mammalian Cell Mammals Measurement Measures Mediating Metabolic Microelectrodes Modeling Molecular Molecular Cloning Molecular Profiling Monitor Morphology Mus National Institute of Diabetes and Digestive and Kidney Diseases Nephrons PHluorin Parents Phase Physiological Physiology Population Postdoctoral Fellow Process Protein Analysis Protein Isoforms Proteins Protocols documentation Recombinant Proteins Regulation Renal function Renal tubule structure Research Personnel Rodent Site Sodium Bicarbonate Sorting Tamoxifen Techniques Testing Time Tissues Transcend Transcription Initiation Site Transfection Transgenic Mice Transgenic Organisms Transport Process Urine Urology Validation Work absorption arm base blood filter cell type design drug mechanism experimental study fly heart function insight kidney cell knock-down novel pH Homeostasis patch clamp promoter protein biomarkers protein expression pup ratiometric red fluorescent protein response sensor solute symporter tool tool development transcription activator-like effector nucleases transcriptome sequencing ultrasound voltage clamp wasting

项目摘要

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.

摘要：使用 nbce1A 启动子和离子传感器检测和控制近端小管肾脏对过滤血液的碳酸氢盐吸收不仅对于血液 pH 稳态至关重要，而且对于近端小管 (PT) 吸收等渗 NaHCO3，产生 70 mM H+。对于缓冲这种酸（CO2/HCO3-）或去除（用可滴定酸的低 pH 尿液）至关重要。 HCO3- 吸收由基底外侧 NBCe1A (SLC4A4-A)（产电的 Na+ 碳酸氢盐）介导肾脏酸排泄的最后阶段是通过顶端 H+ 排出来完成的。 α-闰细胞（αIC）。在过去的几年中，我们专注于使用基因编码的细胞和果蝇实验我们开发了一种新型但明亮的红色荧光蛋白 (pHire)，这是一种 pH 传感器。我们已经在细胞中使用并制造了转基因果蝇，在这里，我们将测试 lox-stop-lox-pHire (LSL-pHire) 小鼠。我们已经制作了（Rosa26 目标）作为一般肾脏 pH 调节实验的工具，我们将指导 pHire。使用 SGLT2-Cre 到 PT 并使用 KitcreERT2/+ 到 αIC，我们将从我们的小鼠杂交中进行显微解剖。 PT 和集合管（用于 αIC）并测试细胞内 pH 调节我们将进一步测试 PT 功能。 SGLT2:pHire ± NBCe1A- 敲除（Nbce1A-KO 小鼠）的差异。在组织特异性验证后，我们将监测小鼠以确保标记蛋白以及基本蛋白肾功能和形态与野生型和 LSL-pHire 亲本菌株相比没有变化，我们将跟踪。在处死之前，我们将进行肾单位节段特异性抗体形态（冰冻切片）的比较。纵向追踪动物：GFR 测量（TKV）和心脏功能；超声波。红色荧光 pHire 将使我们以及肾脏和 KUH 社区的其他人能够快速关注用于 pH 实验 ± 其他基因敲低的特定细胞类型或对细胞进行分类以实现高度富集因此，追求这些目标创造了一套新的酸/碱- 适合整个 KUH 社区的特定评估工具。