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

• 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
• 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

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.

翻译后摘要：测定和控制近端小管使用nbce 1A启动子和离子传感器 肾脏对过滤血液的双氢吸收不仅对血液pH稳态至关重要，而且对血液pH稳态也至关重要。 生活近端小管（PT）吸收等渗NaHCO 3。基础代谢产生70 mM H+，使其 关键是缓冲这种酸（CO2/HCO 3-）或去除（含可滴定酸的低pH尿液）。近端小管 HCO 3吸收由基底外侧NBCe 1A（SLC 4A 4-A）介导， 协同转运蛋白肾脏酸排泄的最后阶段是通过顶H+挤出完成的， α-嵌入细胞（αIC）。 在过去的几年里，我们专注于细胞和果蝇实验， 离子传感器（pH、Cl-、Ca 2+）。我们开发了一种新的但明亮的红色荧光蛋白（pHire），一种pH传感器， 我们已经在细胞中使用，并使转基因苍蝇。在这里，我们将测试lox-stop-lox-pHire（LSL-pHire）小鼠 我们已经使（Rosa 26靶向）作为一般肾脏pH调节实验的工具。我们将直接雇用 使用SGLT 2-Cre测定PT，使用KitcreERT 2/+测定αIC。从我们的老鼠杂交，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 SGLT 2的差异：pHire ± NBCe 1A-敲除（Nbce 1A-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础蛋白。 肾功能和形态与野生型和LSL-pHire亲本菌株相比没有变化。我们将遵循 肾单位节段特异性抗体形态学（冷冻切片）用于比较。在献祭之前，我们会 纵向跟踪动物：GFR测量;总肾脏体积（TKV）和心脏功能， 超声. 红色荧光pHire将使我们和肾脏和KUH社区的其他人能够快速关注 用于pH实验±其他基因敲除的特定细胞类型，或分选细胞以高度富集该基因， 用于RNAseq或蛋白质分析的细胞群。因此，追求这些目标创造了一套新的酸/碱- 为整个KUH社区提供具体的评估工具。