A novel mouse model for an inducible gene modification in the renal thick ascending limb.

IF 3.7 2区 医学 Q1 PHYSIOLOGY American Journal of Physiology-renal Physiology Pub Date : 2023-05-01 DOI:10.1152/ajprenal.00250.2022
Laurent Bourqui, Denise V Winter, Alex Odermatt, Dominique Loffing-Cueni, Johannes Loffing
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Abstract

The thick ascending limb (TAL) is critical for renal control of fluid and ion homeostasis. The function of the TAL depends on the activity of the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2), which is highly abundant in the luminal membrane of TAL cells. TAL function is regulated by various hormonal and nonhormonal factors. However, many of the underlying signal transduction pathways remain elusive. Here, we describe and characterize a novel gene-modified mouse model for an inducible and specific Cre/Lox-mediated gene modification in the TAL. In these mice, tamoxifen-dependent Cre (CreERT2) was inserted into the 3'-untranslated region of the Slc12a1 gene, which encodes NKCC2 (Slc12a1-CreERT2). Although this gene modification strategy slightly reduced endogenous NKCC2 expression at the mRNA and protein levels, the lowered NKCC2 abundance was not associated with altered urinary fluid and ion excretion, urinary concentration, and the renal response to loop diuretics. Immunohistochemistry on kidneys from Slc12a1-CreERT2 mice revealed strong Cre expression exclusively in TAL cells but not in any other nephron portion. Cross-breeding of these mice with the mT/mG reporter mouse line showed a very low recombination rate (∼0% in male mice and <3% in female mice) at baseline but complete (∼100%) recombination after repeated tamoxifen administration in male and female mice. The achieved recombination encompassed the entire TAL and also included the macula densa. Thus, the new Slc12a1-CreERT2 mouse line allows inducible and very efficient gene targeting in the TAL and hence promises to be a powerful tool to advance our understanding of the regulation of TAL function.NEW & NOTEWORTHY The renal thick ascending limb (TAL) is critical for renal control of fluid and ion homeostasis. However, the underlying molecular mechanisms that regulate TAL function are incompletely understood. This study describes a novel transgenic mouse model (Slc12a1-creERT2) for inducible and highly efficient gene targeting in the TAL that promises to ease physiological studies on the functional role of candidate regulatory genes.

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一种新的小鼠肾厚升肢诱导基因修饰模型。
厚升肢(TAL)是肾脏控制液体和离子稳态的关键。TAL细胞的功能取决于布美他尼敏感的Na+- k +- 2cl -共转运体(NKCC2)的活性,而NKCC2在TAL细胞的管腔膜中含量很高。TAL功能受多种激素和非激素因素的调节。然而,许多潜在的信号转导途径仍然难以捉摸。在这里,我们描述和表征了一种新的基因修饰小鼠模型,用于诱导和特异性Cre/ lox介导的TAL基因修饰。在这些小鼠中,他莫昔芬依赖性Cre (CreERT2)被插入Slc12a1基因的3'-未翻译区域,该区域编码NKCC2 (Slc12a1-CreERT2)。尽管这种基因修饰策略在mRNA和蛋白水平上略微降低了内源性NKCC2的表达,但NKCC2丰度的降低与尿液和离子排泄、尿浓度以及肾脏对利尿剂的反应的改变无关。Slc12a1-CreERT2小鼠肾脏的免疫组化显示,Cre仅在TAL细胞中表达,而在其他肾单元部分不表达。这些小鼠与mT/mG报告小鼠系杂交显示,重组率非常低(雄性小鼠约0%),肾厚升肢(TAL)对肾脏控制液体和离子稳态至关重要。然而,调控TAL功能的潜在分子机制尚不完全清楚。本研究描述了一种新的转基因小鼠模型(Slc12a1-creERT2),用于诱导和高效的TAL基因靶向,有望简化候选调节基因功能作用的生理学研究。
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来源期刊
CiteScore
8.40
自引率
7.10%
发文量
154
审稿时长
2-4 weeks
期刊介绍: The American Journal of Physiology - Renal Physiology publishes original manuscripts on timely topics in both basic science and clinical research. Published articles address a broad range of subjects relating to the kidney and urinary tract, and may involve human or animal models, individual cell types, and isolated membrane systems. Also covered are the pathophysiological basis of renal disease processes, regulation of body fluids, and clinical research that provides mechanistic insights. Studies of renal function may be conducted using a wide range of approaches, such as biochemistry, immunology, genetics, mathematical modeling, molecular biology, as well as physiological and clinical methodologies.
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