Laurent Bourqui, Denise V Winter, Alex Odermatt, Dominique Loffing-Cueni, Johannes Loffing
{"title":"A novel mouse model for an inducible gene modification in the renal thick ascending limb.","authors":"Laurent Bourqui, Denise V Winter, Alex Odermatt, Dominique Loffing-Cueni, Johannes Loffing","doi":"10.1152/ajprenal.00250.2022","DOIUrl":null,"url":null,"abstract":"<p><p>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<sup>+</sup>-K<sup>+</sup>-2Cl<sup>-</sup> 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.<b>NEW & NOTEWORTHY</b> 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.</p>","PeriodicalId":7588,"journal":{"name":"American Journal of Physiology-renal Physiology","volume":"324 5","pages":"F446-F460"},"PeriodicalIF":3.7000,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085568/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"American Journal of Physiology-renal Physiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1152/ajprenal.00250.2022","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PHYSIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
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.