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This Article Full Text Full Text (PDF) All Versions of this Article: 296/1/F194    most recent 90495.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hofmeister, M. V. Articles by Praetorius, J. Search for Related Content PubMed PubMed Citation Articles by Hofmeister, M. V. Articles by Praetorius, J.

Fluorescence isolation of mouse late distal convoluted tubules and connecting tubules: effects of vasopressin and vitamin D₃ on Ca²⁺ signaling

Institute of Anatomy and The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark

Submitted 19 August 2008 ; accepted in final form 4 November 2008

The renal late distal convoluted tubules and connecting tubules are sites for the fine regulation of Na⁺ and Ca²⁺ reabsorption. The role of these segments in Na⁺ and K⁺ homeostasis is possibly underestimated, as the tubules are technically difficult to isolate in sufficient numbers and purity for functional analysis. To overcome these difficulties, we have developed a transgenic mouse model expressing enhanced green fluorescent protein in late distal convoluted tubules and connecting tubules. Enhanced green fluorescent protein expression was driven by the promoter for the transient receptor potential subfamily V, member 5. Confocal fluorescence microscopy allowed detection of enhanced green fluorescent protein in living, isolated late distal convoluted tubules and connecting tubules and in the initial cortical collecting ducts. Enhanced green fluorescent protein expression was validated by double- and triple-fluorescence immunolabeling with specific tubule markers. Freshly isolated late distal convoluted tubules and connecting tubules increased their intracellular Ca²⁺ levels in response to the V₂ receptor-specific agonist deamino-Cys,D-Arg⁸-vasopressin (2 x 10^–10 M) after 1 min of superfusion. In addition, both late distal convoluted tubules and connecting tubules displayed a concentration-dependent intracellular Ca²⁺ response to 1,25-dihydroxyvitamin D₃ (range 10^–10 to 10^–8 M). This suggests that 1,25-dihydroxyvitamin D₃ can act through a nongenomic signaling pathway in these tubules. In conclusion, the transgenic mouse model, expressing enhanced green fluorescent protein, is suitable for rapid isolation of viable late distal convoluted tubules, connecting tubules, and initial cortical collecting ducts and provides an ideal tool for a more exhaustive functional characterization of these segments.

transgenic mice; immunohistochemistry; distal nephron; collecting duct; acute signaling