V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice

Abstract

The kidney, epididymis, and lungs are complex organs with considerable epithelial cell heterogeneity. This has limited the characterization of pathophysiological transport processes that are specific for each cell type in these epithelia. The purpose of the present study was to develop new tools to study cell-specific gene and protein expression in such complex tissues and organs. We report the production of a transgenic mouse that expresses enhanced green fluorescent protein (EGFP) in a subset of epithelial cells that express the B1 subunit of vacuolar H(+)-ATPase (V-ATPase) and are actively involved in proton transport. A 6.5-kb portion of the V-ATPase B1 promoter was used to drive expression of EGFP. In two founders, quantitative real-time RT-PCR demonstrated expression of EGFP in kidney, epididymis, and lung. Immunofluorescence labeling using antibodies against the B1 and E subunits of V-ATPase and against carbonic anhydrase type II (CAII) revealed specific EGFP expression in all renal type A and type B intercalated cells, some renal connecting tubule cells, all epididymal narrow and clear cells, and some nonciliated airway epithelial cells. No EGFP expression was detected in collecting duct principal cells (identified using an anti-AQP2 antibody) or epididymal principal cells (negative for V-ATPase or CAII). This EGFP-expressing mouse model should prove useful in future studies of gene and protein expression and their physiological and/or developmental regulation in distinct cell types that can now be separated using fluorescence-assisted microdissection, fluorescence-activated cell sorting, and laser capture microdissection.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers

“…11 The 640-kD V 1 subunit is composed of subunits A-H. Mammals have two B subunits, the ubiquitous B2 isoform and the B1 isoform, which is restricted to specialized epithelia of the inner ear, epididymis, and intercalated cells. 12 Numerous homozygous and compound heterozygous missense, nonsense, frameshift, and splice site mutations along the B1 subunit gene ATP6V1B1 have been reported in individuals with congenital dRTA. 3,13 We previously investigated disease-causing missense B1 subunit mutations in vitro.…”

Overexpression of Pendrin in Intercalated Cells Produces Chloride-Sensitive Hypertension

“…NM_039350) has been successfully used to drive expression of both enhanced green fluorescent protein (EGFP) and the Cre-recombinase (Cre) into renal intercalated cells. 14,15 Importantly, preliminary experiments revealed that the promoter is likely to be insensitive to a high-salt diet (Supplemental Figure 1). We used this promoter fragment to drive the expression of human pendrin (hPDS/SLC26A4) cDNA in intercalated cells of the connecting tubule and the cortical collecting duct (CCD; detailed methods of the transgene construction and genotyping can be found in Supplemental Material and Supplemental Table 1).…”

“…Figure 1C shows that high levels of the human pendrin transcript were detected by RT-PCR in the renal cortex and medulla of transgenic B1-hPDS (Tg B1-hPDS ) mice, whereas it was not present in the wild-type littermates. We previously reported that, beyond expression in renal intercalated cells, our ATP6V1B1 promoter fragment was able to drive expression of EGFP in epididymis, uterus, small and large intestines, and nonciliated airway epithelial cells 15 and Cre-recombinase in the brain. 14 Thus, we tested expression of the transgene in these different tissues ( Figure 1D).…”

“…Ectopic expression of hPDS in a-intercalated cells was expected, because our B1 promoter fragment was shown to drive expression of EGFP and Cre in both intercalated cell subtypes. 14,15 Noteworthy, the expression of the transgene had no particular impact on AE1 or the V H + -ATPase cellular or subcellular localization.…”

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

“…Here the risk of contamination with other segments or cell populations is much higher. Transgenic mouse models expressing EGFP or other fluorescent proteins under the control of cell or segmentspecific promoters have become available over the last few years such as mice expressing EGFP in intercalated cells driven by the promoter for the B1 H + -ATPase subunit 27 . Kidneys from these mice can be used to isolate large quantitities of nephron segments or cells after digestion of the organ using fluorescence-guided sorting of segments or cells (COPAS: complex object parametric analyzer and sorter) 28 .…”