Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct

Vijay Saxena, David Hains, John Ketz, Melinda Chanley, John D. Spencer, X. Brian Becknell, Keith R. Pierce, Raoul D. Nelson, Jeffrey M. Purkerson, George J. Schwartz, Andrew L. Schwaderer

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Saxena V, Hains DS, Ketz J, Chanley M, Spencer JD, Becknell B, Pierce KR, Nelson RD, Purkerson JM, Schwartz GJ, Schwaderer AL. Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct. Am J Physiol Renal Physiol 315: F812–F823, 2018. First published May 3, 2017; doi:10.1152/ajprenal.00512.2016.—The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cretdT mice to fluorescently label ICs and AQP2-cretdT mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Original languageEnglish (US)
Pages (from-to)F812-F823
JournalAmerican Journal of Physiology - Renal Physiology
Volume315
Issue number4
DOIs
StatePublished - Oct 1 2018

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Epithelial Cells
Kidney
Polymerase Chain Reaction
Aquaporin 2
Uropathogenic Escherichia coli
Adenosine Monophosphate
Urinary Tract
Vacuolar Proton-Translocating ATPases
Messenger RNA
Adenosine Triphosphatases

All Science Journal Classification (ASJC) codes

  • Physiology
  • Urology

Cite this

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct. / Saxena, Vijay; Hains, David; Ketz, John; Chanley, Melinda; Spencer, John D.; Becknell, X. Brian; Pierce, Keith R.; Nelson, Raoul D.; Purkerson, Jeffrey M.; Schwartz, George J.; Schwaderer, Andrew L.

In: American Journal of Physiology - Renal Physiology, Vol. 315, No. 4, 01.10.2018, p. F812-F823.

Research output: Contribution to journalArticle

Saxena, V, Hains, D, Ketz, J, Chanley, M, Spencer, JD, Becknell, XB, Pierce, KR, Nelson, RD, Purkerson, JM, Schwartz, GJ & Schwaderer, AL 2018, 'Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct', American Journal of Physiology - Renal Physiology, vol. 315, no. 4, pp. F812-F823. https://doi.org/10.1152/ajprenal.00512.2016
Saxena, Vijay ; Hains, David ; Ketz, John ; Chanley, Melinda ; Spencer, John D. ; Becknell, X. Brian ; Pierce, Keith R. ; Nelson, Raoul D. ; Purkerson, Jeffrey M. ; Schwartz, George J. ; Schwaderer, Andrew L. / Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct. In: American Journal of Physiology - Renal Physiology. 2018 ; Vol. 315, No. 4. pp. F812-F823.
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abstract = "Saxena V, Hains DS, Ketz J, Chanley M, Spencer JD, Becknell B, Pierce KR, Nelson RD, Purkerson JM, Schwartz GJ, Schwaderer AL. Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct. Am J Physiol Renal Physiol 315: F812–F823, 2018. First published May 3, 2017; doi:10.1152/ajprenal.00512.2016.—The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H−-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre−tdT− mice to fluorescently label ICs and AQP2-cre−tdT− mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.",
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AU - Becknell, X. Brian

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AU - Nelson, Raoul D.

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