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Abstract: FR-OR110

The Kidney Makes Gas in Defense against Urinary Bacteria

Session Information

Category: Anemia and Iron Metabolism

  • 201 Anemia and Iron Metabolism: Basic

Authors

  • Xu, Katherine, Columbia University, New York, New York, United States
  • Shen, Tian, Columbia University, New York, New York, United States
  • Levitman, Abraham D., Columbia University, New York, New York, United States
  • Werth, Max, Columbia University, New York, New York, United States
  • Barasch, Jonathan M., Columbia University, New York, New York, United States
Background

The kidney expresses a variety of powerful defense mechanisms to limit urinary infection including iron scavenging, a process called "nutritional immunity". We previously demonstrated that the intercalated cells generate the archetype of the urinary iron defense, a lipocalin called NGAL which binds Enterochelin (Strong, FHCC), a bacterial siderophore. However, because urinary bacteria can evade the bacteriostatic effects of NGAL by modifying Enterochelin and by producing additional siderophores, other mechanisms of "nutritional immunity" are anticipated.

Methods

We utilized a cell type specific method of RNA isolation (adapted from Gay, Oregon). A promiscuous form of phosphoribosyltransferase (UPRT) was cloned into the Rosa26 locus using a floxed-stop design which we activated in a cell specific fashion with HoxB7Cre or Atp6v1b1Cre. Thiouracil was introduced 12hr and 24hrs after infection. Hmox1 was detected with a luciferase based reporter (Contag et al, Stanford). CO was detected with a novel metal-based probe synthesized at Columbia (adapted from Liu, China).

Results

A survey of classical iron transporters revealed segment specific expression of megalin (proximal tubule), TfR1 (TALH), and DMT1 (TALH-DCT). Nonetheless, despite ferritin accumulation, we failed to identify the mechanism of iron import in intercalated cells. Isolation of RNA directly from intercalated and principal cells revealed synthetic (NPAS-BMAL-ALAS), transport (Hrg1, Flvcr1) and metabolic enzymes (Hmox) of heme metabolism. Functional reporters demonstrated Hmox expression and the production of CO gas from cortical>medullary sources in vivo and from AtpCre-mTmG FACS isolated intercalated cells in vitro.

Urinary bacteria (UPEC) with heme transport mutations were not competitive in the colonization of the kidney but conversely were markedly stimulated by heme, suggesting bacterial-host competition for heme capture and metabolism. In fact, infection upregulated both heme synthetic and metabolic genes, suggesting that CO production is induced by infection. Exposure to CO terminated the growth of UPEC.

Conclusion

We have identified an unusual iron trafficking system in the collecting ducts that mirrors the nutritional requirements of UPEC to achieve pyelonephritis. Many of these components are specific to the intercalated cells, consistent with the notion that these cells defend the urinary tract.

Funding

  • NIDDK Support