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Abstract: PO1986

Intercalated Cells Activate Innate Immune Defenses in Response to Uropathogenic Escherichia coli

Session Information

Category: Pediatric Nephrology

  • 1700 Pediatric Nephrology

Authors

  • Linn, Sarah C., Nationwide Children's Hospital, Columbus, Ohio, United States
  • Schwartz, Laura, Nationwide Children's Hospital, Columbus, Ohio, United States
  • Spencer, John David, Nationwide Children's Hospital, Columbus, Ohio, United States
Background

Urinary tract infections (UTI), including pyelonephritis, are common in children. Intercalated cells (IC), positioned in the renal collecting duct, prevent and combat UTI by secreting antimicrobial peptides (AMPs) into the urine. The mechanisms regulating IC AMP production during UTI are unclear. Here, we challenged ICs in vitro with uropathogenic E. coli (UPEC) or bacterial cell membrane components to define the innate immune responses that control AMP production during UTI.

Methods

ICs (Clone C) were infected with a UPEC pyelonephritis strain (CFT073) or challenged with UPEC cell membrane components including lipopolysaccharide (LPS), muramyl dipeptide (MDP) and γ-D-Glu-mDAP (iE-DAP). Following stimulation, IC lysates were collected, and 87 immune genes were profiled using an antimicrobial response PCR array or targeted qRT-PCR. Western blot was performed to identify which innate immune responses are activated.

Results

In response to UPEC, ICs temporally activate immunomodulatory pathways and AMPs. Analysis of the PCR array data via STRING and Ingenuity Pathway Analysis identified 15 upregulated genes associated with Toll-like receptor (TLR), NOD-like receptor (NLR), and NF-kB signaling 4 hours post infection. Immunoblotting confirmed downstream targets in these pathways are activated in response to UPEC. qRT-PCR identified that AMPs like Lcn2 are activated while others, including Rnase8, are suppressed. Upon stimulation with LPS, qRT-PCR showed upregulation of Lcn2, Defb1, and Rnase8 – suggesting that TLR4 activation may regulate the expression of these AMPs. Additionally, qRT-PCR showed Lcn2 is induced in response to the NOD2 agonist, MDP, while AMP expression did not change with the NOD1 agonist, iE-DAP.

Conclusion

During UPEC infection, TLR, NLR, and NF-kB responses are activated in ICs. Activation of TLR and NLR signaling may induce downstream targets like AMPs. Confirmation studies are needed to determine how these pathways regulate AMP expression and their differential regulatory targets. Identification of these nodes may serve as future targets to increase AMP production as an additional means to treat UTIs in children and adults.

Funding

  • NIDDK Support