Kidney Week

Abstract: SA-PO432

Ribonuclease 6 Protects the Kidney from Ascending Infection by Uropathogenic Escherichia coli

Session Information

• Pediatric Nephrology - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Pediatric Nephrology

• 1600 Pediatric Nephrology

Authors

• Cortado, Hanna H., Nationwide Children's Hospital, Columbus, Ohio, United States

Hanna H. Cortado,

• Gupta, Sudipti, Nationwide Childrens Hospital , Columbus, Ohio, United States

Sudipti Gupta,

• Li, Birong, Nationwide Children's Hospital, Columbus, Ohio, United States

Birong Li,

• Jackson, Ashley R., Nationwide Children's Hospital, Columbus, Ohio, United States

Ashley R. Jackson,

• Ching, Christina B., Nationwide Children''s Hospital, Columbus, Ohio, United States

Christina B. Ching,

• Becknell, Brian, Nationwide Children's Hospital, Columbus, Ohio, United States

Brian Becknell,

Background

Ribonuclease 6 (RNase 6) is an evolutionarily-conserved antimicrobial peptide with potent bactericidal activity that is induced during urinary tract infection (UTI). Previously, we demonstrated that human and mouse RNase 6 kills uropathogenic Escherichia coli (UPEC) in vitro at low micromolar concentrations. Here, we investigated the hypothesis that RNase 6 serves an essential protective role in limiting ascending infection by UPEC in vivo.

Methods

We generated a Rnase6^EGFP knock-in allele to (1) identify the cellular sources of RNase 6 in Rnase6^EGFP/+ mice; and (2) to determine the consequence of biallelic Rnase6 deletion (Rnase6^EGFP/EGFP) on host susceptibility to experimental UTI. We evaluated EGFP fluorescence by flow cytometry and epifluorescence microscopy. We evaluated bactericidal activity and nitric oxide production by bone marrow-derived macrophages using gentamicin protection and fluorometric assays, respectively. We transurethrally inoculated Rnase6^EGFP/EGFP, Rnase6^EGFP/+, and control female mice with UPEC. We enumerated bacterial burden in urinary tract tissues by homogenization and serial plating.

Results

Flow cytometry in Rnase6^EGFP/+ mice reveals EGFP expression by circulating Ly6C^hi monocytes which undergo maximal recruitment to the infected bladder by 6 hours post infection. In addition, we identified EGFP expression within resident macrophages of the bladder and kidney. In contrast, the Rnase6 promoter is not active in renal or bladder epithelial cells. We confirmed Rnase6 deletion in Rnase6^EGFP/EGFP mice, which displayed normal urinary tract development, fertility, and hematopoiesis. Rnase6 deficiency did not inhibit nitric oxide production or UPEC killing by bone marrow-derived macrophages. In contrast, Rnase6 deficiency resulted in increased susceptibility to experimental UTI, with significantly higher UPEC burden throughout the urinary tract, compared to control mice.

Conclusion

Use of Rnase6^EGFP/+ mice offers a powerful approach to track cellular sources of RNase 6, as well as a means to screen compounds that regulate Rnase6 promoter activity in the future. Use of Rnase6^EGFP/EGFP mice confirms a critical role for RNase 6 in UPEC clearance in vivo.

Funding

• NIDDK Support