ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

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
  • Gupta, Sudipti, Nationwide Childrens Hospital , Columbus, Ohio, United States
  • Li, Birong, Nationwide Children's Hospital, Columbus, Ohio, United States
  • Jackson, Ashley R., Nationwide Children's Hospital, Columbus, Ohio, United States
  • Ching, Christina B., Nationwide Children''s Hospital, Columbus, Ohio, United States
  • Becknell, Brian, Nationwide Children's Hospital, Columbus, Ohio, United States
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 Rnase6EGFP knock-in allele to (1) identify the cellular sources of RNase 6 in Rnase6EGFP/+ mice; and (2) to determine the consequence of biallelic Rnase6 deletion (Rnase6EGFP/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 Rnase6EGFP/EGFP, Rnase6EGFP/+, and control female mice with UPEC. We enumerated bacterial burden in urinary tract tissues by homogenization and serial plating.

Results

Flow cytometry in Rnase6EGFP/+ mice reveals EGFP expression by circulating Ly6Chi 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 Rnase6EGFP/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 Rnase6EGFP/+ 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 Rnase6EGFP/EGFP mice confirms a critical role for RNase 6 in UPEC clearance in vivo.

Funding

  • NIDDK Support