Abstract: TH-PO684
Insulin Resistance Suppresses Intercalated Cell Antibacterial Defenses
Session Information
- Diabetes Mellitus and Obesity: Basic - Experimental - I
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Diabetes
- 501 Diabetes Mellitus and Obesity: Basic - Experimental
Authors
- Murtha, Matthew J, The Research Institute at Nationwide Children''s Hospital, Columbus Ohio, Pickerington, Ohio, United States
- Eichler, Tad, The Research Institute at Nationwide Childrens Hospital, COLUMBUS, Ohio, United States
- Li, Birong, Nationwide Children's Hospital, Columbus, Ohio, United States
- Becknell, Brian, Nationwide Children's Hospital, Columbus, Ohio, United States
- Spencer, John David, The Research Institute at Nationwide Children's, Columbus, Ohio, United States
Background
Urinary tract infection (UTI) is prevalent in people with diabetes mellitus. New evidence suggests antimicrobial peptides (AMP) play a role in maintaining urine sterility. Data from our lab shows that the kidney’s intercalated cells (IC) secrete AMPs into the urine. Our data also show that insulin induces AMPs to shield the host from uropathogenic E. coli (UPEC). To investigate the impact of insulin resistance on renal antibacterial defense and AMP production, we selectively deleted the insulin receptor (IR) in murine ICs.
Methods
Using a Cre-loxP approach, mice homozygous for the floxed IR gene were crossed with mice expressing Cre recombinase under the ATP6V1B1 promoter (IC-specific isoform of VATPaseB1) to create IC-specific IR knockout mice (IRKO). A Cre-dependent tdTomato fluorescent reporter was incorporated to isolate ICs by fluorescent activated cell sorting (FACS). IRKO mice and Cre-negative littermates (IRflox) were subjected to experimental UTI with UPEC. 24 and 48 hrs post infection, UPEC burden was enumerated. The effects of IR deletion on host defense were confirmed in vitro using siRNA and ex vivo with urine neutralization assays.
Results
Compared to IRflox mice, IRKO mice exhibit a normal phenotype with normal serum insulin/glucose levels and serum/urine pH. qRT-PCR and Western blot confirmed IR mRNA and protein deletion in FACs-isolated ICs. qRT-PCR on FACS-isolated ICs shows that IRKO mice have decreased transcript expression of several AMPs, including RNase 4 and Lipocalin 2. ELISA confirmed lower urinary AMP peptide concentrations in IRKO mice compared to controls. Following experimental UTI, IRKO mice had 2-3 fold greater UPEC burden in urine, bladder, and kidneys. To confirm that suppressed AMP production increases UPEC susceptibility, RNase 4 was silenced in medullary cells in vitro. Cells were challenged with UPEC. With RNase 4 knock-down, UPEC invasion increased. When anti-RNase 4 blocking antibodies were added to mouse urine, UPEC survival increased.
Conclusion
This data suggest that intact IC insulin signaling is critical for UTI defense. Also, they indicate that the hyperglycemic environment alone does not explain diabetes-associated UTI risk. In part, increased UTI risk may be due to decreased expression of AMPs. Studies are needed to develop agents that target IC-specific insulin signaling or AMP production to reduce UTI risk.
Funding
- Other NIH Support