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Abstract: TH-PO491

Acidosis Stimulates SDF/CXCR4 Expression and Antimicrobial Responses via HIF-1α in Mouse Collecting Duct (M-1) Cells

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Peng, Hu, Univeristy of Rochester medical center, Rochester, New York, United States
  • Schwartz, George J., University of Rochester, Rochester, New York, United States
  • Purkerson, Jeffrey M., University of Rochester Medical Center, Rochester, New York, United States
Background

Intercalated cells (ICs) mediate H+ and HCO3 secretion in the kidney collecting duct (CD), We reported that IC adaptation to metabolic acidosis is mediated by SDF1 signaling via CXCR4 (JCI 125:4365, 2015), and that metabolic acidosis stimulates expression of the antimicrobial peptide (AMP) cathelicidin in rabbit urine (AJP 313: F1061, 2017).

Methods

We used M-1 cell line (ATCC CRL-2038), from mouse l (CD), as an ex vivo model. M-1 cells were cultured in normal (25mM NaHCO3) and acidic (10mM NaHCO3, 10uM EIPA) medium±HIF-1α stabilizer (IOX2,100uM), HIF-1α inhibitor PX-478 (60uM), LPS (200ng/ml), and Arginine (5mM) for 24hr. Total RNA was isolated and antimicrobial peptides, cathelicidin (Camp) and beta-defensin 2 (BD2)) and SDF1/CXCR4 mRNA abundance determined by real-time qRT-PCR and the ΔΔCt method. Uropathogenic E. coli (UPEC) were incubated (5 MOI) with M-1 cells for 90min; unbound bacteria removed by washing, intracellular infection of M-1 cells was determined by gentamicin (100ug/ml) treatment, prior to lysis. Dilutions of cell lysates were plated on agar to determine UPEC burden.

Results

Acidosis induced Camp (2.1 fold±0.20, n=3, p< 0.001), BD 2 (2.83 fold±0.26, n=3, p< 0.01), SDF1 (2.44 fold±0.28, n=3, p<0.01), and CXCR4 mRNA (3.1 fold±0.29, n=3, p<0.01); Prior exposure to acid media decreased by 35% the UPEC burden (mean CFU/ml: Normal=1.12±0.07x106; Acidosis= 7.37±0.03x105, p<0.01). IOX2 increased Camp (2.81 fold±0.31, n=6, p< 0.05) and BD2 mRNA abundance (3.80 fold±0.36, n=6, p< 0.01), and PX-478 reduced M-1 cell resistance to infection conferred by acid-medium by 12% (mean CFU/ml: Acidosis=4.5±0.45x104; Acidosis+PX-478=5.3±0.6x104, p<0.05). M-1 cells treated by combination of IOX2, LPS, and Arginine were substantially more resistant to infection (mean CFU/ml: Untreated=6.7±0.55x104; IOX2, LPS,Arg=2.8±0.35x104, p< 0.01), a 58.2% decrease in UPEC burden. LPS induced SDF1 and CXCR4 mRNA in normal (SDF1: 2.67±0.33, n=3, p< 0.01; CXCR4: 2.94±0.38, n=3, p<0.01) or acidic (SDF1: 4.68±0.61, n=3, p< 0.01; CXCR4: 6.47±0.29, n=3, p<0.01) medium.

Conclusion

Acidosis, via activation of HIF-1α, stimulates expression and function of innate immune defense peptides as well as SD1 and CXCR4 in kidney collecting duct cells.