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Abstract: TH-PO138

Ultrasound Reduces Kidney Injury via a Postsynaptic P2X4-Dependent Mechanism in Sepsis-Associated AKI

Session Information

  • AKI: Mechanisms - I
    November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Zheng, Shuqiu, University of Virginia, Charlottesville, Virginia, United States
  • Yao, Junlan, University of Virginia, Charlottesville, Virginia, United States
  • Nash, William, University of Virginia, Charlottesville, Virginia, United States
  • Poudel, Nabin, University of Virginia, Charlottesville, Virginia, United States
  • Goggins, Eibhlin S,, University of Virginia, Charlottesville, Virginia, United States
  • Kuwabara, Shuhei, University of Virginia, Charlottesville, Virginia, United States
  • Okusa, Mark D., University of Virginia, Charlottesville, Virginia, United States
Background

Purinergic X receptor (P2X) receptor subunits are present both in pre- and postsynaptic sites (Gordon, G.R.J et al. 2015). P2X4, one of the most sensitive purinergic ATP receptors, has been reported to exacerbate ischemic acute kidney injury (S.J. Han et al. 2020). We previously showed that pulsed ultrasound (pUS) reduced inflammation and acute kidney injury. However, the exact mechanism has not yet been well defined. Here, we utilized a mouse model of sepsis-associated acute kidney injury (SA-AKI) by injecting lipopolysaccharide (LPS) to investigate the effects of pUS on the expression and trafficking of P2X4 receptor during pathogenesis of SA-AKI.

Methods

C57/BL/6 mice received pUS 24 hours before LPS (5 mg/kg, ip) treatment. The parameters of pUS therapy followed the protocol previously published by us (PMID: 23907510). The expression of P2X4 was measured by immunofluorescence and RT-PCR respectively. ATP levels in the mouse kidney were determined by CellTiter-Glo Luminescent Cell Viability Assay Kit (G7571, Promega, Madison,WI), and the luminescence signal was normalized to the protein concentration of the homogenate. The measured luminescence signal is proportional to the amount of ATP.

Results

In vehicle treated animals, co-labeling with P2X4 and postsynaptic density 95 protein (PSD-95) showed that P2X4 is located mainly in lysosomes and partially colocalized with PSD-95. However, LPS induced injury upregulated P2X4 expression and translocated to the brush border and plasma membrane of proximal tubules, and whereas those issues were decreased in mice pretreated with pUS. In addition, the temporal changes observed with P2X4 mRNA overexpression correlated with a higher ATP release, pUS attenuated the release of ATP and the overexpression of P2X4 mRNA.

Conclusion

Our data suggest that LPS heightened brush border and membrane expression of P2X4 is temporally related to the release of ATP. ATP ligation of P2X4 in proximal tubules may lead to calcium influx and cell death. We believe that the protective mechanism of pUS in response to LPS appears to be associated with attenuated P2X4 receptor expression and signaling in proximal tubules.

Funding

  • NIDDK Support