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Abstract: SA-PO1002

GSTM1 Deficient Kidney Primary Tubular Epithelial Cells Have Augmented NF-kB p65 Signaling in Response to Angiotensin II

Session Information

  • CKD: Pathobiology - II
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Chen, Luojing, University of Rochester, Rochester, New York, United States
  • Beane, Timothy Jason, University of Rochester, Rochester, New York, United States
  • Wang, Yves T., University of Rochester, Rochester, New York, United States
  • Cunningham, Jason J., University of Rochester, Rochester, New York, United States
Background

Glutathione-S-transferase Mu-1 (Gstm1) gene encodes an enzyme that functions in the detoxification of electrophilic compounds. The common GSTM1 deletion variant in humans is associated with increased risks of chronic kidney disease (CKD) progression and incident end stage kidney failure. We previously reported that, in angiotensin II-induced hypertension (AngII-HTN), Gstm1 knockout (KO) mice display increased kidney inflammation, characterized by increased inflammatory cell infiltration (by flow cytometry), and increased kidney expression of CXCL1, MCP-1/CCL2, and IL-6, independent of blood pressure. Moreover, bone marrow cross-transplantation revealed GSTM1 deficiency in the parenchyma, not in bone marrow derived cells, determined kidney inflammation. Much of the proinflammatory effect of AngII is mediated via the canonical NF-kB pathway. We set to determine the influence of GSTM1 on NF-kB p65 signaling pathway in renal tubular cells.

Methods

Primary tubular epithelial cells (PTECs) were isolated from Gstm1 KO and wild-type (WT) kidneys, and grown on pre-coated cover slips until ~ 70% confluent. Cells were then starved for 4 hours before treatment with AgII (100 nM) for 24 hours, fixed, and stained with anti-NF-kB p65 antibody. Images were taken with a fluorescence microscope (Olympus BX51).

Results

NF-kB p65 nuclear staining was quantified as percentage of p65 nuclei-positively stained cells to the total cells. Five random fields were counted for each group and similar results were obtained from two independent experiments. At baseline, there was no statistically significant difference. After 24 hrs of Ang II, there was a significant increase in intensity and localization of p65 in nuclei in KO compared to WT PTECs (% p65 positive cells: WT 9.8 ± 1.6, KO 23.8. ± 2.5; p = 0.0014).

Conclusion

Deletion of Gstm1 augments AngII activation of NF-kB p65 signaling pathway in PTECs. This may explain the increased kidney inflammation in Gstm1 KO mice in Ang II-HTN. Further studies are under way to determine the functional significance, including expression of CXCL1 and MCP-1 in PTECs, and whether this drives migration of inflammatory cells.

Funding

  • NIDDK Support