Abstract: PO0228
Mechanistic Insight for Enhanced Cisplatin (CIS) Nephrotoxicity in Na-H Exchanger Regulatory Factor 1 (NHERF1) Deficiency
Session Information
- AKI Mechanisms - 3
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Bushau-Sprinkle, Adrienne M., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Barati, Michelle T., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Watson, Walter H., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Zheng, Yuxuan, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Gagnon, Kenneth, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Kitterman, Kathleen, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Siskind, Leah J., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Brier, Michael E., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Lederer, Eleanor D., University of Texas Southwestern Medical School, Dallas, Texas, United States
Background
Proximal tubule transport and metabolism of CIS play critical roles in the development of CIS-induced acute kidney injury (AKI). Mice lacking NHERF1 have exacerbated CIS nephrotoxicity. We hypothesize that NHERF1 loss leads to altered oxidative state and/or altered CIS metabolism resulting in increased CIS toxicity.
Methods
We treated 2-4 mo old male wild-type (WT) C57BL/6 and NHERF1 knock out (KO) mice with vehicle or CIS (20 mg/kg IP) for 4, 24, or 72h. Glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), cystine (CySS), cysteine-glutathione disulfide (CySSG) were measured in plasma and kidney cortex by HPLC. Kidney cortex was assayed for γ-glutamyl transferase (GGT) activity, platinum (Pt) levels by ICP-MS, cysteine s-conjugate beta lyase (CCBL1) and GGT by Western blot and immunohistochemistry (IHC); and RT-qPCR for glutathione s-conjugate-translocating ATPase (ABCC1) and γ-glutamylcysteine synthetase (GCLC).
Results
KO mice showed decreased plasma CySS and increased kidney GSSG at baseline. GGT activity was similar 4h after CIS treatment, but significantly greater at 24h in CIS treated WT compared to KO. ABCC1 and GCLC mRNA levels were upregulated 72h post CIS treatment in KO compared to WT. GGT and CCBL1 protein levels were similar by Western blot regardless of genotype or treatment 24h post CIS, but IHC showed greater brush border membrane GGT loss in KO kidneys. CCBL1 localization showed a checkered staining pattern in KO proximal tubules compared to WT linear pattern at baseline. Pt levels were similar 24h post CIS treatment, but decreased significantly more 72h post treatment in KO compared to WT.
Conclusion
We conclude loss of NHERF1 results in increased levels of GSSG, upregulation of genes involved in GSH synthesis in oxidizing conditions, and lower GGT activity that sensitize the kidney to increases in oxidative stress induced by CIS nephrotoxicity. While NHERF1 loss does not affect initial Pt uptake, the aberrant CCBL1 localization in KO proximal tubules suggests a role for enhanced production of toxic CIS metabolite.
Funding
- Veterans Affairs Support