Date on Master's Thesis/Doctoral Dissertation

5-2018

Document Type

Master's Thesis

Degree Name

M.S.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, MS

Committee Chair

Lederer, Eleanor

Committee Co-Chair (if applicable)

Barati, Michelle

Committee Member

Barati, Michelle

Committee Member

Brier, Michael

Committee Member

Merchant, Michael

Committee Member

Siskind, Leah

Author's Keywords

cisplatin nephrotoxicity; acute tubular necrosis; ER stress; apoptosis

Abstract

Background. Acute kidney injury (AKI), an abrupt loss of kidney function which carries a high mortality and confers an increased risk of chronic kidney disease, develops in 30% of patients who receive cisplatin, a widely used chemotherapeutic agent [1], [2], [3]. The sodium hydrogen exchange regulatory factor isoform 1 (NHERF1) is a scaffolding protein that anchors multiple membrane proteins, in renal proximal tubules [4]. NHERF-1 deficient mice have aberrant localization of BBM proteins in intracellular compartments [5]. The investigators have recently demonstrated NHERF1 deficient proximal tubule cells have an underlying increase in PERK phosphorylation and that NHERF1 deficiency results in altered mitochondrial protein expression and function. Since mitochondria and endoplasmic reticulum (ER) are physically and functionally connected, the purpose of this study was to test the hypothesis that NHERF1 loss increases susceptibility to AKI through an underlying ER stress. Methods. Male and female WT C57BL/6J and NHERF1 KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP) and euthanized after 72 hours. Blood was collected for blood urea nitrogen (BUN) levels. Kidneys were harvested for histology (hematoxylin and eosin; periodic acid schiff), TUNEL assay, RT-qPCR of Kidney Injury Molecule-1 (KIM-1), and Western Blot for cleaved caspase 3, p-eIF2α, p58IPK, GRP94 and GRP78. Results. Cisplatin caused significantly greater severity of injury in NHERF1 KO mice compared to WT mice as demonstrated by semi-quantitative injury score and BUN levels (p=0.023). KIM-1 mRNA expression was significantly increased in both WT and NHERF1 KO cisplatin treated mice (p=0.0153) in comparison to vehicle treated mice. TUNEL assay analysis showed significant increases in both NHERF1 KO and WT cisplatin treated mice (pIPK(p=0.20) expression was decreased with cisplatin treatment in WT and NHERF1 KO mice and not different between vehicle treated WT and NHERF1 KO mice. A significant increase in GRP94 expression was seen (p=0.030) in cisplatin treated NHERF1 KO mice compared to cisplatin treated WT mice. Thus, there were no significant gender differences found between WT and NHERF1 KO mice for any of the measured parameters, except for eIF2α phosphorylation and cleavage of caspase 3. Conclusions. These data suggest that an underlying ER stress is not the mechanism of susceptibility in NHERF1 KO mice. In conclusion, this study provides a novel role for NHERF1 and susceptibility to cisplatin-induced AKI.

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