(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

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.