Cisplatin-induced oxidative stress stimulates renal Fas ligand shedding

Abstract

Acute kidney injury (AKI), a significant complication of cisplatin chemotherapy is associated with reactive oxygen species (ROS)-dependent renal cell death, but the cellular targets of ROS in cisplatin nephrotoxicity are not fully resolved. Here, we investigated cisplatin-induced oxidative renal damage and tested the hypothesis that ROS-dependent shedding of death activator Fas ligand (FasL) occurs in cisplatin nephropathy. We show that intraperitoneal injection of sulfobutyl ether-β-cyclodextrin (Captisol™)-solubilized cisplatin elevated the level of lipid peroxidation product malondialdehyde in mouse kidneys and urinary concentration of oxidative DNA damage biomarker 8-hydroxy-2′-deoxyguanosine. Cisplatin increased mouse kidney-to-body weight ratio and the plasma or urinary levels of predictive biomarkers of AKI, including creatinine, blood urea nitrogen, microalbumin, neutrophil gelatinase-associated lipocalin, and cystatin C. Histological analysis and dUTP nick end labeling of kidney sections indicated tubular injury and renal apoptosis, respectively in cisplatin-treated mice. Whereas the plasma concentration of soluble FasL (sFasL) was unaltered, urinary sFasL was increased ∼4-fold in cisplatin-treated mice. Real-time quantitative live-cell imaging and lactate dehydrogenase assay showed that cisplatin stimulated caspase 3/7 activation and cytotoxicity in a human proximal tubule epithelial cell line which were attenuated by inhibitors of the FasL/Fas system and poly [ADP-ribose] polymerase-1. Moreover, TEMPOL, an intracellular free radical scavenger mitigated cisplatin-induced renal oxidative stress and injury, AKI biomarker and urinary sFasL elevation, and proximal tubule cell death. Our findings indicate that cisplatin-induced oxidative stress triggers the shedding of membrane-bound FasL to sFasL in the kidney. We demonstrate that cisplatin elicits nephrotoxicity by promoting FasL/Fas-dependent oxidative renal tubular cell death.

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

“…Cisplatin and DAPT were solubilized in pharmaceutical excipient sulfobutyl ether‐β‐cyclodextrin (20% Captisol) as we have previously described . Mice were randomized into four groups (Figure ; n = 16/group) and housed in ventilated micro‐isolation cages.…”

“…The injection volume was kept at 10 μL/g body weight. On the fifth day, each mouse was weighed and placed on a new 96‐well plate inside an empty box for ~2 hours . Urine samples were collected from the wells and analyzed.…”

“…Kidney sections processed for Periodic acid–Schiff (PAS) staining were analyzed by a semi‐quantitative evaluation of tubular injury by renal pathologists at the Probetex Inc. (San Antonio, TX, USA) as we have previously described . Tubular injury scoring was based on the following scale: 0 = no apparent change; 1+ = focal: few focal areas distributed throughout the section; 2+ = infrequent: up to eight focal areas distributed throughout the section; 3+ = frequent: up to eight tubular profiles per 10× field; 4+ = very frequent: more than eight tubular profiles per 10× field.…”

“…The cell line (CRL‐2190) was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured as we have previously described …”

“…Apoptosis of HK‐2 cells was examined in real time using the CellPlayer caspase‐3/7 reagent, and the IncuCyte ZOOM live content microscopy system (Essen BioScience, Ann Arbor, MI, USA) as we have previously described . Cytotoxicity was also determined using the LDH colorimetric assay kit (Life Technologies).…”