Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury.

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.

The 5-hydroxytryptamine receptor 1F stimulates mitochondrial biogenesis and angiogenesis in endothelial cells.

A hallmark of acute kidney injury (AKI) is vascular rarefication and mitochondrial dysfunction. Promoting vascular recovery following AKI could facilitate kidney repair as the vasculature is responsible for oxygen and nutrient delivery to extravascular tissues. Little is known about mitochondrial biogenesis (MB) in endothelial cells, and the role of 5-HT1F receptor signaling in MB has only been studied in epithelial cells. Our laboratory has shown that stimulating MB through the 5-HT1F receptor promotes recovery from AKI and that 5-HT1F receptor knockout mice have decreased MB and poor renal recovery. We hypothesized that the 5-HT1F receptor plays a role in vascular homeostasis and mediates MB in renal endothelial cells. 5-HT1F receptor knockout mice had decreased renal vascular content, as evidenced by decreased CD31+ endothelial cells and αSMA+ vessels. Human glomerular endothelial cells (HEC) and mouse glomerular endothelial cells (MEC) expressed the 5-HT1F receptor. Treatment of HEC and MEC with 5-HT1F receptor agonists LY344864 or lasmiditan (0-500 nM) induced MB as evidenced by maximal mitochondrial respiration, a marker of MB. HEC and MEC treated with lasmiditan or LY344864 also had increased nuclear- and mitochondrial-encoded proteins (PGC1α, COX-1, and VDAC), and mitochondrial number, confirming MB. Treatment of HEC with LY344864 or lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role for 5-HT1F receptor stimulation in angiogenic pathways. We propose that stimulation of 5-HT1F receptor is involved in MB in endothelial cells and that treatment with 5-HT1F receptor agonists could restore stimulate repair and recovery following kidney injury.

Loss of the Na+/H+ Exchange Regulatory Factor 1 Increases Susceptibility to Cisplatin-Induced Acute Kidney Injury.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase-associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.

Proximal Tubule ß 2-Adrenergic Receptor Mediates Formoterol-Induced Recovery of Mitochondrial and Renal Function after Ischemia-Reperfusion Injury.

Acute kidney injury (AKI) is the rapid loss of renal function after an insult, and renal proximal tubule cells (RPTCs) are central to the pathogenesis of AKI. The ß 2-adrenergic receptor (ß 2AR) agonist formoterol accelerates the recovery of renal function in mice after ischemia-reperfusion injury (IRI) with associated rescue of mitochondrial proteins; however, the cell type responsible for this recovery remains unknown. The role of RPTCs in formoterol-induced recovery of renal function was assessed in a proximal tubule-specific knockout of the ß 2AR (γGT-Cre:ADRB2Flox/Flox). These mice and wild-type controls (ADRB2Flox/Flox) were subjected to renal IRI, followed by once-daily dosing of formoterol beginning 24 hours post-IRI and euthanized at 144 hours. Compared with ADRB2Flox/Flox mice, γGT-Cre:ADRB2Flox/Flox mice had decreased renal cortical mRNA expression of the ß 2AR. After IRI, formoterol treatment restored renal function in ADRB2Flox/Flox but not γGT-Cre:ADRB2Flox/Flox mice as measured by serum creatinine, histopathology, and expression of kidney injury marker-1 (KIM-1). Formoterol-treated ADRB2Flox/Flox mice exhibited recovery of mitochondrial proteins and DNA copy number, whereas γGT-Cre:ADRB2Flox/Flox mice treated with formoterol did not. Analysis of mitochondrial morphology by transmission electron microscopy demonstrated that formoterol increased mitochondrial number and density in ADRB2Flox/Flox mice but not in γGT-Cre:ADRB2Flox/Flox mice. These data demonstrate that proximal tubule ß 2AR regulates renal mitochondrial homeostasis. Formoterol accelerates the recovery of renal function after AKI by activating proximal tubule ß 2AR to induce mitochondrial biogenesis and demonstrates the overall requirement of RPTCs in renal recovery.

Moderate aging does not exacerbate cisplatin-induced kidney injury or fibrosis despite altered inflammatory cytokine expression and immune cell infiltration.

Aging is a risk factor for certain forms of kidney injury due to normal physiological changes, but the role of aging in cisplatin-induced kidney injury is not well defined in humans or animal models of the disease. To improve on current knowledge in this field, we treated 8- and 40-wk-old FVB/n mice with one high dose of cisplatin as a model of acute kidney injury or with repeated low doses of cisplatin (7 mg/kg cisplatin once a week for 4 wk) as a clinically relevant model of chronic kidney disease to determine if aging exacerbates cisplatin-induced kidney injury. Levels of acute kidney injury were comparable in 8- and 40-wk-old mice. In 40-wk-old mice, fibrotic markers were elevated basally, but treatment with cisplatin did not exacerbate fibrosis. We concluded that this may be the result of a decreased inflammatory response in 40-wk-old cisplatin-treated mice compared with 8-wk-old mice. Despite a decreased inflammatory response, the level of immune cell infiltration was greater in 40-wk-old cisplatin-treated mice than 8-wk-old mice. Our data highlight the importance of examining age as a risk factor for cisplatin-induced kidney injury.

The role of sphingolipids in acute kidney injury.

Acute kidney injury (AKI) is most simply defined as the rapid loss of kidney function in a matter of hours to days. AKI can manifest in a number of ways including pre-renal, post-renal, or intrinsic AKI. During acute kidney injury, multiple pathogenic processes are activated including inflammation, cell death, and the generation of reactive oxygen species, just to name a few. Sphingolipids are known to play a role in a number of the pathogenic pathways involved in the pathogenesis of many types of AKI, which suggests a role for sphingolipids in AKI. This short review will discuss the evidence for a role for sphingolipids in AKI.

Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. J. Lipid Res 2017. 58: 1439-1452.

Common cytotoxic chemotherapeutics induce epithelial-mesenchymal transition (EMT) downstream of ER stress.

Endoplasmic reticulum (ER) in eukaryotes is a main organelle involved in a wide variety of functions including calcium storage, lipid biosynthesis, protein folding and protein transport. Disruption of ER homeostasis leads to ER stress and activation of the unfolded protein response (UPR). We and others have previously found that ER stress induces EMT in different cellular systems. Induction of ER stress with chemical modulators of ER homeostasis was sufficient to activate an EMT-like state in all cellular systems tested. Here, we provide evidence for the first time demonstrating that ER stress induces EMT that is neither cancer cell specific nor cell-type specific. In addition, we observed that chemotherapeutic drugs commonly used to treat patients also activate ER stress that is concomitant with activation of an EMT-like state. Interestingly, we find that following removal of ER stress, partial EMT characteristics still persist indicating that ER stress induced EMT is a long-term effect. Induction of mesenchymal characteristics, following chemotherapeutics treatment may be involved in providing cancer stemness and invasiveness in the cellular system. Interestingly, we find that mice treated with cisplatin have elevated level of ER stress and EMT markers in multiple tissues including lung, liver and kidneys. Furthermore, increased ER stress, as demonstrated by increased Bip, Chop, PDI, Ero1α and IRE1, and EMT, as demonstrated by increased Vimentin and Snail, is a hallmark of primary lung adenocarcinoma samples from patients. These observations have potential clinical relevance because overexpression of ER stress and EMT markers might contribute to chemoresistance and poor survival of lung adenocarcinoma patients.

Repeated administration of low-dose cisplatin in mice induces fibrosis.

Cisplatin, a chemotherapeutic used for the treatment of solid cancers, has nephrotoxic side effects leading to acute kidney injury (AKI). Cisplatin cannot be given to patients that have comorbidities that predispose them to an increased risk for AKI. Even without these comorbidities, 30% of patients administered cisplatin will develop kidney injury, requiring the oncologist to withhold or reduce the next dose, leading to a less effective therapeutic regimen. Although recovery can occur after one episode of cisplatin-induced AKI, longitudinal studies have indicated that multiple episodes of AKI lead to the development of chronic kidney disease, an irreversible disease with no current treatment. The standard mouse model of cisplatin-induced AKI consists of one high dose of cisplatin (>20 mg/kg) that is lethal to the animal 3 days later. This model does not accurately reflect the dosing regimen patients receive nor does it allow for the long-term study of kidney function and biology. We have developed a repeated dosing model whereby cisplatin is given once a week for 4 wk. Comparison of the repeated dosing model with the standard dosing model demonstrated that inflammatory cytokines and chemokines were induced in the repeated dosing model, but levels of cell death were lower in the repeated dosing model. The repeated dosing model had increased levels of fibrotic markers (fibronectin, transforming growth factor-ß, and α-smooth muscle actin) and interstitial fibrosis. These data indicate that the repeated dosing model can be used to study the AKI to chronic kidney disease progression as well as the mechanisms of this progression.

Suramin protects from cisplatin-induced acute kidney injury.

Cisplatin, a commonly used cancer chemotherapeutic, has a dose-limiting side effect of nephrotoxicity. Approximately 30% of patients administered cisplatin suffer from kidney injury, and there are limited treatment options for the treatment of cisplatin-induced kidney injury. Suramin, which is Federal Drug Administration-approved for the treatment of trypanosomiasis, improves kidney function after various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin-induced kidney injury. Suramin treatment before cisplatin administration reduced cisplatin-induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin-induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 h after cisplatin also improved kidney function, suggesting that the mechanism of protection is not by inhibition of tubular cisplatin uptake or its metabolism to nephrotoxic species. If suramin is to be used in the context of cancer, then it cannot prevent cisplatin-induced cytotoxicity of cancer cells. Suramin did not alter the dose-response curve of cisplatin in lung adenocarcinoma cells in vitro. In addition, suramin pretreatment of mice harboring lung adenocarcinomas did not alter the initial cytotoxic effects of cisplatin (DNA damage and apoptosis) on tumor cells. These results provide evidence that suramin has potential as a renoprotective agent for the treatment/prevention of cisplatin-induced acute kidney injury and justify future long-term preclinical studies using cotreatment of suramin and cisplatin in mouse models of cancer.

Targeting of BRAF resistant melanoma via extracellular matrix metalloproteinase inducer receptor.

BACKGROUND: The BRAF inhibitor vemurafenib (PLX) has shown promise in treating metastatic melanoma, but most patients develop resistance to treatment after 6 mo. We identified a transmembrane protein, extracellular matrix metalloproteinase inducer (EMMPRIN) as a cell surface receptor highly expressed by PLX-resistant melanoma. Using an S100A9 ligand, we created an EMMPRIN targeted probe and liposome that binds to melanoma cells in vivo, thus designing a novel drug delivery vehicle. METHODS: PLX-resistant cells were established through continuous treatment with PLX-4032 over the course of 1 y. Both PLX-resistant and sensitive melanoma cell lines were evaluated for the expression of unique cell surface proteins, which identified EMMPRIN as an overexpressed protein in PLX0-resistant cells and S100A9 is a ligand for EMMPRIN. To design a probe for EMMPRIN, S100A9 ligand was conjugated to a CF-750 near-infrared (NIR) dye. EMMPRIN targeted liposomes were created to encapsulate CF-750 NIR dye. Liposomes were characterized by scanning electron microscopy, flow cytometry, and in vivo analysis. A2058PLX and A2058 cells were subcutaneously injected into athymic mice. S100A9 liposomes were intravenously injected and tumor accumulation was evaluated using NIR fluorescent imaging. RESULTS: Western blot and flow cytometry demonstrated that PLX sensitive and resistant A2058 and A375 melanoma cells highly express EMMPRIN. S100A9 liposomes were 200 nm diameter and uniformly sized. Flow cytometry demonstrated 100X more intracellular dye uptake by A2058 cells treated with S100A9 liposomes compared with untargeted liposomes. In vivo accumulation of S100A9 liposomes within subcutaneous A2058 and A2058PLX tumors was observed from 6-48 h, with A2058PLX accumulating significantly higher levels (P = 0.001626). CONCLUSIONS: EMMPRIN-targeted liposomes via an S100A9 ligand are a novel, targeted delivery system which could provide improved EMMPRIN specific drug delivery to a tumor.