Expression of Ceramide Synthases in Mice and Their Roles in Regulating Acyl-Chain Sphingolipids: A Framework for Baseline Levels and Future Implications in Aging and Disease.

Sphingolipids are an important class of lipids present in all eukaryotic cells that regulate critical cellular processes. Disturbances in sphingolipid homeostasis have been linked to several diseases in humans. Ceramides are central in sphingolipid metabolism and are largely synthesized by six ceramide synthase (CerS) isoforms (CerS1-6), each with a preference for different fatty acyl chain lengths. Although the tissue distribution of CerS mRNA expression in humans and the roles of CerS isoforms in synthesizing ceramides with different acyl chain lengths are known, it is unknown how CerS expression dictates ceramides and downstream metabolites within tissues. In this study, we analyzed sphingolipid levels and CerS mRNA expression in 3-month-old C57BL/6J mouse brain, heart, kidney, liver, lung, and skeletal muscle. The results showed that CerS expression and sphingolipid species abundance varied by tissue and that CerS expression was a predictor of ceramide species within tissues. Interestingly, although CerS expression was not predictive of complex sphingolipid species within all tissues, composite scores for CerSs contributions to total sphingolipids measured in each tissue correlated to CerS expression. Lastly, we determined that the most abundant ceramide species in mouse tissues aligned with CerS mRNA expression in corresponding human tissues (based on chain length preference), suggesting that mice are relevant preclinical models for ceramide and sphingolipid research. SIGNIFICANCE STATEMENT: The current study demonstrates that ceramide synthase (CerS) expression in specific tissues correlates not only with ceramide species but contributes to the generation of complex sphingolipids as well. As many of the CerSs and/or specific ceramide species have been implicated in disease, these studies suggest the potential for CerSs as therapeutic targets and the use of sphingolipid species as diagnostics in specific tissues.

Mutant and curli-producing E. coli enhance the disease phenotype in a hSOD1-G93A mouse model of ALS.

The gut microbiome is a potential non-genetic contributing factor for Amyotrophic Lateral Sclerosis. Differences in gut microbial communities have been detected between ALS subjects and healthy controls, including an increase in Escherichia coli in ALS subjects. E. coli and other gram-negative bacteria produce curli proteins, which are functional bacterial amyloids. We examined whether long-term curli overexposure in the gut can exacerbate the development and progression of ALS. We utilized the slow-developing hSOD1-G93A mouse model of ALS with their C57BL/6J WT littermate controls, including males and females, with a total of 91 animals. These mice were on a normal chow diet and fed curli-producing or curli-nonproducing (mutant) E. coli in applesauce (vehicle) 3 times/week, from 1 through 7 months of age. Male hSOD1 mice demonstrated gradual slowing in running speed month 4 onwards, while females exhibited no signs of locomotive impairment even at 7 months of age. Around the same time, male hSOD1 mice showed a gradual increase in frequency of peripheral CD19+ B cells. Among the male hSOD1 group, chronic gut exposure to curli-producing E. coli led to significant shifts in α- and ß-diversities. Curli-exposed males showed suppression of immune responses in circulation, but an increase in markers of inflammation, autophagy and protein turnover in skeletal muscle. Some of these markers were also changed in mutant E. coli-exposed mice, including astrogliosis in the brainstem and demyelination in the lumbar spinal cord. Overall, chronic overexposure to a commensal bacteria like E. coli led to distant organ pathology in our model, without the presence of a leaky gut at 6 months. Mechanisms underlying gut-distant organ communication are of tremendous interest to all disciplines.

Lung cancer-kidney cross talk induces kidney injury, interstitial fibrosis, and enhances cisplatin-induced nephrotoxicity.

Patients with cancer represent a unique patient population with increased susceptibility to kidney disease. Drug-induced acute kidney injury (AKI) in patients with cancer is a common problem. Cisplatin is a highly effective treatment used in many solid-organ cancers and causes AKI in 30% of patients, increasing the risk of chronic kidney disease development. Most preclinical cisplatin toxicity studies have been completed in mice without cancer. We believe that the physiology of patients with cancer is not adequately represented in preclinical models, and the objective of this study was to determine how lung cancer will alter the nephrotoxicity of cisplatin. A genetically engineered mouse model and a syngeneic xenograft model of lung cancer were used. Mice were divided into the following four groups: 1) noncancer/vehicle, 2) noncancer/cisplatin, 3) cancer/vehicle, and 4) cancer/cisplatin. Mice were administered cisplatin via intraperitoneal injection once a week for 4 wk. Animals were euthanized 72 h following their final cisplatin injection. Mice with lung cancer had increased renal toxicity, injury, and fibrosis following repeated low doses of cisplatin. In addition, lung cancer alone induced kidney injury and fibrosis in the kidney before cisplatin treatment. In conclusion, this is the first study that we are aware of that assesses the impact of cancer on the kidney in conjunction with the nephrotoxicity of cisplatin. We believe that cancer is providing the first hit to the kidney and the subsequent damage from repeated doses of cisplatin becomes unsurmountable, leading to AKI and progression to chronic kidney disease.NEW & NOTEWORTHY Patients with cancer have impaired kidney function and increased susceptibility to nephrotoxic agents. Cisplatin is a commonly used chemotherapeutic with nephrotoxicity as the dose-limiting side effect. Cisplatin nephrotoxicity is almost exclusively studied in mice without cancer. Our current preclinical models do not adequately represent the complexity of patients with cancer. This study demonstrates increased renal toxicity, injury, and fibrosis in mice with lung cancer, which is exacerbated with cisplatin treatment. These results highlight the necessity of using preclinical models that more accurately capture the altered physiology of patients with cancer treated with cisplatin.

Modeling Cisplatin-Induced Kidney Injury to Increase Translational Potential.

BACKGROUND: Cisplatin-induced kidney injury is a major challenge hindering treatment of cancer patients. Thirty percent of patients treated with cisplatin develop acute kidney injury (AKI). Even patients that do not develop AKI are at risk for long-term decline in renal function and development of chronic kidney disease. Despite researchers' best efforts, no therapeutic agents to treat or prevent cisplatin-induced kidney injury have made it past phase 2 clinical trials. SUMMARY: Modeling cisplatin-induced kidney injury in rodents has primarily been done using a single, high-dose model of injury. Newer models of injury have utilized repeated, low, or intermediate doses of cisplatin to incorporate the study of maladaptive repair processes following a renal insult. We believe that utilization of all these models is important to understand and treat the diverse types of cisplatin-induced kidney injury patients develop in the clinic. Incorporating comorbidities such as cancer and development of large animal models is also vital to increasing the human relevance of our studies. KEY MESSAGES: Utilizing multiple dosing models of cisplatin-induced kidney injury, including relevant comorbidities and biological variables, and development of large animal models will increase the translational potential of preclinical studies.

F4/80hi Resident Macrophages Contribute to Cisplatin-Induced Renal Fibrosis.

Background: Cisplatin-induced kidney injury remains a major obstacle in utilizing cisplatin as a chemotherapeutic for solid-organ cancers. Thirty percent of patients treated with cisplatin develop acute kidney injury (AKI), and even patients who do not develop AKI are at risk for long-term declines in kidney function and development of chronic kidney disease (CKD). Modeling cisplatin-induced kidney injury in mice has revealed that repeated low doses of cisplatin lead to development of kidney fibrosis. This model can be used to examine AKI-to-CKD transition processes. Macrophages play a role in some of these processes, including immune response, wound healing, and tissue remodeling. Depleting macrophage populations in the kidney reduced fibrosis development in other models of renal fibrosis. Methods: We used either C57BL/6 mice with a Ccr2 genetic knockout or liposome encapsulated clodronate (Clodrosome) to deplete macrophage populations during repeated 9 mg/kg cisplatin treatments. We assessed how immune cell populations were altered in the blood and kidney of these mice and how these alterations affected development of renal fibrosis and kidney injury. Results: We found that Clodrosome treatment decreased collagen deposition, myofibroblast accumulation, and inflammatory cytokine production, whereas Ccr2 genetic knockout had no effect on these markers after cisplatin treatment. Additionally, Ccr2-/- mice had decreased levels of F4/80lo infiltrating macrophages in the kidney after cisplatin treatments, but Clodrosome treatment depleted F4/80hi resident and CD206+ M2 macrophages. Conclusions: These data suggest that Clodrosome depletion of F4/80hi and M2 macrophages in the kidney attenuates development of renal fibrosis after repeated low doses of cisplatin.

Pharmacological inhibitors of autophagy have opposite effects in acute and chronic cisplatin-induced kidney injury.

The nephrotoxicity of cisplatin remains a major hurdle in the field of oncology. Thirty percent of patients treated with cisplatin develop acute kidney injury, and all patients are at risk for long-term impacts on kidney function. There are currently no Federal Drug Administration-approved agents to prevent or treat cisplatin-induced kidney injury. The dosing regimen used in preclinical models of nephrotoxicity may impact the success of therapeutic candidates in clinical trials. Here, we demonstrated that pharmacological inhibitors of autophagy have opposite effects when used as interventions in two different models of cisplatin-induced kidney injury. Eight-week-old male C57BL/6 mice were treated with either one dose of 20 mg/kg cisplatin or weekly doses of 9 mg/kg cisplatin for 4 wk or until body weight loss exceeded 30%. Concurrently, mice were administered multiple doses of 60 mg/kg chloroquine or 15 mg/kg 3-methyladenine attempting to globally inhibit autophagy. Mice that received a single high dose of cisplatin had worsened kidney function, inflammation, and cell death with the addition of chloroquine. 3-Methlyadenine did not impact the development of acute kidney injury in this model. In contrast, mice that received repeated low doses of cisplatin showed improved kidney function, reduced inflammation, and reduced fibrosis when treated with either chloroquine or 3-methyladenine. This study highlights how therapeutic candidates can have drastically different effects on the development of cisplatin-induced kidney injury depending on the dosing model used. This emphasizes the importance of choosing the appropriate model of injury for preclinical studies.NEW & NOTEWORTHY This study examined how inhibition of autophagy has opposite effects on the development of acute and chronic kidney injury. Autophagy inhibition exacerbated the development of acute kidney injury following a single high dose of cisplatin but prevented the development of injury and fibrosis following repeated low doses of cisplatin.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

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.

Potential Therapeutic Targets for Cisplatin-Induced Kidney Injury: Lessons from Other Models of AKI and Fibrosis.

The effectiveness of cisplatin, a mainstay in the treatment of many solid organ cancers, is hindered by dose-limiting nephrotoxicity. Cisplatin causes AKI in 30% of patients. Patients who do not develop AKI by clinical standards during treatment are still at risk for long-term decline in kidney function and the development of CKD. The connection between AKI and CKD has become increasingly studied, with renal fibrosis a hallmark of CKD development. To prevent both the short- and long-term effects of cisplatin, researchers must use models that reflect both types of pathology. Although a lot is known about cisplatin-induced AKI, very little is known about the mechanisms by which repeated low levels of cisplatin lead to fibrosis development. In this review, strategies used in various rodent models to prevent kidney injury, its progression to fibrosis, or both, are examined to gain mechanistic insights and identify potential therapeutic targets for cisplatin-induced kidney pathologies. Reviewing the results from these models highlights the diverse and highly complex role of cell death, cell senescence, endoplasmic reticulum stress, autophagy, and immune cell activation in acute and chronic kidney injuries. The use of several models of kidney injury is needed for development of agents that will prevent all aspects of cisplatin-induced kidney injury.

C57BL/6 mice require a higher dose of cisplatin to induce renal fibrosis and CCL2 correlates with cisplatin-induced kidney injury.

C57BL/6 mice are one of the most commonly used mouse strains in research, especially in kidney injury studies. However, C57BL/6 mice are resistant to chronic kidney disease-associated pathologies, particularly the development of glomerulosclerosis and interstitial fibrosis. Our laboratory and others developed a more clinically relevant dosing regimen of cisplatin (7 mg/kg cisplatin once a week for 4 wk and mice euthanized at day 24) that leads to the development of progressive kidney fibrosis in FVB/n mice. However, we found that treating C57BL/6 mice with this same dosing regimen does not result in kidney fibrosis. In this study, we demonstrated that increasing the dose of cisplatin to 9 mg/kg once a week for 4 wk is sufficient to consistently induce fibrosis in C57BL/6 mice while maintaining animal survival. In addition, we present that cohorts of C57BL/6 mice purchased from Jackson 1 yr apart and mice bred in-house display variability in renal outcomes following repeated low-dose cisplatin treatment. Indepth analyses of this intra-animal variability revealed C-C motif chemokine ligand 2 as a marker of cisplatin-induced kidney injury through correlation studies. In addition, significant immune cell infiltration was observed in the kidney after four doses of 9 mg/kg cisplatin, contrary to what has been previously reported. These results indicate that multiple strains of mice can be used with our repeated low-dose cisplatin model with dose optimization. Results also indicate that littermate control mice should be used with this model to account for population variability.