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-β, 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.

The development of nephritis is a leading cause of morbidity and mortality in lupus patients. Although the general pathophysiological progression of lupus nephritis is known, the molecular mediators and mechanisms are incompletely understood. Previously, we demonstrated that the glycosphingolipid (GSL) catabolic pathway is elevated in the kidneys of MRL/lpr lupus mice and human lupus patients with nephritis. Specifically, the activity of neuraminidase (NEU) and expression of Neu1, an enzyme in the GSL catabolic pathway is significantly increased. To better understand the role and mechanisms by which this pathway contributes to the progression of LN, we analyzed the expression and effects of NEU activity on the function of MRL/lpr lupus-prone mesangial cells (MCs). We demonstrate that NEU1 and NEU3 promote IL-6 production in MES13 MCs. Neu1 expression, NEU activity, and IL-6 production are significantly increased in stimulated primary MRL/lpr lupus-prone MCs, and blocking NEU activity inhibits IL-6 production. NEU1 and NEU3 expression overlaps IgG deposits in MCs in vitro and in renal sections from nephritic MRL/lpr mice. Together, our results suggest that NEU activity mediates IL-6 production in lupus-prone MCs possibly through an IgG-receptor complex signaling pathway.

Glycosphingolipids (GSLs) play a role in insulin resistance and diabetes, but their role in diabetic nephropathy (DN) has received limited attention. We used 9- and 17-wk-old nondiabetic db/m and diabetic db/db mice to examine the role of GSLs in DN. Cerebrosides or monoglycosylated GSLs [hexosylceramides (HexCers); glucosyl- and galactosylceramides] and lactosylceramide (LacCers) were elevated in db/db mouse kidney cortices, specifically in glomeruli, and also in urine. In our recent paper (25), we observed that the kidneys exhibited glomerular hypertrophy and proximal tubular vacuolization and increased fibrosis markers at these time points. Mesangial cells contribute to hyperglycemia-induced glomerular hypertrophy in DN. Hyperglycemic culture conditions, similar to that present in diabetes, were sufficient to elevate mesangial cell HexCers and increase markers of fibrosis, extracellular matrix proteins, and cellular hypertrophy. Inhibition of glucosylceramide synthase or lowering glucose levels decreased markers of fibrosis and extracellular matrix proteins and reversed mesangial cell hypertrophy. Hyperglycemia increased phosphorylated (p)SMAD3 and pAkt levels and reduced phosphatase and tensin homolog levels, which were reversed with glucosylceramide synthase inhibition. These data suggest that inhibition of glucosylceramide synthase reversed mesangial cell hypertrophy through decreased pAkt and pSmad3 and increased pathways responsible for protein degradation. Importantly, urinary GSL levels were higher in patients with DN compared with healthy control subjects, implicating a role for these lipids in human DN. Thus, hyperglycemia in type II diabetes leads to renal dysfunction at least in part by inducing accumulation of HexCers and LacCers in mesangial cells, resulting in fibrosis, extracellular matrix production, and hypertrophy.

Sphingolipids are a family of lipids that regulate the cell cycle, differentiation and cell death. Sphingolipids are known to play a role in the induction of apoptosis, but a role for these lipids in necroptosis is largely unknown. Necroptosis is a programmed form of cell death that, unlike apoptosis, does not require ATP. Necroptosis can be induced under a variety of conditions, including nutrient deprivation and plays a major role in ischaemia/reperfusion injury to organs. Sphingolipids play a role in ischaemia/reperfusion injury in several organs. Thus, we hypothesized that sphingolipids mediate nutrient-deprivation-induced necroptosis. To address this, we utilized mouse embryonic fibroblast (MEFs) treated with 2-deoxyglucose (2DG) and antimycin A (AA) to inhibit glycolysis and mitochondrial electron transport. 2DG/AA treatment of MEFs induced necroptosis as it was receptor- interacting protein (RIP)-1/3 kinase-dependent and caspase-independent. Ceramides, sphingosine (Sph) and sphingosine 1-phosphate (S1P) were increased following 2DG/AA treatment. Cells lacking neutral ceramidase (nCDase−/−) were protected from 2DG/AA. Although nCDase−/− cells generated ceramides following 2DG/AA treatment, they did not generate Sph or S1P. This protection was stimulus-independent as nCDase−/− cells were also protected from endoplasmic reticulum (ER) stressors [tunicamycin (TN) or thapsigargin (TG)]. nCDase−/− MEFs had higher autophagic flux and mitophagy than wild-type (WT) MEFs and inhibition of autophagy sensitized them to necroptosis. These data indicate that loss of nCDase protects cells from nutrient-deprivation-induced necroptosis via autophagy, and clearance of damaged mitochondria. Results suggest that nCDase is a mediator of necroptosis and might be a novel therapeutic target for protection from ischaemic injury.

Cancer therapeutics has seen an emergence and re-emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramide's essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre-clinical inhibitor ABT-263, this effect was augmented and production of pro-apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia.

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Pneumocystis pneumonia (PCP) is an opportunistic infection caused by the fungus Pneumocystis jirovecii. Infection with P. jirovecii can result in serious illness in patients with a weakened immune system, and can lead to death if it is not properly diagnosed and treated. Direct detection of P. jirovecii in lower respiratory tract specimens such as bronchoalveolar lavage (BAL) is preferred for rapid diagnosis, a laboratory service currently not available locally. We report here the development of a diagnostic real-time Polymerase Chain Reaction (PCR) assay using BAL specimens to detect P. jirovecii. By targeting the multi-copy mitochondrial large subunit ribosomal RNA gene (mtLSU rRNA) of P. jirovecii, assay sensitivity is increased. Primer pairs were designed to include a fluorescent reporter dye-labeled primer with a unique MultiCode® base pair isoC on the 5'end and one unlabeled primer. The performance characteristics were determined on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. The cassette contains the reagents needed to perform all of the steps including extraction, purification, amplification, and detection, plus a sample processing control. Accuracy, precision, sensitivity, specificity and stability studies were conducted to validate the assay to meet CLIA requirements. The analytical sensitivity was 89.1%, and the analytical specificity was 100%. The assay could reliably detect 200 organisms/ mL, crossing thresholds (Ct) and melt temperatures (Tm) were consistent, and no cross-reactivity was observed with other pathogens known to cause respiratory infections. The results demonstrated that these primers are specific to Pneumocystis jirovecii. The real-time PCR method using the ARIES® system allowed for rapid and sensitive detection of Pneumocystis pneumonia infections with P. jirovecii using clinical respiratory specimens.

Background: Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn), and Legionella pneumophila (Lpn) can cause both epidemic and endemic occurrences of acute respiratory disease and are responsible for up to 22% of cases of community acquired pneumonia. Due to the limited availability of FDA-approved molecular diagnostic assays, we developed and evaluated a multiplexed Real-time PCR assay for the detection of these agents in two respiratory specimen types on the Luminex ARIES ® instrument. The instrument provides for nucleic acid extraction plus PCR amplification and target detection in the same cassette. The ARIES ® instrument generates a cycle threshold value and a confirmatory melt curve value for each reaction, including results for an internal sample processing control. The limit of detection for Mpn, Cpn and Lpn, was 100 CFU/ mL, 1000 CFU/mL and 100 CFU/mL, respectively. In addition, accuracy, precision, specificity and stability studies were conducted to validate the assay for diagnostic use. Between November 2016 and June 2017, a total of 836 patient specimens were processed in our reference laboratory, with six positive Mpn and two positive Lpn. No specimens were positive for Cpn during this time period. The availability of a robust multiplex PCR assay greatly enhances the ability to rapidly diagnose infections caused by these three agents causing atypical pneumonia.