Glycosphingolipid metabolic dysfunction in lupus nephritis.
The long-term goal of this project is to identify and target key molecules leading to reduction of renal damage to slow or prevent the progression of nephritis in lupus. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by increased production of autoantibodies, subsequent deposition of immune complexes in target tissues, inflammation and tissue damage. The most severe complication of lupus is nephritis, which affects up to two-thirds of lupus patients and is associated with high morbidity and mortality. We recently made the novel observation that glycosphingolipid (GSL) metabolism, which is dysfunctional in several kidney diseases, is dysfunctional in mice and humans with lupus nephritis (LN) compared to controls. Specifically, our preliminary data suggest that elevated lactosylceramide (LacCer) levels in the kidney are a result of increased neuraminidase (NEU)-mediated catabolism of gangliosides in a mouse model of human lupus nephritis. We further show that increased NEU activity occurs prior to overt nephritis and the presence of urinary NEU1 may be an earlier or predictive clinical marker of nephritis. Of translational significance, we demonstrated intense LacCer staining in the mesangial region of glomeruli in renal biopsies and elevated LacCer and NEU1 levels in urine samples from human LN patients compared to controls. The cell types and molecular mechanisms responsible for the GSL dysfunction are unknown.
We hypothesize that GSL levels are elevated in mesangial cells due to increased expression/activity of NEUs, contribute to pathogenesis and urinary NEU1 and LacCer are novel, predictive markers of lupus nephritis. To address this hypothesis, we propose two specific aims. Specific Aim 1: Identify the cell types and mechanisms involved in LacCer elevation in lupus mice. Two subaims are proposed. Subaim 1.1- Identify specific cell types expressing increased NEU levels/activity and elevated GSL levels by coupling quantitative measures of GSL levels (using the Lipidomics core facility) and NEU expression/activity levels by western immunoblot/activity assays in total kidneys and in sorted cell types with qualitative measures of GSL levels (LacCer and gangliosides) in specific structures and cell-types in renal tissue sections. Subaim 1.2- Demonstrate that NEU expression/activity drives GSL elevation in renal cells (MCs, endothelial cells, podocytes) and impacts cytokine production. In vivo experiments in the MRL/lpr lupus mouse model will be performed in the absence or presence of the NEU inhibitor Oseltamivir and LacCer levels. In vitro experiments will be performed on MCs (and/or endothelial cells and podocytes) transfected with NEU shRNAs or treated with Oseltamivir and LacCer levels. LacCer levels will be quantified by the Lipidomics core facility for both the in vivo and in vitro experiments. Specific Aim 2: Demonstrate that LacCer and NEU1 levels are elevated prior to urinary albumin and development of nephritis. For this aim we will measure LacCer in longitudinal urine samples from patients in the SLE in Gullah Heath (SLEIGH) cohort at MUSC using the Lipidomics core facility and NEU1 protein levels by western immunoblot. This decade-long longitudinal cohort includes lupus patients without and with nephritis and unaffected controls and paired urine samples. Many lupus patients without nephritis upon enrollment subsequently developed nephritis during the study. Therefore, we can determine when urine NEU1 and/or LacCer levels can be detected in these patients with respect to proteinuria and nephritis development.
Successful completion of this project is critical for competitive VA Merit and R01 submissions planned for March 2015 and November 2014 or February 2015, respectively. The Lipidomics pilot project will allow us to perform targeted in vitro assays to identify specific renal cells, pathway(s) and mechanisms that lead to renal GSL elevation in LN. Specifically, Aim 1 is designed to identify the cell type and mechanism of GSL dysfunction and demonstrate a direct relationship between increased NEU expression/activity and GSL dysfunction. This will provide a focus for proposed molecular mechanisms and demonstrate preliminary efficacy that GSL dysfunction is a pathogenic mechanism and not just a marker of disease. Aim 2 will demonstrate clinical relevance and further support that GSL dysfunction is pathogenic, as well as serve as a marker that could be further developed for clinical use. The aims for the R01/VA Merit applications will be designed to identify the molecular mechanisms by which elevated GSL metabolism affects downstream cellular functions and progression of nephritis. Three FDA approved drugs are available that inhibit enzymes involved in GSL metabolism and are currently being used/tested in the treatment of other diseases. Therefore, our studies could support a rapid repurposing of these drugs for the treatment of LN.
This project is a multi disciplinary collaboration between Dr. Tammy Nowling (PI) in the Dept. of Medicine, an expert in molecular immunology, Dr. Ashely Cowart, expert in the biochemistry and signaling of bioactive lipids, Dr. Rick Drake, expert in MALDI-IMS imaging and Dr. Jim Oates, Rheumatologist and expert in lupus nephritis. In addition, Dr. Leah Siskind, expert in GSL biochemistry in kidney, will serve as a consultant for the cell culture experiments. Dr. Drake, Dr. Oates and Dr. Siskind collaborated with Dr. Nowling in collecting the preliminary data.