Role of Glycosphingolipids in Kidney Disease in Diabetes and Obesity

Obesity and diabetes continue to be leading causes of chronic kidney disease (CKD). In our models of diabetes and obesity, we have found increased renal glycosphingolipids which are associated with impaired mitochondrial function, increased inflammatory T cells (Th17 cells), and progression of renal disease. Hypothesis: We will test the hypothesis that increases in glucosylceramide composition play an important role in the pathogenesis and progression of kidney disease in obesity and diabetes. We propose that these lipids mediate impaired mitochondrial function and inflammation, which are important mediators of kidney disease. In Specific Aim 1, we will test the hypothesis that inhibition of glucosylceramide synthesis by employing a) podocyte or b) renal tubule specific inducible inhibition of glucosylceramide synthase (UGCG: UDP-Glucose Ceramide Glucosyltransferase) will prevent the progression of kidney disease in mouse models of A) obesity and B) diabetes. We will determine bulk lipid composition by LC-MS/MS and glomerular and tubular lipid alterations with fluoMALDI and CARS-SRS. We will determine and localize glomerular and tubular cell diversity, cell state, and gene regulation by multiomic interrogation with snRNA-seq and ATAC-seq integrated with spatial transcriptomics. In Specific Aim 2, we will test the hypothesis that reductions glucosylceramide levels will improve mitochondrial function (oxidative phosphorylation and/or fatty acid oxidation rate) and dynamics (fusion/fission and/or mitophagy) in mouse models of obesity and diabetes. We will determine mitochondrial function by measurement of oxidative phosphorylation, extracellular acidification, and fatty acid oxidation. We will determine ultrastructural changes in mitochondria by superresolution fluorescence microscopy and electron microscopy (TEM and SEM). We will determine mitochondrial fusion/fission by utilizing PhAMexcised mice and mitophagy by utilizing mito-QC mice. In Specific Aim 3, we will test the hypothesis that inhibition of proinflammatory TH17 cell-specific glucosylceramide synthase will decrease inflammation and result in improved kidney function in mouse models of obesity and diabetes. We will determine proinflammatory immune cells including Th17 cells by high- dimensional spectral flow cytometry.