Recent progress in the field

Recent progress in the field of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has shed light on understanding the distribution of analytes in various tissues. MALDI-MSI is a powerful tool that can determine the distribution of hundreds of ionized analytes based on their molecular masses in a single measurement and enables the acquisition of multiple images as 2-dimensional (D) and 3-D ion density maps (Walch et al., 2008; Watrous et al., 2013). MALDI-MSI enables the visualization of individual molecules on tissue sections, without antibodies, staining, or complicated pretreatment steps. Thus, MALDI-MSI may be a suitable tool for investigating the distribution of analytes in kidney tissues without major processing that could interfere with relevant in vivo regulation of metabolites. MALDI-MSI requires a matrix, a small acidic aromatic molecule that absorbs Lenalidomide at the wavelength of the irradiating laser (Walch et al., 2008). By applying matrix onto the tissue sections using either sprayer or sublimation, the analytes are extracted from the tissue and form co-crystals with matrix. As the size of the co-crystals is one of the important determinants of spatial resolution, especially in the high resolution MSI analysis, particle size of matrix should be as small as possible. In addition, it is crucial to deposit matrix homogeneously across the tissue section without any migration of analytes (Collins et al., 2010). The matrix-coated slide is then submitted to very short laser pulses which allow optimal desorption and ionization of the analytes, and ionized analytes are detected and classified by the difference of mass-to-charge ratio (m/z) (Walch et al., 2008). It has been reported that MALDI-MSI can be an applicable method for the accurate quantification of nucleotides in tissue sections (Hattori et al., 2010), thus we applied Fourier transform ion cyclotron resonance (FTICR)-MALDI-MSI at high spatial resolution to localize ATP, AMP, and ADP in the kidney microstructure of wild-type (WT) and diabetic mice. For the screening of metabolites and lipids that regulate renal nucleotide levels, we performed untargeted MALDI-MSI followed by the identification by the ambient ionization interfaced with a microscope and mass spectrometry (AMM). We demonstrated that one of the sphingomyelin (SM) species may play a key role in the regulation of ATP levels and the AMPK pathway.
Material and Methods
Results
Discussion In the present study, using MALDI-MSI and AMM approaches, we show that 1) the ATP/AMP and ATP/ADP ratio are increased in the glomeruli of diabetic mice compared with WT mice, 2) SM(d18:1/16:0) is preferentially distributed in the glomeruli in normal human kidney, and 3) SM(d18:1/16:0) is increased in the glomeruli of type 1 diabetic and HFD-fed mice compared with controls. We also demonstrate by in vitro studies using mesangial cells that AMPK–PGC1α is reduced and ATP production is increased by the treatment with liposomal SM(d18:1/16:0). We have previously demonstrated that renal AMPK activity is reduced in mouse models of type 1 diabetes and obesity-related kidney disease (conditions characterized by caloric excess states), and that AMPK activation inhibits the progression of chronic kidney damage (Decleves et al., 2011; Dugan et al., 2013). We have also reported that activation of AMPK has a potent role in blocking profibrotic signaling via inhibition of high-glucose induced Smad4 nuclear translocation in cultured murine mesangial cells (Zhao et al., 2015). In addition, we have recently shown that the reduced mitochondrial function in diabetic kidneys is associated with reduction of AMPK–PGC1α signaling (Dugan et al., 2013). Although AMPK is a key player in regulating energy balance and related to many signaling pathways in the progression of DKD, little is known about the relationship of AMPK with ATP levels in vivo, due to the difficulty in measurements in tissues. In addition, as kidneys are complex organs and consist of many cell types and each cell type may differentially regulate energy balance, the evaluation of ATP/ADP and ATP/AMP ratios is difficult to accurately assess in specific compartments, such as the glomerulus. We show here that with FTICR-MALDI-MSI, there is an increase in the ATP, ATP/AMP and ATP/ADP ratio in diabetic glomeruli compared with WT glomeruli. It has been reported that, in cultured rat mesangial cells, the extracellular ATP levels are increased by high glucose, and in addition, fibrosis-related markers including fibronectin and TGF-β are significantly increased by the addition of ATP (Solini et al., 2005). ATP levels are now considered to be a promoter of inflammation (Cauwels et al., 2014), and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), which is expressed in the glomeruli and hydrolyze ATP and ADP to AMP, is reported to prevent renal inflammation in the kidney of type 1 diabetic mice (Friedman et al., 2007). Based on our results it is likely that enhanced ATP production is a characteristic of diabetic glomerular disease.