Bimodal Effect of Advanced Glycation End Products on Mesangial Cell Proliferation Is Mediated by Neutral Ceramidase Regulation and Endogenous Sphingolipids

Abstract

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 M) induced a significant increase (؉26%) of MC proliferation, whereas higher concentrations (10 M) markedly reduced it (؊24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 M)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.

Identification of a novel amidase motif in neutral ceramidase

“…It was shown that neutral CDase activity increased and ceramide level decreased after IL-1β stimulation of rat mesangial cells [19], whereas NO led to degradation of neutral CDase [20,21]. In mesangial cells, neutral CDase could also mediate the effect of advanced glycation end-products (generated during chronic hyperglycaemia) on cell proliferation [22]. In Drosophila, mutation of neutral CDase causes synaptic dysfunction with impaired vesicle fusion and trafficking [23].…”

Glomerular proliferation during early stages of diabetic nephropathy is associated with local increase of sphingosine‐1‐phosphate levels

“…HPLC assay was used to quantitate the released S1P and Sph, as described by Min et al [14] and applied in our laboratory [7]. Glomeruli pellets were resuspended in 100 ll phosphate-buffered saline (PBS) and an aliquot was kept for protein determination.…”

“…Sphingosine kinase activity was measured as described by Olivera et al [13] with slight modifications as reported [7]. Briefly, glomeruli pellets were homogenized in 300 ll of lysis/reaction buffer (20 mM Tris-pH 7.4, 20% glycerol, 1 mM b-mercaptoethanol, 1 mM EDTA, 1 mM sodium vanadate, 15 mM sodium fluoride, 40 mM glycerophophate, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM 4-deoxypyridoxine, 10 ll/ml protease inhibitors cocktail).…”

“…In this way, CDase activity may be a key step in determining the intracellular levels of Cer and Sph/S1P, thus playing a crucial role in the regulation of cell survival or death in response to external stimuli [11]. A previous study from our laboratory [7] showed that CDase modulation is implicated in the bimodal effects of advanced glycation end-products (AGE) on MC proliferation. Results showed that proliferation induced by low AGE concentrations (<1 lM) was paralleled by CDase and SphK activation and increased S1P production.…”

“…In addition, Cer is a key intermediate in the formation of biologically important sphingolipids, among which, sphingosine (Sph) and sphingosine-1-phosphate (S1P). In contrast to the growth inhibitory and pro-apoptotic effects of Cer and Sph [4], S1P accumulation has been shown to play opposing roles by promoting cell growth in various cell types including MC [5][6][7]. Therefore, the relative levels of these sphingolipid metabolites have been pointed out as a sensitive rheostat controlling growth and death of various cell types [8,9].…”