Sphingolipids in the gut? Which are the important issues?

Glycosphingolipids and sphingomyelin (SM) are important components of the apical brush border and the Golgi and endocytic vesicles of the gut epithelium. In particular, glucosylceramide is abundant in the microvilli. Synthesis and degradation of mucosal sphingolipids and targeting of sphingolipids to distinct cell compartments during cell differentiation are thus important features of intestinal lipid metabolism. Sphingolipids are also present in the ordinary Western diet, and sphingolipid‐rich formulations of dairy, plant or yeast origin are now available for studies of their biological effects in animals and humans. Since sphingolipids account for a large part of the polar lipids in milk, their digestion and effects in the suckling infant is of particular interest. Dietary sphingolipids are slowly digested and the exposure of the whole gut to sphingolipids and their metabolites can be increased by dietary supply. Metabolites from dietary sphingolipids may have anti‐inflammatory and anticarcinogenic effects and undigested sphingolipids may be protective. Dietary sphingolipids inhibit cholesterol absorption and may have beneficial metabolic effects. Some mucosal sphingolipids have blood group A, B and H reactivity and some act as receptors for bacterial toxins and virus. Sphingolipid signaling triggered by mucosal‐bacterial interaction may be important in both the gut and the bronchi.     

Neurodegenerative Disorders: Spotlight on Sphingolipids

Neurodegenerative diseases are incurable diseases of the nervous system that lead to a progressive loss of brain areas and neuronal subtypes, which is associated with an increase in symptoms that can be linked to the affected brain areas. The key findings that appear in many neurodegenerative diseases are deposits of proteins and the damage of mitochondria, which mainly affect energy production and mitophagy. Several causative gene mutations have been identified in various neurodegenerative diseases; however, a large proportion are considered sporadic. In the last decade, studies linking lipids, and in particular sphingolipids, to neurodegenerative diseases have shown the importance of these sphingolipids in the underlying pathogenesis. Sphingolipids are bioactive lipids consisting of a sphingoid base linked to a fatty acid and a hydrophilic head group. They are involved in various cellular processes, such as cell growth, apoptosis, and autophagy, and are an essential component of the brain. In this review, we will cover key findings that demonstrate the relevance of sphingolipids in neurodegenerative diseases and will focus on neurodegeneration with brain iron accumulation and Parkinson’s disease.     

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes—including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides—were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.     

Chemical tools to investigate sphingolipid metabolism and functions

Sphingolipids comprise an important group of biomolecules, some of which have been shown to play important roles in the regulation of many cell functions. From a structural standpoint, they all share a long 2-amino-1,3-diol chain, which can be either saturated (sphinganine), hydroxylated at C4 (phytosphingosine), or unsaturated at C4 (sphingosine) as in most mammalian cells. N-acylation of sphingosine leads to ceramide, a key intermediate in sphingolipid metabolism that can be enzymatically modified at the C1-OH position to other biologically important sphingolipids, such as sphingomyelin or glycosphingolipids. In addition, both ceramide and sphingosine can be phosphorylated at C1-OH to give ceramide-1-phosphate and sphingosine-1-phosphate, respectively. To better understand the biological and biophysical roles of sphingolipids, many efforts have been made to design synthetic analogues as chemical tools able to unravel their structure-activity relationships, and to alter their cellular levels. This last approach has been thoroughly studied by the development of specific inhibitors of some key enzymes that play an important role in biosynthesis or metabolism of these intriguing lipids. With the above premises in mind, the aim of this review is to collect, in a systematic way, the recent efforts described in the literature leading to the development of new chemical entities specifically designed to achieve the above goals.     

Novelty of Sphingolipids in the Central Nervous System Physiology and Disease: Focusing on the Sphingolipid Hypothesis of Neuroinflammation and Neurodegeneration

For decades, lipids were confined to the field of structural biology and energetics as they were considered only structural constituents of cellular membranes and efficient sources of energy production. However, with advances in our understanding in lipidomics and improvements in the technological approaches, astounding discoveries have been made in exploring the role of lipids as signaling molecules, termed bioactive lipids. Among these bioactive lipids, sphingolipids have emerged as distinctive mediators of various cellular processes, ranging from cell growth and proliferation to cellular apoptosis, executing immune responses to regulating inflammation. Recent studies have made it clear that sphingolipids, their metabolic intermediates (ceramide, sphingosine-1-phosphate, and N-acetyl sphingosine), and enzyme systems (cyclooxygenases, sphingosine kinases, and sphingomyelinase) harbor diverse yet interconnected signaling pathways in the central nervous system (CNS), orchestrate CNS physiological processes, and participate in a plethora of neuroinflammatory and neurodegenerative disorders. Considering the unequivocal importance of sphingolipids in CNS, we review the recent discoveries detailing the major enzymes involved in sphingolipid metabolism (particularly sphingosine kinase 1), novel metabolic intermediates (N-acetyl sphingosine), and their complex interactions in CNS physiology, disruption of their functionality in neurodegenerative disorders, and therapeutic strategies targeting sphingolipids for improved drug approaches.     

Human Glucose Transporters in Renal Glucose Homeostasis

The kidney plays an important role in glucose homeostasis by releasing glucose into the blood stream to prevent hypoglycemia. It is also responsible for the filtration and subsequent reabsorption or excretion of glucose. As glucose is hydrophilic and soluble in water, it is unable to pass through the lipid bilayer on its own; therefore, transport takes place using carrier proteins localized to the plasma membrane. Both sodium-independent glucose transporters (GLUT proteins) and sodium-dependent glucose transporters (SGLT proteins) are expressed in kidney tissue, and mutations of the genes coding for these glucose transporters lead to renal disorders and diseases, including renal cancers. In addition, several diseases may disturb the expression and/or function of renal glucose transporters. The aim of this review is to describe the role of the kidney in glucose homeostasis and the contribution of glucose transporters in renal physiology and renal diseases.     

The quantitative analysis of sphingolipids by determination of long chain base as the trinitrobenzene sulfonic acid derivative

A spectrophotometric procedure has been developed for the determination of microquantities of sphingolipids. The assay system involved includes the methanolysis of the fatty acid moiety of the sphingolipid with boron trifluoride in methanol to yield a long chain base containing a free amino group. The long chain amine, sphingosine, is then extracted into an organic phase and reacted with aqueous trinitrobenzene sulfonic acid to yield a product with an absorption maximum at 340 mμ. Lipid peroxidation products and silicic acid do not seriously interfere in the 340 region. A wide variety of pure sphingolipids yielded equivalent optical densities per mole of sphingolipid. Sphingolipids assayed included cerebroside, cerebroside sulfate, ganglioside and sphingomyelin. Presented at the AOCS Meeting, New Orleans, April 1970.     

Are Alterations in DNA Methylation Related to CKD Development?

The modifications in genomic DNA methylation are involved in the regulation of normal and pathological cellular processes. The epigenetic regulation stimulates biological plasticity as an adaptive response to variations in environmental factors. The role of epigenetic changes is vital for the development of some diseases, including atherogenesis, cancers, and chronic kidney disease (CKD). The results of studies presented in this review have suggested that altered DNA methylation can modulate the expression of pro-inflammatory and pro-fibrotic genes, as well those essential for kidney development and function, thus stimulating renal disease progression. Abnormally increased homocysteine, hypoxia, and inflammation have been suggested to alter epigenetic regulation of gene expression in CKD. Studies of renal samples have demonstrated the relationship between variations in DNA methylation and fibrosis and variations in estimated glomerular filtration rate (eGFR) in human CKD. The unravelling of the genetic–epigenetic profile would enhance our understanding of processes underlying the development of CKD. The understanding of multifaceted relationship between DNA methylation, genes expression, and disease development and progression could improve the ability to identify individuals at risk of CKD and enable the choice of appropriate disease management.     

Extracellular Vesicles Tune the Immune System in Renal Disease: A Focus on Systemic Lupus Erythematosus,Antiphospholipid Syndrome,Thrombotic Microangiopathy and ANCA-Vasculitis

Extracellular vesicles (EV) are microparticles released in biological fluids by different cell types, both in physiological and pathological conditions. Owing to their ability to carry and transfer biomolecules, EV are mediators of cell-to-cell communication and are involved in the pathogenesis of several diseases. The ability of EV to modulate the immune system, the coagulation cascade, the angiogenetic process, and to drive endothelial dysfunction plays a crucial role in the pathophysiology of both autoimmune and renal diseases. Recent studies have demonstrated the involvement of EV in the control of renal homeostasis by acting as intercellular signaling molecules, mediators of inflammation and tissue regeneration. Moreover, circulating EV and urinary EV secreted by renal cells have been investigated as potential early biomarkers of renal injury. In the present review, we discuss the recent findings on the involvement of EV in autoimmunity and in renal intercellular communication. We focused on EV-mediated interaction between the immune system and the kidney in autoimmune diseases displaying common renal damage, such as antiphospholipid syndrome, systemic lupus erythematosus, thrombotic microangiopathy, and vasculitis. Although further studies are needed to extend our knowledge on EV in renal pathology, a deeper investigation of the impact of EV in kidney autoimmune diseases may also provide insight into renal biological processes. Furthermore, EV may represent promising biomarkers of renal diseases with potential future applications as diagnostic and therapeutic tools.     

Kidney Injuries and Evolution of Chronic Kidney Diseases Due to Neonatal Hyperoxia Exposure Based on Animal Studies

Preterm birth interrupts the development and maturation of the kidneys during the critical growth period. The kidneys can also exhibit structural defects and functional impairment due to hyperoxia, as demonstrated by various animal studies. Furthermore, hyperoxia during nephrogenesis impairs renal tubular development and induces glomerular and tubular injuries, which manifest as renal corpuscle enlargement, renal tubular necrosis, interstitial inflammation, and kidney fibrosis. Preterm birth along with hyperoxia exposure induces a pathological predisposition to chronic kidney disease. Hyperoxia-induced kidney injuries are influenced by several molecular factors, including hypoxia-inducible factor-1α and interleukin-6/Smad2/transforming growth factor-β, and Wnt/β-catenin signaling pathways; these are key to cell proliferation, tissue inflammation, and cell membrane repair. Hyperoxia-induced oxidative stress is characterized by the attenuation or the induction of multiple molecular factors associated with kidney damage. This review focuses on the molecular pathways involved in the pathogenesis of hyperoxia-induced kidney injuries to establish a framework for potential interventions.     

Ceramide and Sphingosine-1-Phosphate in Neurodegenerative Disorders and Their Potential Involvement in Therapy

Neurodegenerative disorders (ND) are progressive diseases of the nervous system, often without resolutive therapy. They are characterized by a progressive impairment and loss of specific brain regions and neuronal populations. Cellular and animal model studies have identified several molecular mechanisms that play an important role in the pathogenesis of ND. Among them are alterations of lipids, in particular sphingolipids, that play a crucial role in neurodegeneration. Overall, during ND, ceramide-dependent pro-apoptotic signalling is promoted, whereas levels of the neuroprotective spingosine-1-phosphate are reduced. Moreover, ND are characterized by alterations of the metabolism of complex sphingolipids. The finding that altered sphingolipid metabolism has a role in ND suggests that its modulation might provide a useful strategy to identify targets for possible therapies. In this review, based on the current literature, we will discuss how bioactive sphingolipids (spingosine-1-phosphate and ceramide) are involved in some ND (Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis) and their possible involvement in therapies.     

Macrophage Migration Inhibitory Factor (MIF) as a Stress Molecule in Renal Inflammation

Renal inflammation is an initial pathological process during progressive renal injury regardless of the initial cause. Macrophage migration inhibitory factor (MIF) is a truly proinflammatory stress mediator that is highly expressed in a variety of both inflammatory cells and intrinsic kidney cells. MIF is released from the diseased kidney immediately upon stimulation to trigger renal inflammation by activating macrophages and T cells, and promoting the production of proinflammatory cytokines, chemokines, and stress molecules via signaling pathways involving the CD74/CD44 and chemokine receptors CXCR2, CXCR4, and CXCR7 signaling. In addition, MIF can function as a stress molecule to counter-regulate the immunosuppressive effect of glucocorticoid in renal inflammation. Given the critical position of MIF in the upstream inflammatory cascade, this review focuses on the regulatory role and molecular mechanisms of MIF in kidney diseases. The therapeutic potential of targeting MIF signaling to treat kidney diseases is also discussed.     

Role of Klotho in Hyperglycemia: Its Levels and Effects on Fibroblast Growth Factor Receptors,Glycolysis, and Glomerular Filtration

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.     

Role of Arachidonic Acid and Its Metabolites in the Biological and Clinical Manifestations of Idiopathic Nephrotic Syndrome

Studies concerning the role of arachidonic acid (AA) and its metabolites in kidney disease are scarce, and this applies in particular to idiopathic nephrotic syndrome (INS). INS is one of the most frequent glomerular diseases in childhood; it is characterized by T-lymphocyte dysfunction, alterations of pro- and anti-coagulant factor levels, and increased platelet count and aggregation, leading to thrombophilia. AA and its metabolites are involved in several biological processes. Herein, we describe the main fields where they may play a significant role, particularly as it pertains to their effects on the kidney and the mechanisms underlying INS. AA and its metabolites influence cell membrane fluidity and permeability, modulate platelet activity and coagulation, regulate lymphocyte activity and inflammation, preserve the permeability of the glomerular barrier, influence podocyte physiology, and play a role in renal fibrosis. We also provide suggestions regarding dietary measures that are able to prevent an imbalance between arachidonic acid and its parental compound linoleic acid, in order to counteract the inflammatory state which characterizes numerous kidney diseases. On this basis, studies of AA in kidney disease appear as an important field to explore, with possible relevant results at the biological, dietary, and pharmacological level, in the final perspective for AA to modulate INS clinical manifestations.     

Stereoselective Synthesis of Novel Sphingoid Bases Utilized for Exploring the Secrets of Sphinx

Sphingolipids are ubiquitous in eukaryotic plasma membranes and play major roles in human and animal physiology and disease. This class of lipids is usually defined as being derivatives of sphingosine, a long-chain 1,3-dihydroxy-2-amino alcohol. Various pathological conditions such as diabetes or neuropathy have been associated with changes in the sphingolipidome and an increased biosynthesis of structurally altered non-canonical sphingolipid derivatives. These unusual or non-canonical sphingolipids hold great promise as potential diagnostic markers. However, due to their low concentrations and the unavailability of suitable standards, the research to explore the secret of this class of ‘Sphinx’ lipids is ultimately hampered. Therefore, the development of efficient and facile syntheses of standard compounds is a key endeavor. Here, we present various chemical approaches for stereoselective synthesis and in-depth chemical characterization of a set of novel sphingoid bases which were recently utilized as valuable tools to explore the metabolism and biophysical properties of sphingolipids, but also to develop efficient analytical methods for their detection and quantification.     

Ceramide and Related Molecules in Viral Infections

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.     

Effect of Sterol Carrier Protein-2 Expression on Sphingolipid Distribution in Plasma Membrane Lipid Rafts/Caveolae

Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.     

Study of sphingolipids oxidation by ESI tandem MS

Sphingolipids are a class of lipids that play important cellular roles both as structural components of membranes and as signaling molecules. They have also an active participation in apoptosis and inflammation processes which are associated with oxidative stress conditions. However, no studies so far accomplished the oxidative modification of sphingolipids under oxidative stress conditions. In the present study, different sphingolipids, ((d18:1/16:0) sphingomyelin (N‐palmitoyl‐D ‐erythro‐sphingosylphosphorylcholine, SM), (d18:1) sphingosylphosphorylcholine (a lysosphingolipid, SPC), and (d18:1/18:0) ceramide (Cer)) were oxidized by the hydroxyl radical, generated under Fenton reaction conditions, and the oxidation reaction was monitored by ESI‐MS in positive mode. No oxidation products were identified for Cer under the oxidative conditions used, while ESI‐MS spectra of SPC and SM after oxidation show some oxidation products that were analyzed by ESI‐MS/MS. This approach allowed identifying hydroxyl and keto derivatives of SPC and acetaldehydephosphorylcholine derivative (m/z 226). SM oxidation occurs exclusively in sphingosine backbone with formation of SPC, hydroxyl, and keto derivatives of SPC and the oxidation product at m/z 226. This study may give new insight and could help to understanding the behavior and biological roles of the sphingolipids under oxidative stress conditions.     

Differential Expression of Sphingolipid Metabolizing Enzymes in Spontaneously Hypertensive Rats: A Possible Substrate for Susceptibility to Brain and Kidney Damage

Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis of hypertension-related cerebrovascular and renal damage. In this study, we evaluated the existence of possible abnormalities related to the sphingolipid metabolism in the brain and kidneys of two well validated spontaneously hypertensive rat strains, the stroke-prone (SHRSP) and the stroke-resistant (SHRSR) models, as compared to the normotensive Wistar Kyoto (WKY) rat strain. Our results showed a global alteration in the metabolism of sphingolipids in both cerebral and renal tissues of both hypertensive strains as compared to the normotensive rat. However, few defects, such as reduced expression of enzymes involved in the metabolism/catabolism of sphingosine-1-phosphate and in the de novo biosynthetic pathways, were exclusively detected in the SHRSP. Although further studies are necessary to fully understand the significance of these findings, they suggest that defects in specific lipid molecules and/or their related metabolic pathways may likely contribute to the pathogenesis of hypertensive target organ damage and may eventually serve as future therapeutic targets to reduce the vascular consequences of hypertension.     

Role of Rho GTPase Interacting Proteins in Subcellular Compartments of Podocytes

The first step of urine formation is the selective filtration of the plasma into the urinary space at the kidney structure called the glomerulus. The filtration barrier of the glomerulus allows blood cells and large proteins such as albumin to be retained while eliminating the waste products of the body. The filtration barrier consists of three layers: fenestrated endothelial cells, glomerular basement membrane, and podocytes. Podocytes are specialized epithelial cells featured by numerous, actin-based projections called foot processes. Proteins on the foot process membrane are connected to the well-organized intracellular actin network. The Rho family of small GTPases (Rho GTPases) act as intracellular molecular switches. They tightly regulate actin dynamics and subsequent diverse cellular functions such as adhesion, migration, and spreading. Previous studies using podocyte-specific transgenic or knockout animal models have established that Rho GTPases are crucial for the podocyte health and barrier function. However, little attention has been paid regarding subcellular locations where distinct Rho GTPases contribute to specific functions. In the current review, we discuss cellular events involving the prototypical Rho GTPases (RhoA, Rac1, and Cdc42) in podocytes, with particular focus on the subcellular compartments where the signaling events occur. We also provide our synthesized views of the current understanding and propose future research directions.