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Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins

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Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins G C A T T A C G G C A T genes Article Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins Samar M. Hammad 1,*, Waleed O. Twal 1 , Ehtesham Arif 2, Andrea J. Semler 3, Richard L. Klein 3,4 and Deepak Nihalani 2 1 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA; [email protected] 2 Division of Nephrology, Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA; [email protected] (E.A.); [email protected] (D.N.) 3 Division of Endocrinology, Metabolism, and Medical Genetics, Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA; [email protected] (A.J.S.); [email protected] (R.L.K.) 4 Research Service, Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, SC 29401, USA * Correspondence: [email protected] Received: 23 January 2020; Accepted: 4 February 2020; Published: 7 February 2020 Abstract: Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases. Keywords: RNA-Seq; podocyte; ceramide; lipoprotein; sphingolipids; mTOR 1. Introduction Sphingolipids are both structural lipids and signaling molecules with significant physiological functions. Sphingolipids are found associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated to maintain homeostasis [1,2]. A classic form of chronic renal disease, Fabry disease, results from the genetic impairment of the glycosphingolipid metabolism [3]. Furthermore, diseases associated with kidney dysfunction—including diabetic nephropathy, polycystic kidney disease, and renal cell carcinoma—have been associated with changes in renal glycosphingolipid levels and the associated metabolism [4–6]. However, little is known about how changes in plasma sphingolipids and the sphingolipid composition of lipoproteins may Genes 2020, 11, 178; doi:10.3390/genes11020178 www.mdpi.com/journal/genes Genes 2020, 11, x FOR PEER REVIEW 2 of 19 carcinoma—have been associated with changes in renal glycosphingolipid levels and the associated Genes 2020, 11, 178 2 of 18 metabolism [4–6]. However, little is known about how changes in plasma sphingolipids and the sphingolipid composition of lipoproteins may contribute to the development of renal disease. Urine contributeprotein metabolites to the development in patients with of renal diabetic disease. kidn Urineey disease protein provided metabolites some in biochemical patients with insights diabetic for kidneyidentifying disease novel provided candidate some biomarkers biochemical for insightsthe disease for [7]; identifying however, novel the information candidate biomarkers regarding forplasma the diseaseand urine [7]; however,lipidomics the is information still scarce. regarding Sphingolipids plasma are and rapidly urine lipidomicsemerging isas still clinically scarce. Sphingolipidsimportant biomolecules are rapidly due emerging to their as clinicallyeffects on important multiple biomoleculesmetabolic pathways due to their [1]. Unlike effects onother multiple lipid metabolicmolecules pathways(e.g., cholesterol) [1]. Unlike small other changes lipid moleculesin the levels (e.g., of cholesterol)sphingolipids small can changeselicit potent in the responses levels of sphingolipidsand it is becoming can elicit increasingly potent responses clear andthat itdysfunctional is becoming increasingly lipid metabolism clear that extends dysfunctional far beyond lipid metabolismcholesterol and extends triglycerides far beyond [8,9]. cholesterol and triglycerides [8,9]. ThereThere isis growing evidence that sphingolipid metabolism is altered in diabetes [10,11]; [10,11]; however, thethe majoritymajority ofof evidenceevidence comescomes fromfrom studiesstudies inin animalanimal modelsmodels [[12–14]12–14] withwith littlelittle attentionattention givengiven toto thethe deliverydelivery ofof plasmaplasma sphingolipidssphingolipids toto renalrenal cells.cells. WeWe previouslypreviously showedshowed thatthat sphingolipidomicssphingolipidomics cancan predict predict the the risk risk of ofdeveloping developing nephropathy nephropathy in type in type1 diabetes 1 diabetes patients patients [15]. We [15 measured]. We measured plasma plasmaconcentrations concentrations of ceramide of ceramide species species to investigate to investigate their their association association with with the the development development ofof albuminuriaalbuminuria in type 1 patientspatients inin thethe DiabetesDiabetes ControlControl andand ComplicationsComplications TrialTrial/Epidemiology/Epidemiology ofof DiabetesDiabetes Interventions and ComplicationsComplications (DCCT(DCCT/EDIC)/EDIC) study during 14–20 years of follow-up [15]. [15]. TheThe resultsresults demonstrateddemonstrated that increased plasma levels of C16 ceramide and very long (C20, C20:1, C22:1,C22:1, C24:1,C24:1, C26,C26, and and C26:1) C26:1) chain chain ceramide ceramide species species measured measured at DCCT at DCCT baseline baseline were were associated associated with awith decreased a decreased likelihood likelihood of developing of developing macroalbuminuria macroalbuminuria [15]. Other [15]. Other investigators investigators found found higher higher levels oflevels ceramide of ceramide in HDL inparticles HDL particles of obese of patientsobese patients who were who insulinwere insulin insensitive insensitive [13]; in [13]; the in small the small HDL particlesHDL particles (HDL3) (HDL3) of type of 2type diabetes 2 diabetes patients patients with with dyslipidemia dyslipidemia [16], [16], and and in HDL in HDL after after ingestion ingestion of aof high a high fat fat diet diet [17 [17].]. CeramidesCeramides are anan integralintegral partpart ofof cellcell membranesmembranes associatedassociated withwith macrodomainsmacrodomains regulatingregulating surfacesurface receptorsreceptors andand signalingsignaling pathwayspathways [[18–23].18–23]. PreviousPrevious studies addressing the role ofof ceramideceramide inin diabetesdiabetes focusedfocused onon insulininsulin resistanceresistance [[12,1312,13,,24,25]24,25] andand diddid notnot addressaddress diabeticdiabetic complicationscomplications includingincluding nephropathy.nephropathy. ThereThere isis anan increasingincreasing bodybody ofof evidenceevidence suggestingsuggesting thatthat injuryinjury toto podocytespodocytes leadsleads toto proteinuria proteinuria and and lipiduria lipiduria (lipuria) (lipuria) in nephropathy in nephropathy [26–28 [26–28]] (Figure (Figure1). In this 1). study,In this we study, exposed we culturedexposed cultured human podocytes human podocytes to C16 ceramide-enriched to C16 ceramide-enriched lipoproteins lipoproteins (low density (low density lipoproteins lipoproteins (LDL), high-density(LDL), high-density lipoproteins lipoproteins 2 and 3 (HDL2 2 and and 3 (HDL2 HDL3) andand performedHDL3) and a comprehensive performed a transcriptomicscomprehensive analysis.transcriptomics The results analysis. revealed The possibleresults revealed metabolic possible and signaling metabolic pathways and signaling through whichpathways extracellular through sphingolipidswhich extracellular carried sphingolipids on lipoprotein carried particles on can lipopro regulatetein the particles expression can ofregulate genes encoding the expression podocyte of functionalitygenes encoding and podocyte pathophysiology. functionality Special and focus pathophysiology. is on the mTOR Special signaling focus pathway,is on the which mTOR is wellsignaling known pathway, to regulate which the is expressionwell known of to slitregulate diaphragm the expression proteins of and slit cytoskeleton diaphragm proteins structure and in podocytescytoskeleton [29 structure–31]. in podocytes [29–31]. Figure 1. Cartoon depicting the microstructure of the glomerulus. Diagram showing the transfer of albumin and lipoproteins including their sphingolipid cargo from the capillary lumen through fenestration of endothelial cells to the podocytes. Genes 2020, 11, 178 3 of 18 2.
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