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Article Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 -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, , 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: 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 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 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 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 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 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. Materials and Methods

2.1. Cells and Reagents Human immortalized podocytes were originally obtained from Dr. Moin Saleem [32]. Tissue culture flasks and plates were obtained from Greiner bio-one, Inc., and tissue culture reagents were from Gibco. Lipoproteins were isolated from healthy volunteers as per our routine procedure [33]. Prior to enrollment in the study, written informed consent was obtained from each volunteer. Blood collection for lipoprotein preparation was approved by the ethics committee of the Medical University of South Carolina (MUSC) and was performed according to the protocols approved by our institutions’ institutional review board (IRB), (Protocol number: Pro00070819). All volunteer data were analyzed anonymously. Blood was collected after a 12 h fast from healthy subjects who were normolipemic, not receiving prescription medication for any acute or chronic condition, and without family history of coronary artery disease, peripheral vascular disease, or stroke; none of the subjects were receiving antioxidant therapy. The blood was pooled from 3 or 4 donors in the presence of a lipoprotein preservative cocktail consisting of ethylenediaminetetraacetic acid (EDTA) (0.1% w/v), chloramphenicol (20 µg/mL), gentamycin sulfate (50 µg/mL), epsilon amino-caproic acid (0.13% w/v), and dithiobisnitrobenzoic acid (0.04% w/v) to inhibit the activity of lecithin-cholesterol acyltransferase, a major on HDL and LDL that converts free cholesterol into cholesteryl ester. Blood was then centrifuged, plasma was obtained, and lipoproteins fractioned by preparative ultracentrifugation as described previously [33]. Lipoprotein fractions were dialyzed against saline/EDTA (150 mM NaCl, 300 µM EDTA, pH 7.4), sterilized by filtering through a 0.22 µm membrane, and stored at 4 ◦C until used. Protein levels in lipoprotein preparations were measured using the Lowry method. Podocytes were plated at 0.5–0.6 106 cell in 10-cm2 collagen-treated plates in RPMI 1640 media × supplemented with 10% fetal bovine serum (FBS) (Corning), 200 units/mL Penicillin/Streptomycin (Roche Applied Science), and 1% insulin-transferrin-sodium selenite (ITS) (Sigma), and incubated overnight at 33 ◦C, 5% CO2. Media was then changed but without the inclusion ITS, and cells were transferred to a 37 ◦C-incubator. Cells were grown for 14 days to be differentiated and the growth media were changed every 3–4 days.

2.2. Ceramide Enrichment of Lipoprotein Particles Stock solution of each lipoprotein: LDL, HDL2 and HDL3 [2 mL of 4113 µg protein/mL in phosphate buffered saline (PBS)] was prepared [33] and used fresh for enrichment procedure. We have selected C16 and C24 ceramide species as representatives of long and very long chain ceramide species, respectively, and because they were previously shown to be negatively associated with the likelihood to develop macroalbuminuria in type 1 diabetes patients [15]. were synthesized at the Synthesis Unit of the MUSC Lipidomics Shared Resource. Stock solutions of sphingolipids: D-e-C16 ceramide (C16 ceramide, MW 538.2), D-e-C24 ceramide (C24 ceramide, MW 650.2), and D-e- hydroxy C16 ceramide (2OH C16-Ceramide, MW 554) were prepared in 100% methanol at 100 µM each. Duplicate aliquots from each lipoprotein (100 µg, 24 µL each) were analyzed at the Analytical Unit of the MUSC Lipidomics Shared Resource for sphingolipid content as previously described [33]. All preparations were performed in foil wrapped sterile glass tubes under yellow light. To each sterile tube, 50 µL of each synthetic ceramide solution was added and evaporated onto the side of the tube using nitrogen. The remaining volume of each 2 mL sterile lipoprotein solution with additional 50 µL of each ceramide solution were added to the tube. Each tube was then sealed under nitrogen and incubated for 24 h at 37 ◦C. Duplicate aliquots of each ceramide-enriched lipoprotein (100 µg, 24 µL each) were frozen in glass tube until analysis at the MUSC Lipidomics Shared Resource. The enriched lipoprotein solution was dialyzed against 0.01% EDTA in PBS, and aliquots (100 µg) of each ceramide-enriched lipoprotein were analyzed for sphingolipid content. Control (native) lipoproteins were not supplemented with ceramide but were subjected to a similar processing protocol. Genes 2020, 11, 178 4 of 18

2.3. RNA Sequencing (RNA-Seq) Analysis Podocytes were differentiated as described above. Media were changed 24 h before the start of the experiment, then lipoproteins with and without enrichment were added to a final concentration of 200 µg/mL. After 7 h of incubation at 37 ◦C, 5% CO2, the plates were washed with PBS, and stored at 80 C wrapped with aluminum foil. − ◦ RNA was extracted from frozen cells in the stored plates using Qiagen’s RNeasy kit according to manufacturer protocol and concentration quantified. An amount of 100–200 ng of total RNA was used to prepare RNA-Seq libraries using the TruSeq RNA Sample Prep Kit V2 (Illumina, San Diego, CA, USA), following the protocol described by the manufacturer. High-throughput single read (1 50 cycles) sequencing (HTS) was performed using an Illumina HiSeq2500 (v4 chemistry) with × each sample sequenced to a minimum depth of ~50 million reads. Data were subjected to Illumina quality control (QC) procedures (>80% of the data yielded a Phred score of 30). Secondary analysis was carried out on an OnRamp Bioinformatics Genomics Research Platform (OnRamp Bioinformatics, San Diego, CA, USA) (Codexis, Redwood City, CA, USA) [34,35]. OnRamp’s advanced Genomics Analysis Engine utilized an automated RNAseq workflow to process the data, including (i) data validation and quality control, (ii) read alignment to the (hg19) using TopHat2 [36], which revealed 96.9% mapping of the single end-reads, and (iii) generation of -level count data with HTSeq. The resulting SAM files were sorted and inputted into the Python package HTSeq to generate count data for gene-level differential expression analyses [Genomics Research Platform with RNAseq workflow v1.0.1, including FastQValidator v0.1.1a, Fastqc v0.11.3, Bowtie2 v2.1.0, TopHat2 v2.0.9, HTSeq v0.6.0]. In order to infer differential signal within the data sets with robust statistical power, we utilized the paired-T test on Limma [37]. The data was first converted to log2 with the “voom” function and then fitted to a linear model by “lm.fit” [37]. Fold-change (FC) estimation and hypothesis testing for differential expression were performed using the lm.fit and ebays function [37]. The transcript expression data from Limma analysis of the samples were sorted according to their adjusted p-value or q-value, which is the smallest false discovery rate (FDR) at which a transcript is called significant. FDR is the expected fraction of false positive tests among significant tests and was calculated using the Benjamini–Hochberg multiple testing adjustment procedure. The statistical analysis of pathways and terms were carried out using this sorted transcript list as described previously [38,39] and using iPathwayGuide (Advaita Bioinformatics, Plymoth, MI, USA) [40].

2.4. Multiplex Analysis of MTOR Signaling Pathway Intermediates Cells were plated and differentiated in 6-well collagen-treated plates at 3.0 105 cells per well as × described above. Media were changed 24 h before the start of the experiment, then lipoproteins with and without enrichment were added to a final concentration of 200 µg/mL. After 2 h of incubation at 37 ◦C, 5% CO2, cells were washed with cold PBS and extracted using the extraction buffer of the mTOR signaling Millipex kit from Millipore (Billerica, MA, USA) with added enzyme inhibitors (Roche). Extracts were stored at 80 C until use. Cell lysates were cleared by centrifugation at 16,000 g for − ◦ × 15 min, then by passing in a Costar 0.22 µm spin-x filter unit (Cambridge, MA, USA). A total of 25 µL of cell extract containing 11.5 µg of protein in assay buffer was used for each Millipex assay well. The Millipex mTOR signaling kit contains eleven antibodies against the following phosphorylated intermediates: GSK3B, IGFR1, IRS1, AKT, mTOR, P70S6K, IR, PTEN, GSK3a, TSC2, and RPS6. The kit antibodies were validated by the manufacturer for lack of cross reactivity. The assay was performed according to the manufacturer’s instructions and using the Biorad Bio-Plex 200 Multiplex System (Bio-Rad) at the MUSC Proteogenomics facility. All treatments were performed in duplicate wells and the cell extract from each well was analyzed in duplicates. Results from treatments with ceramide-enriched lipoprotein were compared to those with control lipoproteins using Student t-test at p 0.05. ≤ Genes 2020, 11, 178 5 of 18

3. Results and Discussion We previously demonstrated that increased plasma levels of baseline C16 ceramide and very long (C20–C26) chain ceramide species were associated with decreased likelihood to develop macroalbuminuria after several years of follow-up [15]. On the other hand, higher levels of circulating long and very long chain ceramides were reported in systemic lupus erythematous patients with confirmed renal involvement [41]. In the present study, we aimed at determining whether lipoproteins enriched with C16 ceramide species could induce critical metabolic and signaling pathways in cultured human podocytes.

3.1. Ceramide Enrichment of Lipoprotein Particles We previously determined levels of sphingolipid species in isolated lipoprotein classes in healthy human subjects using mass spectroscopy [33]. The smallest lipoprotein particles, HDL3 were found to be the major carriers of 1-phosphate (S1P), dihydrosphingosine 1-phosphate, and sphingosine. HDL3 particles contain the lowest levels of and ceramide; however, HDL2 and HDL3 particles have similar sphingomyelin/ceramide ratios (72.9% and 78.9%, respectively) despite the difference in their particle size (8.5–13 and 7.3–8.5 nm, respectively) [33]. The results of the analysis of the ceramide species in lipoprotein particles showed that the concentration of C24 ceramide is the highest, followed by C24:1, C 22, C20, C16, and C18 ceramide species [33]. Ståhlman et al. found that small HDL-particles predominated in dyslipidemic subjects, with and without diabetes, compared to respective normolipidemic controls, and were distinguished as the primary carrier of ceramides, which is known for promoting inflammation and insulin resistance [16]. In healthy individuals, LDL particles are typically the major carriers of ceramide compared to VLDL and HDL particles [33,42]. In the current study, when lipoprotein particles were incubated in vitro with different ceramide species, C16 ceramide had the highest level of incorporation into all lipoproteins (LDL, HDL2, HDL3) (Figure2). 2OH C16 ceramide had lower incorporation (Figure2), whereas the very long-chain C24 ceramide was not incorporated in any lipoprotein incorporation (Data not shown). In vivo, the main tissue sources for circulating sphingolipids, their flux rate and half-life remain unclear. Clues to the origin of sphingolipids in the circulation have come from the recent studies, which identified microsomal triglyceride transfer protein (MTP) and ATP binding cassette family A protein 1 (ABCA1) as critical determinants of sphingolipid levels in lipoproteins [43,44]. A possible reason why the very long chain ceramide (C24) was not incorporated into the lipoprotein particles in vitro is probably the lack of an active process that requires MTP, similar to the naturally occurring process in the intracellular in vivo system [43].

3.2. Signaling and Metabolic Pathways Induced by C16 Ceramide-Enriched Lipoproteins The signaling and metabolic pathways that were affected by exposing cultured human podocytes to ceramide-enriched lipoproteins for seven hours were examined using RNAseq analysis. The pathways that were significantly (p < 0.05) affected by the incubation of ceramide-enriched LDL, HDL2 and HDL3 are presented in Tables S1–S3, respectively. Treatment of podocytes with C16 ceramide-enriched LDL showed that the metabolic and signaling pathways were most affected when compared to C16 ceramide-enriched HDL2 or C16 ceramide-enriched HDL3, each relative to its corresponding control (native) lipoprotein. We focused primarily on the pathways that were significantly affected by C16 ceramide-enriched LDL and are pertinant to kidney functions. Thus, the sphingolipid metabolic pathway, glycophospholipid biosynthesis-ganglio series pathway, sphingolipid signaling pathway, adherens junctions pathway, mTOR signaling pathway, focal adhesion pathway, and apopotosis pathway were studied (Table1). Genes 2020, 11, 178 6 of 18 Genes 2020, 11, x FOR PEER REVIEW 6 of 19

Figure 2. Enrichment of the lipoprotein particles with ceramide. Lipoproteins isolated from healthy Figure 2. Enrichment of the lipoprotein particles with ceramide. Lipoproteins isolated from healthy volunteers were incubated with 100 µM of ceramides. After incubation for 24 h at 37 C, the lipoproteins volunteers were incubated with 100 µM of ceramides. After incubation for◦ 24 h at 37 °C, the were dialyzed against PBS and samples of before and after dialysis were analyzed for lipoprotein lipoproteinscontent. were D-e-C16 dialyzed ceramide against had the PBS highest and samples incorporation of before while and D-e-2OH after dialysis C16 ceramide were analyzed had low for lipoproteinincorporation content. and D-e-C16 D-e-C24 ceramideceramide had had no the incorporation highest incorporation (Data not shown). while D-e-2OH C16 ceramide had low incorporation and D-e- C24 ceramide had no incorporation (Data not shown). Table 1. C16 ceramide-enriched LDL regulate of metabolic and signaling pathways in human podocytes. 3.2. Signaling and Metabolic Pathways Induced by C16 Ceramide-Enriched Lipoproteins Rank Pathway p Value * Number of Genes The signaling and metabolic pathways that were affected by exposing cultured human 1 Metabolic pathways 1.1 10 10 348 podocytes to ceramide-enriched lipoproteins for seven× hours− were examined using RNAseq 2 Lysosome 2.2 10 09 52 × − analysis. The pathways3 that Adherens were junction significantly (p3.3 < 100.05)09 affected by41 the incubation of × − ceramide-enriched4 LDL, Ubiquitin HDL2 and mediated HDL3 proteolysis are presented5.2 in 10Tables08 S1–S3, respectively.50 Treatment of × − 5 Endocytosis 5.0 10 07 87 podocytes with C16 ceramide-enriched LDL showed that× the− metabolic and signaling pathways 6 Spliceosome 5.6 10 05 44 × − were most affected whenAGE-RAGE compared signaling to C16 pathway ceramide-enriched HDL2 or C16 ceramide-enriched 7 0.0003 45 HDL3, each relative to its incorresponding diabetic complications control (native) lipoprotein. We focused primarily on the pathways that were8 significantlySphingolipid signalingaffected pathway by C16 ceramide-enriched0.0004 LDL 51 and are pertinant to 12 Insulin signaling pathway 0.0007 57 kidney functions.14 Thus, mTOR signalingthe sphingolipid pathway metabolic 0.0008 pathway, 31 glycophospholipid biosynthesis-ganglio17 series pathway, Focal adhesion sphingolipid signaling 0.0010 pathway, adherens 91 junctions pathway, mTOR signaling18 pathway, PI3K-Akt focal ad signalinghesion pathwaypathway, and apopotosis 0.0013 pathway 114were studied (Table 1). 20 TNF signaling pathway 0.0018 47 * Bonferroni test-validated p values. Table 1. C16 ceramide-enriched LDL regulate gene expression of metabolic and signaling pathways in human podocytes. 3.2.1. Sphingolipid Metabolism Pathway Rank Our data show that genesPathway regulating the sphingolipid metabolismp Value pathway * (Figure Number S1) of partly Genes 1regulate the sphingolipid Metabolic signaling pathways pathway (Figure S2). This is not 1.1 × surprising, 10–10 since348 several 2sphingolipid metabolites (e.g., Lysosome S1P, sphingosine, ceramide, ceramide 2.2 1-phosphate) × 10–09 are bioactive52 3signaling molecules. C16 ceramide-enriched Adherens junction LDL induced the upregulation 3.3 × 10 of–09 sphingosine kinase41 1 4and 2 (SPHK1 & SPHK2 Ubiquitin), ceramide mediated synthase proteolysis 1 ((CERS1 ), generates C18 5.2 ceramide), × 10–08 ceramide50 kinase 5( CERK). On the other hand, C16Endocytosis ceramide-enriched LDL elicited the 5.0 downregulation × 10–07 of alkaline87 (ACER2), delta desturase sphingolipid 2 (DEGS2), ceramide glucosyltransferase enzyme 6 Spliceosome 5.6 × 10–05 44 (UGCG), serine palmitoyltransferase long chain subunits 1, 2 and 3 (SPTL1, 2, 3), and ceramide synthase 7 AGE-RAGE signaling pathway in diabetic complications 0.0003 45 6 ((CERS6), generates C16 ceramide) (Figure3A and Table S4). Interestingly, the downregulation of 8 Sphingolipid signaling pathway 0.0004 51 12 Insulin signaling pathway 0.0007 57 14 mTOR signaling pathway 0.0008 31 17 Focal adhesion 0.0010 91 18 PI3K-Akt signaling pathway 0.0013 114 20 TNF signaling pathway 0.0018 47 * Bonferroni test-validated p values.

Genes 2020, 11, x FOR PEER REVIEW 7 of 19

3.2.1. Sphingolipid Metabolism Pathway Our data show that genes regulating the sphingolipid metabolism pathway (Figure S1) partly regulate the sphingolipid signaling pathway (Figure S2). This is not surprising, since several sphingolipid metabolites (e.g., S1P, sphingosine, ceramide, ceramide 1-phosphate) are bioactive signaling molecules. C16 ceramide-enriched LDL induced the upregulation of 1 and 2 (SPHK1 & SPHK2), ceramide synthase 1 ((CERS1), generates C18 ceramide), ceramide kinase (CERK). On the other hand, C16 ceramide-enriched LDL elicited the downregulation of alkaline ceramidase (ACER2), delta desturase sphingolipid 2 (DEGS2), ceramide glucosyltransferase enzyme Genes 2020, 11, 178 7 of 18 (UGCG), serine palmitoyltransferase long chain subunits 1, 2 and 3 (SPTL1, 2, 3), and ceramide synthase 6 ((CERS6), generates C16 ceramide) (Figure 3A and Table S4). Interestingly, the downregulationCERS6 gene expression of CERS6 and gene the likelyexpression decrease and in the ceramide likely decrease synthase in 6 enzymeceramide suggests synthase a rate 6 enzyme limiting suggestsprocess a regulating rate limiting this proc enzyme.ess regulating this enzyme.

Figure 3. The sphingolipid metabolism mRNA transcripts regulated in response to C16 ceramide-enriched Figure 3. The sphingolipid metabolism mRNA transcripts regulated in response to C16 LDL in human podocytes. Podocytes were induced with 200 µg/mL C 16 ceramide-enriched or native ceramide-enriched LDL in human podocytes. Podocytes were induced with 200 µg/mL C 16 LDL and incubated for 7 h at 37 ◦C, 5% CO2; RNA was extracted and RNA-seq assay was performed ceramide-enriched or native LDL and incubated for 7 h at 37 °C, 5% CO2; RNA was extracted and as described under Methods. (A) Sphingolipid metabolism; (B) synthesis. Figure RNA-seq assay was performed as described under Methods. (A) Sphingolipid metabolism; (B) generated using iPathwayGuide (Advaita Bioinformatics) software; log FC, logarithm of fold change of glycosphingolipids synthesis. Figure generated using iPathwayGuide (Advaita Bioinformatics) gene expression. Box and whisker plot: The ends of the box are the upper and lower quartiles, box spans software; log FC, logarithm of fold change of gene expression. Box and whisker plot: The ends of the the interquartile range. Horizontal line inside the box denotes the median and the whiskers are the two box are the upper and lower quartiles, box spans the interquartile range. Horizontal line inside the lines outside the box extend to the highest and lowest observations. box denotes the median and the whiskers are the two lines outside the box extend to the highest and lowestA key observations. regulatory enzyme in lipid metabolism, phosphotidate phosphatase 2C (PPAP2C) was upregulated in response to C16 ceramide-enriched LDL. Additionally, SMPD1 and 4 which encode lysosomalA key regulatory acid sphingomyelinase enzyme in lipid and meta neutralbolism, membrane phosphotidate sphingomyelinase, phosphatase respectively, 2C (PPAP2C were) was also upregulatedupregulated. in Recentresponse findings to C16 stronglyceramide-enriched support that LDL. altered Additionally, circulating SMPD1 sphingolipids and 4 which are associated encode lysosomalwith the developmentacid sphingomyelinase of nephropathy and neutral in diabetes membra [15,45ne] sphingomyelinase, and lupus [41,46]. How respectively, different were circulating also upregulated.sphingolipids Recent influence findings renal strongly sphingolipid support metabolism that altered and circulating signalingremains sphingolipids to be investigated. are associated with the development of nephropathy in diabetes [15,45] and lupus [41,46]. How different circulating3.2.2. Glycosphingolipids sphingolipids influence Biosynthesis-Ganglio renal sphingolipid Series Pathway metabolism and signaling remains to be investigated. Glycosphingolipids are particularly abundant in the kidney and play a critical role in kidney function [4]. It has been established that sphingolipid enzyme replacement therapy ameliorates kidney disease progression in sphingolipid-associated genetic disorders such as Gaucher and Fabry disease [47]. However, whether circulating sphingolipids regulate renal glycosphingolipid metabolism is still unknown. Our data showed that all genes in the glycospingolipid biosynthesis-ganglio series pathway that were induced by C16 ceramide-enriched LDL were upregulated (Figure S3), with ST3 β-galactoside α-2,3-sialyltransferase 1 (ST3GAL1, 5) (Figure3B and Table S5) being the most upregulated gene. ST3GAL1 is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates. This sialyltransferase enzyme is believed to be a target for cancer treatment to prevent metastasis [48]. ST3GAL isoform 5 causes GM3 synthase deficiency, which Genes 2020, 11, x FOR PEER REVIEW 8 of 19

3.2.2. Glycosphingolipids Biosynthesis-Ganglio Series Pathway Glycosphingolipids are particularly abundant in the kidney and play a critical role in kidney function [4]. It has been established that sphingolipid enzyme replacement therapy ameliorates kidney disease progression in sphingolipid-associated genetic disorders such as Gaucher and Fabry disease [47]. However, whether circulating sphingolipids regulate renal glycosphingolipid metabolism is still unknown. Our data showed that all genes in the glycospingolipid biosynthesis-ganglio series pathway that were induced by C16 ceramide-enriched LDL were upregulated (Figure S3), with ST3 β-galactoside α-2,3-sialyltransferase 1 (ST3GAL1, 5) (Figure 3B and Table S5) being the most upregulated gene. ST3GAL1 is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates. This sialyltransferase enzyme is believed to be a target for cancer treatment to prevent metastasis [48]. Genes 2020, 11, 178 8 of 18 ST3GAL isoform 5 causes GM3 synthase deficiency, which results in the upregulation of ST3GAL1, 5 in podocytes contributing to kidney diseases associated with changes in renal glycosphingolipid levelsresults [4–6]. in the upregulation of ST3GAL1, 5 in podocytes contributing to kidney diseases associated with changesAdditionally, in renal glycosphingolipid the transcripts levels of [ST64–6 ]. N-acetylgalactosaminide α-2,6-sialyltransferase 4 (ST6GALNAC4Additionally,) and the isoform transcripts 6 (ST6GALNAC6 of ST6 N-acetylgalactosaminide) were upregulated α-2,6-sialyltransferasein response to C16 4 ceramide-enriched(ST6GALNAC4) and LDL isoform (Figure 6 (ST6GALNAC6 3B and Table) were S5). upregulated This enzyme in response is involved to C16 in ceramide-enriched the synthesis of disialylgalactosLDL (Figure3B andylgloboside Table S5). (DSGG This enzyme) from monosialylga is involved inlactosylgloboside the synthesis of disialylgalactosylgloboside(MSGG) in the kidney [49], and(DSGG the) fromeffect monosialylgalactosylgloboside of its upregulation on renal glycosphingolipid (MSGG) in the kidney levels [49], and and associated the effect of kidney its upregulation diseases mayon renal possibly glycosphingolipid be relevant. levels and associated kidney diseases may possibly be relevant.

3.2.3. Sphingolipid Sphingolipid Signaling Signaling Pathway Pathway The sphingolipid signaling pathway genes genes th thatat were were regulated in response to C16 ceramide-enriched LDL LDL are are shown in Figure 44,, TableTable S6 S6 and and Figure Figure S2. S2. InIn additionaddition toto SPHK1 & SPHK2,, sphingosinesphingosine 1 1 phosphate phosphate receptor receptor 1 ( S1PR11 (S1PR1), CERS1,), CERS1, CERS CERS 6, mitogen-activated 6, mitogen-activated protein protein kinase kinase3 (MAPK3 3 (),MAPK3 ras-related), ras-related C3 botulinum C3 toxinbotulinum substrate1 toxin (RAC1 substrate1) and phosphatidylinositol (RAC1) and phosphatidylinositol 3-kinase subunit 3-kinaseβ (PIK3R2 subunit) were upregulated.β (PIK3R2) were In contrast, upregulated. C16 ceramide-enriched In contrast, C16 ceramide-enriched LDL elicited the downregulation LDL elicited the of downregulationDEGS2, ACER2, sphingosineof DEGS2 1, phosphateACER2, receptorsphingosine 3 (S1PR3 1 ),phosphate and mitogen-activated receptor 3 protein(S1PR3 kinase), and 8, 10mitogen-activated and 13 (MAPK 8,protein 10, 13). kinase Additionally, 8, 10 and phosphatase 13 (MAPK and 8, 10, tensin 13). homolog Additionally, (PTEN phosphatase), Rho associated and tensinprotein homolog kinase2 ( (PTENROCK2), Rho), serine associated/threonine protein protein kinase2 kinase (ROCK2 B (AKT3), serine/threonine) and phosphatidylinositol protein kinase 4,5 B (bisphosphateAKT3) and 3-kinasephosphatidylinositol subunit β (PIK3CB 4,5 ) werebisphosphate downregulated. 3-kinase It remains subunit to beβ determined(PIK3CB) whichwere downregulated.of these downstream It remains signaling to be molecules determined that which were of induced these downstream by circulating signaling sphingolipids molecules could that be weretargeted induced to modify by circulating podocyte sphingolipids pathophysiology. could be targeted to modify podocyte pathophysiology.

Figure 4. TheThe sphingolipid sphingolipid signaling signaling pathway pathway mRNA mRNA tr transcriptsanscripts regulated regulated in in response response to C16 ceramide-enriched LDLLDL inin humanhuman podocytes. podocytes. Podocytes Podocytes were were induced induced with with C C 16 16 ceramide-enriched ceramide-enriched or ornative native LDL LDL and and RNA-seq RNA-seq assay assay was was performed performed as describedas described in Figurein Figure3 legend. 3 legend. Figure Figure generated generated by byiPathwayGuide iPathwayGuide (Advaita (Advaita Bioinformatics) Bioinformatics) software; software; log FC, log logarithm FC, logarithm of fold change of fold of change gene expression. of gene expression.Box and whisker Box and plot: whisker the ends plot: of the the box ends are of the the upper box andare the lower upper quartiles, and lower box spans quartiles, the interquartile box spans therange. interquartile Horizontal range. line inside Horizontal the box line denotes inside the th mediane box denotes and the the whiskers median are and the the two whiskers lines outside are the twobox extendlines outside to the the highest box extend and lowest to the observations. highest and lowest observations. 3.2.4. Adherens Junction Pathway 3.2.4. Adherens Junction Pathway The effects of C16 ceramide-enriched LDL on podocyte gene expression relating to the adherens The effects of C16 ceramide-enriched LDL on podocyte gene expression relating to the adherens junction pathway are presented in Figure5, Table S7, and Figure S4. The glomerular filtration slit junction pathway are presented in Figure 5, Table S7, and Figure S4. The glomerular filtration slit (slit diaphragm) is considered as a modified adherens junction [50]. After the metabolic and lysosome (slit diaphragm) is considered as a modified adherens junction [50]. After the metabolic and pathways, the adherens junction pathway was the third pathway that was significantly affected in lysosome pathways, the adherens junction pathway was the third pathway that was significantly response to C16 ceramide-enriched LDL (p = 3.3 10 09) (Table1). Genes that are involved in encoding × − the formation of adherens junctions [51] and were downregulated in response to C16 ceramide-enriched

LDL include nectin 3 (PVRL3), nectin 4 (PVRL4), β catenin (CTNNB1) and delta catenin (CTNND1) (Figure5 and Table S7). In contrast, nectin 2 ( PVRL2), and the actin genes ACTB and ACTG1 were upregulated along with the actin crosslinking genes α-actinin 1 and 4 (ACTN1 and ACTN4). It has been shown that mutations in ACTN4 are involved in focal segmental glomerulosclerosis (FSGS), which is a leading cause of kidney failure [52]. Mutations in this protein result in increased affinity for actin binding, the formation of intracellular aggregates, and decreased protein half-life, which—as a result of altering podocyte actin cytoskeleton—may induce toxic effects on podocytes. Notably, the α-actinin (ACTN2) gene, which is involved in actin cytoskeleton reorganization, was the most downregulated gene in the adherens junction pathway. The gene encoding the tight junction protein 1/zonula occludens 1 (TJP1/ZO1) was also downregulated (Figure5 and Table S7). However, the gene Genes 2020, 11, x FOR PEER REVIEW 9 of 19 affected in response to C16 ceramide-enriched LDL (p = 3.3 × 10–09) (Table 1). Genes that are involved in encoding the formation of adherens junctions [51] and were downregulated in response to C16 ceramide-enriched LDL include nectin 3 (PVRL3), nectin 4 (PVRL4), β catenin (CTNNB1) and delta catenin (CTNND1) (Figure 5 and Table S7). In contrast, nectin 2 (PVRL2), and the actin genes ACTB and ACTG1 were upregulated along with the actin crosslinking genes α-actinin 1 and 4 (ACTN1 and ACTN4). It has been shown that mutations in ACTN4 are involved in focal segmental glomerulosclerosis (FSGS), which is a leading cause of kidney failure [52]. Mutations in this protein result in increased affinity for actin binding, the formation of intracellular aggregates, and decreased protein half-life, which—as a result of altering podocyte actin cytoskeleton—may induce toxic effects on podocytes. Notably, the α-actinin (ACTN2) gene, which is involved in actin cytoskeleton reorganization, was the most downregulated gene in the adherens junction pathway. The gene Genes 2020, 11, 178 9 of 18 encoding the tight junction protein 1/zonula occludens 1 (TJP1/ZO1) was also downregulated (Figure 5 and Table S7). However, the gene encoding vinculin (VCL), another key protein involved in theencoding formation vinculin of the (VCL tight), anotherjunction key complex protein was involved upregulated in the formationin response of to the C16 tight ceramide-enriched junction complex LDL.was upregulated in response to C16 ceramide-enriched LDL.

Figure 5. The adherens junction pathway mRNA transcripts regulated in response to C16 Figure 5. The adherens junction pathway mRNA transcripts regulated in response to C16 ceramide-enriched LDL in human podocytes. Podocytes were induced with C 16 ceramide-enriched or ceramide-enriched LDL in human podocytes. Podocytes were induced with C 16 ceramide-enriched native LDL and RNA-seq assay was performed as described in Figure3 legend. Figure generated by or native LDL and RNA-seq assay was performed as described in Figure 3 legend. Figure generated iPathwayGuide (Advaita Bioinformatics) software; log FC, logarithm of fold change of gene expression. by iPathwayGuide (Advaita Bioinformatics) software; log FC, logarithm of fold change of gene Box and whisker plot: the ends of the box are the upper and lower quartiles, box spans the interquartile expression. Box and whisker plot: the ends of the box are the upper and lower quartiles, box spans range. Horizontal line inside the box denotes the median and the whiskers are the two lines outside the the interquartile range. Horizontal line inside the box denotes the median and the whiskers are the box extend to the highest and lowest observations. two lines outside the box extend to the highest and lowest observations. Genes encoding the transforming growth factor β 1 (TGF-β1) signaling pathway components suchGenes as TGFbR1 encodingand SMAD the transforming 2, 3, 4 were allgrowth downregulated factor β 1 in(TGF- responseβ1) signaling to C16 ceramide-enriched pathway components LDL such as TGFbR1 and SMAD 2, 3, 4 were all downregulated in response to C16 ceramide-enriched (Figure5 and Table S7). Interestingly, the gene encoding epidermal growth factor receptor ( EGFR) LDL (Figure 5 and Table S7). Interestingly, the gene encoding epidermal growth factor receptor was downregulated, whereas the gene encoding fibroblast growth factor receptor 1 (FGFR1) was (EGFR) was downregulated, whereas the gene encoding fibroblast growth factor receptor 1 (FGFR1) upregulated. It has been suggested that such interactions might serve as negative feedback loops to was upregulated. It has been suggested that such interactions might serve as negative feedback limit the extent of cellular activation [53]. loops to limit the extent of cellular activation [53]. The gene encoding SNAI2—a zinc finger protein commonly known as SLUG—was downregulated The gene encoding SNAI2—a zinc finger protein commonly known as SLUG—was in response to C16 ceramide-enriched LDL (Figure5 and Table S7). This protein is a transcriptional downregulated in response to C16 ceramide-enriched LDL (Figure 5 and Table S7). This protein is a repressor which downregulates the expression of E-cadherin, involved in epithelial-mesenchymal transcriptional repressor which downregulates the expression of E-cadherin, involved in transitions, and has anti-apoptotic activity [54]. The downregulation of this protein might have epithelial-mesenchymal transitions, and has anti-apoptotic activity [54]. The downregulation of this a positive effect on the continued functionality of healthy podocytes. protein might have a positive effect on the continued functionality of healthy podocytes. 3.2.5. MTOR Signaling Pathway 3.2.5. MTOR Signaling Pathway The treatment of podocytes for 7 h with C16 ceramide-enriched LDL upregulated several intermediatesThe treatment in the of mTOR podocytes signaling for 7 pathway h with (FigureC16 ceramide-enriched S5). These include LDL the upregulated genes MAPK3, severalthe intermediatesregulatory-associated in the mTOR protein signaling of mTOR pathway (RPTOR) ,(Figureproline-rich S5). AKT1These substrateinclude the 1 ( AKT1S1genes MAPK3,), ribosomal the regulatory-associatedprotein S6 kinase β 1 andprotein 2 (RPS6KB1, of mTOR 2) and(RPTOR the proline-rich), proline-rich AKT AKT1 substrate substrate of 40 kDa1 ( (AKT1S1PRAS40),) ribosomal(Figure6A andprotein Table S6 S8). kinase In contrast, β 1 and several 2 (RPS6KB1, intermediates 2) and in the the proline-rich mTOR signaling AKT pathway—includingsubstrate of 40 kDa (insulin-likePRAS40) (Figure growth 6A factor and 1Table (IGF1 )S8)., phosphoinositide In contrast, several 3 kinase intermediates catalytic subunit in theα mTOR(PIK3CA signaling, a class pathway—includingI PI3K), the proto-oncogene insulin-like B-RAF growth (BRAF factor), AKT3,1 (IGF1 PTEN,), phosphoinositidetuberous sclerosis 3 kinase 1 catalytic (TSC1) andsubunit the α rapamycin-insensitive (PIK3CA, a class I PI3K) companion, the proto-oncogene of mammalian targetB-RAF of ( rapamycinBRAF), AKT3, (RICTOR PTEN,)—were tuberous downregulated sclerosis 1 (inTSC1 response) and to C16the ceramide-enrichedrapamycin-insensitive LDL (Figurecompanion6A, Table of S8,mammalian and Figure S5).target of rapamycin (RICTORWhen)—were human downregulated podocytes were in incubated response for to7 C16 h with ceramide-enriched C16 ceramide-enriched LDL (Figure HDL2, 6A, the onlyTable gene S8, andthat Figure was downregulated S5). (by over two folds) was the protein kinase C β (PRKCβ) (Figure6B, Table S8, and Figure S6). It is possible that after incubation with HDL2 for 7 h, the activation signal went back to background level, whereas that of LDL was still measurable after 7 h. This could be due to the ‘short’ time needed for HDL particle docking to its receptor(s) versus LDL holoparticle endocytosis via the LDL receptor and delivery of its sphingolipid cargo. The mTOR signaling pathway is known to be a major regulatory pathway in the kidney [29–31]; therefore, this particular pathway was selected to test the phosphorylated intermediates (Section 3.3 below). Since signaling pathways are activated by phosphorylation and not by gene expression levels per se, the determination of gene regulation was performed at 7 h post incubation with C16 ceramide-enriched lipoproteins, whereas the mTOR signaling pathway multiplex experiment was performed at 2 h post incubation for the early detection of induction effects. How mTOR signaling pathway components induced by circulating sphingolipids affect podocyte pathophysiology certainly warrants further investigation. Genes 2020, 11, 178 10 of 18 Genes 2020, 11, x FOR PEER REVIEW 10 of 19

Figure 6. The mTOR signaling pathway mRNA transcripts regulated in response to C16 ceramide-enriched Figurelipoproteins 6. The in humanmTOR podocytes.signaling Podocytespathway weremRNA induced transcr withipts Cregulated 16 ceramide-enriched in response orto native C16 ceramide-enrichedlipoproteins and RNA-seq lipoproteins assay wasin human performed podocytes. as described Podocytes in Figure were3 legend. induced ( A) Cellswith induced C 16 ceramide-enrichedwith ceramide-enriched or native or nativelipoproteins LDL; and (B) cellsRNA- inducedseq assay with was ceramide-enriched performed as described or native in Figure HDL2. 3 legend.Figuregenerated (A) Cells by iPathwayGuideinduced with (Advaitaceramide-enriched Bioinformatics) or software;native LDL; log FC, (B logarithm) cells induced of fold change with ceramide-enrichedof gene expression. Boxor native and whisker HDL2. plot: Figure the endsgenera ofted the boxby iPathwayGuide are the upper and (Advaita lower quartiles, Bioinformatics) box spans software;the interquartile log FC, range. logarithm Horizontal of fold linechange inside of thegene box expression. denotes the Box median and whisker and the plot: whiskers the ends are the of twothe boxlines are outside the upper the box and extend lower to quartiles, the highest box and spans lowest the observations. interquartile range. Horizontal line inside the box denotes the median and the whiskers are the two lines outside the box extend to the highest and 3.2.6.lowest Focal observations. Adhesion Pathway The focal adhesion-related (cytoskeleton reorganization) genes that were differentially regulated in responseWhen human to C16 podocytes ceramide-enriched were incubated LDL treatment for 7 h with of podocytes C16 ceramide-enriched are shown in Figure HDL2,7, Tablethe only S9, geneand Figurethat was S7. downregulated The genes encoding (by over filamin two (FLNAfolds) ),was paxilin the protein (PXN), vinculinkinase C (VCLβ (PRKC) andβ) actin (Figure (ACTB 6B,) Tablewere allS8, upregulated,and Figure S6). which It is canpossible result that in reorganization after incubation of with focal HDL2 adhesion for and7 h, increasethe activation cell motility. signal wentCyclin back D, whichto background regulates level, cell cyclewhereas progression, that of LDL drives was still the measurable G1/S phase after transition, 7 h. This and could is involved be due toin the cell ‘short’ growth time [55] needed was also for upregulated HDL particle in responsedocking to to its C16 receptor(s) ceramide-enriched versus LDL LDL holoparticle (Figure7, endocytosisTable S9). The via Rho the pathway LDL receptor genes, includingand delivery Rho-Associated, of its sphingolipid Coiled-Coil-Containing cargo. The mTOR Protein signaling Kinase pathway1 and 2 (ROCK1is known and to 2be) anda major Rho regulatory GTPase Activating pathway in Protein the kidney 5 (ARHGAP5 [29–31]; ),therefore, which are this involved particular in pathwayactin polymerization was selected and to test the the formation phosphorylated of filipodia intermediates and lamellipodia (Section were 3.3 downregulated below). Since signaling (Figure7, pathwaysTable S9) [ 56are]. Inactivated contrast, extracellularby phosphorylation matrix (ECM) and receptornot by genesgene andexpression ECM protein levels genes per including se, the determinationintegrin α subunits of gene 4 andregulation V (ITGA was 4, Vpe),rformed integrin atβ 7subunits h post incubation 3, 8 and A11 with (ITGB C16 ceramide-enriched 3, 8 and ITGA11 , lipoproteins,respectively), whereas non-collagenous the mTOR ECM signaling protein pathway (COMP), multip collagenlex 4experimentα 5 (COL4A5 was), collagen performed 6 α 3 at (COL6A3 2 h post), incubationand collagen for 9 αthe2 (earlyCOL9A2 detection) were downregulatedof induction effects. in response How mTOR to C16 ceramide-enrichedsignaling pathway LDL components (Figure7, inducedTable S9). byThe circulating downregulation sphingolipids of these affect ECM podo proteinscyte pathophysiology suggests that C16 certainly ceramide-enriched warrants further LDL investigation.treatment may be involved in inducing podocyte dysfunction (e.g., effacement) which needs further investigation. Additionally, the genes encoding the cytokine receptors vascular endothelial growth 3.2.6. Focal Adhesion Pathway factor receptor 2 and 3 (VEGFR 2, 3), and epidermal growth factor receptor (EGFR) were downregulated (FigureThe7 , Tablefocal S9).adhesion-related The e ffect of these (cytoskeleton receptors onreorganization) focal adhesion andgenes migration that were of endothelial differentially cells regulatedhas been studiedin response extensively to C16 [ceramide-enriched57]; however, their LDL effects treatment on podocytes of podocytes in the context are shown of kidney in Figure disease 7, Tableprocesses S9, and remains Figure unknown. S7. The genes encoding filamin (FLNA), paxilin (PXN), vinculin (VCL) and actin (ACTB) were all upregulated, which can result in reorganization of focal adhesion and increase cell motility.3.2.7. Apoptosis Cyclin D Pathway, which regulates cell cycle progression, drives the G1/S phase transition, and is involvedIt is in well-known cell growth that[55] was podocyte also upregulated effacement in is response a major to outcome C16 ceramide-enriched of glomerular diseasesLDL (Figure and 7,is Table associated S9). The with Rho podocyte pathway loss genes, leading including to renal Rho-Associated, dysfunction. Whether Coiled-C programmedoil-Containing cell Protein death Kinasemechanisms 1 and contribute2 (ROCK1 toand podocyte 2) and Rho loss GTPase remains Activating contentious, Protein in part 5 because(ARHGAP5 the), detection which are of apoptosisinvolved inand actin other polymerization pathways of programmed and the formation cell death of filipodia in podocytes and lamellipodia has been technically were downregulated challenging. Our (Figure data 7,show Table that S9) C16 [56]. ceramide-enriched In contrast, extracellular LDL induced matrix the(ECM) upregulation receptor gene of thes and apoptosis-related ECM protein genes genes α includingencoding αintegrin-tubulin ( TUBAsubunits), ACTN 4 andand V ( laminITGA (4,LMNA V), integrin) (Figure β8 subunits, Table S10, 3, and8 and Figure A11 ( S8),ITGB which 3, 8 and are ITGA11, respectively), non-collagenous ECM protein (COMP), collagen 4 α 5 (COL4A5), collagen 6 α 3 (COL6A3), and collagen 9 α 2 (COL9A2) were downregulated in response to C16

Genes 2020, 11, 178 11 of 18 known to cause microtubule dysfunction [58], shrinkage of cells [59] and loss of nuclear membrane stability [60], respectively. In contrast, the Fas ligand (FasLG) gene, which drives cells into apoptosis [61], was downregulated (Figure8 and Table S10). Comprehensive understanding of the mechanisms leading to podocyte loss are of particular interest due to the need for developing novel therapeutic strategies targeting these mechanisms to prevent glomerular dysfunction.

3.3. C16 Ceramide-Enriched Lipoproteins Regulate Levels of Phosphorylated Intermediates in the MTOR Signaling Pathway The mTOR signaling pathway is a well-described pathway in kidney cell biology, and the phosphorylation of its intermediates is a key step in the activation of the pathway. To further validate our RNAseq data, we employed multiplex technology to detect the regulation (phosphorylation) of mTOR signaling intermediates in response to C16 ceramide-enriched LDL, HDL2 and HDL3 treatments of podocytes. Unlike the qualitative/semi quantitative immunoblot system, the multiplex assay used here offered a quantitative determination of phosphoproteins detected. Each antibody used in this assay was validated and all the antibodies in the provided kits do not cross-react with each other. We found that podocytes treated for two hours with C16 ceramide-enriched HDL2 resulted in downregulation of all phosphorylated intermediates of the mTOR signaling pathway except IGFR1, AKT, and insulin receptor (IR), in comparison to treatment with native HDL2 (Figure9A). In contrast, C16 ceramide-enriched HDL3 induced the upregulation of GSK3B, AKT and p70S6K phosphorylation, in comparison to treatment with native HDL3 (Figure9B). It is well-known that not all proteins need to be phosphorylated to be active and that transcript levels do not always translate to protein levels; however, the mTOR pathway after 2 h of incubation with C16 ceramide-enriched HDL2 showed the downregulation of phosphorylation of most activated intermediates measured. The C16 ceramide-enriched LDL treatment induced downregulation of phosphorylated GSK3a and GSK3B signaling molecules in comparison to treatment with native LDL (Figure9C). The data thus showed that the effect of C16 ceramide-enriched HDL2 (Figure9A) and C16 ceramide-enriched LDL (Figure9C) on the downregulation of phosphorylated GSK3B and GSK3a is similar. This similarity could be attributed to factors relevant to the modification of particle structure and/or size after C16 ceramide-enrichment. Intriguingly, 2OH C16 ceramide-enriched HDL3 induced upregulation of IR phosphorylation compared to native HDL3, whereas 2OH C16 ceramide-enriched LDL induced upregulation of phosphorylated AKT, mTOR and IR. The pathophysiological relevance of 2OH C16 ceramide modification of lipoproteins is currently unknown. Genes 2020, 11, x FOR PEER REVIEW 12 of 19

GenesGenes2020 2020, 11, 11, 178, x FOR PEER REVIEW 12 of 1219 of 18

Figure 7. The focal adhesion pathway mRNA transcripts regulated in response to C16 ceramide-enriched LDL in human podocytes. Podocytes were induced with C 16 ceramide-enriched or native LDL and RNA-seq assay was performed as described in Figure 3 legend. Figure generated by iPathwayGuide (Advaita Bioinformatics)FigureFigure 7. 7.The The focal focal software; adhesion adhesion log pathway FC,pathway logarith mRNA mRNAm of transcripts transcriptsfold change regulated regulated of genein inexpression. response responseto toBoxC16 C16 andceramide-enriched ceramide-enriched whisker plot: the LDLLDL ends inin humanofhuman the bo podocytes.podocytes.x are the upper PodocytesPodocytes and werewelowerre inducedinduced quartiles, withwith box C spans16C ceramide-enriched 16 the ceramide-enriched interquartile or range. native or Horizontalnative LDL andLDL RNA-seq line and inside RNA-seq assay the wasbox assay de performednotes was theperforme asmedian describedd asand described inthe Figure whiskers 3in legend. Figure are the Figure 3 two lege generatedlinesnd. Figureoutside by generated iPathwayGuidethe box extend by iPath to (Advaita thewayGuide highest Bioinformatics) and(Advaita lowest observations.software;Bioinformatics) log FC, logarithmsoftware; log of foldFC, changelogarith ofm geneof fold expression. change of Box gene and expression. whisker plot: Box theand ends whisker of the plot: box the are theends upper of the and box lower are the quartiles, upper and box spanslower thequartiles, interquartile box range.spans Horizontalthe interquartile line inside range. the Horizontal box denotes line theinside median the box and de thenotes whiskers the median are the and two the lines whiskers outside arethe the boxtwoextend lines outside to the highestthe box extend and lowest to the observations. highest and lowest observations.

Figure 8. The apoptosis pathway mRNA transcripts regulated in response to C16 ceramide-enriched LDL in human podocytes. Podocytes were induced with C 16 Figure 8. The apoptosis pathway mRNA transcripts regulated in response to C16 ceramide-enriched LDL in human podocytes. Podocytes were induced with C 16 ceramide-enriched or native LDL and RNA-seq assay was performed as described in Figure3 legend. Figure generated by iPathwayGuide (Advaita Bioinformatics) ceramide-enriched or native LDL and RNA-seq assay was performed as described in Figure 3 legend. Figure generated by iPathwayGuide (Advaita Bioinformatics) software; log FC, logarithm of fold change of gene expression. Box and whisker plot: the ends of the box are the upper and lower quartiles, box spans the interquartile software;Figure 8. log The FC, apoptosis logarithm pathway of fold mRNA change transcripts of gene regulaexpression.ted in responseBox and towhisker C16 ceramide-enriched plot: the ends of LDL the in box human are thepodocytes. upper andPodocytes lower werequartiles, induced box with spans C 16the range. Horizontal line inside the box denotes the median and the whiskers are the two lines outside the box extend to the highest and lowest observations. interquartileceramide-enriched range. orHorizontal native LDL line and inside RNA-seq the boxassay denotes was performed the median as described and the inwhiske Figurers 3 arelegend. the Figuretwo lines generated outside by the iPathwayGu box extenided to (Advaita the highest Bioinformatics) and lowest observations.software; log FC, logarithm of fold change of gene expression. Box and whisker plot: the ends of the box are the upper and lower quartiles, box spans the interquartile range. Horizontal line inside the box denotes the median and the whiskers are the two lines outside the box extend to the highest and lowest observations.

Genes 2020, 11, 178 13 of 18 Genes 2020, 11, x FOR PEER REVIEW 14 of 19

Figure 9. The mTOR signaling pathway phosphorylated proteins regulated in response to C16 ceramide-Figure 9. orThe 2OH mTOR C16 ceramide-enrichedsignaling pathway lipoproteins phosphorylated in human proteins podocytes. regulated Podocytes in response were inducedto C16 withceramide- 200 µ gor/mL 2OH ceramide-enriched C16 ceramide-enriched or with corresponding lipoproteins native in human lipoproteins podocytes. (HDL2, Podocytes HDL3, or LDL),were

andinduced incubated with for200 2 hµg/mL at 37 ◦ceramide-enrichedC, 5% CO2. Cells were or extractedwith corresponding and processed native using lipoproteins the mTOR signaling (HDL2, MillipexHDL3, or kitLDL), (Millipore). and incubated A total for of2 h 11.5 at 37µ g°C, protein 5% CO2. in 25CellsµL were assay extracted buffer was and usedprocessed for each using assay the sample.mTOR signaling The assay Millipex was analyzed kit (Millipore). using the A total Bio-Plex of 11.5 200 µg Multiplex protein in System 25 µL (Bio-Rad).assay buffer All was treatments used for wereeach assay performed sample. in duplicatesThe assay was and analyzed the cell extract using fromthe Bio-Plex each treatment 200 Multiplex was analyzed System (Bio-Rad). in duplicates. All (treatmentsA) Treatment were with performed C16 ceramide-enriched in duplicates and HDL2, the cell (B) treatmentextract from with each C16 treatment ceramide-enriched was analyzed HDL3, in (duplicates.C) treatment (A with) Treatment C16 ceramide-enriched with C16 ceramide-enriched LDL. Results from HDL2, treatments (B) withtreatment ceramide-enriched with C16 lipoproteinsceramide-enriched were compared HDL3, (C to) treatment those with with corresponding C16 ceramide-enric native lipoproteinshed LDL. Results using from Student treatmentst-test at pwith0.05. ceramide-enriched lipoproteins were compared to those with corresponding native lipoproteins ≤ using Student t-test at p ≤ 0.05. 4. Conclusions TheIntriguingly, transcriptomics 2OH C16 findings ceramide-enriched of this study HDL3 suggest induced that the upregulation analyses of of sphingolipid IR phosphorylation profiles ofcompared lipoprotein to native particles, HDL3, although whereas more 2OH laborious, C16 ceramide-enriched have the potential LDL forinduced an added upregulation value over of plasmaphosphorylated/serum sphingolipidomics AKT, mTOR and in providingIR. The pathop morehysiological specific information relevance about of not2OH only C16 biomarkers ceramide ofmodification disease but of also lipoprotei mechanisticns is currently insights aboutunknown. the pathology of the disease and potential drug target molecules. Sphingolipids are non-soluble molecules and the cell membrane is impermeable to these molecules,4. Conclusions and in the circulation they are carried and transported by lipoproteins. Mechanistically, our findingsThe suggesttranscriptomics that whereas findings LDL of particles—including this study suggest theirthat the bioactive analyses sphingolipid of sphingolipid cargo—are profiles likely of internalizedlipoprotein andparticles, metabolized although upon more their contactlaborious, with have podocytes, the potential HDL particles for an (mainly added HDL2) value remain over plasma/serum sphingolipidomics in providing more specific information about not only biomarkers of disease but also mechanistic insights about the pathology of the disease and potential drug target molecules. Sphingolipids are non-soluble molecules and the cell membrane is impermeable to these

Genes 2020, 11, 178 14 of 18 in contact with the cell surface capable of altering the structure of membrane signaling domains and triggering downstream signaling pathways (e.g., mTOR signaling pathway). For future research on the effect of sphingolipid-altered lipoproteins on the functionality of renal cells (podocytes, mesangial cells, or tubular epithelial cells), other ceramide or sphingolipid species could be incorporated into lipoproteins in vitro, as described here. For sphingolipids containing very long-chain fatty acids (and therefore not easily incorporated in lipoproteins in vitro), fabricated lipoprotein model particles with incorporated sphingolipids could be utilized to investigate the potential for defined sphingolipid species to modify cell membrane sphingolipid composition and alter the cell signaling cascades involved with the pathophysiology of renal cells. Uncovering the role of lipoprotein sphingolipids in the development and progression of kidney disease could completely change the paradigm of intervention to prevent/delay the development or reduce the progression of nephropathy.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/2/178/s1. Figure S1: The sphingolipid metabolism pathway showing genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S2: The glycosphingolipid biosynthesis-ganglio series pathway showing genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S3: The sphingolipid signaling pathway showing the genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S4: The adherens junction pathway showing the genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S5: The mTOR signaling pathway showing genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S6: The mTOR signaling pathway showing the gene regulated in response to C16 ceramide-enriched HDL2 in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S7: The focal adhesion pathway showing genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Figure S8: The apoptosis pathway showing genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S1: Metabolic and signaling pathways that were significantly (p < 0.05) affected by incubation of human podocytes with C16 ceramide-enriched LDL (iPathwayGuide, Advaita Bioinformatics), Table S2: Metabolic and signaling pathways that were significantly (p < 0.05) affected by incubation of human podocytes with C16 ceramide-enriched HDL2 (iPathwayGuide, Advaita Bioinformatics), Table S3: Metabolic and signaling pathways that were significantly (p < 0.05) affected by incubation of human podocytes with C16 ceramide-enriched HDL3 (iPathwayGuide, Advaita Bioinformatics), Table S4: The sphingolipid metabolism genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S5: The glycosphingolipids synthesis genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S6: The sphingolipid signaling pathway genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S7: The adherens junction pathway genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S8: The mTOR signaling pathway genes regulated in response to C16 ceramide-enriched LDL or HDL2 in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S9: The focal adhesion pathway genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S10: The apoptosis pathway genes regulated in response to C16 ceramide-enriched LDL in human podocytes (iPathwayGuide, Advaita Bioinformatics), Table S11: C16 ceramide LDL responsive genes (iPathwayGuide, Advaita Bioinformatics). Author Contributions: S.M.H., R.L.K. and D.N. conceptualized the study. S.M.H. designed and directed the experiments, wrote the original draft of the manuscript, and reviewed, edited and approved the final version. W.O.T. performed and analyzed the multiplex experiment, and wrote several sections of the manuscript. D.N. and E.A. provided, maintained, and differentiated the cells. E.A. and A.J.S. treated the cells and extracted the RNA. R.L.K. and A.J.S. prepared the lipoproteins and enriched them with ceramide species. S.M.H. and W.O.T. analyzed and revised the data. W.O.T., D.N. and E.A. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Department of Regenerative Medicine and Cell Biology (S.M.H.), R01 DK087956 (D.N.), and the VA Merit Review Program (R.L.K.). Sphingolipid reagents and analyses supported in part by the Lipidomics Core in the SC Lipidomics and Pathobiology COBRE (P20 RR017677 from NCRR) and Lipidomics Shared Resource, Hollings Cancer Center, MUSC (P30 CA138313 and P30 GM103339). Services of the MUSC Proteogenomics Core Facility supported by NIHNIGMS P30 GM103342 to the SC COBRE for Developmentally Based Cardiovascular Diseases. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Genes 2020, 11, 178 15 of 18

Acknowledgments: We thank the MUSC Hollings Cancer Center Genomics & MUSC Bioinformatics Cores for RNA-Seq data processing and analyses, particularly Gary Hardiman. We also thank J. Barth (MUSC) for helpful discussions. Contents do not represent the views of Department of VA or US Government. Conflicts of Interest: The authors declare no conflict of interest.

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