Journal of Clinical Medicine

Article The Effect of Interleukin-4 and Dexamethasone on RNA-Seq-Based Transcriptomic Profiling of Human Podocytes: A Potential Role in Minimal Change Nephrotic Syndrome

Jiwon M. Lee 1,† , Younhee Ko 2,†, Chul Ho Lee 3,4, Nara Jeon 5, Keum Hwa Lee 3 , Jun Oh 6 , Andreas Kronbichler 7 , Moin A. Saleem 8, Beom Jin Lim 5,* and Jae Il Shin 3,9,10,*

1 Department of Pediatrics, Chungnam National University Hospital and College of Medicine, Daejeon 35015, Korea; [email protected] 2 Division of Biomedical Engineering, Hankuk University of Foreign Studies, Gyeonggi-do 17035, Korea; [email protected] 3 Department of Pediatrics, Yonsei University College of Medicine, Seoul 03722, Korea; [email protected] (C.H.L.); [email protected] (K.H.L.) 4 Division of Clinical Genetics, Severance Children’s Hospital, Seoul 03722, Korea 5 Department of Pathology, Yonsei University College of Medicine, Seoul 03722, Korea; [email protected] 6 Department of Pediatrics, University Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] 7 Department of Internal Medicine IV (Nephrology and Hypertension), Medical University Innsbruck, 6020 Innsbruck, Austria; [email protected] 8 Children’s and Renal Unit and Bristol Renal, University of Bristol, Bristol BS2 8BJ, UK; [email protected] 9 Division of Pediatric Nephrology, Severance Children’s Hospital, Seoul 03722, Korea 10 Institute of Kidney Disease Research, Yonsei University College of Medicine, Seoul 03722, Korea



 * Correspondence: [email protected] (B.J.L.); [email protected] (J.I.S.) † These authors contributed equally to the work. Citation: Lee, J.M; Ko, Y.; Lee, C.H.; Jeon, N.; Lee, K.H.; Oh, J.; Abstract: Interleukin-4 (IL-4) expression is implicated in the pathogenesis of nephrotic syndrome Kronbichler, A.; Saleem, M.A; Lim, (NS). This study aimed to investigate the changes in the transcriptomes of human podocytes in- B.J.; Shin, J.I. The Effect of duced by IL-4 treatment and to analyze whether these changes could be affected by simultaneous Interleukin-4 and Dexamethasone on steroid treatment. Three groups of human podocytes were treated with control, IL-4, and IL-4 plus RNA-Seq-Based Transcriptomic dexamethasone (DEX), respectively. We performed whole-transcriptome sequencing to identify Profiling of Human Podocytes: A Potential Role in Minimal Change differentially expressed genes (DEGs) between the groups. We investigated relevant biological path- Nephrotic Syndrome. J. Clin. Med. ways using Gene Ontology (GO) enrichment analyses. We also attempted to compare and validate 2021, 10, 496. https://doi.org/ the DEGs with the genes listed in PodNet, a literature-based database on mouse podocyte genes. 10.3390/jcm10030496 A total of 176 genes were differentially expressed among the three groups. GO analyses showed that pathways related to cytoskeleton organization and cell signaling were significantly enriched. Among Received: 22 August 2020 them, 24 genes were listed in PodNet, and 12 of them were previously reported to be associated with Accepted: 20 January 2021 IL-4-induced changes in human podocytes. Of the 12 genes, the expression levels of BMP4, RARB, Published: 01 February 2021 and PLCE1 were reversed when podocytes were simultaneously treated with DEX. In conclusion, this study explored changes in the transcriptome profiles of human podocytes treated with IL-4. Few Publisher’s Note: MDPI stays neutral genes were reported in previous studies and were previously validated in experiments with human with regard to jurisdictional claims in podocytes. We speculate that IL-4 may exert pathogenic effects on the transcriptome of human published maps and institutional affil- podocytes, and a few genes may be involved in the pathogenesis. iations.

Keywords: nephrotic syndrome; transcriptome; RNA-sequencing; interleukin; gene; podocytes

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Minimal change nephrotic syndrome (MCNS) is the major form of nephrotic syn- conditions of the Creative Commons drome (NS) reported in children, which includes steroid-sensitive NS (SSNS) [1–3]. As Attribution (CC BY) license (https:// most primary MCNS cases exhibit a high response rate to corticosteroids, its pathogenesis creativecommons.org/licenses/by/ has been speculated to be immune-mediated, especially involving T cell-associated mecha- 4.0/). nisms [1,4]. Therefore, multiple studies have been conducted to explore the immunological

J. Clin. Med. 2021, 10, 496. https://doi.org/10.3390/jcm10030496 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 496 2 of 18

basis of NS [5–13], and several studies specifically suggested T-helper 2 lymphocyte (Th2)- dependency by demonstrating over-activity of type 2 helper T cells (Th2) and decreased Th1/Th2 ratios, thus highlighting Th2 predominance in NS [8,14,15]. Therefore, more studies have been performed with an aim to validate the role of Th2 cytokines such as interleukin (IL)-4, IL-5, and IL-13 in NS [16–24]. Among these cytokines, the expression of only IL-4 and IL-13 has mostly been explored because IL-5 expression is reportedly eosinophil-specific. Yap et al. examined the mRNA expression profiles of IL-2, interferon-γ, and IL-13 in 55 children with steroid-sensitive NS and reported increased cytoplasmic IL-13 expression in patients during relapse compared to remission [23]. Further, van den Berg et al. examined the role of IL-4 and IL-13 in human and rat glomerular visceral epithelial cells (GVECs) and showed that IL-4 and IL-13 could exert direct effects on podocytes by binding to specific IL-4 and IL-13 receptors. They also decreased transepithelial electrical resistance of monolayers of rat GVEC to approximately 30% and 40% of baseline values, respectively, in a dose-dependent manner [20]. Moreover, the increased activity of IL-4 has been reported in correlation with the activity of NS [14,21]. Youssef et al. investigated cytokine profiles in 52 children with steroid-sensitive NS and found that serum levels of IL-4, along with immunoglobulin (Ig) E, IL-13, and tumor necrosis factor (TNF)-α were significantly increased during the active phase of proteinuria compared to the remission phase [14]. Prasad et al. studied the T-cell phenotypes and their respective cytokine profiles in children with NS and discovered that the levels of suppressive cytokines, such as IL-10 and transforming growth factor (TGF)-β, decreased in the stimulated peripheral blood lymphocytes at relapse of NS and that these levels increased during remission [25]. Conversely, the expression of stimulatory cytokines, such as IL-4 and interferon (IFN)-γ, increased during relapse and decreased during remission [25]. This study also suggested the role of regulatory T cells (Tregs) by demonstrating that the frequency of Tregs decreased at relapse and increased at remission [25]. Youn et al. also reported increased serum levels of IL-4 and IL-5 in 32 children with NS, which decreased to normal levels after steroid therapy [26], suggesting their pathogenic role in the development of NS. Moreover, a recent in vivo study showed that the overexpression of IL-4 in mice was sufficient to induce kidney injury and result in proteinuria [17]. In this study, the transfer of antigen-specific B cells induced glomerular injury and caused proteinuria, but the transfer of IL-4-deficient B cells did not lead to the development of proteinuria, demonstrating an important role of IL-4 in the pathogenesis of NS. This study also presented evidence that IL-4 induced podocyte membrane ruffling and widespread foot process retraction in murine podocytes [17]. Based on the accumulated evidence on the pathogenic role of IL-4 in NS, we speculated that IL-4 might induce changes in the protein expression in human podocytes. First, we examined the effect of IL-4 on intracytoplasmic actin filaments by immunofluorescence mi- croscopy. We then performed transcriptome analysis in human podocytes and investigated the changes in gene expression levels induced by IL-4. We also examined whether these changes could be reversed by simultaneous steroid treatment. To our knowledge, this is the first study to explore the effect of IL-4 on human podocytes by transcriptome profiling.

2. Materials and Methods 2.1. Cell Culture of Human Podocytes Conditionally immortalized human podocytes (AB8/23), primarily cloned from hu- man glomerular cultures, were characterized and generously provided by Dr. Moin A. Saleem (University of Bristol, Bristol, UK). These human podocytes were maintained in the RPMI 1640 medium (Gibco™) supplemented with 10% heat-inactivated fetal bovine serum (FBS), insulin-transferrin-selenium-pyruvate supplement (ITSP; Gibco™), and antibiotics. Fresh media was provided once every two days. To induce differentiation, podocytes were maintained at 37 ◦C without ITPS (non-permissive conditions) for at least 2 weeks, and 0.05% trypsin was used to detach cells from the culture dishes [27]. J. Clin. Med. 2021, 10, 496 3 of 18

2.2. Podocyte Treatment with IL-4 and Visualization and Quantification of Intracytoplasmic Actin Filaments To identify whether IL-4 at a dose of 10 ng/mL could induce changes in intracyto- plasmic actin filaments in podocytes, which is the main hallmark of MCNS pathogenesis, cytoplasmic actin filaments were visualized by immunofluorescence microscopy. To quan- titatively compare the differences in expression, the number of actin filaments was counted in cells from each group (IL-4-treated, IL4+DEX-treated, vehicle-treated controls, and non-treated controls). Podocytes treated with IL-4 were compared with vehicle-treated podocytes and human podocytes at 37 ◦C without any treatment for 6 h. The cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature (RT). Cells were permeabilized using 0.3% Triton X-100 in PBS for 10 min at RT. After adding PBS and subjecting the cells to washing steps, the cells were stained with rhodamine-phalloidin (1:500, Invitrogen) by incubating for 30 min at RT. Thereafter, PBS was used to conduct the washing steps again, and the nuclei were stained with DAPI (1:5000, Sigma-Aldrich, St. Louis, MO, USA) and mounted. Actin filaments and nuclei were visualized using fluorescence microscopy. Images were acquired, and the number of actin filaments per cell was counted by the point-counting method using the Image J software (version 1.50i; National Institutes of Health, Bethesda, MD, USA).

2.3. Podocyte Treatment with IL-4 with or without Dexamethasone Podocytes were divided into three groups. The first group was treated with IL-4, the second group was treated with IL-4 plus dexamethasone (DEX), and the third group was control (non-treated podocytes). The cells were then incubated for 6 h. Each experiment was performed in triplicate. In the first group, IL-4 (Peprotech Inc., Rocky Hill, NJ, USA) was administered to hu- man podocytes at a dose of 10 ng/mL in serum-free RPMI 1640 at 37 ◦C. As the dissolving vehicle for IL-4, 1% bovine serum albumin (BSA) was used following the manufacturer’s instructions. The dose of IL-4 used herein was 10 ng/mL, which was used in a previous animal model in which 10 ng/mL IL-4 induced podocyte effacement as detected under electron microscopy [17]. In the second group, IL-4 was treated in the same manner as the first group, and DEX (Sigma-Aldrich, Saint Louis, MO, USA) was simultaneously added and incubated together. DEX was dissolved in distilled water according to the manufac- turer’s instructions. Detailed quantitative assessments were performed by Xing et al. using various concentrations of DEX in which DEX concentrations between 10−7 and 10−5 M equated to therapeutic concentrations in vivo. Therefore, 10−6 M DEX was used to treat podocytes in our study [28]. The third group, considered as a control, was incubated for 6 h in 1% BSA, the vehicle for IL-4, because IL-4 and cytokines are already sent as BSA as a routinely used carrier protein.

2.4. Whole Transcriptome Sequencing RNA sequencing of the three groups of podocytes was performed, and the differen- tially expressed genes (DEGs) between the groups were analyzed. Podocytes after treatment and incubation were suspended in RNAlater® (Invitrogen, Carlsbad, CA, USA) and stored at −20 ◦C until further analysis to prevent RNA degrada- tion. Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and DNase digestion was performed to prevent genomic DNA contamination. RNA integrity and quantity of each sample were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Using mRNA captured from total RNA by using oligo-dT beads, a library was constructed using the NEXTFLEX® Rapid RNA-Seq Kit (PerkinElmer, Inc., Waltham, MA, USA), whereas the small RNA-Seq library was pre- pared using the NEXTFLEX® Small RNA-Seq Kit (PerkinElmer Inc., Waltham, MA, USA) following the manufacturer’s instructions. Paired-end sequencing was performed using Illumina Nexseq550 (Illumina, San Diego, CA, USA) with a 100-bp read length after the J. Clin. Med. 2021, 10, 496 4 of 18

quality control process, and the quantity of the libraries was assessed using the Agilent 2100 Bioanalyzer.

2.5. Data Analysis and Statistical Modeling 2.5.1. Principal Component Analysis Principal component analysis (PCA) was used as a statistical procedure to simplify the complexity of high-dimensional data while maintaining trends and patterns. PCA was performed for each of the individual samples to determine the overall patterns of the responding genes based on the RNA-seq expression profiles.

2.5.2. Identification of DEGs The primary analysis of high-throughput sequencing data was performed using TopHat, htseq, and DEGseq2 (version 1.10.0) [29]. In our study, raw FASTQ files were mapped to the UCSC hg19 reference genome using the TopHat software (version 1.4.0) and Bowtie software (version 2.3.5) [30]. Once the sequence reads were successfully mapped to the reference genome, the htSeq program, which contains powerful options for analyzing high-throughput sequencing data, was used to count the reads mapped to each gene. Once the count of the reads for each gene was calculated, the tags with markedly low counts were filtered out to prevent the intervention of weakly expressed tags. Then, a normalization process was applied to account for the differences in the number of reads produced in different sequencing runs, as well as technical biases caused by the library preparation protocols, sequencing platforms, and nucleotide compositions. To identify the DEGs, the DESeq2 package was used. Using the negative bionomical assumption, the coefficient of variance for each gene was estimated. Pairwise comparisons were performed between the control group and the two treatment groups to identify significant DEGs (adjusted p-value < 0.1).

2.5.3. Construction of Protein-Protein Interaction Networks The STRING 9.1 network database is one of the largest databases of direct (i.e., phys- ical) and indirect (i.e., functional) protein–protein interactions and contains data from various sources, including genomic context predictions, high-throughput experiments, co-expression analyses, and existing databases [31]. The database comprises data on 9.6 mil- lion proteins obtained from more than 2031 organisms. This database was used to identify protein-protein interactions covering the identified DEGs from different comparisons. As our gene sets were represented by gene symbols, the Ensembl protein identifiers in the original STRING database were converted into gene symbols using mapping information in EntrezID.

2.5.4. Gene Ontology Enrichment Analysis Gene ontology (GO) enrichment analysis was performed for all DEGs obtained from each comparison. The enriched GO terms were identified using hypergeometric tests between DEGs from each comparison and GO-annotated biological pathway genes with a p-value of 0.05.

2.5.5. Selection of the Genes of Potential Relevance To identify the critical genes involved in IL-4-induced podocyte changes, DEGs were compared with those reported in previous investigations and databases. PodNet [31], a literature-based, expert-curated database of mouse podocytes, was used. Although PodNet is based on data obtained from mouse podocyte analyses, it has been regarded as a valuable tool for conducting interactome analysis in podocytes and for interpretation of podocyte expression data [31]. J. Clin. Med. 2021, 10, 496 5 of 18 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 19

3. 3.Results Results 3.1.3.1. Visualization Visualization of ofIntracytoplasmic Intracytoplasmic Actin Actin Filaments Filaments in inHuman Human Podocytes Podocytes WeWe investigated investigated the the effect effect of ofIL-4 IL-4 on on intracytoplasmic intracytoplasmic actin actin filaments filaments by byimmunoflu- immunoflu- orescenceorescence microscopy. microscopy. The The four four groups groups of ofhu humanman podocytes, podocytes, namely namely IL-4-treated IL-4-treated cells, cells, IL-4+DEX-treatedIL-4+DEX-treated cells, cells, vehicle-treated vehicle-treated controls, controls, andand non-treatednon-treated controls, controls, were were compared. com- pared.The IL-4-treatedThe IL-4-treated cells cells showed showed a decreased a decrea amountsed amount of intracytoplasmic of intracytoplasmic actin actin filaments fila- compared to the vehicle-treated and non-treated controls (Figure1A–E). ments compared to the vehicle-treated and non-treated controls (Figure 1A–E).

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FigureFigure 1. 1.IntracytoplasmicIntracytoplasmic actin actin filaments filaments in in podocytes. podocytes. Rhodamine-Phalloidin Rhodamine-Phalloidin stain stain for for six hours revealed intracytoplasmicintracytoplas- micactin actin filaments filaments (red) (red) in (inA )(A cultured) cultured human human podocytes podocytes without without any any treatment, treatment, (B) vehicle-treated(B) vehicle-treated control control podocytes, podocytes, (C) IL- (C) IL-4-treated podocytes, and (D) IL-4+dexamethasone-treated cells. (E) shows the average number of actin filaments 4-treated podocytes, and (D) IL-4+dexamethasone-treated cells. (E) shows the average number of actin filaments counted counted from each group. Statistically significant results are shown for p value. Blue marks indicate the nuclei. Abbrevia- from each group. Statistically significant results are shown for p value. Blue marks indicate the nuclei. Abbreviations: IL-4, tions: IL-4, interleukin-4; DEX, dexamethasone. interleukin-4; DEX, dexamethasone.

J. Clin. Med. 2020, 9, x FOR PEER REVIEW 6 of 19 J. Clin. Med. 2021, 10, 496 6 of 18 3.2. PCA and Analysis of DEGs 3.2. PCABased and on Analysis the data of obtained DEGs from the above-mentioned experiment, which indicated that IL-4Based expression on the data induced obtained changes from in the the above-mentioned major structural proteins experiment, of human which podocytes, indicated wethat performed IL-4 expression transcriptome induced changesanalysis. in We the studied major structuralpodocytes proteins in three of groups, human namely podocytes, IL- 4-treatedwe performed cells, vehicle-treat transcriptomeed controls, analysis. and We podocytes studied podocytes co-treated inwith three IL-4 groups, and DEX. namely The resultsIL-4-treated of PCA cells, showed vehicle-treated that IL-4-treated controls, podocytes and podocytes were highly co-treated distinct with from IL-4 the and other DEX. groups,The results and ofover PCA 90% showed of their that variance IL-4-treated was explained podocytes by were PC1 highly(Figure distinct 2A). The from assessment the other ofgroups, DEGs andamong over the 90% three of their groups variance of podocytes was explained revealed by PC1the (Figureinvolvement2A). The of assessment3368 DEGs betweenof DEGs IL-4-treated among the threeand groupsIL-4+DEX-treat of podocytesed podocytes revealed theand involvement3633 DEGs ofbetween 3368 DEGs IL- 4+DEX-treatedbetween IL-4-treated and control and IL-4+DEX-treated groups (adjusted podocytes p-value < and 0.1). 3633 Between DEGs betweenIL-4-treated IL-4+DEX- podo- cytestreated and and controls, control 380 groups DEGs (adjusted were identifiedp-value <(Figure 0.1). Between 2B). A total IL-4-treated of 176 genes podocytes varied and at thecontrols, intersection 380 DEGs of the were three identified groups. (Figure 2B). A total of 176 genes varied at the intersection of the three groups.

Figure 2. Principal component analysis (PCA) and differentially expressed genes (DEGs) between the podocyte groups. Figure(A) PCA 2. Principal of human component podocytes analysis whole-transcriptome (PCA) and differentially treated with expressed interleukin-4 genes (red), (DEGs) interleukin-4 between the plus podocyte dexamethasone groups. (A) PCA of human podocytes whole-transcriptome treated with interleukin-4 (red), interleukin-4 plus dexamethasone (green), and control (blue), and (B) the DEGs. Abbreviations: Ctrl, vehicle-treated control; IL-4, interleukin-4; DEX, (green), and control (blue), and (B) the DEGs. Abbreviations: Ctrl, vehicle-treated control; IL-4, interleukin-4; DEX, dexa- dexamethasone. methasone.

3.3.3.3. GO GO Pathways Pathways Related Related to to the the DEGs DEGs ToTo investigate investigate thethe clinicalclinical significance significance of of the the DEGs DEGs among among the the three three groups groups of podocytes, of podo- cytes,we performed we performed GO enrichment GO enrichment analysis. analysis Among. Among the 380the DEGs380 DEGs between between IL-4-treated IL-4-treated and andcontrol control groups, groups, positively positively regulated regulated pathways pathways were were related related to angiogenesis, to angiogenesis, cell signaling, cell sig- naling,regulation, regulation, and migration and migration (Figure3 A),(Figure while 3A), negatively while negatively regulated pathwaysregulated were pathways involved were in involvedrenal development in renal development and morphogenesis and morphogenesis (Figure3B).Figure (Figure3C 3B). illustrates Figure the3C pathwaysillustrates forthe DEGs observed between IL-4-treated and IL-4+DEX-treated podocytes, thereby indicating the pathways for DEGs observed between IL-4-treated and IL-4+DEX-treated podocytes, effects of DEX addition. Here, the pathways most differentially affected by the addition of thereby indicating the effects of DEX addition. Here, the pathways most differentially af- DEX were those involved in the regulation of apoptosis, actin cytoskeleton organization, and fected by the addition of DEX were those involved in the regulation of apoptosis, actin cell regulation (Figure3C). Likewise, between the control podocytes and those co-treated with cytoskeleton organization, and cell regulation (Figure 3C). Likewise, between the control IL-4 plus DEX, the considerably affected pathways comprised those involved in the regulation podocytes and those co-treated with IL-4 plus DEX, the considerably affected pathways of apoptosis, cytoskeleton organization, and cell regulation (Figure3D). Further, the pathways comprised those involved in the regulation of apoptosis, cytoskeleton organization, and that were commonly different in the three comparison groups were related to morphogene- cell regulation (Figure 3D). Further, the pathways that were commonly different in the sis, angiogenesis, and several signaling pathways involving extracellular signal-regulated three comparison groups were related to morphogenesis, angiogenesis, and several sig- kinase (ERK), Notch, and mitogen-activated protein kinase (MAPK) (Figure3E). naling pathways involving extracellular signal-regulated kinase (ERK), Notch, and mito- gen-activated protein kinase (MAPK) (Figure 3E).

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-logp value 0123456 Angiogenesis Protein kinase C-activating G-protein coupled receptor signaling pathway Positive regulation of endothelial cell migration (A) Chronic inflammatory response Transforming growth factor beta receptor signaling pathway Cell migration Activation of MAPKK activity MAPK cascade Sphingosine-1-phosphate signaling pathway

-logp value 012345678910 Regulation of kidney development (B) Kidney epithelium development Metanephric nephron development Epithelial cell differentiation involved in kidney development

-logp value 02468101214 Positive regulation of apoptotic process (C) Positive regulation of angiogenesis Regulation of cell proliferation Intrinsic apoptotic signaling pathway in response to DNA damage Microtubule cytoskeleton organization J. Clin. Med. 2020, 9, x FOR PEER REVIEW 8 of 19

-logp value 012345678910 Negative regulation of apoptotic process Actin cytoskeleton organization (D) Intrinsic apoptotic signaling pathway in response to DNA damage Negative regulation of cell migration Vascular endothelial growth factor receptor signaling pathway Angiogenesis Epidermal growth factor receptor signaling pathway

-logp value 012345 Positive regulation of ERK1 and ERK2 cascade Branching involved in ureteric bud morphogenesis Angiogenesis Positive regulation of angiogenesis Positive regulation of branching involved in ureteric bud morphogenesis (E) Transforming growth factor beta receptor signaling pathway Positive regulation of GTPase activity Protein kinase C-activating G-protein coupled receptor signaling pathway Notch signaling pathway Negative regulation of pathway-restricted SMAD protein phosphorylation Sphingosine-1-phosphate signaling pathway Positive regulation of MAPK cascade Negative regulation of apoptotic process MAPK cascade

Figure 3. Gene ontology (GO) enriched pathyway analyses. (A) positively regulated DEGs between IL-4-treated podocytes and controls, (B) negatively regulated Figure 3.DEGsGene between ontology IL-4-treated (GO) podocytes enriched and controls, pathyway (C) most differentially analyses. expressed (A) DEGs positively between IL regulated-4-treated andDEGs IL-4+DEX-treated between podocytes, IL-4-treated (D) most differ- podocytes entially expressed DEGs between controls and IL-4+DEX-treated podocytes, and (E) most differentially expressed DEGs among the 176 overlapping DEGs in the and controls,three groups. (B) Abbreviations: negatively DEG, regulated differentially DEGs expressed between genes; IL-4, IL-4-treated interleukin-4; DEX, podocytes dexamethasone. and controls, (C) most differentially expressed DEGs between IL-4-treated and IL-4+DEX-treated podocytes, (D) most differentially expressed DEGs between controls and IL-4+DEX-treated podocytes, and (E) most differentially expressed DEGs among the 176 overlapping DEGs in the three groups. Abbreviations: DEG, differentially expressed genes; IL-4, interleukin-4; DEX, dexamethasone.

The heatmap of genes that showed the highest variance among the three groups (IL-4/IL-4+DEX/control) is presented in Supplemental Figure S1. The top ranked posi- tively or negatively regulated DEGs observed between IL-4-treated and vehicle-treated podocytes, and between IL-4-treated and IL-4+DEX-treated podocytes are summarized in Supplemental Figures S2 and S3. Although not all DEGs have been clearly elucidated for their roles exhibited in podocytes, the expression of a few genes has been previously reported to be associated with podocytes (Table1). J. Clin. Med. 2021, 10, x FOR PEER REVIEW 1 of 19

The heatmap of genes that showed the highest variance among the three groups (IL- 4/IL-4+DEX/control) is presented in Supplemental Figure S1. The top ranked positively or negatively regulated DEGs observed between IL-4-treated and vehicle-treated podocytes, and between IL-4-treated and IL-4+DEX-treated podocytes are summarized in Supple- mental Figures S2 and S3. Although not all DEGs have been clearly elucidated for their roles exhibited in podocytes, the expression of a few genes has been previously reported to be associated with podocytes (Table 1). J. Clin. Med. 2021, 10, 496 8 of 18 Table 1. Genes associated with podocytes in the main Gene Ontology (GO) pathways.

Gene Ontology (GO) Pathway Genes Table 1. Genes associated with podocytes in the main Gene Ontology (GO) pathways. Positive Regulation of ERK1 and ERK2 FGFR2, FGF2, PDGFB, ICAM1, GCNT2, CCL2, CD44, SEMA7A, PDGFRB, cascade Gene Ontology (GO) PathwayCX3CL1 Genes Positive Regulation of MAPK cascade FGFR2, PDGFB,FGFR2, C1QTNF1, FGF2, PDGFB, WWC1, ICAM1, NGFR GCNT2, CCL2, CD44, SEMA7A, Positive Regulation of ERK1 and ERK2 cascade PDGFRB, PAK2, CD44, SPHK1, PRKCI, MALT1,PDGFRB, NGFR, CX3CL1 RARB, GREM1, Negative Regulation of apoptotic process Positive Regulation of MAPK cascadeAPBB2, FGFR2, SOX9 PDGFB, C1QTNF1, WWC1, NGFR Abbreviations: APBB2, amyloid beta A4 precursor protein-binding family B member 2; C1QTNF1, complement C1q tumor PDGFRB, PAK2, CD44, SPHK1, PRKCI, MALT1, NGFR, RARB, GREM1, necrosis factor-relatedNegative protein Regulation 1; CCL2, of C-C apoptotic Motif Chemokine process Ligand 2; CD44, cluster of differentiation 44; CX3CL1, C- APBB2, SOX9 X3-C Motif Chemokine Ligand 1; FGF2, fibroblast growth factor 2; FGFR2, fibroblast growth factor receptor 2; GCNT2, glucosaminyl (NAbbreviations:-acetyl) transferase APBB2, 2; amyloid GREM1, beta Gr A4emlin precursor 1; ICAM1, protein-binding Intercellular family Adhesion B member Molecule 2; C1QTNF1, 1; MALT1, complement mucosa- C1q tumor necrosis factor-related protein 1; CCL2, C-C Motif Chemokine Ligand 2; CD44, cluster of differentiation 44; CX3CL1, C-X3-C Motif Chemokine associated lymphoidLigand tissue 1; FGF2, lymphoma fibroblast translocation growth factor 2;1; FGFR2,NGFR, fibroblastNerve Growth growth Factor factor Receptor; receptor 2; PAK2, GCNT2, p21-activated glucosaminyl kinase (N-acetyl) transferase 2; 2; PDGFB, plateletGREM1, derived Gremlin growth 1; ICAM1, factor Intercellular subunit B; AdhesionPDGFRB, Molecule platelet 1;derived MALT1, growth mucosa-associated factor receptor lymphoid subunit tissue B; lymphoma PRKCI, translocation 1; Protein kinase CNGFR, iota; NerveRARB, Growth retinoic Factor acid Receptor; receptor PAK2, beta;p21-activated SEMA7A, Semaphorin kinase 2; PDGFB, 7A; plateletSOX9, SRY-Box derived growth Transcription factor subunit Factor B; PDGFRB, platelet 9; SPHK1, Sphingosinederived growth kinase factor 1; WWC receptor1, WW subunit domain-containing B; PRKCI, Protein kinaseprotein C iota;1. RARB, retinoic acid receptor beta; SEMA7A, Semaphorin 7A; SOX9, SRY-Box Transcription Factor 9; SPHK1, Sphingosine kinase 1; WWC1, WW domain-containing protein 1. We investigated DEGs in the protein-protein interaction (PPI) network. Figure 4 shows the interactionWe of investigated the 380 DEGs DEGs between in the protein-protein IL-4-treated podocytes interaction and (PPI) controls network. (Fig- Figure4 shows ure 4A) and ofthe 176 interaction common DEGs of the in 380 the DEGs three between groups (Figure IL-4-treated 4B). podocytes and controls (Figure4A) and of 176 common DEGs in the three groups (Figure4B).

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Figure 4. Cont.

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(B) (B) Figure 4. Protein-protein interaction (PPI) netwotk of differentially expressed genes (DEGs) (A) 380 DEGs between IL-4- Figure 4. Protein-proteinFigure 4. Protein-proteininteraction (PPI) interactionnetwotk of(PPI) differentially netwotk expressed of differentially genes expressed(DEGs) (A) genes 380 DEGs (DEGs) between (A) 380 IL-4- DEGs between IL- treated podocytes and controls, and (B) 176 common DEGs among the three podocyte groups. Abbreviations: DEG, dif- treated podocytes4-treated and controls, podocytes and and(B) 176 controls, common and DEGs (B) 176 among common the three DEGs podocy amongte the groups. three Abbrevia podocytetions: groups. DEG, Abbreviations: dif- DEG, ferentially expressedferentially genes; expressed IL-4, interleukin genes; IL-4, -4. positively interleukin regulated. -4. positively regulated. differentially expressed genes; IL-4, interleukin -4. positively regulated. 3.4. Selection of3.4. the SelectionGenes of Potentialof the Genes Relevance of Potential to IL-4-Induced Relevance to Pathogenesis IL-4-Induced of PathogenesisNS of NS To identify3.4. the SelectionTo critical identify of genes the the Genes involvedcritical of Potential genes in IL-4-induced involved Relevance in to podocyteIL-4-induced IL-4-Induced changes, Pathogenesispodocyte we com- changes, of NS we com- pared the identifiedparedTo theDEGs identify identified with the those critical DEGs reported geneswith involvedthose in previous reported in IL-4-induced studies in previous and podocyte those studies existing changes, and thosein we existing compared in databases. Wedatabases.the first identified compared We DEGs first the withcompared176 thosecommonly reportedthe 176 varied commonly in previous DEGs withvaried studies 315 DEGs and listed those with genes existing 315 in listed in databases. genes in PodNet [31]. WePodNetWe found first [31]. compared 22 overlappingWe found the 176 22 gene commonlyoverlappings (7 upregulated varied genes DEGs (7 and upregulated with15 downregulated 315 listedand 15 genes downregulated ex- in PodNet [31ex-]. pression levels;pressionWe Supplemental found levels; 22 overlapping SupplementalTable S1). genes The Table gene (7 upregulated S1).expression The gene andpatterns expression 15 downregulated and thepatterns protein- expressionand the protein- levels; protein interactionproteinSupplemental network interaction Tableof these network S1). 22 Thegene of genes these are expressionpresented 22 genes arein patterns Supplemental presented and thein SupplementalFigure protein-protein S4A– Figure interaction S4A– C). Second, forC).network the Second, overlapping of these for the 22 22 overlapping genes genes, are we presented performed 22 genes, in Supplemental we a literature performed search Figure a literature to S4A–C). explore search Second, the to forexplore the over- the functional relevancefunctionallapping reported 22 genes,relevance in we previous performedreported po indocyte a literatureprevious models. searchpodocyte The to exploreoverall models. theselection The functional overall process relevance selection reportedprocess is summarizedisin in summarized previous Figure 5. podocyte in Figure models. 5. The overall selection process is summarized in Figure5.

Figure 5. OverallFigure flow 5. Overall of sortingFigure flow out of5. the Overallsorting possibly outflow relevant the of possiblysorting genes out relevant related the possibly togenes IL-4-induced relatedrelevant to pathogenesisgenes IL-4-induced related of topathogen- nephrotic IL-4-induced syndrome. pathogen- Abbreviations:esis Ctrl, of nephrotic control;esis IL-4,syndrome of interkleukin-4; nephrotic. Abbreviations: syndrome DEX, dexamethasone;. Ctrl,Abbreviations: control; IL-4, DEGs,Ctrl, interkleukin-4; control; differentially IL-4, DEX,interkleukin-4; expressed dexamethasone; genes; DEX, NS, dexamethasone; nephrotic syndrome, PodNet,DEGs, differentially a protein-proteinDEGs, expressed differentially interaction genes; expressed network NS, nephrotic in genes; mouse syndrome, NS, podocyte, nephrotic PodNet, published syndrome, a protein-protein in 2013 PodNet, [31]. a protein-proteininterac- interac- tion network in tionmouse network podocyte, in mouse published podocyte, in 2013 published [31]. in 2013 [31].

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Twelve out of the twenty-two DEGs were underpinned by previous studies using podocyteTwelve injury out ofmodels. the twenty-two These DEGs DEGs incl wereuded underpinnedPRKCI, PARD2, by previousTLN2, WWC1, studies CASK, using Twelve out of the twenty-two DEGs were underpinned by previous studies using podocyteARHGAP2, injury PTGER4, models. CAMK2B, These CDKN1A, DEGs included PLCE1, PRKCI, RARB, PARD2, and BMP4 TLN2, (Figure WWC1, 6A). Among CASK, podocyte injury models. These DEGs included PRKCI, PARD2, TLN2, WWC1, CASK, ARHGAP2,them, the expression PTGER4, CAMK2B,levels of PTGER4 CDKN1A, and PLCE1, CDKN1A RARB, were and upregulated, BMP4 (Figure while6A). Amongthose of ARHGAP2, PTGER4, CAMK2B, CDKN1A, PLCE1, RARB, and BMP4 (Figure 6A). Among them,the remaining the expression were downregulated. levels of PTGER4 The and heatmap CDKN1A of were the 12 upregulated, genes graphically while those illustrates of the them, the expression levels of PTGER4 and CDKN1A were upregulated, while those of remainingtheir expression were downregulated.(Figure 6B), and Thethe PPI heatmap network of the for 12 these genes 12 graphicallyDEGs depicts illustrates their interac- their the remaining were downregulated. The heatmap of the 12 genes graphically illustrates expressiontion (Figure (Figure 6C). All6B), 12 and genes the showed PPI network positive for theseor negative 12 DEGs expression depictstheir induced interaction by IL-4 their expression (Figure 6B), and the PPI network for these 12 DEGs depicts their interac- (Figuretreatment6C). (Supplementary All 12 genes showed Figure positive S5A–L). or negative expression induced by IL-4 treatment tion (Figure 6C). All 12 genes showed positive or negative expression induced by IL-4 (Supplementary Figure S5A–L). treatment (Supplementary Figure S5A–L).

(A) (B) (C)

Figure 6. Features of(A the) twelve common differentrially expressed(B genes) (DEGs). (A) Expression profiles(C (red,) positively regulated; green, negatively regulated), (B) Heat map, and (C) protein-protein interaction network of the 12 common Figure 6. Features of the twelve common differentrially expressed genes (DEGs). (A) Expression profiles (red, positively FigureDEGs. 6. Features of the twelve common differentrially expressed genes (DEGs). (A) Expression profiles (red, positively regulated; green,green, negatively negatively regulated), regulated), (B ()B Heat) Heat map, map, and and (C) ( protein-proteinC) protein-protein interaction interaction network network of the of 12 the common 12 common DEGs. DEGs. Thereafter, we compared the expression of the 12 DEGs in the three podocyte groups and Thereafter,investigated we the compared genes that the showed expression IL-4 of-induced the 12 DEGs effects in thereversed three podocyteby co-treatment groups Thereafter, we compared the expression of the 12 DEGs in the three podocyte groups andwith investigated DEX. This process the genes helped that showed us to obtain IL-4-induced data on effects three reversedgenes, namely by co-treatment BMP4, RARB, with and investigated the genes that showed IL-4-induced effects reversed by co-treatment DEX.and PLCE1 This process (Figure helped 7). These us togenes obtain were data either on threepositive genes, in the namely negative BMP4, direction RARB, of and ex- with DEX. This process helped us to obtain data on three genes, namely BMP4, RARB, PLCE1pression (Figure when7 exposed). These genesto IL-4 were or exhibited either positive opposite in thedirectional negative expression direction of when expression treated andwhen PLCE1 exposed (Figure to IL-4 7). These or exhibited genes were opposite either directional positive in expression the negative when direction treated of with ex- with simultaneous DEX and IL-4. pressionsimultaneous when DEX exposed and IL-4.to IL-4 or exhibited opposite directional expression when treated

with simultaneous DEX and IL-4.

(A) (B) (C)

FigureFigure 7.7. Gene expression(A) of three genes which showed reversedrevers(B) ed expression when treatedtreated with dexamethasone(C) (A)) BMP4, (B) CAMK2B, and (C) PLCE1 (cyan: vehicle-treated control; yellow: IL-4 treated; red:IL-4+DEX-treated). Abbreviations: (FigureB) CAMK2B, 7. Gene andexpression (C) PLCE1 of three (cyan: genes vehicle-treated which showed control; revers yellow:ed expression IL-4 treated; when trea red:IL-4+DEX-treated).ted with dexamethasone Abbreviations: (A) BMP4, DEG, differentially expressed genes; IL-4, interleukin -4; DEX, dexamethasone. DEG,(B) CAMK2B, differentially and ( expressedC) PLCE1 genes;(cyan: IL-4,vehicle-treated interleukin control; -4; DEX, yellow: dexamethasone. IL-4 treated; red:IL-4+DEX-treated). Abbreviations: DEG, differentially expressed genes; IL-4, interleukin -4; DEX, dexamethasone. Overall, 12 genes—PRKCI,genes—PRKCI, PARD2,PARD2, TLN2,TLN2, WWC1,WWC1, CASK,CASK, ARHGAP2,ARHGAP2, PTGER4,PTGER4, CAMK2B, CDKN1A, PLCE1, RARB, and BMP4—were differentially expressed when CAMK2B,Overall, CDKN1A, 12 genes—PRKCI, PLCE1, RARB, PARD2, and TLN2, BMP4—were WWC1, differentiallyCASK, ARHGAP2, expressed PTGER4, when treated with IL-4. These genes were shown to be related to podocytopathy in previous CAMK2B,treated with CDKN1A, IL-4. These PLCE1, genes RARB, were shown and BMP4—were to be related differentially to podocytopathy expressed in previous when studies using human podocyte models and were also listed in PodNet. Moreover, three of treatedstudies usingwith IL-4. human These podocyte genes modelswere shown and were to be also related listed to in podocytopathy PodNet. Moreover, in previous three of them seemed to exhibit reversed expression when co-treated with DEX. studiesthem seemed using human to exhibit podocyte reversed models expression and were when also co-treated listed in withPodNet. DEX. Moreover, three of Additionally, to elucidate whether the subsequent signaling pathways were acti- themAdditionally, seemed to exhibit to elucidate reversed whether expression the subsequent when co-treated signaling with pathways DEX. were activated vated after IL-4 administration, the expression levels of IL-4 receptorα α subunit α(IL-4Rα), afterAdditionally, IL-4 administration, to elucidate the expression whether levelsthe subsequent of IL-4 receptor signalingsubunit pathways (IL-4R were), Janus acti- vatedkinase after 3 (JAK3), IL-4 administration, and the signal transducerthe expression and levels activator of IL-4 of transcription receptor α subunit 6 (STAT6) (IL-4R wereα),

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Janus kinase 3 (JAK3), and the signal transducer and activator of transcription 6 (STAT6) werefurther further analyzed analyzed in the in three the groupsthree groups (IL-4-treated, (IL-4-treated, control, control, and IL-4- and plus IL-4- DEX-co-treated plus DEX-co- treatedgroups). groups). There was There no differencewas no difference in the IL-4a in genethe IL-4a expression gene expression among the among three groups, the three but groups,JAK3 was but significantly JAK3 was significantly upregulated upregulated (activated) in(activated) the IL-4-treated in the IL-4-treated group compared group tocom- the paredcontrol to group,the control and thisgroup, upregulation and this upregulation was suppressed was suppressed by DEX addition. by DEX However,addition. How- there ever,was nothere difference was no indifference the STAT6 in genethe STAT6 expression gene (Figureexpression8A–C). (Figure 8A–C).

(A) (B) (C)

Figure 8. Gene expression of three genes which were involved in in the signaling process process of of interleukin-4 (IL-4) (IL-4) ( (AA)) IL-4R IL-4Rαα,, (B) JAK3, and ( C) STAT6 (cyan:(cyan: vehicle-treated control; yellow:yellow: IL-4 treated;treated; red: IL-4+DEX-treated). Abbreviations: Abbreviations: DEG, DEG, differentially expressed genes; IL-4, interleukin-4; DEX, dexamethasone; IL-4Rα, IL-4 receptor α subunit. differentially expressed genes; IL-4, interleukin-4; DEX, dexamethasone; IL-4Rα, IL-4 receptor α subunit.

4. Discussion 4. Discussion In this study, we analyzed the transcriptome of human podocytes and explored In this study, we analyzed the transcriptome of human podocytes and explored DEGs between the three groups treated with IL-4, IL-4 plus DEX, and the control group, DEGs between the three groups treated with IL-4, IL-4 plus DEX, and the control group, respectively. Further, we performed GO analysis to investigate the pathways related to respectively. Further, we performed GO analysis to investigate the pathways related to the the DEGs. Among the DEGs, we investigated those that were supported by a literature- DEGs. Among the DEGs, we investigated those that were supported by a literature-based based database and experimental results reported by studies conducted using human po- database and experimental results reported by studies conducted using human podocyte docytemodels. models. We also We examined also examined whether thewhether IL-4-induced the IL-4-induced expression expression levels of these levels genes of these were genesreversed were when reversed the podocytes when the were podocytes co-treated were with co-treated DEX. To with our knowledge,DEX. To our this knowledge, is the first thisstudy is the to examine first study the to effects examine of IL-4 the treatment effects of onIL-4 the treatment transcriptome on the of transcriptome human podocytes. of hu- man podocytes.We identified various biological pathways activated in podocytes after IL-4 treatment. A fewWe notable identified cell-signaling various biological pathways, pathways including activated extracellular in podocytes signal-regulated after IL-4 kinasetreat- ment.(ERK), A a few member notable of cell-signaling the family of pathways, mitogen-activated including protein extracellular kinases signal-regulated along with c-Jun ki- naseN-terminal (ERK), a kinase, member were of the identified. family of The mitogen-activated ERK pathway has protein been kinases implicated along in with podocyte c-Jun N-terminalinjury in the kinase, progression were ofidentified. various glomerulopathiesThe ERK pathway [32 has]. One been study implicated examined in podocyte the phos- injuryphorylation in the progression of ERK in rat of puromycinvarious glomerul aminonucleosideopathies [32]. nephropathy One study examined (PAN) model the phos- and phorylationmouse podocytes of ERKin in vitro rat andpuromycin concluded aminonucleoside that the sustained nephropathy activation (PAN) of ERK model was a and cru- mousecial process podocytes in infliction in vitro ofand podocyte concluded injury that [th33e]. sustained In our study, activation genes of regulating ERK was a the crucial ERK processpathway in wereinfliction positively of podocyte expressed injury in [33]. the IL-4-treatedIn our study, podocytes genes regulating compared the toERK controls, path- waysupporting were positively the results expressed of a previous in the study IL-4-treated [32], which podocytes suggested compared that ERK to activation controls, might sup- portingbe an important the results mechanism of a previous resulting study in [32], IL-4-induced which suggested podocyte that damage. ERK Similarly,activation TGF- mightβ1 besignaling an important was also mechanism shown to resulting be increased in IL-4 in-induced podocytes podocyte from PAN damage. rats, suggestingSimilarly, TGF- that βTGF-1 signalingβ1 might was be also involved shown in to the be increased pathogenic inmechanisms podocytes from of podocytopathies PAN rats, suggesting [33]. Ourthat TGF-resultsβ1 weremight in be line involved with those in the reported pathogenic by a previousmechanisms study, of podocytopathies which stated that [33]. TGF-ß Our 1 resultssignaling were increased in line with in IL-4-treated those reported cells by compared a previous to controls.study, which Notably, stated the that expression TGF-ß 1 signalinglevels of theseincreased two pathwaysin IL-4-treated were cells upregulated compared compared to controls. to controlsNotably, in the both expression IL-4-treated lev- elsgroups of these with two or without pathways DEX were addition, upregulated indicating compared that DEX to might controls not in have both inhibited IL-4-treated these groupschanges. with The or common without pathwaysDEX addition, related indicating to DEGs that between DEX might the three not groupshave inhibited included these the changes.Notch signaling The common pathway, pathways which isrelated related toto DEGs cytoskeleton between organization.the three groups The included activation the of Notchthe Notch signaling pathway pathway, in podocytes which is has related been to suggested cytoskeleton to be organization. involved in glomerular The activation injury. of Niranjan et al. found that expression levels of genes belonging to the Notch pathway

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J. Clin. Med. 10 2021, , 496 the Notch pathway in podocytes has been suggested to be involved in glomerular injury.12 of 18 Niranjan et al. found that expression levels of genes belonging to the Notch pathway were regulated in patients and animal models of renal disease [34], showing that mice with were regulated in patients and animal models of renal disease [34], showing that mice conditionalwith conditional expression expression of active of Notch active 1 Notchprotein 1 presented protein presented with massive with albuminuria, massive albumin- glo- merulosclerosis,uria, glomerulosclerosis, and renal andfailure renal [34]. failure In support [34]. In of support this, the of this,IL-4-treated the IL-4-treated group, com- group, paredcompared to controls, to controls, showed showed negatively negatively expressed expressed DEGs DEGsin the inpathways the pathways associated associated with renalwith development renal development and morphogenesis, and morphogenesis, such as such nephron as nephron and tubule and tubule morphogenesis morphogenesis and cytoskeletonand cytoskeleton organization. organization. Our results Our imply results that imply IL-4 that may IL-4 induce may podocytopathy induce podocytopathy involv- inginvolving the activation the activation of the Notch of the signaling Notch signaling pathway, pathway, which might which downregulate might downregulate the func- the tionsfunctions associated associated with cytoskeleton with cytoskeleton structur structures.es. Our experimental Our experimental study studyon intracytoplas- on intracyto- micplasmic actin filaments actin filaments in podocytes in podocytes treated treated with IL-4 with also IL-4 supports also supports the results the resultsobtained obtained from GOfrom pathway GO pathway analysis, analysis, in that inIL-4 that treatment IL-4 treatment may affect may the affect levels the levelsof major of majorproteins proteins that constitutethat constitute the cytoskeleton. the cytoskeleton. ByBy comparing comparing the the common common DEGs DEGs with with genes genes listed listed in in the the PodNet PodNet database database and and those those previouslypreviously reported reported in in relation relation to to podocytopathy, podocytopathy, we we finally finally selected selected 12 12 genes, genes, namely namely PTGER4PTGER4, ,CDKN1ACDKN1A,, PARD3PARD3, PRKCI, PRKCI, WWC1, WWC1, CASK, CASK, TLN2, ,TLN2PLCE1, ,PLCE1RARB,, BMP4RARB, ARHGAP2, BMP4, , ARHGAP2and CAMK2B, and. TheCAMK2B expression. The levelsexpression of the levels first two of the genes, firstPTGER4 two genes,and CDKN1APTGER4 ,and were CDKN1Aupregulated,, were and upregulated, those of the and remaining those of werethe re downregulated.maining were downregulated. Figure9 summarizes Figure the 9 summarizesproposed mechanism the proposed of IL-4-induced mechanism podocyteof IL-4-induced injury involving podocyte these injury genes involving and pathways these genesimplicated and pathways in GO analyses. implicated in GO analyses.

FigureFigure 9. 9.ProposedProposed mechanisms mechanisms of IL4-induce of IL4-inducedd podocyte podocyte injury. injury. Abbreviations: Abbreviations: α-ACT,α-ACT, alpha alpha ac- tinin;actinin; ARHGAP24, ARHGAP24, Rho RhoGTPase-activating GTPase-activating protein protein 24; Akt, 24; Protein Akt, Protein kinase kinase B; BMP4, B; BMP4, Bone Morpho- Bone Mor- geneticphogenetic Protein Protein 4; CAMK2B, 4; CAMK2B, Calcium/Calmodulin Calcium/Calmodulin Dependent Dependent Protein Kinase Protein II KinaseBeta; CASK, II Beta; periph- CASK, eralperipheral plasma membrane plasma membrane protein; protein;CDK1A, CDK1A, cyclin-dep cyclin-dependentendent kinase inhibitor kinase inhibitor 1A; EMT, 1A; epithelial-mes- EMT, epithelial- enchymal-transition; ERK, extracellular signal-regulated kinases; GTP, guanosine triphosphate; IL- mesenchymal-transition; ERK, extracellular signal-regulated kinases; GTP, guanosine triphosphate; 4, interleukin-4; IL-4Rα, IL-4 receptor alpha; JAK, Janus kinase; MAGI2, membrane associated α guanylateIL-4, interleukin-4; kinase inverted-2; IL-4R , MAPK, IL-4 receptor mitogen-acti alpha;vated JAK, Janusprotein kinase; kinase; MAGI2, mTOR, membrane mammalian associated target ofguanylate rapamycin; kinase PARD3, inverted-2; partitioning MAPK, defective mitogen-activated 3; PI3k, phosphoinositide protein kinase; 3- mTOR,kinase; mammalian PLCE1, phospho- target of lipaserapamycin; C epsilon-1; PARD3, PTGER4, partitioning prostaglandin defective E 3; receptor PI3k, phosphoinositide 4; RARB, retinoic 3-kinase; acid receptor PLCE1, beta; phospholipase STAT6, signalC epsilon-1; transducer PTGER4, and activa prostaglandintor of transcription E receptor 6; 4; TGF- RARB,β, transforming retinoic acid growth receptor factor beta; STAT6,beta; VEGF, signal vasculartransducer endothelial and activator growth of factor; transcription WWC1, 6; WW TGF- doβ,main-containing transforming growth protein factor 1 (a.k.a. beta; kidney VEGF, vascu-and brainlar endothelialscaffolding growthprotein factor;(KIBRA). WWC1, WW domain-containing protein 1 (a.k.a. kidney and brain scaffolding protein (KIBRA).

Of the two positively expressed DEGs, PTGER4 (prostaglandin E receptor 4) encodes for prostaglandin (PG) E4, and PGE2 is known to induce cyclooxygenase (COX)-2 expres- sion in podocytes via the PGE4 receptor [35,36]. Enhanced expression of E-prostanoid receptor 4 (EP4) in cultured podocytes was shown to negatively affect podocyte adapta-

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tion to mechanical stretch [37], while the inhibition of EP4 attenuated the development of nephropathy in rodent models [38,39]. Additionally, EP4 inhibition prevented the TGF-ß1-induced dedifferentiation of glomerular podocytes [39]. In our study, the marked upregulation of PTGER4 expression suggests that IL-4 may have triggered the EP4-induced pathogenic process. CDKN1A encodes cyclin-dependent kinase inhibitor 1A. In the kid- ney, the expression of cyclin I and p35 is specifically restricted to podocytes, and they limit podocyte apoptosis by activating cyclin-dependent kinase (Cdk)-5 [40,41]. Cyclin I functions as a cell survival factor by limiting apoptotic cell death in podocytes following injury [41]. Taniguchi et al. investigated their roles in a double cyclin I-p35 null murine model and demonstrated that the mutants showed markedly increased podocyte apoptosis when exposed to stress, which resulted in podocyte loss, proteinuria, and glomerulosclero- sis [40]. They suggested that the activators of Cdk5, p35, and cyclin I might play a critical role in maintaining podocyte survival during stress. In support of this, our results showed the upregulation of CDKN1A expression, suggesting that increased cyclin I expression might be considered as a response to IL-4-induced stress injury. We also observed that expression levels of 10 DEGs were downregulated. Partitioning defective 3 (PARD3) encodes polarity protein partitioning defective-3 protein, and polarity signaling through the atypical protein kinase C (aPKC), a par-polarity complex, is essential for the development and maintenance of podocyte architecture [42]. PARD3 has been sug- gested to play a functional role in the slit diaphragm [42], and its defect causes podocyte detachment [43]. In our study, PARD3 expression was downregulated by IL-4 treatment, which suggested that IL-4 might cause a loss of polarity in podocytes. Along with PARD3, the aPKC complex is an essential regulator of podocyte morphology [43]. Protein kinase C Iota (PRKCI), which encodes protein kinase C iota, is an aPKC enzyme that contributes to cell proliferation [44]. As mentioned, structural maintenance of the slit diaphragm seems to be tightly regulated by polarity signaling pathways, and aPKC is an essential regulator of podocyte morphology [45]. It has been shown that aPKC interacts with the slit diaphragm complex and plays a critical role in the maintenance of the slit diaphragm and podocyte foot process [46]. The downregulation of PRKCI expression along with PARD3 expression in our study suggests that IL-4 may induce dysregulation of critical polarity regulators. WW domain-containing protein 1 (WWC1), also known as kidney and brain scaffolding protein (KIBRA), is predominantly expressed in the kidney [47]. The knockdown of WWC1 in an in vitro model promoted podocyte apoptosis by inducing nuclear relocation of den- drin protein [48] and impaired directed cell migration [49]. Conversely, KIBRA has also been shown to promote podocyte injury by inhibiting YAP signaling, by disrupting actin cytoskeletal dynamics [50], and by interacting with synaptopodin [51]. While the published results on the role of WWC1 in podocytes appear contradictory, WWC1 expression was downregulated in the present study. CASK is a calcium/calmodulin-dependent serine protein kinase (CASK), a scaffolding protein that participates in the maintenance of po- larized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton [52]. CASK has been shown to induce proteinuria and podocyte foot process effacement in a mouse model [53]. Circulating CASK has also been associated with post-transplantation recurrence of focal segmental glomerulosclerosis (FSGS) [53]. It may be interpreted that the downregulation of CASK expression by IL-4 treatment results in the impaired maintenance of epithelial structures. TLN2 is one of the two talin genes that vertebrates possess, encoding talin-2 [54]. Talin-1 and talin-2 are cytoplasmic adapter proteins that are essential for integrin-mediated cell adhesion to the extracellular matrix and signaling via interaction with kidney ankyrin repeat-containing protein (KANK) [54]. Talins have been proposed to activate integrins and link them to the actin cytoskeleton. Mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells of these animals exhibited severe cytoskeletal adhesion, and polarity defects [55]. We speculate that the downregulation of TLN2 expression by IL-4 treatment in this study supports the previous observations and our experiment, which highlights a decreased number of actin filaments after IL-4 treatment. ARHGAP24 is a kidney structure-related J. Clin. Med. 2021, 10, 496 14 of 18

gene encoding Rho-GAP 24, and its expression is upregulated during podocyte differentia- tion [56]. Knockdown of ARHGAP24 results in increased levels of active Rac1 and Cdc42, which consequently induces NS [56]. It could be suggested that IL-4 treatment decreased the expression of ARHGAP24 in this study and caused podocyte injury. The gene CAMK2B encodes calcium/calmodulin-dependent protein kinase 2 (CaMK II), which participates in calcium signaling in podocyte injury [57]. CaMK II is involved in angiotensin (Ang) II-induced podocyte injury [58]. Specifically, Ang II can promote canonical Wnt upregula- tion, which is mediated by CaMK II/CREB signaling activation, and the inhibition of this signaling cascade ameliorated Ang II-induced podocyte injury in vitro and in vivo [58]. In this study, CAMK2B expression was most remarkably downregulated. Notably, the IL-4-induced expression of three genes (PLCE1, RARB, and BMP4) was reversed when podocytes were co-treated with DEX. PLCE1 encodes phospholipase C epsilon. The gene has been identified to cause monogenic NS, and affected individuals present with histologic characteristics of FSGS or diffuse mesangial sclerosis (DMS) [59,60]. PLCE1 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is known to directly interact and co-localize with nephrin in podocytes [59]. The absence of PLCE1 expression halted glomerular development in a rodent model [59]. The downregulation of PLCE1 expression reported in our study supports previous studies on exploration of podocyte changes, and IL-4 treatment may have induced this process. RARB encodes the retinoic acid receptor (RAR) beta, and nuclear receptors, including retinoic acid receptor (RAR), have been proposed to play a protective role in podocytes [61]. Using an RAR ß2 agonist improved podocyte effacement in a diabetic nephropathy model [62]. In our study, the downregulated expression of RAR during IL-4 treatment supported this finding. BMP4 encodes bone morphogenetic protein 4, which exerts multiple biological effects on the morphogenesis of the kidney and ureters [63]. Podocyte-derived BMP reportedly plays an important role in glomerular capillary formation [63]. In our study, IL-4 decreased BMP4 expression. The fact that PLCE1 expression was reversed by steroid treatment is of clinical interest because PLCE1 expression is known to cause monogenic NS, which is theoretically not immune-based [59]. Downregulated expression of RARB, which plays a protective role in podocytes, and BMP4, which is involved in kidney morpho- and angiogenesis, were also reversed by DEX treatment. Our findings suggest that these three DEGs could be implicated in the pathophysiology of NS in response to steroids. Our study has several limitations. First, as our study was not a proof-of-concept study for targeted genes with a particular hypothesis but was rather a screening for the potential genes expressed in human podocytes affected by IL-4 treatment, the results of RNA sequencing were not confirmed by targeted qPCR and/or western blotting. This process of validation is warranted to elucidate the functional significance of the genes. The present study can only propose that these genes are possibly implicated in the IL- 4-induced pathogenesis of NS. Second, IL-4 and DEX were not investigated for dose or time-dependence. We used a dose of 10 ng/mL for IL-4, which is known to cause NS in animal models, but higher doses of IL-4 might have led to the induction of pronounced changes in DEGs in podocytes. Likewise, the protective effect of DEX on IL-4-induced changes may have resulted in different outcomes at different doses. Additionally, the podocytes were uniformly incubated for 6 h after treatment in this study. Analyses at different time points would have yielded different results. Nevertheless, this study demonstrated a negative effect of IL-4 exerted on human podocytes, which supported its potentially pathogenic role in NS, as suggested in the literature. First, immunohistochemistry revealed that incubation with IL-4 for 6 h led to a decrease in the number of actin filaments. Second, transcriptome analysis showed that IL-4 induced changes in gene expression. Third, genes that were supported by previous evidence were identified. The expression of three of these genes could be reversed by simultaneous steroid treatment. Since MCNS is usually responsive to steroid treatment, we suggest that these three genes may play a role in its pathogenesis. Further in vitro J. Clin. Med. 2021, 10, 496 15 of 18

and in vivo studies are necessary to investigate the mechanisms by which the aberrant expression of these genes induces podocytopathy.

5. Conclusions In this study, we propose few genes of interest that have possible implications in IL-4-induced pathogenesis of NS. RNA sequencing revealed that IL-4 induced changes in the expression of genes in human podocytes. GO analysis revealed certain pathways of relevance in NS, such as pathways related to podocyte cytoskeleton organization. Based on the DEG analysis, 12 genes were found to be involved in IL-4-induced changes in podocytes, and 3 genes, BMP4, RARB, and PLCE1, showed reversed expression when the podocytes were co-treated with IL-4 and DEX. The clinical significance of these genes remains to be explored; however, they might be involved in the induction of MCNS, as their expression levels were reversed by steroid treatment. This study suggests that molecular players may be involved in the induction of IL-4 mediated podocyte changes in an in vitro setting.

Supplementary Materials: The following are available online at https://www.mdpi.com/2077-0 383/10/3/496/s1, Table S1: 22 genes overlapping with 380 genes differentially identified between the IL-4- and vehicle-treated groups and with genes listed in PodNet; Figure S1: Heatmap of human podocytes whole-transcriptome sequencing of genes which showed top variance between the IL-4/IL- 4+DEX treated and control groups of podocytes; Figure S2: Top-listed DEGs that were (A) negatively and (B) positively regulated in IL-4-treated-podocytes compared to vehicle-treated controls; Figure S3: Top-listed DEGs which were (A) negatively and (B) positively changed in IL-4 treated-podocytes compared to IL-4 plus dexamethasone treated podocytes; Figure S4: (A) Heatmap of 22 genes overlapping with 176 common differentially identified genes (DEGs) and genes listed in the PodNet, (B) positive- or negative expression of the 22 DEGs, (C) Protein-protein interaction network for the 22 DEGs; Figure S5: Gene expression levels of the following 12 DEGs among the three groups. Author Contributions: Conceptualization, J.I.S.; methodology, J.I.S., Y.K., C.H.L., N.J., B.J.L.; soft- ware, Y.K. and C.H.L.; validation, J.I.S. and B.J.L.; formal analysis, Y.K. and J.M.L.; resources, M.A.S. and J.I.S.; data curation, Y.K. and J.M.L.; writing—original draft preparation, J.M.L.; writing—review and editing, J.I.S., J.O., A.K., M.A.S., K.H.L., J.M.L.; supervision, J.I.S. and B.J.L.; funding acquisition, J.I.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a faculty research grant from Yonsei University College of Medicine for 2015 (6-2015-0042, to J.I.S.), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03032457) and the Hankuk University of Foreign Studies Research Fund (to Y.K.). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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