Epigenetic regulation of RCAN1 expression in kidney disease and its role in podocyte injury

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Epigenetic regulation of RCAN1 expression in kidney disease and its role in podocyte injury Huilin Li1,2,5, Weijia Zhang1,5, Fang Zhong1,5, Gokul C. Das3, Yifan Xie1, Zhengzhe Li1, Weijing Cai1, Gengru Jiang2, Jae Choi3, Mohamad Sidani3, Deborah P. Hyink3, Kyung Lee1, Paul E. Klotman3, and John Cijiang He1,4 1 Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA; 2Division of Nephrology, Department of Medicine, Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China; 3 Division of Nephrology, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA; and 4Kidney Center at James J. Peters VA Medical Center, Bronx, New York, USA

Mounting evidence suggests that epigenetic modification is important in kidney disease pathogenesis. To determine whether epigenetic regulation is involved in HIV-induced kidney injury, we performed genome-wide methylation profiling and transcriptomic profiling of human primary podocytes infected with HIV-1. Comparison of DNA methylation and RNA sequencing profiles identified several genes that were hypomethylated with corresponding upregulated RNA expression in HIV-infected podocytes. Notably, we found only one hypermethylated gene with corresponding downregulated RNA expression, namely regulator of calcineurin 1 (RCAN1). Further, we found that RCAN1 RNA expression was suppressed in glomeruli in human diabetic nephropathy, IgA nephropathy, and lupus nephritis, and in mouse models of HIV-associated nephropathy and diabetic nephropathy. We confirmed that HIV infection or high glucose conditions suppressed RCAN1 expression in cultured podocytes. This suppression was alleviated upon pretreatment with DNA methyltransferase inhibitor 5-Aza-20 -deoxycytidine, suggesting that RCAN1 expression is epigenetically suppressed in the context of HIV infection and diabetic conditions. Mechanistically, increased expression of RCAN1 decreased HIV- or high glucose–induced nuclear factor of activated T cells (NFAT) transcriptional activity. Increased RCAN1 expression also stabilized actin cytoskeleton organization, consistent with the inhibition of the calcineurin pathway. In vivo, knockout of RCAN1 aggravated albuminuria and podocyte injury in mice with Adriamycin-induced nephropathy. Our findings suggest that epigenetic suppression of RCAN1 aggravates podocyte injury in the setting of HIV infection and diabetic nephropathy. Kidney International (2018) j.kint.2018.07.023

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https://doi.org/10.1016/

Correspondence: John Cijiang He, Division of Nephrology, Box 1243, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA. E-mail: [email protected]; or Paul E. Klotman, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: [email protected] 5

HL, WZ, and FZ contributed equally to the work.

Received 23 September 2017; revised 26 June 2018; accepted 19 July 2018 Kidney International (2018) -, -–-

KEYWORDS: calcineurin inhibitors; diabetic nephropathy; HIV-1; NFAT; podocyte; RCAN1 Copyright ª 2018, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

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ecent studies suggest that epigenetic regulation, such as DNA methylation, histone modification, and microRNA-mediated gene regulation, play a major role in the pathogenesis of kidney diseases, such as diabetic nephropathy (DN).1 Epigenetic alterations in DN are supported by the Diabetes Control and Complications Trial/ Epidemiology of Diabetes Interventions and Complications study, in which participants were followed up for more than 30 years after being previously assigned to conventional therapy or intensive therapy for diabetes.2 Participants assigned to conventional therapy continued to experience complications at significantly higher rates than the intensive therapy group, despite nearly similar HbA1c levels. This persistence of benefit from early therapy suggested “metabolic memory,” potentially as a consequence of epigenetic modifications induced by transitory hyperglycemia. More recent study further demonstrated that DNA methylation differences during the Diabetes Control and Complications Trial stage persisted at certain loci associated with hyperglycemia for several years during the Epidemiology of Diabetes Interventions and Complications study, further supporting the involvement of epigenetic regulation in metabolic memory.3 Similarly, HIV-infected patients undergoing antiretroviral therapy continue to experience chronic diseases, including kidney disease, potentially as a consequence of persistent chronic inflammation.4 One mechanism of chronic inflammation may stem from the HIV-induced epigenetic changes during the acute infection phase.5–7 In support of this notion, Chandel et al8 showed that HIV infection leads to the repression of vitamin D receptor expression in podocytes, which was associated with hypermethylation of the vitamin D receptor promoter. However, the exact mechanism of epigenetic regulation in HIV kidney disease remains unclear. To better understand the epigenetic changes associated with podocyte injury in HIV kidney disease, we performed genome-wide DNA methylation profiling arrays in 1

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conjunction with transcriptomic analysis of HIV-1–infected primary human podocytes. Among the differentially expressed genes, we identified regulator of calcineurin-1 (RCAN1), which was the only hypermethylated gene with decreased RNA expression after the HIV infection. RCAN1 belongs to the RCAN family of small evolutionarily conserved proteins made up of RCAN1, RCAN2, and RCAN3 that directly bind to and inhibit calcineurin.9 Calcineurin is a calcium- and calmodulin-dependent serine/ threonine phosphatase that participates in a number of cellular processes including T-cell activation, neuronal apoptosis, and remodeling of cardiomyocytes.10,11 Calcineurin-dependent signals are mainly transduced to the nucleus by NFAT, which upon dephosphorylation by calcineurin translocates to the nucleus to mediate the transcription of downstream genes. RCAN1 inhibits the calcineurin/NFAT signaling pathway by binding to and blocking the catalytic activity of calcineurin and by dephosphorylation of NFAT.12 Calcineurin is also an important modulator of podocyte and glomerular function. Calcineurin inhibitors have been used to treat primary glomerular disease.13 In addition to their immunosuppressive effects, inhibition of calcineurin has a direct antiproteinuric effect through stabilization of the podocyte actin cytoskeleton.14 In addition, a critical role of NFAT in podocyte injury and kidney disease has been reported recently.15–18 Thus we sought to better understand the role of RCAN1, a key inhibitor of calcineurin and NFAT, in podocyte injury and glomerular disease. Our data suggest that RCAN1 expression is epigenetically suppressed in podocytes in glomerular disease and that decreased RCAN1 contributes to podocyte injury through the dysregulation of the calcineurin-NFAT pathway. This finding is further supported by in vivo evidence in which genetic ablation of Rcan1 in mice aggravates podocyte and glomerular injury in doxorubicin (Adriamycin)-induced glomerulosclerosis. RESULTS HIV-1 infections alter the DNA methylation profiles in primary human podocytes

To characterize HIV-1–induced epigenetic and transcriptional dysregulation in podocytes, podocyte-enriched primary cell cultures established from nephrectomy samples (n ¼ 6) were infected with replication-incompetent HIV-1 virus (vesicular stomatitis virus [VSV]-pseudotyped pNL4-3:DG/P-EGFP) or control enhanced green fluorescent protein (EGFP) virus.19 Genomic DNA was extracted from the infected cells, and methylation profiling was performed using the Infinium HumanMethylation450K BeadChip (Illumina, San Diego, CA; “chip”). A total of 703 differentially methylated sites

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(515 hypermethylated and 188 hypomethylated) on the autosomal chromosomes were identified in HIV-infected versus control virus-infected podocyte cultures using a paired LIMMA test with a P value <0.05 (Figure 1a). Compared with the distribution of all methylation sites on genomic loci represented on the chip, the hypermethylated sites were enriched in the gene body region (51% vs. 22%, Figure 1b). The functions of hypomethylated genes included cell cycle, division, regulation of transcription, and DNA repair (Figure 1c), all of which are critical pathways known to be altered in HIV-associated nephropathy (HIVAN).20,21 Hypermethylated gene functions included vesicle-mediated transport, exocytosis, and lipid transport (Figure 1d), which are important features of podocyte function.22,23 HIV-1 infection alters the transcriptomic profiles in primary human podocytes

To correlate the DNA methylation changes with mRNA expression levels, transcriptomic profiling was also performed on mRNAs isolated from aforementioned podocyte cultures 48 hours after infection with either HIV-1 or control virus. Differential analysis with a paired LIMMA test (with false discovery rate–adjusted P value <0.05) identified 663 dysregulated genes (564 upregulated and 99 downregulated genes; Figure 2a and Supplementary Data Files S1 and S2). Top upregulated genes included sialic acid–binding Ig-like lectin 15 (SIGLEC15) involved in immune response and ret proto-oncogene (RET) and epithelial membrane protein 1 (EMP1), which are involved in cell proliferation and growth. Top downregulated genes included endothelin 2 (END2), encoding a member of the endothelin protein family of secretory vasoconstrictive peptides,24 and transforming growth factor-b2 (TGFB2) (Figure 2b). Gene ontology and canonical pathway enrichment analyses both indicated that the genes involved in lipid and fatty acid metabolism were upregulated in HIV-1–infected podocytes (Figure 2c and d and Supplementary Data Files S3 and S4). Other enriched functional categories included cell growth, activation of protein kinase activity, extracellular matrix organization, and cell differentiation (Figure 2c and Supplementary Data File S3), all of which are key features involved in the pathogenesis of HIVAN.25 The canonical pathways of adipocytokine, integrin, and peroxisome proliferator–activated receptor signaling pathways were also enriched (Figure 2d and Supplementary Data File S4). The adipocytokine and peroxisome proliferator–activated receptor signaling pathways are highly regulated in DN.26,27 Together with the aforementioned metabolic pathways, our results suggest an interaction between HIV infection and metabolic dysregulation as observed in diabetes-induced podocyte and kidney injury.

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Figure 1 | Profiling of DNA methylation in HIV-infected podocytes. Identification of differentially methylated sites on autosomal chromosomes in HIV-infected primary podocytes by Infinium HumanMethylation450K BeadChip (“chip”). (a) Heatmap of hypo- and hypermethylated genes in HIV-1 infected podocytes compared with green fluorescent protein control virus–infected podocytes. (b) Distribution of genomic location of hypomethylated sites (top), hypermethylated sites (middle), or all sites (bottom). (c,d) Gene ontology enrichment analysis of genes harboring the hypomethylated (c, green bar) or hypermethylated (d, red bar) sites. X axis denotes the –log10 of enrichment P values by Fisher exact test. Kidney International (2018) -, -–-

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Figure 2 | Profiling of mRNA expression in HIV-1 infected podocytes. Identification of dysregulated genes in HIV-1-infected podocytes by RNA sequencing. (a) Heatmap of up- and downregulated genes in HIV-1–infected podocytes (H1-H6) compared with enhanced green fluorescent protein (eGFP) control virus–infected podocytes (E1-E6). (b) Volcano plot of dysregulated genes showing top up- and downregulated genes (the X axis denotes the log2 fold change of gene expression in HIV infection compared with the GFP control virus, and the Y axis denotes the false discovery rate adjusted P values). (c) Gene ontology enrichment analysis of dysregulated genes is shown. The X axis denotes the –log10 enrichment P values by Fisher exact test. The pink and green bars represent the portion of up- and downregulated genes. (d) Canonical pathway enrichment analysis of dysregulated genes. The pathways were compiled from multiple sources including KEGG, Reactome, Biocarta, WiKi, PID Ingenuity, and Panther databases. 4

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Comparison of transcriptomic and DNA methylation profiles in HIV-infected podocytes reveals suppression of RCAN1 expression

We next identified genes with a correlation of DNA methylation to mRNA expression in HIV-1– infected podocytes. The criteria for selection of genes for this correlation were as follows: (i) at least 10% methylation level change, (ii) LIMMA

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Figure 3 | Comparison between DNA methylation and mRNA expression profiles in HIV-1 infected podocytes. Correlation of gene expression dysregulation and epigenetic changes in HIV infected podocytes. (a) The heatmap of 20 hypomethylated and upregulated genes and 1 hypermethylated and downregulated gene (methylation on the left panel and gene expression on the right panel). (b) Integrated Genomics Viewer display of epigenetic changes for RCAN1. The heatmap of hypermethylation in the promoter and gene body is shown in red vertical bars. The bottom panel shows the read coverage of RNA sequencing of an HIV-infected sample. (c) Real-time polymerase chain reaction analysis of RCAN1 expression in human podocytes infected with control enhanced green fluorescent protein (EGFP) lentivirus or with NL43Dgag-pol HIV-1 pseudovirus. The P value from the unpaired 2-tailed t test is indicated (n ¼ 6). Kidney International (2018) -, -–-

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macrophages, and monocytes.28 Notably, only 1 gene was found to be hypermethylated and correspondingly downregulated at the RNA level: regulator of calcineurin-1 (RCAN1; Figure 3a). In HIV-infected primary podocytes, RCAN1 was hypermethylated in multiple sites in the promoter and in the gene body region (Figure 3b). Because calcineurin pathway plays a critical role in podocyte biology and kidney disease,14 we selected RCAN1 for further studies. The reduction of RCAN1 mRNA levels in HIV-infected human podocytes was further confirmed by real-time polymerase chain reaction analysis (Figure 3c).

infection or a high level of glucose can suppress RCAN1 expression in cultured human podocytes.31 High glucose treatment indeed reduced RCAN1 mRNA and protein levels in comparison with normal glucose or high mannitol treatment (Figure 5a, b, and c), whereas pretreatment with DNA methyltransferase inhibitor 5-Aza-20 -deoxycytidine restored its expression. Similarly, transient infection of HIV-1 in podocytes suppressed RCAN1 expression, which was restored by pretreatment with 5-Aza-20 -deoxycytidine (Figure 5d and e). These results are consistent with the suppression of RCAN1 expression via DNA methylation.

RCAN1 expression is decreased in glomerular diseases

RCAN1 regulates NFAT activity and its downstream gene expression in podocytes

To determine whether the changes in RCAN1 expression occur in human glomerular diseases, its mRNA expression was queried through the Nephroseq database (nephroseq. org). Indeed, RCAN1 expression was reduced in the glomeruli in the context of multiple kidney diseases including DN, IgA nephropathy, and lupus nephritis (Supplementary Figure S1A, B, and C). In addition, Rcan1 expression was reduced in isolated glomeruli of diabetic db/db mice in comparison with the control db/m mice29 (Nephroseq dataset; Supplementary Figure 1D). Consistent with this, Rcan1 mRNA was also reduced in isolated podocytes from streptozotocin-induced diabetic endothelial nitric oxide synthase-/- mice according to our previous RNA sequencing data30 (Figure 4a). Although human transcriptomic data are not yet available for HIVAN kidneys, we observed that Rcan1 mRNA expression was suppressed in the glomeruli of Tg26 HIVAN mice by quantitative polymerase chain reaction (Figure 4b). Immunofluorescence staining in the frozen sections from nephrectomy samples showed that much of RCAN1 expression co-localized with podocyte marker synaptopodin (Figure 4c). Moreover, consistent with aforementioned RNA data, RCAN1 expression was diminished in glomeruli of patient biopsy samples of DN, IgA nephropathy, and lupus nephritis, and in HIVAN (Figure 4d and e). Together, these data show that decreased RCAN1 expression is associated with the development and progression of multiple primary and secondary glomerular diseases.

To determine the role of RCAN1 in kidney cells, we increased expression of RCAN1 with overexpression of FLAG-tagged hRCAN1 (RCAN1OE) or decreased its expression with short hairpin RNA–mediated knockdown (RCAN1KD) in HEK293T cells (Figure 6a). Overexpression of control EGFP protein (EGFPOE) or transfection of scrambled shRNA expressing vector was used as negative control (ScrKD). Cells were then transfected with an NFAT-luciferase reporter and treated with normal glucose, high mannitol, or high glucose; or infected with HIV-1 or control virus. Measurement of NFAT-luciferase activity confirmed that both HIV infection and high glucose induced NFAT transcriptional activity, which was inhibited by overexpression of RCAN1 but was further enhanced by knockdown of RCAN1 expression (Figure 6a and b; NFATLuciferase activity in EGFPOE and ScrKD were indistinguishable from other control cells, and thus only 1 representative “control” is shown). Next we determined NFAT target gene expression by real-time polymerase chain reaction in HIV-1 infected or high glucose–treated human podocytes with RCAN1 overexpression or knockdown. Consistent with the aforementioned luciferase data, RCAN1 overexpression suppressed NFAT target gene expression of CXCL2, NFAT, and Wnt6 in HIV-1–infected or high glucose–treated podocytes (Figure 6d), and knockdown of RCAN1 further enhanced their expression (Figure 6e). These data suggest that RCAN1 regulates HIV-1– and high glucose–induced NFAT transcriptional activation in podocytes.

RCAN1 is epigenetically regulated by HIV-1 and high glucose in podocytes

RCAN1 regulates actin cytoskeleton in podocytes

Because the aforementioned data showed that RCAN1 expression is decreased in both diabetic and HIV-1– infected kidneys, we next determined whether HIV-1

It was previously shown that calcineurin inhibitors help stabilize actin cytoskeleton in injured podocytes.14 Therefore, we determined whether RCAN1 could stabilize the podocyte actin

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Figure 4 | RCAN1 is reduced in various glomerular diseases. (a) RCAN1 mRNA is reduced in sorted podocytes from diabetic endothelial nitric oxide synthase (eNOS)–null mice compared with nondiabetic podocytes according to our previous RNA sequencing dataset30 (n ¼ 3 samples, where each sample is pooled from 3–4 mice). (b) Quantitative polymerase chain reaction analysis shows reduced Rcan1 mRNA in the glomeruli of HIV-1 transgenic mice (Tg26) compared with glomeruli of control wild-type (WT) littermates (n ¼ 7). (c) Immunofluorescence staining for RCAN1 and podocyte marker synaptopodin in the kidney sections from normal nephrectomy samples. DNA is counterstained with 40 6-diamidino-2-phenylindole (DAPI). Bar ¼ 50 mm. (d,e) Representative immunostaining of RCAN1 (d) shows reduced RCAN1 expression in the kidney sections of patients with diabetic nephropathy (DN), IgA nephropathy (IgAN), lupus nephritis (LN), and HIV-associated nephropathy (HIVAN) compared with kidneys of normal nephrectomy samples (original magnification 400; bar ¼ 50 mm). Semi-quantification of immunostaining is shown in (e) (n ¼ 5, *P < 0.05, ***P < 0.001, and ****P < 0.0001 compared with nephrectomy control). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International (2018) -, -–-

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organization through the inhibition of the calcineurin pathway. Both HIV-1 infection and high glucose treatment reduced actin stress fibers in cultured podocytes (Figure 7a and b). Overexpression of RCAN1 restored stress fiber formation, whereas RCAN1 knockdown further reduced stress fiber formation in HIV-1–infected or high glucose–treated enriched podocytes. These data are consistent with stabilization of the actin cytoskeleton by RCAN1 by suppression of the calcineurin signaling pathway. Apoptosis also increased in podocytes cultured in high glucose conditions, which was attenuated in RCAN1 overexpressing podocytes but further exacerbated in RCAN1 knockdown cells, as assessed by active Caspase-3 concentration (Figure 7c). RCAN1 loss aggravates glomerular disease and podocyte injury in vivo

Loss of RCAN1 expression in Rcan1-null mice (Rcan1-/-) was confirmed by Western blot analysis of kidney cortices and immunofluorescence staining of RCAN1 (Figure 8a and b). Rcan1-/- mice did not exhibit any obvious abnormal renal phenotypes at baseline when checked up to 6 months of age. To ascertain the effect of RCAN1 loss in Rcan1-/- mice, we induced Adriamycin nephropathy (ADRN)32 in Rcan1-/- and their wildtype littermates (Rcan1þ/þ). We found that Rcan1-/- mice experienced worsened albuminuria and glomerulosclerosis in response to ADRN compared with Rcan1þ/þ mice (Figure 8c and d). Transmission electron microscopy analysis revealed increased podocyte foot process effacement (Figure 9a and b) and greater podocyte loss in Rcan1-/- mice than in Rcan1þ/þ mice in response to ADRN (Figure 9c and d). Taken together, loss of RCAN1 significantly worsened glomerular disease and podocyte loss in the setting of glomerulosclerosis. DISCUSSION

Study of epigenetic regulation occurring specifically in kidney cells is hampered by the heterogeneity of cell types present in kidney cortices and even in isolated glomeruli. To overcome this limitation, a recent study analyzed the DNA methylation patterns in microdissected kidney tubular epithelial cells, which represented mainly the proximal tubules.33 It showed significant cytosine methylation profiles for chronic kidney disease and showed that differentially methylated loci are enriched in kidney-specific enhancer regions. To determine the podocyte-specific epigenetic changes in HIV kidney disease, we utilized primary podocyte cultures infected with or without HIV-1. We found that HIV-1 infection modified the DNA methylation patterns of genes involved in inflammation,

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cellular metabolism, cell proliferation, and actin cytoskeleton. Our findings are consistent with previously shown mechanisms of podocyte injury in HIVAN,25 suggesting that the observed methylation modifications also occur in vivo. The correlation analysis between DNA methylation and transcriptomic profiles identified only 20 hypomethylated genes and only 1 hypermethylated gene, RCAN1. The fact that we used the criterion of at least 10% methylation level change in our analysis is likely the major factor in identifying only 1 hypermethylated gene that correlated with the downregulated gene expression. However, if we loosened the criteria to all levels of methylation change, the analysis would yield an additional 12 genes, including RCAN1: ADAM10, ADAMDEC1, C6orf170, CLSTN2, CLUAP1, GLIS3, GRID2, MYT1L, NXN, PLXDC2, PRPS1L1, and PSD3. Because RCAN1 is a critical regulator of the calcineurin pathway that plays an important role in kidney disease and podocyte function,9,13,14 we decided to focus on this gene for further analysis. We confirmed that the expression of RCAN1 was indeed reduced in the glomeruli of HIVAN kidneys, as well as in murine model of HIVAN, Tg26. Notably, reduced RCAN1 expression was observed in the glomeruli of several human kidney diseases, including DN, IgA nephropathy, and lupus nephritis, suggesting a broad role of RCAN1 in glomerular disease. We further observed that RCAN1 expression is downregulated by high glucose conditions and HIV infection in cultured podocytes, which was relieved by 5-Aza-20 -deoxycytidine, suggesting that the reduced RCAN1 expression in DN and in HIVAN is a result of epigenetically regulation. However, we were not able to confirm these changes of DNA methylation sites by bisulfite sequencing because of technical limitations. Our data indicated that RCAN1 is a regulator of highglucose activation of the calcineurin-NFAT pathway in podocytes. Several studies suggest that activation of the NFAT pathway leads to the development of glomerular diseases such as in focal segmental glomerulosclerosis and DN.15–17 Therefore, RCAN1 may be an important regulator of kidney disease progression. Calcineurin inhibitors have been widely used to treat primary glomerular disease such as focal segmental glomerulosclerosis and membranous nephropathy.34 However, only a portion of patients respond to treatment. It would be important to assess whether DNA methylation changes or expression of RCAN1 determine the response of these patients to calcineurin inhibitors. Our data suggest that alteration of RCAN1 expression affects podocyte cytoskeleton in diabetic or HIV infected

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Figure 5 | High glucose and HIV-1 infection suppresses RCAN1 expression in human podocytes in vitro. (a–c) Conditionally immortalized human podocytes were incubated with media containing normal glucose (NG; 5 mM), high glucose (HG; 30 mM) or high mannitol (HM; 5 mM glucose þ 25 mM mannitol), with or without DNA methyltransferase inhibitor 5-Aza-20 -deoxycytidine (1 mM) for 72 hours. RCAN1 expression was assessed by Western blot and quantitative polymerase chain reaction (qPCR). Western blot using b-actin as a loading control is shown in (a) and densitometric analysis in (b). qPCR of RCAN1 mRNA is shown in (c). (d,e) Human podocytes were infected with HIV-1 (HIV) or control virus (enhanced green fluorescent protein); 72 hours after infection, cells were harvested for Western blot analysis of RCAN1. Western blot using b-actin as loading control is shown in (d) and densitometric analysis in (e). *P < 0.05, **P < 0.01, and ***P < 0.001 between indicated groups, n ¼ 3. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

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Figure 6 | RCAN1 regulates nuclear factor of activated T cells (NFAT) transcriptional activity in kidney cells. (a–c) To assess the effects of RCAN1 expression on downstream NFAT signaling, HEK293T cells were transfected with FLAG-tagged RCAN1 overexpression plasmid (RCAN1OE) or control enhanced green fluorescent protein (EGFP) overexpression plasmid (EGFPOE); similarly, HEK293T cells were transfected with RCAN1 short hairpin (sh)RNA-expressing knockdown plasmid (RCAN1KD) or scrambled shRNA control plasmid (ScrKD). (Continued) 10

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conditions, indicating a major role of RCAN1 in maintaining normal podocyte function. Previous studies showed that calcineurin inhibition stabilizes the podocyte actin cytoskeleton through protein kinase A–mediated phosphorylation of synaptopodin,14 and protein kinase A activation protects against podocyte injury in glomerular disease.35,36 Interestingly, it was reported that RCAN1 expression could also be regulated by protein kinase A.37 Our data also suggest that RCAN1 reduces podocyte apoptosis under high glucose conditions. Consistent with our findings, it has been shown that calcineurin activation promotes podocyte apoptosis through activation of NFAT.38 In addition, we showed that RCAN1 loss led to aggravated podocyte injury and glomerular disease in mice with ADRN, further confirming an in vivo role of RCAN1 in maintaining normal podocyte function. However, because we used global RCAN1 knockout mice in our study, we cannot rule out the systemic effects of RCAN1 loss on podocyte injury. The effects of RCAN1 on immune cells also may contribute to the development and progression of kidney disease. RCAN1 is also implicated in atherosclerosis progression.34 Furthermore, it has been shown that RCAN1 is regulated by vascular endothelial growth factor A,39 and in turn RCAN1 has been shown to inhibit vascular endothelial growth factor–mediated endothelial cell proliferation and angiogenesis in primary human endothelial cells.40 Thus it is plausible that RCAN1 also may have an important role in glomerular endothelial cell homeostasis. Future studies with conditional null allele of Rcan1 are required to examine the kidney cell–specific contribution of RCAN1 in the setting of kidney disease. In 2 longitudinal studies, we and other investigators have confirmed an additive effect of HIV-1 infection and diabetes in promoting chronic kidney disease progression in the U.S. veteran population.41,42 Our recent murine studies suggest that an upregulation of local inflammation induced by HIV-1 aggravates diabetic kidney disease.43 Our current study now indicates that both HIV-1 infection and a high glucose level may induce similar epigenetic changes, causing podocyte injury and kidney disease. Therefore, diabetes and HIV infection may share similar mechanisms that cause kidney disease. As summarized in the schematics in Figure 10, our study demonstrates that RCAN1 is downregulated in podocytes of patients with glomerular disease, in part through an epigenetic regulation of RCAN1. Together, our study suggests that

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RCAN1 may be a potential drug target to treat glomerular disease. MATERIALS AND METHODS Cell culture Enriched human podocyte primary cultures were established from nephrectomy material (“enriched podocytes”). Glomeruli were isolated using differential sieving and then plated onto collagen-coated plates to promote outgrowth of podocytes.44 Cultures were expanded up to 4 times in podocyte medium. Podocyte enrichment of >90% was determined by morphology and immunofluorescent positivity for podocyte marker synaptopodin (catalog #NBP2-39100, Novus Biologicals, Littleton, CO). Conditionally immortalized human podocytes from Dr. Moin Saleem were cultured as previously reported.45 In brief, podocytes were cultured in podocyte medium made from Roswell Park Memorial Institute 1640 media (RPMI 1640) (Gibco, Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum and penicillin/streptomycin (pen/strep) with iron-transferrin-selenium (Gibco) at 33  C for proliferation. To induce differentiation, podocytes were maintained at 37  C for 10 days. Before each experiment, cell culture medium was replaced by RPMI 1640 medium containing 0.1% fetal bovine serum for 24 hours. Treatments were administered for 48 hours. Cells were exposed to either normal glucose (5 mM), high mannitol hyperosmolar control (5 mM glucose þ 25 mM mannitol), or high glucose (30 mM) with or without DNA methyltransferase activity inhibitor 5-Aza-20 -deoxycytidine (1 mM, Sigma-Aldrich, St. Louis, MO). HEK293T cells were cultured in 10% fetal bovine serum in Dulbecco’s modified Eagle’s medium (Life Technologies, Carlsbad, CA). HIV-1 infection The enriched podocytes were transduced for 48 hours with vesicular stomatitis virus–pseudotyped pNL4-3:DeltaG/P-EGFP (HIV-1) or control pseudotyped green fluorescent protein.19 Virally transduced podocytes from each condition were used to isolate RNA for RNA sequencing, and DNA for DNA methylation array. Conditionally immortalized human podocytes were maintained in RPMI 1640 medium. Cells were cultivated at 33  C for propagation. HIV or other study relative plasmids were transient transfected by using ViaFect transfection reagent (Promega, Madison, WI). After 48 hours of transfection, cells were removed to 37  C for differentiation. RNA Sequencing Total RNA was isolated from virally transduced podocytes using TRIzol reagent (Ambion, Thermo Fisher Scientific). Paired-end sequencing with 100 base pairs read length was carried out on the HiSeq 2000 sequencing system (Illumina, San Diego, CA). The reads with good quality were firstly aligned to several human reference databases including hg19 human genome, exon, splicing junction segment and contamination databases, including

= Figure 6 | (Continued) Transfected cells were infected with control or HIV-1 virus. At 72 hours after infection, RCAN1 expression was determined by Western blot analysis (a). (b,c) HEK293T cells were co-transfected with NFAT-luciferase reporter plasmid, p3xNFAT-GL, and TK-Renilla control plasmid in conjunction with the aforementioned overexpression or knockdown plasmids. At 48 hours after transfection, cells were incubated with high glucose (HG) (b) or infected with HIV-1 (c) for an additional 24 hours. Cells were harvested for NFAT-luciferase activity. Control untransfected cells, EGFPOE, and ScrKD were indistinguishable, and only 1 control is shown for simplicity. *P < 0.05, **P < 0.01 and ***P < 0.001 between indicated groups, n ¼ 3. (d,e) Immortalized human podocytes were transfected with plasmids above and quantitative polymerase chain reaction analysis of NFAT target genes were determined after 48 hours of HG treatment (d) or HIV-1 infection (e). *P < 0.05 and ***P < 0.001 compared with normal glucose (NG) or control virus; #P < 0.05 and ###P < 0.001 compared with HG or HIV-infected cells, n ¼ 3. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International (2018) -, -–-

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Figure 7 | RCAN1 regulates cytoskeletal structures in podocytes. (a) Immortalized human podocytes transfected with RCAN1OE, RCAN1KD, or control plasmids were infected with control or HIV-1 virus. At 48 hours after infection, F-actin staining was performed with Cy5-conjugated phalloidin. Representative images of 3 independent experiments are shown (original magnification 630; bar ¼ 10 mm). (b,c) Immortalized human podocytes transfected with RCAN1OE, RCAN1KD, or control plasmids were cultured under high mannitol (HM) or high glucose (HG) conditions for 48 hours. (b) F-actin staining was performed with rhodamine-conjugated phalloidin. Representative images of 3 independent experiments are shown (original magnification 630; bar ¼ 10 mm). (c) Active Caspase-3 concentration in cultured podocytes. ****P < 0.0001

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ribosome and mitochondria sequences using the Burrows-Wheeler alignment algorithm. After filtering reads mapped to the contamination database, the reads that were uniquely aligned to the exon and splicing-junction segments with a maximal 2 mismatches for each transcript were then counted as expression level for the corresponding transcript. The read counts were log2 transformed and normalized at an equal global median value in order to compare transcription level across samples. The differential analysis by paired LIMMA was performed to identify significantly dysregulated genes in primary podocytes after HIV infection at adjusted false discovery rate P value <0.05, which were then subjected to gene ontology function and canonical pathway enrichment analysis by Fisher exact test. Methylation profiling Total DNA from virally transduced podocytes (HIV and EGFP) was prepared by using spin columns (Qiagen, Hilden, Germany) and was analyzed on an Illumina 450K methylation array by the Laboratory for Translational Genomics at Baylor College of Medicine, Houston, Texas. The methylation data were processed with open source R package Illumination Methylation Analyzer (IMA) and MADAM (Meta-Analysis Data Aggregation Methods): first, after data filtering, only the sites on autosomal chromosomes with a significant detection P value <0.001 were kept for downstream analysis, and then the beta was converted to log2 ratio of beta value to (1-beta) value and further smoothed along the genome coordinates to reduce data variation. The differentially methylated sites in HIV-infected primary podocytes were identified by paired LIMMA test at P value <0.05. The distribution of differentially methylated (hypo- or hyper-) sites located in various genomic regions was investigated and the functions of genes that harbor these methylation sites were evaluated with gene ontology enrichment analysis. Lastly, the gene expression data were correlated with methylation data to identify the differentially expressed genes with corresponding differential methylation. Western blot analysis Cell proteins were extracted by lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein lysates were separated on sodium dodecylsulfate polyacrylamide gel by electrophoresis and transferred to polyvinylidine difluoride membranes (Millipore, Burlington, MA). The membranes were probed with the following antibodies: rabbit anti-RCAN1 (Abcam, ab140131), mouse anti-FLAG (Sigma-Aldrich, F3165), and mouse anti-beta-actin (Sigma-Aldrich, A5316). Quantitative real-time polymerase chain reaction Cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). First strand cDNA was reverse-transcribed from a total of 1 mg RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). A total of 2 mg of cDNA products was amplified in a 20-ml reaction system containing 10 ml iQ SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) and 400 nmol/l primer mixture. The following primers were used: hRCAN1 (forward:50 -AACCTACAGCCTCTTGGAAAG-30 ;

= versus control; ###P < 0.001 versus HG. DAPI, 40 6-diamidino-2phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

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Figure 8 | Loss of RCAN1 results in aggravated Adriamycin nephropathy (ADRN) in mice. (a) Western blot of RCAN1 in kidney cortices of Rcan1þ/þ and Rcan1-/- mice. (b) Immunofluorescence staining for RCAN1 and podocyte marker synaptopodin in the kidney sections of Rcan1þ/þ and Rcan1-/- mice. (c) Urinary albumin-to-creatinine ratio (UACR) Rcan1þ/þ and Rcan1-/- mice with or without Adriamycin (ADR) injection. (d) Albuminuria was also measured in 12-hour urine collection. (e) Representative images of kidneys of Rcan1þ/þ and Rcan1-/- mice stained with periodic acid–Schiff. Bar ¼ 50 mm. (f) Glomerulosclerosis scoring of Rcan1þ/þ and Rcan1-/- mouse kidneys (**P < 0.01 and ****P < 0.0001 when compared with –ADR mice; # P < 0.01 when compared with Rcan1þ/þ mice with ADRN; n ¼ 6). DAPI, 40 6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

reverse: 50 -TCATACGTCCTAAAGAGGGAC-30 ), hWNT6 (forward: 50 -TTGGTGCAACTGCACAACAAC-30 ; reverse: 50 -GCGAAATGG AGGCAGCTTCT-3); hCXCL2 (forward:50 -TGCAGGGAATTCA Kidney International (2018) -, -–-

CCTCAAG-30 ; reverse: 50 -TCTGCCCATTCTTGAGTGTG-30 ), hNFATc (forward: 50 -ATTGAGGTGCAGCCCAAGTC-30 ; reverse: 50 -GCGGCTCATTCTCCAAGTAG-30 ), and hGAPDH (forward 13

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Figure 9 | Loss of RCAN1 results in increased podocyte injury and loss in Adriamycin nephropathy (ADRN) mice. (a) Representative transmission electron microscopy images of Rcan1þ/þ and Rcan1-/- mice. Bar ¼ 1 mm. (b) Quantification of podocyte foot process (FP) effacement. (c) Representative immunofluorescence staining of WT-1 in Rcan1þ/þ and Rcan1-/- mouse kidneys. Bar ¼ 50 mm. (d) Quantification of WT-1þ podocyte number per glomerular cross sections (**P < 0.01 and ****P < 0.0001 when compared with –Adriamycin [ADR] mice; #P < 0.05 when compared with Rcan1þ/þ mice with ADRN; n ¼ 6). DAPI, 40 6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. 14

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reporter assay system (Promega) according to the manufacturer’s instructions. Expression of Renilla luciferase was used as an internal control to normalize the luciferase activities of pGL3-NFAT. Each assay was performed in duplicates, and all results are shown as mean  SEM for at least 3 independent experiments.

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Immunofluorescence staining Differentiated human podocytes were cultured on collagencoated glass coverslips. Cells were fixed with 3% paraformaldehyde solution in phosphate-buffered saline solution at room temperature for 10 minutes and treated with the following buffer (10% normal Horse serum, 0.3% Triton X-100 in phosphate-buffered saline solution) to block nonspecific reaction at room temperature for 45 minutes. Slides were incubated with 0.1 mg/ml rhodamine-phalloidin (Sigma-Aldrich) for 30 minutes at room temperature in the dark. Nuclear staining was performed with 0.1 mg/ml 40 6-diamidino-2-phenylindole (Sigma-Aldrich).

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Apoptosis analysis Caspase-3 activity was measured in human podocytes with use of a Human Active Caspase-3 Immunoassay Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol.

Nucleus

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Figure 10 | Summary schematics of the RCAN1 function in podocytes. NFAT, nuclear factor of activated T cells; pNFAT, phosphorylated (active) NFAT.

50 -AATTGAGCCCCGCAGCCTCCC-30 ; reverse: 50 -CCAGGCGCCCA ATACGACCA-30 ). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The 2-DDCt method was used for the analysis of relative gene expression. Plasmid transfection FLAG-tagged pCMV-mRCAN1 was purchased from Addgene, Cambridge, Massachusetts (catalog #65413), and short hairpin RNA plasmid for human RCAN1 was purchased from Santa Cruz Biotechnology, Dallas, Texas (catalog #SC-45480-SH). Scrambled short hairpin RNA plasmid was used as a negative experimental control. ViaFect Transfection Reagent (Promega, Madison, WI) was used for transfection of podocytes according to the manufacturer’s instructions. Luciferase assay pGL3-NFAT luciferase reporter vector was purchased from Addgene (catalog #17870). pGL3-Basic3 was used as a negative control. EGFPC1-huNFATc1EE-WT was purchased from Addgene (catalog #24219) and used as positive control. Luciferase reporter vectors, pRL-TK, and Renilla Luciferase Control Reporter Vectors was purchased from Promega (catalog #E2241). Transfections were carried out in HEK293T cells using PolyJet transfection reagent (SignaGen Laboratories, Rockville, MD). Twenty-four hours after transfection, cells were treated with either high glucose or normal glucose for another 48 hours. Luciferase activities were measured by using the dual-luciferase

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Immunofluorescence staining of human kidney samples Archival human biopsy specimens of healthy donor nephrectomies and patients with DN, IgA nephropathy, lupus nephritis, and HIVAN were collected at Mount Sinai Hospital, Icahn School of Medicine at Mount Sinai, New York, under a protocol approved by the Institutional Review Board. Kidney sections from human kidney biopsies were prepared accordingly. Immunostaining was performed using rabbit anti-RCAN1 (Abcam, Cambridge, United Kingdom, ab185931), mouse anti-synaptopodin (R&D Systems, MAB8977), and 40 6-diamidino-2-phenylindole (Sigma-Aldrich, D9542). After washing, sections were incubated with a fluorophore-linked secondary antibody (Alexa Fluor 488 antirabbit IgG and Alexa Fluor 568 anti-mouse IgG from Invitrogen). After staining, slides were mounted in Aqua Poly/Mount (Polysciences, Inc., Warrington, PA) and photographed under an AxioVision IIe microscope with a digital camera. Immunohistochemistry Specimens were initially baked for 20 minutes in a 55  C to 60  C oven and then processed as described below. Briefly, formalin-fixed and paraffin-embedded sections were deparaffinized, and endogenous peroxidase was inactivated with hydrogen peroxide. Sections were then blocked in 2% goat serum in phosphate-buffered saline solution for 1 hour at room temperature and then incubated with a rabbit anti-RCAN1 antibody (1:1000, GenScript, Piscataway, NJ) at 4  C overnight. The next day, sections were washed 3 times with phosphatebuffered saline solution and then incubated with secondary antibody for 30 minutes. Positive staining was revealed by peroxidase-labeled streptavidin and diaminobenzidine substrate with a fixed exposure time of 3 minutes for all experiments among the groups. The control included a section stained with only secondary antibody. Quantification of immunostaining Immunostained images with a final magnitude of approximately 400 were obtained. ImageJ 1.26t software (National Institutes of Health, Bethesda, MD, rsb.info.nih.gov) was used to

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measure the level of immunostaining in the glomeruli. First, the images were converted to 8-bit grayscale. Glomerular regions were selected for measurement of area and integrated density, and background intensity was measured by selecting 3 distinct areas in the background with no staining. The corrected optical density (COD) was determined as follows: COD ¼ ID  ðA  MGV Þ where ID is the integrated density of the selected glomerular region, A is the area of the selected glomerular region, and MGV is the mean gray value of the background readings. ADRN murine models Rcan1-/- mice in the B6/129 background were obtained from The Jackson Laboratory, Bar Harbor, Maine (stock #009072). ADRN was induced by injection of Rcan1-/- mice and their control littermates at the age of 8 weeks at 22 mg/kg via tail vein as described previously.46 Proteinuria was monitored every week, and the mice were killed 28 days after injection. Measurement of urine albumin and creatinine Urine albumin was quantified by enzyme-linked immunosorbent assay using a kit from Bethyl Laboratories, Inc., Houston, Texas. Urine creatinine levels were measured in the same samples using a QuantiChrom creatinine assay kit (DICT-500; BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions. The urine albumin excretion rate was expressed as the ratio of albumin to creatinine. Twelve-hour urine collections in the metabolic cages were also used for determination of urinary albumin excretion. Kidney histology Kidneys were removed and fixed with 4% paraformaldehyde for 16 hours at 4  C. The 4 mm sections were cut from paraffinembedded kidney tissues. Sections were stained with periodic acid–Schiff for histologic analysis. Assessment of the mesangial and glomerular cross-sectional areas was performed by pixel counts on a minimum of 10 glomeruli per section in a blinded fashion under 400 magnification (Zeiss AX10 microscope, Carl Zeiss Canada, Toronto, Ontario, Canada). Renal histologic abnormalities were scored as previously described.47 The glomerulosclerosis was graded on a semiquantitative scale (0 to 3þ): 0 (absent), 1þ (involving 1%–25% of all glomeruli sampled), 2þ (involving 26%– 50% of glomeruli), and 3þ (involving >50% of glomeruli) as described. An average of 50 glomeruli was sampled per animal. Immunofluorescence staining of kidney sections from these mice was performed with mouse anti-WT1 antibodies (Santa Cruz Biotechnology, sc-192) for counting podocyte number per glomerular cross section. Transmission electron microscopy Tissues were fixed in 2.5% glutaraldehyde with 0.1 M sodium cacodylate (pH 7.4) for 72 hours at 4  C. Samples were further incubated with 2% osmium tetroxide and 0.1 M sodium cacodylate (pH 7.4) for 1 hour at room temperature. Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a Hitachi H7650 microscope. Briefly, negatives were digitized, and images with a final magnitude of up to 10,000 were obtained. ImageJ 1.26t software was used to measure the length of the peripheral glomerular basement membrane (GBM), and the number of slit pores overlying

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this GBM length was counted. The arithmetic mean of the foot process width (WFP) was calculated as follows: P p GBM LENGTH P W FP ¼  silts 4 where Sslits indicates the total number of slits counted, SGBM LENGTH indicates the total GBM length measured in 1 glomerulus, and p/4 is the correction factor for the random orientation by which the foot processes were sectioned.48 Statistical analyses Analysis and graphing of data were performed with Prism version 6.0 (GraphPad Software). All data were reported as mean  SEM. Analysis of variance with Bonferroni correction was used to evaluate statistical difference between groups. The unpaired Student t-test was used to compare experimental and control groups. A P value <0.05 was considered statistically significant. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENT

The authors would like to thank Dr. Alejandro R. Gener for critical reading and editing of the manuscript. KL is supported by NIH R01DK117913; JCH, WZ, and PEK are supported by NIH P01DK056492 and NIH RC4 DK090860. AUTHOR CONTRIBUTIONS

JCH and PEK conceived and designed the experiments; HL, WZ, GCD, ZL, WC, GJ, JC, and MS performed the experiments; HL, WZ, FZ, YX, DPH, KL, PEK, and JCH analyzed the data; HL, WZ, DPH, KL, and JCH wrote and edited the manuscript. SUPPLEMENTARY MATERIAL

Figure S1. Human RCAN1 mRNA expression data from Nephroseq datasets (nephroseq.org). Data File S1. Upregulated genes in HIV-infected podocytes. Data File S2. Downregulated genes in HIV-infected podocytes. Data File S3. Gene ontology plot. Data File S4. Pathway plot. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Kato M, Natarajan R. Diabetic nephropathy—–emerging epigenetic mechanisms. Nature Rev Nephrol. 2014;10:517–530. 2. Nathan DM, Group DER. The diabetes control and complications trial/ epidemiology of diabetes interventions and complications study at 30 years: overview. Diabetes Care. 2014;37:9–16. 3. Chen Z, Miao F, Paterson AD, et al. Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort. Proc Natl Acad Sci U S A. 2016;113:E3002–E3011. 4. Erlandson KM, Campbell TB. Inflammation in chronic HIV infection: what can we do? J Infect Dis. 2015;212:339–342. 5. Zhang X, Justice AC, Hu Y, et al. Epigenome–wide differential DNA methylation between HIV–infected and uninfected individuals [e-pub ahead of print]. Epigenetics. 2016;12:1–11. 6. Desplats P, Dumaop W, Cronin P, et al. Epigenetic alterations in the brain associated with HIV–1 infection and methamphetamine dependence. PLoS One. 2014;9:e102555. 7. Verma M. Epigenetic regulation of HIV, AIDS, and AIDS–related malignancies. Methods Mol Biol. 2015;1238:381–403.

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8. Chandel N, Ayasolla KS, Lan X, et al. Epigenetic modulation of human podocyte vitamin D receptor in HIV milieu. J Mol Biol. 2015;427:3201–3215. 9. Davies KJ, Ermak G, Rothermel BA, et al. Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin. FASEB J. 2007;21:3023– 3028. 10. Rusnak F, Mertz P. Calcineurin: form and function. Physiol Rev. 2000;80: 1483–1521. 11. Aramburu J, Heitman J, Crabtree GR. Calcineurin:a central controller of signalling in eukaryotes. EMBO Rep. 2004;5:343–348. 12. Martinez–Martinez S, Genesca L, Rodriguez A, et al. The RCAN carboxyl end mediates calcineurin docking–dependent inhibition via a site that dictates binding to substrates and regulators. Proc Natl Acad Sci U S A. 2009;106:6117–6122. 13. Laurin LP, Gasim AM, Poulton CJ, et al. Treatment with glucocorticoids or calcineurin inhibitors in primary FSGS. Clin J Am Soc Nephrol. 2016;11: 386–394. 14. Faul C, Donnelly M, Merscher–Gomez S, et al. The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A. Nat Med. 2008;14:931–938. 15. Wang Y, Jarad G, Tripathi P, et al. Activation of NFAT signaling in podocytes causes glomerulosclerosis. J Am Soc Nephrol. 2010;21:1657– 1666. 16. Nijenhuis T, Sloan AJ, Hoenderop JG, et al. Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT– mediated positive feedback signaling pathway. Am J Pathol. 2011;179:1719–1732. 17. Wang L, Hitron JA, Wise JT, et al. Ethanol enhances arsenic–induced cyclooxygenase–2 expression via both NFAT and NF–kappaB signalings in colorectal cancer cells. Toxicology Appl Pharmacol. 2015;288:232–239. 18. Pedigo CE, Ducasa GM, Leclercq F, et al. Local TNF causes NFATc1– dependent cholesterol–mediated podocyte injury. J Clin Invest. 2016;126: 3336–3350. 19. Husain M, Gusella GL, Klotman ME, et al. HIV–1 Nef induces proliferation and anchorage–independent growth in podocytes. J Am Soc Nephrol. 2002;13:1806–1815. 20. Rosenstiel PE, Chan J, Snyder A, et al. HIV–1 Vpr activates the DNA damage response in renal tubule epithelial cells. AIDS. 2009;23:2054–2056. 21. Barisoni L, Mokrzycki M, Sablay L, et al. Podocyte cell cycle regulation and proliferation in collapsing glomerulopathies. Kidney Int. 2000;58: 137–143. 22. Rastaldi MP, Armelloni S, Berra S, et al. Glomerular podocytes contain neuron–like functional synaptic vesicles. FASEB J. 2006;20:976–978. 23. Soda K, Balkin DM, Ferguson SM, et al. Role of dynamin, synaptojanin, and endophilin in podocyte foot processes. J Clin Invest. 2012;122:4401–4411. 24. Davenport AP, Hyndman KA, Dhaun N, et al. Endothelin. Pharmacol Rev. 2016;68:357–418. 25. Medapalli RK, He JC, Klotman PE. HIV–associated nephropathy: pathogenesis. Curr Opin Nephrol Hypertens. 2011;20:306–311. 26. Yang J, Zhou Y, Guan Y. PPARgamma as a therapeutic target in diabetic nephropathy and other renal diseases. Curr Opin Nephrol Hypertens. 2012;21:97–105. 27. Christou GA, Kiortsis DN. The role of adiponectin in renal physiology and development of albuminuria. J Endocrinol. 2014;221:R49–R61. 28. Laguette N, Sobhian B, Casartelli N, et al. SAMHD1 is the dendritic– and myeloid–cell–specific HIV–1 restriction factor counteracted by Vpx. Nature. 2011;474:654–657.

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