Alternative Splicing in CKD

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Alternative Splicing in CKD Megan Stevens*† and Sebastian Oltean*† *School of Physiology and Pharmacology, Faculty of Biomedical Sciences, and †Academic Renal Unit, School of Clinical Sciences, Faculty of Health Sciences, University of Bristol, Bristol, United Kingdom

ABSTRACT Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with $94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. J Am Soc Nephrol 27: 1596–1603, 2016. doi: 10.1681/ASN.2015080908

The human genome consists of approximately 20,000 genes, however, the human proteome is estimated to be far greater, formed of hundreds of thousands proteins.1 One of the main processes that accounts for this increase in diversity is alternative splicing (AS) through which a single transcript gives rise to multiple proteins, thus increasing the coding capacity of a gene.2 We now know that .94% of human genes can be alternatively spliced.3,4 This is not consistent between all vertebrates. The more evolved the species, the higher the percentage of genes that undergo AS. For example, although there is a high conservation at DNA level between human and mouse, only approximately 30% of AS events are conserved.5 AS is a highly regulated process. Any fault in this regulation can result in cellular dysfunction and disease. There 1596

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are .2000 splicing mutation disease entities known, resulting in 370 diseases and involving 303 genes.6 The disease most commonly linked to deregulation of AS is cancer,7–9 however, AS has also been implicated in diseases ranging from Parkinson 10 to dilated cardiomyopathy11 or diabetes.12,13 An increasing number of splice isoforms have been recently reported to be associated with various CKDs. This review recapitulates the basics of AS, highlights some of the most interesting examples of faulty splicing associated with CKD, and discusses possible ways to manipulate AS for therapeutic benefit. We want to emphasize that we are not referring here to “aberrant splicing,” i.e., mutations in splice sites that cause disease (which are usually covered in genetic diseases reviews), but rather to “abnormal regulation of AS” in disease

(e.g., through abnormal expression of splice factors or their regulators such as splicing-specific kinases). MECHANISMS OF AS The Splicing Reaction

Removal of the introns (noncoding regions) from pre-mRNA and joining of the exons (coding regions) is accomplished by the spliceosome, a macromolecular assembly composed of small nuclear ribonucleoproteins and their associated accessory proteins.14 The spliceosome assembles on splice sites, which refer to conserved sequences present in the premRNA transcript at the exon–intron junction. Splicing occurs through two transesterification reactions involving a particular intronic sequence called the branch point, and the 59 and 39 splice sites. Another important sequence is the polypyrimidine tract, a region rich in pyrimidine nucleotides that promotes spliceosome assembly through binding of various splice factors (Figure 1). Modes of AS

AS is the process through which whole exons or parts of exons/introns may be

Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Sebastian Oltean, School of Physiology and Pharmacology, Faculty of Biomedical Sciences, University of Bristol, Dorothy Hodgkin Building, Bristol, BS1 3NY, UK. Email: [email protected] Copyright © 2016 by the American Society of Nephrology

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Figure 1. Basic cis- and trans-acting elements involved in splicing of an intron. Positions and consensus sequences of 59 and 39 splice sites, branch point and polypyrimidine tract are shown. Auxilliary sequences: ESE, ESS, ISE, and ISS. Binding of a splice factor (SF) to an ESE might activate usage of a neighboring splice site while binding to a ISI may repress it. Blue and orange represent different SFs; red denotes a silencer element and green an enhancer element.

included or excluded in the final transcript. The main categories are (Figure 2): (1) cassette exon, where a certain exon is either skipped or retained in the transcript; (2) intron retention, when an intron remains in the mature transcript; (3) alternative 59 or 39 splice sites, when different splice sites are used in exons; and (4) mutually exclusive exons, two exons alternate in their inclusion/exclusion in the mRNA depending on cell type or various conditions. AS occurs either in the open reading frame (affecting the protein encoded) or in the 59 and 39 untranslated regions

(with effects on mRNA localization, stability, or translation). Regulation of AS by Splice Factors and Intronic/Exonic Elements

Cis-acting regulatory elements (auxiliary sequences) may influence the splicing outcome. They can be divided into four subgroups; exonic and intronic sequence enhancers (ESE and ISE), and exonic and intronic sequence silencers (ESS and ISS) (Figure 1). They function by recruiting trans-acting splice factors to either activate of suppress certain steps of RNA splicing.2

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There are several RNA-binding proteins that are classified as splice factors. One of the most common groups are the serine/arginine-rich (SR) proteins.15 They contain an Arg/Ser-rich domain and combine with small nuclear ribonucleoproteins to form and mediate the action of the spliceosome. SR proteins generally activate splicing of a cassette exon by binding to ESEs and aiding the recruitment of core splice factors to the polypyrimidine tract. Another key splicing regulatory family of proteins are the heterogeneous nuclear ribonucleoproteins, which are classically viewed as being responsible for suppressing RNA splicing at a particular exon by blocking access of the spliceosome to the polypyrimidine tract. 16,17 However, there are exceptions to the general rules. For instance, recent literature has shown that heterogeneous nuclear ribonucleoproteins may also be activators of exon inclusion with their activity being dependent on context or whether they bind an exon or an intron. 16 Aside from these two big classes of splice factors, there are many other RNA-binding proteins with the same function. Interestingly, several of them have been shown to be cell- and tissue-specific, and act as master regulators of several physiologic processes. A few examples are: Nova, an RNA-binding protein that specifically regulates AS of several proteins involved in neuronal synapses18; ESRP1 and -2, epithelial splicing regulatory proteins 1 and 2 that are thought to be crucial in defining the epithelial state of a cell by coordinating the exon content of hundreds of transcripts 19,20; and Rbm24 and RbFox1, RNA-binding proteins that control muscle-specific AS. 21 This raises the possibility that kidney development and/or function may also have a yet uncharacterized master splice factor.

E Integration into Cell Signaling and Gene Regulatory Networks

Figure 2. Various modes of AS. (A) Cassette exon. (B) Intron retention. (C) 59 alternative splice site. (D) 39 alternative splice site. (E) Mutually exclusive exons. Various colors denote different exons. J Am Soc Nephrol 27: 1596–1603, 2016

AS is heavily regulated within cell homeostasis. Similar to transcription factors, splice factors are also integral parts of various cellular signaling pathways. Intracellular and extracellular signals Alternative Splicing in CKD

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modify splice factor availability and activity, which induce changes in the splice isoform composition of several transcripts, and therefore changes in the protein repertoire and finally cell functions. For instance, SR proteins, usually inactive in the cytoplasm, are phosphorylated by protein kinases in response to a stimulus and become active. This induces shuttling (often accompanied by a chaperone) into the nucleus where they modify AS patterns (Figure 3). In general, protein kinases are not very specific and have many substrates; however, several kinases are now widely accepted to be more frequently involved in splicing regulation than other processes, e.g., SR-protein kinases (SRPKs) or cyclin-dependent-like kinases.22,23 Virtually all canonical signaling pathways may be involved in splice factor regulation and isoform choice.7 However, a recent report has shown a crucial role of the (EGF/EGFR– phosphatidylinositol 3-kinase–Akt) axis in regulating SRPK1 and SR-protein phosphorylation and thus having a rather comprehensive effect on AS modulation.22

SPLICING ISOFORMS IN CKD

In this section, far from being exhaustive, we highlight some of the most well studied examples of splice isoforms of genes known to be involved in the pathology of CKD. VEGF‑A165b

The human VEGF‑A gene, a major regulator of angiogenesis and vessel permeability, consists of eight exons and seven introns.24 Depending on the inclusion/ exclusion of various exons, AS of VEGF‑A gives rise to a family of isoforms (e.g., VEGF‑A121, VEGF‑A145, VEGF‑A165, VEGF‑A 189 , and VEGF‑A 206 , generically known as VEGF‑Axxx), where the number depicts the number of amino acids, all of which were described as proangiogenic.25 In 2002, it was discovered that exon 8 of VEGF‑A could be differentially spliced by using an alternative 39 splice site to give rise to a functionally different family of isoforms, which was denoted VEGF‑A xxx b. 26 The resultant

Figure 3. Activation of SR proteins by signaling. In unstimulated cells (upper) SR proteins are concentrated mostly in cytoplasm. When stimulated by EGF for instance, kinases such as SRPK1 are activated, and SR proteins are phosphorylated and move into the nucleus where they bind and change the splicing pattern of various transcripts (lower). P, denotes phosphorylated state.

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VEGF‑Axxx b proteins have an altered C‑terminal sequence differing from VEGF‑Axxx by only six amino acids (Figure 4). However, this small alternative sequence results in VEGF‑Axxxb having radically different properties from those of VEGF‑Axxx. Unlike VEGF‑Axxx, VEGF‑Axxxb is not able to efficiently autophosphorylate vascular endothelial growth factor receptor 2 (VEGFR‑2).27 The result is poor activation of the kinase domain and weak, transient phosphorylation of the downstream targets.28 Within the glomerulus, VEGF‑A is a key regulator of normal function. High levels of VEGF‑A are expressed by mature podocytes,29 which crosses the glomerular basement membrane to signal to glomerular endothelial cells expressing VEGFR‑2. However, despite the high expression of VEGF‑A from the podocytes, there is no angiogenesis in the mature renal cortex. One of the reasons for this is that podocytes express a balance of the proangiogenic VEGF‑Axxx and antiangiogenic VEGF‑Axxxb isoforms in order to maintain normal functioning of the glomerular filtration barrier.30 Podocytes express both VEGF‑A 165 and VEGF‑A165b mRNA and protein when differentiated in culture.31 Both excessive and reduced expression of VEGF‑A in the glomerulus has been implicated in several human kidney diseases.32,33 A recent report has highlighted a strong correlation between the expression of VEGF‑A and eGFR in CKD patients.34 In addition, there has been evidence that diabetic nephropathy (DN) is associated with a switch in VEGF‑A splice isoform expression. In late DN in humans, there is an increase in the expression of VEGF xxx mRNA over VEGFxxxb mRNA in the glomerulus, however during the early stages of DN, when the GFR is still relatively high, there is an increase in the expression of the VEGFxxxb isoforms, presumably offering protection to the glomerular filtration barrier.35 sVEGFR-1 (Soluble Flt-1)

VEGF‑A signals through two main receptor tyrosine kinases; Flk‑1 (VEGFR‑2) and Flt‑1 (VEGFR-1). Flk‑1 signaling results J Am Soc Nephrol 27: 1596–1603, 2016

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Figure 4. VEGF‑A splice variants. VEGF‑A has eight exons and depending on their inclusion/ exclusion there are several isoforms with different amino acid length. An alternative 39 splice site in the terminal exon results in a new family of isoforms, xxxb, with the same number of amino acids but a different sequence at the C‑terminus. DSS, distal splice site; PSS, proximal splice site; UTR, untranslated region. Red and green denote different parts of exon 8.

in the main functions of VEGF‑A, which includes endothelial cell migration, proliferation, and survival. However, relatively little is understood about the function of Flt‑1. There are two major splice variants of the Flt‑1 gene which encode the full-length transmembrane Flt‑1 (tmFlt‑1), and the truncated soluble Flt‑1 (sFlt‑1).36 tmFlt-1 mRNA consists of 30 exons, whereas sFlt‑1 only contains the first 13–14 exons, following intron 13 retention and usage of a new alternative polyadenylation signal and new stop codon. The resulting translated protein lacks the transmembrane and C‑terminal kinases domains. sFlt‑1 negatively regulates the action of VEGF‑A by binding free circulating VEGF‑A and preventing it from binding to its functionally active receptor, Flk‑1. Therefore sFlt‑1 acts as an antiangiogenic factor, with VEGF‑A and sFlt‑1 constituting a balance of pro- and anti-angiogenic factors within the circulation. An imbalance between VEGF‑A and sFlt‑1 has been reported in many diseases, such as preeclampsia, diabetic retinopathy, and hypertension.37–39 In a study on 130 patients with CKD stages 3 to 5, it was found that plasma sFlt‑1 is increased compared with matched controls and correlates with reduced renal function and expression of markers of endothelial dysfunction.40 J Am Soc Nephrol 27: 1596–1603, 2016

Podocytes themselves have recently been shown to express sFlt‑1.41 sFlt‑1 was shown to act in an autocrine manner to control podocyte behavior through binding to glycosphingolipid GM3 in lipid rafts on the podocyte cell surface, promoting adhesion and actin reorganization. IgA nephropathy is commonly associated with endothelial dysfunction. The underlying mechanism has recently been proposed to be due to an imbalance of VEGF‑A/sFlt‑1, where sFlt‑1 levels correlated with proteinuria, hypertension, and von Willebrand factor levels in these IgA nephropathy patients.42 Soluble Klotho

Klotho is a transmembrane protein with an important role in maintaining mineral homeostasis. In addition to the fulllength Klotho, there is a soluble form (sKlotho) that is generated via AS through intron 3 retention and introduction of an early stop codon. The highest level of Klotho expression is within the kidney, particularly within the cells of the proximal and distal convoluted tubules, with a substantial portion of circulating sKlotho being nephrogenic in origin. The main function of transmembrane Klotho is as a coreceptor for fibroblast growth factor‑23, however, sKlotho appears to

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have several paracrine and endocrine functions, including modulation of ion transport, Wnt signal transduction, antirenin–angiotensin system, antisenescence, and antioxidation.43 Human patients with CKD in general exhibit reduced renal expression of Klotho,44 as well as reductions in sKlotho. In an apparent controversy, a recent study demonstrated that plasma and urinary levels of sKlotho are significantly elevated in patients with type 2 diabetes and preserved renal function. However, plasma sKlotho decreased in proportion to increasing urinary albumin excretion, suggesting a possible initial adaptive increase to preserve kidney function.45 In animal models of CKD, reduced levels of renal, plasma, and urinary Klotho have been detected. Overexpression or supplementation with sKlotho have resulted in improved renal function and ameliorated renal histologic changes, thus suggesting that sKlotho may be renoprotective by reducing oxidative stress, cell senescence, and apoptosis.44 Soluble Erythropoietin Receptor

Erythropoietin (Epo) is a hormone induced in the kidney by hypoxia. Epo binds to the erythropoietin receptor (EpoR), a membrane receptor located on erythroblasts, and stimulates transcription of antiapoptotic genes. This results in an improvement in anemia via the production of new red blood cells. Epo treatment is now widely used in patients with renal failure. A subset of dialysis patients develop resistance to Epo and require increases in dosage in order to maintain their hemoglobin levels. A potential cause of this is suggested to be a change in the splicing of the EpoR gene to upregulate the alternatively spliced soluble EpoR (sEpoR).46 The EpoR gene consists of eight exons and seven introns. Exons 1–5 encode the extracellular domain, exon 6 encodes the membrane-spanning region, and exons 7 and 8 encode the intracellular/cytoplasmic domain. Several splice variants of EpoR have been described; intron 5 retention or skipping of exon 6 introduce novel stop codons, generating truncated proteins as sEpoR. 47 The Alternative Splicing in CKD

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function of sEpoR is not completely understood, although levels of circulating sEpoR appear to correlate with the amount of erythropoiesis, suggesting a potential physiologic role.48 In patients with ESRD, higher serum sEpoR levels correlated with increased erythropoietin requirements and inhibited Epo-mediated Stat5 phosphorylation of EpoR, thus contributing to Epo resistance.46 It is hypothesized that sEpoR production may be mediated by proinflammatory cytokines. Fibronectin

The majority of CKDs progress toward sclerosis both in the glomerular and tubular compartments, with deposition of extracellular matrix proteins. One of the most abundant of these proteins is fibronectin (Fn). Fn is a large glycoprotein with several exons being regulated by AS to result in variation of the composition of amino acids in the extradomain 1 (EDA), extradomain 2, or type 3 connecting segment.49,50 The splice variant containing EDA is virtually absent from extracellular matrix composition in normal kidneys, but very abundant in renal fibrosis, as well as skin, lung, or liver fibrosis.51 The presence of the EDA region induces conformational changes in the protein and modifies its properties by increasing adhesion to integrins and promoting cell migration, proliferation, and adhesion. One of the most important profibrotic cytokines, TGF‑b, induces EDA inclusion in human tubular cells.52 A recent study has shed light into the regulation of the EDA region with the involvement of the splice factor SRp40 (or SRSF5) and signaling by the phosphatidylinositol 3-kinase–Akt pathway, providing a rationale for developing new drugs against renal fibrosis by targeting Fn exon composition.51

splice factor kinases. A few examples are: RBM20, thought to be a major regulator of cardiac-specific AS and mutated in cardiomyopathies;11 MBNL and CELF proteins, which play a crucial role in coordinating AS in heart development53 but have also been implicated in the pathogenesis of myotonic dystrophy; and MBNL‑1, which is sequestered by the specific expanded repeats in the 39 untranslated region of the DMPK gene.54,55 The lower availability results in alterations in AS of specific genes regulated by MBNL, which contribute to myotonic dystrophy pathogenesis. SRPK1, a kinase that phosphorylates SR-proteins (the class of splice factors mentioned above), is overexpressed in several cancers56–58 and also in Denys– Drash syndrome, associated renal failure, and increased incidence of Wilms tumors.59 SRPK1 is a determinant of angiogenesis through modulation of VEGF‑A splicing and its abnormal expression maintains a pathologic loop by promoting proangiogenic and propermeability VEGF‑A isoforms. Similar to physiology, there is the possibility that a certain splice factor or class of splice factors is predominantly involved in CKDs. Identification of these

molecules would provide new therapeutic targets. MANIPULATION OF AS AS A POTENTIAL THERAPEUTIC AVENUE IN CKD

Given the extent of AS both in physiology and disease, the question arises whether we can manipulate AS for therapeutic benefit. The general idea is to try and switch the splicing of an isoform that has been identified as deleterious and promoting disease pathogenesis, toward a beneficial isoform. The strategy most commonly used involves splicing-switching oligonucleotides (SSOs) (Figure 5A). The general idea is to design oligonucleotides that bind either exon-intron junctions or regulatory sequences like ESE/ESS or ISE/ISS, therefore affecting the splice outcome of the targeted event. To date, SSOs have proved very promising, with several of them being used in clinical trials, e.g., for Duchenne muscular dystrophy or spinal muscular atrophy.60 An increasing number of smallmolecule splicing modulators (smSM) have been described recently. An interesting

SPLICE FACTORS OR SPLICINGSPECIFIC KINASES ARE DEREGULATED IN PATHOLOGIC STATES

An increasing number of reports describe pathologies associated with abnormal expression of splice factors or 1600

Figure 5. Ways in which splicing-based therapeutics may be designed. (A) Splicingswitching oligonucleotides. (B) Small molecule splicing-modulators. P, denotes phosphorylated state. *represents smSM. Purple and green represent different SFs.

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example is amiloride, a diuretic with the main mechanism of action through effects on the ion pumps in the renal tubules. However, it has been discovered in a screen to potently affect splicing of several apoptotic genes and to be able to decrease tumor growth in animal models.61,62 Recently, a class of small molecule compounds that inhibit SRPK1 (SRPIN340, SPHINX), a major regulator of AS through SR-protein phosphorylation, has been shown to switch VEGF‑A splicing toward the VEGF‑Axxxb isoforms, and therefore inhibit angiogenesis in several mouse models including ocular neovascularization,63 melanoma xenografts,64 and orthotopic prostate cancer.65 Administration of rhVEGF165b is therapeutic in mouse models of DN.35 It would, therefore, be interesting to test whether this may also be accomplished by using SRPK1 inhibitors. We may envision other types of molecules that could act as splicing modulators, e.g., small molecules that interfere with splice factors assembly at splice sites, splice factor/RNA interactions, or molecules that affect directly the tertiary structure of a particular splice junction (Figure 5B). Recombinant B-type natriuretic peptide has clinically been used to treat heart failure due to its vasodilatory properties, however, the use has been limited due to observed declines in renal function and hypotension.66 An alternatively spliced transcript of B-type natriuretic peptide (ASBNP) results from intron 2 retention and was found to be expressed in failing human hearts. 67 Modification of this ASBNP peptide to delete the C‑terminus (ASBNP.1) resulted in the ASBNP peptide that lacked systemic vascular effects while having renal effects that resulted in GFR enhancement. This is an example of intelligent use of splicing knowledge to artificially design molecules that have better therapeutic properties. Similar too many other drug classes the question arises whether splicing modulators would be specific. This will probably not be a concern for SSOs, which bind to unique RNA sequences, although challenges with delivery and toxicity still need to be solved. J Am Soc Nephrol 27: 1596–1603, 2016

smSMs could affect several other splice junctions regulated by the same splicing kinase or splicing factor intended to be modulating. However, it is important that the manipulation of the main targeted splice event is dominant functionally in the system/cell line of interest (i.e., the other splice events affected do not result in major unwanted effects). Interestingly, a recent paper reports novel smSMs developed for survival motor neuron splicing and treatment of spinal muscular atrophy.68 The compounds resulted from a screen using a splicing reporter that followed the endogenous splicing event. RNAseq was performed to inquire specificity. It was found that very few splice junctions aside from the intended one are modified, therefore suggesting that specificity in splicing therapeutics using small molecules is possible. DISCUSSION

The explosion of literature in the recent years reporting faulty AS in several diseases, including CKD, warrants further investigation in an area little explored in terms of its contribution to pathogenesis. We envision that future studies will elucidate even more of the mechanistic aspects of deregulated AS in disease and form the basis of development of a novel class of drugs–splicing-modifying therapeutics.

ACKNOWLEDGMENTS Funding for this study was supported by the British Heart Foundation (grant no.: PG/15/ 53/31371 to S.O.) and by the Richard Bright VEGF Research Trust (award to S.O.).

DISCLOSURES None.

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