MicroRNAs: a new avenue to understand, investigate and treat immunoglobulin A nephropathy?

MicroRNAs: a new avenue to understand, investigate and treat immunoglobulin A nephropathy? IgA nephropathy (IgAN) is the most common cause of primary glomerulonephritis worldwide. Up to 30% of cases develop the progressive form of the disease, eventually requiring renal replacement therapy. Diagnosis and risk stratification relies on an invasive kidney biopsy and management options are limited, with recurrence following renal transplantation being common. Thus the quest to understand the pathophysiology of IgAN has been one of great importance. MicroRNAs (miRs) are short nucleotides that suppress gene expression by hybridizing to the 3 untranslated region of messenger RNA (mRNAs), promoting mRNA degradation or disrupting translation. First discovered in 1993, miRs have since been implicated in a number of chronic conditions, including cancer, heart disease and kidney disease. The mounting interest in the field of miRs has led to fascinating developments in the field of nephrology, ranging from their roles as biomarkers for disease to the development of miR antagonists as avenues for treatment. The translational potential for miRs in IgAN is thus well grounded and may represent a paradigm shift in current approaches to the disease. This review aims to summarize the literature with regard to miRs and their roles in IgAN. Key words: biomarkers, epigenetic, IgA nephropathy, microRNA, therapy Immunoglobulin A nephropathy (IgAN) is a primary kidney dis- this modelis anincrease inpoorly O-galactosylated IgA1 O-glyco- ease characterized by the deposition of IgA in the glomerulus. forms in the serum displaying low levels of O-linked galactose at With an incidence of at least 2.5 per 100,000/year, it is the most the IgA1 hinge [7]. A systemic IgG antibody response is directed common cause of primary glomerulonephritis worldwide [1]. The against this altered hinge region, generating IgA–IgG antibody condition exacts a substantial socio-economic cost, with up to complexes, which are then deposited in the kidneys, triggering an 30% of cases developing a progressive phenotype characterized inflammatory cascade eventually leading to chronic kidney dis- by glomerular or interstitial fibrosis, eventually requiring renal ease (CKD). Indeed, a proportion of patients with increased levels replacement therapy [2]. Diagnosis and risk stratification relies of poorly O-galactosylated IgA1 O-glycoforms never develop the on an invasive kidney biopsy [3] and management options are disease [8]and in vitro experiments suggest mesangial cells of pa- limited, with recurrence following renal transplantation being tients with IgAN may be ‘primed’ to respond to IgG–IgA complexes common [4, 5]. Thus the quest to understand the pathophysi- [9], highlighting the necessity of multiple hits in the pathophysi- ology of IgAN has been one of the great importance. ology of IgAN. Furthermore, it is possible that each ‘hit’ may be The prevalent model for the pathogenesis of IgAN is the ‘four- mediated by several independent variables, such as genetic, epi- hit hypothesis’, which postulates that the kidney is an innocent genetic and environmental factors, including exposure to mucosal bystander of an otherwise systemic disease [6]. Fundamental to infections [10]. Thus there is still much to be clarified about this Received: June 12, 2017. Editorial decision: July 20, 2017 V C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 30 | H. Selvaskandan et al. enigmatic disease, with the roles of antibodies, complement, and form double-stranded RNA [19]. A helicase then separates the increasingly microRNAs (miRs), being explored. two strands to form a passenger strand, which is degraded, and a miRs are short nucleotides that suppress gene expression by functional guide strand. The latter is loaded onto an RNA- hybridizing to the 3 untranslated region of messenger RNA induced silencing complex (RISC) and the functional strand (mRNA), promoting mRNA degradation or disrupting translation guides the RISC to potential mRNA targets [20]. The action by [11]. First discovered in 1993 [12], miRs have since been impli- which the RISC silences gene expression is determined by the de- cated in a number of chronic conditions, including cancer, heart gree of complementarity between the functional strand’s ‘seed disease and kidney disease [13–15]. The biogenesis of miRs is region’, a six to eight nucleotide long region usually beginning at now well elucidated (Fig. 1A). A primary miR transcript is gener- Position 2, and the target mRNA [21]. Complete complementarity ated from nuclear DNA through the action of RNA polymerases. results in the argonaute protein of the RISC promoting mRNA This hairpin transcript undergoes editing within the nucleus degradation, whereas partial complementarity promotes disrup- through the action of the nuclease Drosha, which cleaves the 5 tion of translation [21]. In this manner, miRs can control the ex- and 3 regions of the transcript to form precursor miR (pre-miR) pression of many genes, playing vital roles in the regulation of [16]. The pre-miR is shuttled out of the nucleus through coupling several crucial cell processes. with an exportin 5–ran-GTP complex [17], which also shields the Intriguingly, miRs were recently found to exist in stable edited transcript from degradation [18]. In the cytoplasm, the forms outside of the cell [22]. These miRs are durable even in pre-miR undergoes further processing through the action of the relatively harsh conditions, including extremes of temperatures RNAse Dicer, removing the loop region of the hairpin structure to and pH, and are resistant to immediate degradation from Fig. 1. (A) The biogenesis of miRs. (1) The biogenesis of miRs begins in the nucleus, with the production of a hairpin loop transcript known as a primary miR (pri-miR), under the action of RNA polymerase. (2) The pri-miR undergoes post-transcriptional editing by the action of Drosha, an RNAse, supported by the action of DGCR8. This 0 0 process cleaves the pri-miR at its 5 and 3 regions to produce a precursor miR (pre-miR). (3) The pre-miR is exported out of the cell in complex with exportin 5–Ran- GTP, which also prevents degradation of the edited transcript. (4) The pre-miR then undergoes further editing under the action of Dicer, which cleaves the loop struc- ture to produce a double-stranded RNA. (5) The strands are separated by helicases to produce a passenger strand that is left exposed in the cytoplasm and is thus swiftly degraded. (6) The remaining functional strand is the final mature miR. (7) The mature miR is then complexed with argonaute proteins to form the RISC. This complex moves on to degrade mRNA or disrupt translation, guided by the complementarity of the mature miR within the complex. (B) Extracelluar miRs. Extracellular miRs can be found within apoptotic bodies (released during cell apoptosis) or exosomes or in complex with argonaute proteins or high-density lipoproteins (HDLs). Of the extracellular miRs, 95–99% are argonaute bound, making this the predominant miR complex in biological fluids. Extracellular miRs can thus travel to target cells ei- ther locally within the tissue space or to distant target cells via the vasculature or lymphatic system. Here, they are able to silence gene expression following uptake into target cells. The mechanisms of secretion and uptake of extracellular miRs are yet to be clarified, although several hypotheses exist. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 31 nucleases [23]. There is a growing body of evidence to suggest Urine that extracellular miRs are selectively secreted [24] and owe Wang et al. [33] used microarray analysis to investigate miR their stability to complexes with high-density lipoproteins, expression in the urinary sediment of IgAN patients and com- argonaute proteins or their containment within exomes (Fig. 1B) pared these to that seen in disease controls, which included [25]. In vitro experiments have demonstrated that extracellular membranous nephropathy (MN) and minimal change disease miRs are capable of being taken up by ‘target’ cells, where they (MCD) [33]. While no miR was significantly dysregulated com- are then able to silence gene expression and thus influence the pared with all three controls, subgroup analysis by histolo- phenotype [26]. This has raised the possibility of these nucleo- gical severity of IgAN (Lee’s grade [34]) revealed four were tides playing a role in cell-to-cell communication, acting as dysregulated in the Grade I/II group, six were dysregulated in endocrine or paracrine agents. the Grade III group (with the down-regulation of miR-3613-3p The increasing interest in miRs has led to a number of devel- and 4668-5p being common to both) and none were dysregu- opments in the field of nephrology, ranging from their roles as lated in the Grade IV/V group compared with all controls. biomarkers for disease [27] to the development of miR antagon- These findings suggest that miR profiles in IgAN may be dy- ists as avenues for treatment [28]. The translational potential for namic, varying with the time course and severity of the dis- miRs in IgAN is thus well grounded and may represent a para- ease. A similar study reported by Konta et al. [35]that digm shift in current approaches to the disease. This review aims included diabetic nephropathy (DN) and crescentic glomer- to summarize the literature with regard to miRs and their role in ulonephritis as disease controls also found no single miR to IgAN. We begin by exploring the broad differential expression of be differentially expressed, although subgroup analysis was miRs in IgAN and provide an overview of associations between not performed. individual miRs and clinical correlates, exploring their potential Current evidence would suggest that there is generally a good roles in diagnosis and monitoring. Finally, the pathophysiological correlation between urinary and renal miR expression [35]. roles of miRs in IgAN are explored and their potentials as targets However, in IgAN this is not always the case, with miRs-150-5p for therapy are discussed. and 223-3p abundantly expressed in urinary sediment [33]but down-regulated in renal tissue [29]. One possible explanation in IgAN is that urinary miRs may be arriving as passengers in miRs are differentially expressed in the blood, erythrocytes [36]. Duan et al. [36] found 112 miRs to be differen- urine and renal tissue in IgAN tially expressed in the urinary sediment of IgAN patients by microarray analysis and validated the three highest expressed Demonstrating differential expression of miRs in IgAN com- miRs (25-3p, 144-3p and 486-5p) in a cohort of IgAN patients pared with other types of kidney disease and in healthy subjects against disease controls (MN, MCD, focal segmental glomerular is fundamental to establishing a precedent for further investiga- sclerosis, Henoch–Scho¨nlein nephritis, renal amyloidosis) and tion of miRs in IgAN. Several small studies have been conducted healthy controls. Curiously, expression of these miRs was great- using microarray analysis to measure miR expression, with est in urinary sediment containing erythrocytes compared to the real-time quantitative polymerase chain reaction (qPCR) to val- urinary sediment without (erythrocytes were removed using idate differentially expressed miRs. While microarray analysis immunomagnetic seperation). Furthermore, expression was has a role in profiling the differential expression of miRs, its highest within urinary erythrocytes compared with other urinary main disadvantage is that novel miRs are inevitably omitted cell types. This ‘loading’ of miRs appeared to be occurring within from detection. This problem is overcome by next-generation the kidney, as no difference in expression was identified between sequencing (NGS). However, the technique is expensive and the blood erythrocytes of IgAN patients and healthy controls [36]. time consuming; and as a consequence, there are limited data All of these observational cross-sectional studies demon- from NGS in IgAN. strate a differential expression of miRs in IgAN and highlight the variation in miR profiles with tissue type, disease severity The kidney and ethnicity. Although no single miR was differentially ex- pressed across all of these studies, five dysregulated miRs (134, Dai et al. [29] demonstrated, by microarray analysis, reduced ex- 185, 233, 615p and let-7a) were common to renal tissue and ei- pression of 31 miRs and elevated levels of 34 miRs in renal biop- ther PMBCs or cultured HMCs in IgAN (Table 1). sies of IgAN patients compared with healthy subjects [29]. However, no statistical analysis was performed to demonstrate significance. Tan et al. [30] used NGS to profile the renal tissue miR expression in blood, urine and renal of six IgAN patients and compared this with healthy subjects, tissue correlates with histological and clinical and identifying 84 miRs that were differentially expressed. parameters in IgAN Additionally, seven novel miRs were detected in the IgAN co- hort. Twenty of the differentially expressed miRs were common Diagnosis and risk stratification of IgAN currently relies on kid- to other microarray studies in IgAN (Table 1). However, the co- ney biopsy and histopathological assessment, which is inva- hort studied was small, no disease controls were included and sive, time consuming and expensive. miRs can be remarkably the results were not validated in a separate cohort. Consistent stable [37] and are easily obtained from blood and urine, with alteration of miR expression in the kidney in IgAN, human providing readily available sources in the clinical setting for mesangial cells (HMCs) exposed to salivary secretory IgA from diagnosis and monitoring [27]. As such, clinical and histo- IgAN patients differentially expressed 56 miRs [31]. pathological correlations between miR expression in renal tis- sue, urine and plasma have been investigated. The miRs selected for these studies have been based on earlier studies Peripheral blood mononuclear cells (PBMCs) reporting differential expression using microarray or NGS or A single study has shown differential expression of 37 miRs in because of their prominent roles in inflammation and fibrosis PBMCs in IgAN [32]. in settings outside of IgAN. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 32 | H. Selvaskandan et al. Table 1. miRs differentially expressed in at least two different studies in IgA N a b c d e f Tissue Renal tissue , Renal tissue , PMBCs , SIgA HRMCs , Urinary sediment , Urinary sediment , control used healthy healthy healthy healthy healthy þ disease healthy þ disease 15b "# 17 #" 23a "# 30a-5p "# 30d ## 98 "" 99a ## 128 #" 133a #" 133b #" 134 "" 148b #" 150 #" " " 185 "" 195 #" 199b-3p #" 221 #" 223 ## " 374b #" 486 #" " 502-3p #" 572 "" 615 ## 625 #" 628 "# 3613-3p ## let-7a "# " let-7c #" let-7d #" miRs differentially regulated in at least two microarray/RNA sequencing studies. ", increased expression compared with controls (2.0 fold); #, reduced expression compared with controls (0.5 fold). Dai et al. [29]; microarray analysis of IgAN renal tissue compared with healthy controls. Tan et al. [30]; RNA sequencing of IgAN renal tissues compared with healthy controls. Serino et al. [32]; microarray analysis of IgAN PMBCs compared with healthy controls. Liang et al. [31]); microarray analysis of HRMCs stimulated with secretory IgA from patients compared to stimulation with secretory IgA from healthy controls. Wang et al. [33]; microarray analysis of IgAN urinary sediment compared with healthy and disease controls. Duan et al. [36]; microarray analysis of IgAN urinary sediment compared with healthy and disease controls. Correlation of renal miR expression with renal histology Correlation of urinary miR expression with renal and clinical outcome histology and clinical outcome Four studies have explored 12 intrarenal miRs and their associ- Four studies from a group in Hong Kong have investigated ations with clinical features, histological findings and mRNA urinary miR profiles in IgAN and their clinical correlates. markers of fibrosis [38–41]. miRs 21-5p, 155, 199a-5p, 205 and 214- miRs 93 and 429 were associated with glomerulosclerosis [43, 3p correlated with the extent of interstitial fibrosis, miRs 21-5p 44], while miRs 217 and 377 were associated with tubulo- and 214-3p were associated with an increased risk of renal failure interstitial scarring [45]. miRs-200 b inversely correlated with {hazard ratio [HR] 4.08 [95% confidence interval (CI) 1.32–12.55] and the rate of GFR decline [44], and various correlations with HR 3.81 (95% CI 1.06–13.74), respectively} and miR-192 was associ- markers for EMT (SMAD3, vimentin and ZEB2) and cytokines ated with an increased rate of estimated glomerular filtration rate [interleukin 1b (IL-1b), Interleukin 6 (IL6) and tumour necrosis decline. Of note, levels of miR-141 and -200c expression in the kid- factor a (TNF-a)] were reported (Table 2). Most of these stud- ney correlated with vimentin and E-cadherin mRNA, respectively, ies, like those investigating renal miR expression, are limited both of which are markers of epithelial-to-mesenchymal transi- by the lack of disease controls. It is therefore not possible to tion (EMT), which is often observed in CKD (Table 2). determine if the changes in miR expression are specific for Although many of these miRs were also noted to be differen- IgAN or simply generic to CKD. The study by Szeto et al.[45] tially regulated using NGS [30], it is noteworthy that none were included diabetic and hypertensive nephropathy as disease found to be specific for IgAN when studies included disease controls, but no glomerulonephritis controls, and found controls. Dysregulation of these miRs is thus likely to be as a re- miR-17 to be overexpressed in IgAN (although healthy con- sult of generic renal fibrosis pathways common to CKD; indeed, trols were not included), but found no specific correlation miR-21-5p alone has been linked to renal fibrosis secondary to between any of the eight other miRs investigated and clinical many pathologies, including DN [42]. correlates. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 33 Table 2. miR expression in IgAN with clinical correlates Proteinuria Histopathology Renal failure Other mRNA Study Tissue miR eGFR correlation correlation associations risk correlations Hennino Kidney 21-5p Interstitial/glomeru- HR 4.08 (95% CI et al. [40] lar fibrosis 1.32–12.55) 199a-5p 214-3p HR 3.81 (95% CI 1.06–13.74) Wang et al. [38] Kidney 200c Positive Correlated with E-cadherin 205 Positive Tubular interstitial fibrosis 192 Correlated with Glomerular fibrosis rate of decline 141 Inverse correlation with vimentin Wang et al. [39] Kidney 146a Inverse Positive 155 Inverse Positive Tubular interstitial fibrosis Bao et al. [41] Kidney 21 Interstitial/glomeru- lar fibrosis Wang et al. [39] Urinary 146a Positive Inverse correlation with sediment urinary IL-1b, IL-6 and TNF-a. Positive correl- ation with RANTES 155 Positive Inverse correlation with urinary IL-1b and TNF-a. Positive correl- ation with RANTES and FOXP3 Szeto et al. [45] Urinary 15 Positive Inverse sediment 17 Positive 192 Positive Inverse 216a Inverse 217 Positive Tubular interstitial fibrosis (inverse correlation) 377 Tubular interstitial fibrosis Wang et al. [43] Urinary 21 Positive Positive correlation with sediment SMAD3 29b Positive Positive Positive correlation with SMAD3 29c Positive Positive correlation with SMAD3 93 Glomerular fibrosis Positive correlation with SMAD3 Wang et al. [44] Urinary 200a Positive Inverse correlation with sediment vimentin 200b Correlated with Positive Inverse correlation with rate of decline vimentin, ZEB2 429 Positive Positive Glomerular fibrosis Inverse correlation with (inverse vimentin correlation) Hu et al. [46] Bcells 374b Positive Correlates with MEST score Bao et al. [47] GECs 233 Positive Correlation with glomerular endo- thelial proliferation GECs, Glomerular Endothelial Cells; MEST, mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibro- sis; RANTES, regulated on activation, normal T cell expressed and secreted. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 34 | H. Selvaskandan et al. Correlation of blood miR expression with renal histology and higher serum levels of poorly O-galactosylated IgA1 O- glycoforms. and clinical outcome These studies demonstrate the regulatory potential of miRs Hu et al. [46] and Bao et al. [47] reported that expression levels of in IgA1 O-glycosylation, a fundamental component in the devel- miRs 374b and 233 in B-lymphocytes and circulating endothelial opment of IgAN (Fig. 2). Although each study restricted their pa- cells, respectively, correlated with histological findings in IgAN tient cohorts to a single ethnic group, Serino et al. [48] (Table 2). Serino et al. [32] noted that miRs let-7b and 148 were subsequently demonstrated dysregulation of miRs 148b and let- up-regulated in PMBCs using microarray, and in a retrospective 7b in an East Asian cohort as well as in Caucasians, providing multicentre study, it was demonstrated that the two miRs com- some evidence that these miRs may have a universal role in the bined could discriminate between IgAN patients, healthy sub- pathophysiology of IgAN. jects and disease controls (MCD, MN, FSGS). The combined miR biomarkers also held their diagnostic validity across two ethnic cohorts (Caucasians and East Asians) [48]. The kidney: dysregulated miRs lead to activation of local Although these data represent an exciting first step towards inflammatory pathways in IgAN a non-invasive diagnostic test for IgAN, it is yet to be validated There is also evidence that specific miR networks may be im- prospectively by a separate research group or in further cohorts portant in the development of inflammatory and profibrotic of patients. Such steps are crucial, particularly as IgAN miR pro- changes in the glomeruli and tubulointerstitium in IgAN. files can be variable even within the same ethnic group at differ- There is convincing evidence that secretory IgA (SIgA) is an ent stages of development. Indeed, two groups from China important component of the pathogenic fraction of circulating demonstrated that the presence of a polymorphism in pre-miR- IgA in IgAN [55]. Liang et al. [31, 56] examined the effect of expos- 146a was associated with IgAN susceptibility in paediatric pa- ing HMCs to purified SIgA and showed that SIgA from IgAN pa- tients of the Chinese Han population but not in adults of the tients resulted in release of several pro-inflammatory cytokines same ethnicity [49, 50]. from HMC, including IL-6, IL-8 and IL-1b, compared with SIgA from healthy subjects. Subsequent in silica analysis predicted IL- 6, IL-8 and IL-1b to be targets for miRs 16, 100-3p and 877-3p, re- miRs have demonstrable roles in the spectively. Of note, these three miRs were down-regulated in pathophysiology of IgAN IgAN on both NGS and microarray compared with healthy sub- The role of miRs in the pathogenesis of IgAN is an area of grow- jects [30, 31]. This down-regulation was validated and transfec- ing interest, fuelled by evidence of miR involvement in a spec- tion experiments with miR mimics confirmed miRs 16, 100-3p trum of other chronic diseases, including cancer and heart and 877-3p to be negative regulators of IL-6, IL-8 and IL-1b,re- failure [13, 14], and in the development of miR-based treatment spectively. Of note, the three miRs had no effect on their respect- strategies such as miR-21 antagonism for the treatment of ive cytokines at an mRNA level, suggesting the miRs disrupt Alport’s disease. translation instead of triggering mRNA degradation [31, 56]. Xing et al. [57] conducted a similar set of experiments using a human embryonic kidney cell line (HEK293), focusing on PBMCs: miRs 148b, 374b and let-7b regulate IgA1 miR-29 b-3p, which had been documented to be down-regu- O-glycosylation lated in previous NGS experiments [30]. Transfection with The extent of O-galactosylation of IgA1 is widely accepted as miR-29 b-3p down-regulated CDK6, a p65 kinase, at a protein being an important contributor to the development of IgAN. level, and inhibiting miR-29 b-3p increased TNF-a-induced p65 IgA1 hinge region O-glycosylation involves the initial addition phosphorylation, leading to an increase in TNF-a-induced IL-8 of N-acetylgalactosamine (GalNAc) to threonine or serine expression [57]. residues, catalysed by N-acetylgalactosaminyltransferase 2 There have been two laser capture microdissection studies (GALNT2) [51]. GalNAc is subsequently O-galactosylated by (LCMS) using renal biopsies to investigate the effects of miRs core 1 b1,3-galactosyltransferase (C1GALT1) acting with a within specific compartments of the nephron [41, 47]. In one chaperone, Cosmc [52, 53]. Three studies have investigated the study, Bao et al. [46] sought to clarify the roles of miRs in glomer- roles of miRs 148b, 374b and let-7b in regulating glycosylation uli displaying endothelial proliferation/hypercellularity (the E1 le- of IgA1 in PBMCs. sion of the Oxford Classification), a histological lesion associated Serino et al. [32, 54] identified up-regulation of miRs 148 and with an increased risk of progression to fibrosis and possibly re- let-7b in PMBCs of Caucasian IgAN patients and in silica analysis sponsiveness to immunosuppression in IgAN. Bao et al. [47]found revealed C1GALT1 and GALNT2 to be targets for miRs 148b and that miR-233 was down-regulated in glomeruli with hypercellu- let-7b, respectively. Indeed, both these enzymes were expressed larity compared with those without. Subsequently, through an less in PMBCs of IgAN patients compared with healthy subjects extended series of experiments, IgA from these patients was and levels negatively correlated with their respective associated shown to trigger IL-6 release in cultured HMC, resulting in a re- miRs. Subsequent transfection experiments with miR mimics duction of miR-233 levels in cultured glomerular endothelial cells and inhibitors demonstrated miRs 148b and let-7b significantly (GEnCs). This reduction caused GEnC proliferation, monocyte– suppressed expression of C1GALT1 and GALNT2, both at an endothelial adhesion and the expression of intracellular adhesion mRNA and protein level. The group also found a positive correl- molecule 1 (responsible for the migration of inflammatory cells) ation between miR-148b and the serum levels of poorly O-galac- through the upregulation of importin a5and importin a4, causing tosylated IgA1 O-glycoforms. In parallel, Hu et al. [46] showed increased p65 and STAT3 activity, respectively [47]. All of these that miR-347b was overexpressed in B-lymphocytes of Chinese changes are consistent with down-regulation of miR-233, playing IgAN patients, and following transfection with miR-347b there some role in the development of endocapillary hypercellularity in was suppression of Cosmc and a proliferation pathway regula- susceptible patients with IgAN. tor [phosphatase and tensin homolog (PTEN)] at both an mRNA In a separate study, Bao et al. [41] used LCMS to investigate and a protein level, leading to greater proliferation of B-cells the role of miR-21, an miR associated with a number of CKDs, in Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 35 Fig. 2. MicroRNAs and the four-hit hypothesis. (A) IgA1 is O-galactosylated in B cells under the action of C1GALT, GALNT2 and the chaperone protein Cosmc. miRs 148b, let-7b and 347b suppress these proteins, respectively, and thus their increased expression generates poorly O-galactosylated IgA1 O-glycoforms. (B) Hit 1 and Hit 2. The first hit of the four-hit hypothesis is the generation of poorly O-galactosylated IgA1, the second is the generation of anti-IgA1 hinge IgG antibodies. (C) Hit 3. As poorly O-galactosylated IgA1 is targeted by anti-IgA1, IgA1–IgG immune complexes are formed in circulation. (D) Hit 4. Deposition of IgA–IgG immune complexes in the kidneys results in an inflammatory process leading to glomerulonephritis, fibrosis and eventually CKD. miRs in mesangial cells: miRs 16, 100-3p and 877-3p are down- regulated in mesangial cells stimulated by secretory IgA taken from IgAN patients [31, 56]. These miRs suppress IL-6, IL-8 and IL-1b, respectively, resulting in overactiv- ity of these cytokines in IgAN. miRs in endothelial cells: IL-6 produced by mesangial cells suppresses miR-233 in GEnCs, increasing p65 and STAT3 activity, promoting local inflammatory effects [47]. miRs in podocytes: miR-26a expression is low in the glomeruli of IgAN patients. Its expression positively correlates with markers of healthy podocyte function [60]. miRs in proximal tubular cells: miR-29c, a known suppressor of extracellular matrix proteins, is reduced in expression in IgAN [58, 59]. miRs of the interstitium: miR-21 increases in IgAN, promoting EMT [41]. IgAN. The group found miR-21 expression was significantly ele- Animal studies: can they help us delineate the role for vated in both the glomeruli and tubulointerstitium in IgAN miRs in IgAN? compared with healthy subjects. In vitro, HMC exposed to IgA While there remains controversy regarding the applicability of from IgAN secreted higher levels of TGF-b1 and TNF-a compared current animal models of IgAN to human disease, acceptable with those stimulated with IgA from healthy subjects. Addition animal models of CKD do exist. As the glomerular and tubulo- of these cytokines to podocytes and a proximal tubule epithelial interstitial fibrosis seen in IgAN is likely to share many common cell line (HK2 cells) significantly increased levels of miR-21, pathways with other types of CKDs, these models may provide which was absent when neutralizing antibodies were intro- useful avenues to investigate the pathophysiological role of duced. Anti-miR-21 reduced the expression of EMT markers and miRs in IgAN. PTEN activation [41], suggesting a downstream role of miR-21 in Fang et al. [58] demonstrated that miR-29c, an miR known to IgAN fibrosis. supress extracellular matrix proteins [57], was down-regulated In contrast to the IgAN-specific effect of miRs 148b, 374b and in a rat model of tubulo-interstitial fibrosis and in human kid- let-7b in regulating O-glycosylation of IgA1 in PBMCs, it is likely neys with progressive IgAN. Having previously demonstrated a that the data generated from miR expression in renal tissue are protective effect of hypoxia inducible factor (HIF) on fibrosing likely to represent pathways common to all fibrosing renal dis- renal disease, the group postulated an interaction between HIF eases. What these data do highlight, however, is the complex and miR-29c. The group used an established model of CKD in interaction between miRs and inflammatory cytokines in IgAN, rats (5/6 nephrectomy) and exposed the rats to L-mimosine, an with multiple miRs regulating a single cytokine (as with IL-8) inducer of HIF, after which the renal tissue and blood samples and single miRs potentially regulating a multitude of inflamma- of the rats were compared with control animals. The group tory pathways (as with miR-233). Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 36 | H. Selvaskandan et al. found that miR-29c expression decreased following nephrec- References tomy and increased in response to L-mimosine. This increase 1. McGrogan A, Franssen CFM, de Vries CS. The incidence of correlated with HIF levels and inversely correlated with intersti- primary glomerulonephritis worldwide: a systematic review tial fibrosis. 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Cell 2004; 116: 281–297 ous cancers [61]. While it is unlikely that targeting a single miR 12. Lee RC, Feinbaum RL, Ambros V. The C. elegans hetero- will be effective for the treatment of IgAN, it is possible that a chronic gene lin-4 encodes small RNAs with antisense com- multi-antago-miR approach may be efficacious to target differ- plementarity to lin-14. Cell 1993; 75: 843–854 ent elements of the disease. For instance, blocking the actions 13. Jansson MD, Lund AH. MicroRNA and cancer. Mol Oncol 2012; of miRs let-7b, 374b and 148b could conceivably blunt the ‘first 6: 590–610 hit’ of IgAN by reducing circulating poorly O-galactosylated IgA1 14. Vegter EL, van der Meer P, de Windt LJ et al. MicroRNAs in O-glycoform levels, while antagonizing miR-21 may be a suit- heart failure: from biomarker to target for therapy. Eur J able target to reduce downstream fibrosis developing as a con- Heart Fail 2016; 18: 457–468 sequence of glomerular injury and the development of 15. 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Nucleic It is clear from the literature that while we have a tantalizing Acids Res 2004; 32: 4776–4785 early insight into the role of miRs in IgAN, it is still too early for 19. Zhang H, Kolb FA, Brondani V et al. Human Dicer preferen- tially cleaves dsRNAs at their termini without a requirement concrete conclusions. We need properly validated studies, spanning different ethnic groups and encompassing diverse for ATP. EMBO J 2002; 21: 5875–5885 20. Pratt AJ, MacRae IJ. The RNA-induced silencing complex: a versa- populations before we can reliably comment on the role of par- ticular miRs in IgAN. The role of miRs in regulating IgA1 O-gly- tile gene-silencing machine. JBiolChem 2009; 284: 17897–17901 21. Mullany LE, Herrick JS, Wolff RK et al. MicroRNA seed region cosylation is currently the most convincing data we have; however, this is likely to change in the coming years as large length impact on target messenger RNA expression and sur- vival in colorectal cancer. PLoS One 2016; 11: e0154177 studies report on the value of serum and urine miRs and miR expression in the kidney in IgAN and other forms of kidney 22. Turchinovich A, Weiz L, Burwinkel B. Extracellular miRNAs: disease. the mystery of their origin and function. Trends Biochem Sci 2012; 37: 460–465 23. Turchinovich A, Weiz L, Langheinz A et al. Characterization Conflict of interest statement of extracellular circulating microRNA. Nucleic Acids Res 2011; 39: 7223–7233 None declared. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 37 44. Wang G, Kwan BC-H, Lai FM-M et al. Expression of 24. Pigati L, Yaddanapudi SCS, Iyengar R et al. Selective release of microRNA species from normal and malignant mammary microRNAs in the urinary sediment of patients with IgA epithelial cells. PLoS One 2010; 5: e13515 nephropathy. Dis Markers 2010; 28: 79–86 25. 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Anti–microRNA- importin a4 and a5 in IgA nephropathy. Kidney Int 2014; 85: 21 oligonucleotides prevent Alport nephropathy progression 624–635 48. Serino G, Pesce F, Sallustio F et al. In a retrospective interna- by stimulating metabolic pathways. J Clin Invest 2015; 125: 141–156. tional study, circulating miR-148b and let-7b were found to 29. Dai Y, Sui W, Lan H et al. Microarray analysis of micro- be serum markers for detecting primary IgA nephropathy. ribonucleic acid expression in primary immunoglobulin A Kidney Int 2016; 89: 683–692 nephropathy. Saudi Med J 2008; 29: 1388–1393 49. Lin J, Huang Y, Zhang X et al. Association of miR-146a 30. Tan K, Chen J, Li W et al. Genome-wide analysis of rs2910164 with childhood IgA nephropathy. Pediatr Nephrol microRNAs expression profiling in patients with primary IgA 2014; 29: 1979–1986 nephropathy. Genome 2013; 56: 161–169 50. Yang B, Wei W, Shi Y et al. Genetic variation in miR-146a is 31. Liang Y, Zhao G, Tang L et al. MiR-100-3p and miR-877-3p not associated with susceptibility to IgA nephropathy in regulate overproduction of IL-8 and IL-1b in mesangial cells adults from a Chinese Han population. PLoS One 2015; 10: activated by secretory IgA from IgA nephropathy patients. e0139554 Exp Cell Res 2016; 347: 312–321 51. Iwasaki H, Zhang Y, Tachibana K et al. Initiation of O-glycan 32. Serino G, Sallustio F, Cox SN et al. Abnormal miR-148b ex- synthesis in IgA1 hinge region is determined by a single pression promotes aberrant glycosylation of IgA1 in IgA enzyme, UDP-N-acetyl-alpha-D-galactosamine:polypeptide nephropathy. J Am Soc Nephrol 2012; 23: 814–824 N-acetylgalactosaminyltransferase 2. J Biol Chem 2003; 278: 33. Wang N, Bu R, Duan Z et al. Profiling and initial validation of 5613–5621 urinary microRNAs as biomarkers in IgA nephropathy. PeerJ 52. Gale DP, Molyneux K, Wimbury D et al. Galactosylation of 2015; 3: e990 IgA1 Is associated with common variation in C1GALT1. JAm 34. 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Hennino M, Buob D, Van der Hauwaert C et al. miR-21-5p interstitial fibrosis in humans and rats and restored by HIF-a renal expression is associated with fibrosis and renal sur- activation. Am J Physiol Renal Physiol 2013; 304: F1274–F1282 vival in patients with IgA nephropathy. Sci Rep 2016; 6: 27209 59. Kriegel AJ, Liu Y, Fang Y et al. The miR-29 family: genomics, 41. Bao H, Hu S, Zhang C et al. Inhibition of miRNA-21 prevents cell biology, and relevance to renal and cardiovascular in- fibrogenic activation in podocytes and tubular cells in IgA jury. Physiol Genomics 2012; 44: 237–244 nephropathy. Biochem Biophys Res Commun 2014; 444: 455–460 60. Ichii O, Otsuka-Kanazawa S, Horino T et al. Decreased miR- 42. McClelland AD, Herman-Edelstein M, Komers R et al. miR-21 26a expression correlates with the progression of podocyte promotes renal fibrosis in diabetic nephropathy by targeting injury in autoimmune glomerulonephritis. PLoS One 2014; 9: PTEN and SMAD7. Clin Sci 2015; 129: 1237–1249 e110383 43. Wang G, Kwan BC-H, Lai FM-M et al. Urinary miR-21, miR-29, 61. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a and miR-93: novel biomarkers of fibrosis. Am J Nephrol 2012; new era for the management of cancer and other diseases. Nat Rev Drug Discov 2017; 16: 203–222 36: 412–418 Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Kidney Journal Oxford University Press

MicroRNAs: a new avenue to understand, investigate and treat immunoglobulin A nephropathy?

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Abstract

IgA nephropathy (IgAN) is the most common cause of primary glomerulonephritis worldwide. Up to 30% of cases develop the progressive form of the disease, eventually requiring renal replacement therapy. Diagnosis and risk stratification relies on an invasive kidney biopsy and management options are limited, with recurrence following renal transplantation being common. Thus the quest to understand the pathophysiology of IgAN has been one of great importance. MicroRNAs (miRs) are short nucleotides that suppress gene expression by hybridizing to the 3 untranslated region of messenger RNA (mRNAs), promoting mRNA degradation or disrupting translation. First discovered in 1993, miRs have since been implicated in a number of chronic conditions, including cancer, heart disease and kidney disease. The mounting interest in the field of miRs has led to fascinating developments in the field of nephrology, ranging from their roles as biomarkers for disease to the development of miR antagonists as avenues for treatment. The translational potential for miRs in IgAN is thus well grounded and may represent a paradigm shift in current approaches to the disease. This review aims to summarize the literature with regard to miRs and their roles in IgAN. Key words: biomarkers, epigenetic, IgA nephropathy, microRNA, therapy Immunoglobulin A nephropathy (IgAN) is a primary kidney dis- this modelis anincrease inpoorly O-galactosylated IgA1 O-glyco- ease characterized by the deposition of IgA in the glomerulus. forms in the serum displaying low levels of O-linked galactose at With an incidence of at least 2.5 per 100,000/year, it is the most the IgA1 hinge [7]. A systemic IgG antibody response is directed common cause of primary glomerulonephritis worldwide [1]. The against this altered hinge region, generating IgA–IgG antibody condition exacts a substantial socio-economic cost, with up to complexes, which are then deposited in the kidneys, triggering an 30% of cases developing a progressive phenotype characterized inflammatory cascade eventually leading to chronic kidney dis- by glomerular or interstitial fibrosis, eventually requiring renal ease (CKD). Indeed, a proportion of patients with increased levels replacement therapy [2]. Diagnosis and risk stratification relies of poorly O-galactosylated IgA1 O-glycoforms never develop the on an invasive kidney biopsy [3] and management options are disease [8]and in vitro experiments suggest mesangial cells of pa- limited, with recurrence following renal transplantation being tients with IgAN may be ‘primed’ to respond to IgG–IgA complexes common [4, 5]. Thus the quest to understand the pathophysi- [9], highlighting the necessity of multiple hits in the pathophysi- ology of IgAN has been one of the great importance. ology of IgAN. Furthermore, it is possible that each ‘hit’ may be The prevalent model for the pathogenesis of IgAN is the ‘four- mediated by several independent variables, such as genetic, epi- hit hypothesis’, which postulates that the kidney is an innocent genetic and environmental factors, including exposure to mucosal bystander of an otherwise systemic disease [6]. Fundamental to infections [10]. Thus there is still much to be clarified about this Received: June 12, 2017. Editorial decision: July 20, 2017 V C The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 30 | H. Selvaskandan et al. enigmatic disease, with the roles of antibodies, complement, and form double-stranded RNA [19]. A helicase then separates the increasingly microRNAs (miRs), being explored. two strands to form a passenger strand, which is degraded, and a miRs are short nucleotides that suppress gene expression by functional guide strand. The latter is loaded onto an RNA- hybridizing to the 3 untranslated region of messenger RNA induced silencing complex (RISC) and the functional strand (mRNA), promoting mRNA degradation or disrupting translation guides the RISC to potential mRNA targets [20]. The action by [11]. First discovered in 1993 [12], miRs have since been impli- which the RISC silences gene expression is determined by the de- cated in a number of chronic conditions, including cancer, heart gree of complementarity between the functional strand’s ‘seed disease and kidney disease [13–15]. The biogenesis of miRs is region’, a six to eight nucleotide long region usually beginning at now well elucidated (Fig. 1A). A primary miR transcript is gener- Position 2, and the target mRNA [21]. Complete complementarity ated from nuclear DNA through the action of RNA polymerases. results in the argonaute protein of the RISC promoting mRNA This hairpin transcript undergoes editing within the nucleus degradation, whereas partial complementarity promotes disrup- through the action of the nuclease Drosha, which cleaves the 5 tion of translation [21]. In this manner, miRs can control the ex- and 3 regions of the transcript to form precursor miR (pre-miR) pression of many genes, playing vital roles in the regulation of [16]. The pre-miR is shuttled out of the nucleus through coupling several crucial cell processes. with an exportin 5–ran-GTP complex [17], which also shields the Intriguingly, miRs were recently found to exist in stable edited transcript from degradation [18]. In the cytoplasm, the forms outside of the cell [22]. These miRs are durable even in pre-miR undergoes further processing through the action of the relatively harsh conditions, including extremes of temperatures RNAse Dicer, removing the loop region of the hairpin structure to and pH, and are resistant to immediate degradation from Fig. 1. (A) The biogenesis of miRs. (1) The biogenesis of miRs begins in the nucleus, with the production of a hairpin loop transcript known as a primary miR (pri-miR), under the action of RNA polymerase. (2) The pri-miR undergoes post-transcriptional editing by the action of Drosha, an RNAse, supported by the action of DGCR8. This 0 0 process cleaves the pri-miR at its 5 and 3 regions to produce a precursor miR (pre-miR). (3) The pre-miR is exported out of the cell in complex with exportin 5–Ran- GTP, which also prevents degradation of the edited transcript. (4) The pre-miR then undergoes further editing under the action of Dicer, which cleaves the loop struc- ture to produce a double-stranded RNA. (5) The strands are separated by helicases to produce a passenger strand that is left exposed in the cytoplasm and is thus swiftly degraded. (6) The remaining functional strand is the final mature miR. (7) The mature miR is then complexed with argonaute proteins to form the RISC. This complex moves on to degrade mRNA or disrupt translation, guided by the complementarity of the mature miR within the complex. (B) Extracelluar miRs. Extracellular miRs can be found within apoptotic bodies (released during cell apoptosis) or exosomes or in complex with argonaute proteins or high-density lipoproteins (HDLs). Of the extracellular miRs, 95–99% are argonaute bound, making this the predominant miR complex in biological fluids. Extracellular miRs can thus travel to target cells ei- ther locally within the tissue space or to distant target cells via the vasculature or lymphatic system. Here, they are able to silence gene expression following uptake into target cells. The mechanisms of secretion and uptake of extracellular miRs are yet to be clarified, although several hypotheses exist. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 31 nucleases [23]. There is a growing body of evidence to suggest Urine that extracellular miRs are selectively secreted [24] and owe Wang et al. [33] used microarray analysis to investigate miR their stability to complexes with high-density lipoproteins, expression in the urinary sediment of IgAN patients and com- argonaute proteins or their containment within exomes (Fig. 1B) pared these to that seen in disease controls, which included [25]. In vitro experiments have demonstrated that extracellular membranous nephropathy (MN) and minimal change disease miRs are capable of being taken up by ‘target’ cells, where they (MCD) [33]. While no miR was significantly dysregulated com- are then able to silence gene expression and thus influence the pared with all three controls, subgroup analysis by histolo- phenotype [26]. This has raised the possibility of these nucleo- gical severity of IgAN (Lee’s grade [34]) revealed four were tides playing a role in cell-to-cell communication, acting as dysregulated in the Grade I/II group, six were dysregulated in endocrine or paracrine agents. the Grade III group (with the down-regulation of miR-3613-3p The increasing interest in miRs has led to a number of devel- and 4668-5p being common to both) and none were dysregu- opments in the field of nephrology, ranging from their roles as lated in the Grade IV/V group compared with all controls. biomarkers for disease [27] to the development of miR antagon- These findings suggest that miR profiles in IgAN may be dy- ists as avenues for treatment [28]. The translational potential for namic, varying with the time course and severity of the dis- miRs in IgAN is thus well grounded and may represent a para- ease. A similar study reported by Konta et al. [35]that digm shift in current approaches to the disease. This review aims included diabetic nephropathy (DN) and crescentic glomer- to summarize the literature with regard to miRs and their role in ulonephritis as disease controls also found no single miR to IgAN. We begin by exploring the broad differential expression of be differentially expressed, although subgroup analysis was miRs in IgAN and provide an overview of associations between not performed. individual miRs and clinical correlates, exploring their potential Current evidence would suggest that there is generally a good roles in diagnosis and monitoring. Finally, the pathophysiological correlation between urinary and renal miR expression [35]. roles of miRs in IgAN are explored and their potentials as targets However, in IgAN this is not always the case, with miRs-150-5p for therapy are discussed. and 223-3p abundantly expressed in urinary sediment [33]but down-regulated in renal tissue [29]. One possible explanation in IgAN is that urinary miRs may be arriving as passengers in miRs are differentially expressed in the blood, erythrocytes [36]. Duan et al. [36] found 112 miRs to be differen- urine and renal tissue in IgAN tially expressed in the urinary sediment of IgAN patients by microarray analysis and validated the three highest expressed Demonstrating differential expression of miRs in IgAN com- miRs (25-3p, 144-3p and 486-5p) in a cohort of IgAN patients pared with other types of kidney disease and in healthy subjects against disease controls (MN, MCD, focal segmental glomerular is fundamental to establishing a precedent for further investiga- sclerosis, Henoch–Scho¨nlein nephritis, renal amyloidosis) and tion of miRs in IgAN. Several small studies have been conducted healthy controls. Curiously, expression of these miRs was great- using microarray analysis to measure miR expression, with est in urinary sediment containing erythrocytes compared to the real-time quantitative polymerase chain reaction (qPCR) to val- urinary sediment without (erythrocytes were removed using idate differentially expressed miRs. While microarray analysis immunomagnetic seperation). Furthermore, expression was has a role in profiling the differential expression of miRs, its highest within urinary erythrocytes compared with other urinary main disadvantage is that novel miRs are inevitably omitted cell types. This ‘loading’ of miRs appeared to be occurring within from detection. This problem is overcome by next-generation the kidney, as no difference in expression was identified between sequencing (NGS). However, the technique is expensive and the blood erythrocytes of IgAN patients and healthy controls [36]. time consuming; and as a consequence, there are limited data All of these observational cross-sectional studies demon- from NGS in IgAN. strate a differential expression of miRs in IgAN and highlight the variation in miR profiles with tissue type, disease severity The kidney and ethnicity. Although no single miR was differentially ex- pressed across all of these studies, five dysregulated miRs (134, Dai et al. [29] demonstrated, by microarray analysis, reduced ex- 185, 233, 615p and let-7a) were common to renal tissue and ei- pression of 31 miRs and elevated levels of 34 miRs in renal biop- ther PMBCs or cultured HMCs in IgAN (Table 1). sies of IgAN patients compared with healthy subjects [29]. However, no statistical analysis was performed to demonstrate significance. Tan et al. [30] used NGS to profile the renal tissue miR expression in blood, urine and renal of six IgAN patients and compared this with healthy subjects, tissue correlates with histological and clinical and identifying 84 miRs that were differentially expressed. parameters in IgAN Additionally, seven novel miRs were detected in the IgAN co- hort. Twenty of the differentially expressed miRs were common Diagnosis and risk stratification of IgAN currently relies on kid- to other microarray studies in IgAN (Table 1). However, the co- ney biopsy and histopathological assessment, which is inva- hort studied was small, no disease controls were included and sive, time consuming and expensive. miRs can be remarkably the results were not validated in a separate cohort. Consistent stable [37] and are easily obtained from blood and urine, with alteration of miR expression in the kidney in IgAN, human providing readily available sources in the clinical setting for mesangial cells (HMCs) exposed to salivary secretory IgA from diagnosis and monitoring [27]. As such, clinical and histo- IgAN patients differentially expressed 56 miRs [31]. pathological correlations between miR expression in renal tis- sue, urine and plasma have been investigated. The miRs selected for these studies have been based on earlier studies Peripheral blood mononuclear cells (PBMCs) reporting differential expression using microarray or NGS or A single study has shown differential expression of 37 miRs in because of their prominent roles in inflammation and fibrosis PBMCs in IgAN [32]. in settings outside of IgAN. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 32 | H. Selvaskandan et al. Table 1. miRs differentially expressed in at least two different studies in IgA N a b c d e f Tissue Renal tissue , Renal tissue , PMBCs , SIgA HRMCs , Urinary sediment , Urinary sediment , control used healthy healthy healthy healthy healthy þ disease healthy þ disease 15b "# 17 #" 23a "# 30a-5p "# 30d ## 98 "" 99a ## 128 #" 133a #" 133b #" 134 "" 148b #" 150 #" " " 185 "" 195 #" 199b-3p #" 221 #" 223 ## " 374b #" 486 #" " 502-3p #" 572 "" 615 ## 625 #" 628 "# 3613-3p ## let-7a "# " let-7c #" let-7d #" miRs differentially regulated in at least two microarray/RNA sequencing studies. ", increased expression compared with controls (2.0 fold); #, reduced expression compared with controls (0.5 fold). Dai et al. [29]; microarray analysis of IgAN renal tissue compared with healthy controls. Tan et al. [30]; RNA sequencing of IgAN renal tissues compared with healthy controls. Serino et al. [32]; microarray analysis of IgAN PMBCs compared with healthy controls. Liang et al. [31]); microarray analysis of HRMCs stimulated with secretory IgA from patients compared to stimulation with secretory IgA from healthy controls. Wang et al. [33]; microarray analysis of IgAN urinary sediment compared with healthy and disease controls. Duan et al. [36]; microarray analysis of IgAN urinary sediment compared with healthy and disease controls. Correlation of renal miR expression with renal histology Correlation of urinary miR expression with renal and clinical outcome histology and clinical outcome Four studies have explored 12 intrarenal miRs and their associ- Four studies from a group in Hong Kong have investigated ations with clinical features, histological findings and mRNA urinary miR profiles in IgAN and their clinical correlates. markers of fibrosis [38–41]. miRs 21-5p, 155, 199a-5p, 205 and 214- miRs 93 and 429 were associated with glomerulosclerosis [43, 3p correlated with the extent of interstitial fibrosis, miRs 21-5p 44], while miRs 217 and 377 were associated with tubulo- and 214-3p were associated with an increased risk of renal failure interstitial scarring [45]. miRs-200 b inversely correlated with {hazard ratio [HR] 4.08 [95% confidence interval (CI) 1.32–12.55] and the rate of GFR decline [44], and various correlations with HR 3.81 (95% CI 1.06–13.74), respectively} and miR-192 was associ- markers for EMT (SMAD3, vimentin and ZEB2) and cytokines ated with an increased rate of estimated glomerular filtration rate [interleukin 1b (IL-1b), Interleukin 6 (IL6) and tumour necrosis decline. Of note, levels of miR-141 and -200c expression in the kid- factor a (TNF-a)] were reported (Table 2). Most of these stud- ney correlated with vimentin and E-cadherin mRNA, respectively, ies, like those investigating renal miR expression, are limited both of which are markers of epithelial-to-mesenchymal transi- by the lack of disease controls. It is therefore not possible to tion (EMT), which is often observed in CKD (Table 2). determine if the changes in miR expression are specific for Although many of these miRs were also noted to be differen- IgAN or simply generic to CKD. The study by Szeto et al.[45] tially regulated using NGS [30], it is noteworthy that none were included diabetic and hypertensive nephropathy as disease found to be specific for IgAN when studies included disease controls, but no glomerulonephritis controls, and found controls. Dysregulation of these miRs is thus likely to be as a re- miR-17 to be overexpressed in IgAN (although healthy con- sult of generic renal fibrosis pathways common to CKD; indeed, trols were not included), but found no specific correlation miR-21-5p alone has been linked to renal fibrosis secondary to between any of the eight other miRs investigated and clinical many pathologies, including DN [42]. correlates. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 33 Table 2. miR expression in IgAN with clinical correlates Proteinuria Histopathology Renal failure Other mRNA Study Tissue miR eGFR correlation correlation associations risk correlations Hennino Kidney 21-5p Interstitial/glomeru- HR 4.08 (95% CI et al. [40] lar fibrosis 1.32–12.55) 199a-5p 214-3p HR 3.81 (95% CI 1.06–13.74) Wang et al. [38] Kidney 200c Positive Correlated with E-cadherin 205 Positive Tubular interstitial fibrosis 192 Correlated with Glomerular fibrosis rate of decline 141 Inverse correlation with vimentin Wang et al. [39] Kidney 146a Inverse Positive 155 Inverse Positive Tubular interstitial fibrosis Bao et al. [41] Kidney 21 Interstitial/glomeru- lar fibrosis Wang et al. [39] Urinary 146a Positive Inverse correlation with sediment urinary IL-1b, IL-6 and TNF-a. Positive correl- ation with RANTES 155 Positive Inverse correlation with urinary IL-1b and TNF-a. Positive correl- ation with RANTES and FOXP3 Szeto et al. [45] Urinary 15 Positive Inverse sediment 17 Positive 192 Positive Inverse 216a Inverse 217 Positive Tubular interstitial fibrosis (inverse correlation) 377 Tubular interstitial fibrosis Wang et al. [43] Urinary 21 Positive Positive correlation with sediment SMAD3 29b Positive Positive Positive correlation with SMAD3 29c Positive Positive correlation with SMAD3 93 Glomerular fibrosis Positive correlation with SMAD3 Wang et al. [44] Urinary 200a Positive Inverse correlation with sediment vimentin 200b Correlated with Positive Inverse correlation with rate of decline vimentin, ZEB2 429 Positive Positive Glomerular fibrosis Inverse correlation with (inverse vimentin correlation) Hu et al. [46] Bcells 374b Positive Correlates with MEST score Bao et al. [47] GECs 233 Positive Correlation with glomerular endo- thelial proliferation GECs, Glomerular Endothelial Cells; MEST, mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibro- sis; RANTES, regulated on activation, normal T cell expressed and secreted. Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 34 | H. Selvaskandan et al. Correlation of blood miR expression with renal histology and higher serum levels of poorly O-galactosylated IgA1 O- glycoforms. and clinical outcome These studies demonstrate the regulatory potential of miRs Hu et al. [46] and Bao et al. [47] reported that expression levels of in IgA1 O-glycosylation, a fundamental component in the devel- miRs 374b and 233 in B-lymphocytes and circulating endothelial opment of IgAN (Fig. 2). Although each study restricted their pa- cells, respectively, correlated with histological findings in IgAN tient cohorts to a single ethnic group, Serino et al. [48] (Table 2). Serino et al. [32] noted that miRs let-7b and 148 were subsequently demonstrated dysregulation of miRs 148b and let- up-regulated in PMBCs using microarray, and in a retrospective 7b in an East Asian cohort as well as in Caucasians, providing multicentre study, it was demonstrated that the two miRs com- some evidence that these miRs may have a universal role in the bined could discriminate between IgAN patients, healthy sub- pathophysiology of IgAN. jects and disease controls (MCD, MN, FSGS). The combined miR biomarkers also held their diagnostic validity across two ethnic cohorts (Caucasians and East Asians) [48]. The kidney: dysregulated miRs lead to activation of local Although these data represent an exciting first step towards inflammatory pathways in IgAN a non-invasive diagnostic test for IgAN, it is yet to be validated There is also evidence that specific miR networks may be im- prospectively by a separate research group or in further cohorts portant in the development of inflammatory and profibrotic of patients. Such steps are crucial, particularly as IgAN miR pro- changes in the glomeruli and tubulointerstitium in IgAN. files can be variable even within the same ethnic group at differ- There is convincing evidence that secretory IgA (SIgA) is an ent stages of development. Indeed, two groups from China important component of the pathogenic fraction of circulating demonstrated that the presence of a polymorphism in pre-miR- IgA in IgAN [55]. Liang et al. [31, 56] examined the effect of expos- 146a was associated with IgAN susceptibility in paediatric pa- ing HMCs to purified SIgA and showed that SIgA from IgAN pa- tients of the Chinese Han population but not in adults of the tients resulted in release of several pro-inflammatory cytokines same ethnicity [49, 50]. from HMC, including IL-6, IL-8 and IL-1b, compared with SIgA from healthy subjects. Subsequent in silica analysis predicted IL- 6, IL-8 and IL-1b to be targets for miRs 16, 100-3p and 877-3p, re- miRs have demonstrable roles in the spectively. Of note, these three miRs were down-regulated in pathophysiology of IgAN IgAN on both NGS and microarray compared with healthy sub- The role of miRs in the pathogenesis of IgAN is an area of grow- jects [30, 31]. This down-regulation was validated and transfec- ing interest, fuelled by evidence of miR involvement in a spec- tion experiments with miR mimics confirmed miRs 16, 100-3p trum of other chronic diseases, including cancer and heart and 877-3p to be negative regulators of IL-6, IL-8 and IL-1b,re- failure [13, 14], and in the development of miR-based treatment spectively. Of note, the three miRs had no effect on their respect- strategies such as miR-21 antagonism for the treatment of ive cytokines at an mRNA level, suggesting the miRs disrupt Alport’s disease. translation instead of triggering mRNA degradation [31, 56]. Xing et al. [57] conducted a similar set of experiments using a human embryonic kidney cell line (HEK293), focusing on PBMCs: miRs 148b, 374b and let-7b regulate IgA1 miR-29 b-3p, which had been documented to be down-regu- O-glycosylation lated in previous NGS experiments [30]. Transfection with The extent of O-galactosylation of IgA1 is widely accepted as miR-29 b-3p down-regulated CDK6, a p65 kinase, at a protein being an important contributor to the development of IgAN. level, and inhibiting miR-29 b-3p increased TNF-a-induced p65 IgA1 hinge region O-glycosylation involves the initial addition phosphorylation, leading to an increase in TNF-a-induced IL-8 of N-acetylgalactosamine (GalNAc) to threonine or serine expression [57]. residues, catalysed by N-acetylgalactosaminyltransferase 2 There have been two laser capture microdissection studies (GALNT2) [51]. GalNAc is subsequently O-galactosylated by (LCMS) using renal biopsies to investigate the effects of miRs core 1 b1,3-galactosyltransferase (C1GALT1) acting with a within specific compartments of the nephron [41, 47]. In one chaperone, Cosmc [52, 53]. Three studies have investigated the study, Bao et al. [46] sought to clarify the roles of miRs in glomer- roles of miRs 148b, 374b and let-7b in regulating glycosylation uli displaying endothelial proliferation/hypercellularity (the E1 le- of IgA1 in PBMCs. sion of the Oxford Classification), a histological lesion associated Serino et al. [32, 54] identified up-regulation of miRs 148 and with an increased risk of progression to fibrosis and possibly re- let-7b in PMBCs of Caucasian IgAN patients and in silica analysis sponsiveness to immunosuppression in IgAN. Bao et al. [47]found revealed C1GALT1 and GALNT2 to be targets for miRs 148b and that miR-233 was down-regulated in glomeruli with hypercellu- let-7b, respectively. Indeed, both these enzymes were expressed larity compared with those without. Subsequently, through an less in PMBCs of IgAN patients compared with healthy subjects extended series of experiments, IgA from these patients was and levels negatively correlated with their respective associated shown to trigger IL-6 release in cultured HMC, resulting in a re- miRs. Subsequent transfection experiments with miR mimics duction of miR-233 levels in cultured glomerular endothelial cells and inhibitors demonstrated miRs 148b and let-7b significantly (GEnCs). This reduction caused GEnC proliferation, monocyte– suppressed expression of C1GALT1 and GALNT2, both at an endothelial adhesion and the expression of intracellular adhesion mRNA and protein level. The group also found a positive correl- molecule 1 (responsible for the migration of inflammatory cells) ation between miR-148b and the serum levels of poorly O-galac- through the upregulation of importin a5and importin a4, causing tosylated IgA1 O-glycoforms. In parallel, Hu et al. [46] showed increased p65 and STAT3 activity, respectively [47]. All of these that miR-347b was overexpressed in B-lymphocytes of Chinese changes are consistent with down-regulation of miR-233, playing IgAN patients, and following transfection with miR-347b there some role in the development of endocapillary hypercellularity in was suppression of Cosmc and a proliferation pathway regula- susceptible patients with IgAN. tor [phosphatase and tensin homolog (PTEN)] at both an mRNA In a separate study, Bao et al. [41] used LCMS to investigate and a protein level, leading to greater proliferation of B-cells the role of miR-21, an miR associated with a number of CKDs, in Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Role of miRs in IgA Nephropathy | 35 Fig. 2. MicroRNAs and the four-hit hypothesis. (A) IgA1 is O-galactosylated in B cells under the action of C1GALT, GALNT2 and the chaperone protein Cosmc. miRs 148b, let-7b and 347b suppress these proteins, respectively, and thus their increased expression generates poorly O-galactosylated IgA1 O-glycoforms. (B) Hit 1 and Hit 2. The first hit of the four-hit hypothesis is the generation of poorly O-galactosylated IgA1, the second is the generation of anti-IgA1 hinge IgG antibodies. (C) Hit 3. As poorly O-galactosylated IgA1 is targeted by anti-IgA1, IgA1–IgG immune complexes are formed in circulation. (D) Hit 4. Deposition of IgA–IgG immune complexes in the kidneys results in an inflammatory process leading to glomerulonephritis, fibrosis and eventually CKD. miRs in mesangial cells: miRs 16, 100-3p and 877-3p are down- regulated in mesangial cells stimulated by secretory IgA taken from IgAN patients [31, 56]. These miRs suppress IL-6, IL-8 and IL-1b, respectively, resulting in overactiv- ity of these cytokines in IgAN. miRs in endothelial cells: IL-6 produced by mesangial cells suppresses miR-233 in GEnCs, increasing p65 and STAT3 activity, promoting local inflammatory effects [47]. miRs in podocytes: miR-26a expression is low in the glomeruli of IgAN patients. Its expression positively correlates with markers of healthy podocyte function [60]. miRs in proximal tubular cells: miR-29c, a known suppressor of extracellular matrix proteins, is reduced in expression in IgAN [58, 59]. miRs of the interstitium: miR-21 increases in IgAN, promoting EMT [41]. IgAN. The group found miR-21 expression was significantly ele- Animal studies: can they help us delineate the role for vated in both the glomeruli and tubulointerstitium in IgAN miRs in IgAN? compared with healthy subjects. In vitro, HMC exposed to IgA While there remains controversy regarding the applicability of from IgAN secreted higher levels of TGF-b1 and TNF-a compared current animal models of IgAN to human disease, acceptable with those stimulated with IgA from healthy subjects. Addition animal models of CKD do exist. As the glomerular and tubulo- of these cytokines to podocytes and a proximal tubule epithelial interstitial fibrosis seen in IgAN is likely to share many common cell line (HK2 cells) significantly increased levels of miR-21, pathways with other types of CKDs, these models may provide which was absent when neutralizing antibodies were intro- useful avenues to investigate the pathophysiological role of duced. Anti-miR-21 reduced the expression of EMT markers and miRs in IgAN. PTEN activation [41], suggesting a downstream role of miR-21 in Fang et al. [58] demonstrated that miR-29c, an miR known to IgAN fibrosis. supress extracellular matrix proteins [57], was down-regulated In contrast to the IgAN-specific effect of miRs 148b, 374b and in a rat model of tubulo-interstitial fibrosis and in human kid- let-7b in regulating O-glycosylation of IgA1 in PBMCs, it is likely neys with progressive IgAN. Having previously demonstrated a that the data generated from miR expression in renal tissue are protective effect of hypoxia inducible factor (HIF) on fibrosing likely to represent pathways common to all fibrosing renal dis- renal disease, the group postulated an interaction between HIF eases. What these data do highlight, however, is the complex and miR-29c. The group used an established model of CKD in interaction between miRs and inflammatory cytokines in IgAN, rats (5/6 nephrectomy) and exposed the rats to L-mimosine, an with multiple miRs regulating a single cytokine (as with IL-8) inducer of HIF, after which the renal tissue and blood samples and single miRs potentially regulating a multitude of inflamma- of the rats were compared with control animals. The group tory pathways (as with miR-233). Downloaded from https://academic.oup.com/ckj/article-abstract/11/1/29/4191363 by Ed 'DeepDyve' Gillespie user on 16 March 2018 36 | H. Selvaskandan et al. found that miR-29c expression decreased following nephrec- References tomy and increased in response to L-mimosine. This increase 1. McGrogan A, Franssen CFM, de Vries CS. The incidence of correlated with HIF levels and inversely correlated with intersti- primary glomerulonephritis worldwide: a systematic review tial fibrosis. 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Clinical Kidney JournalOxford University Press

Published: Feb 1, 2018

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