Epigenetic Mechanisms and Nephrotic Syndrome: A Systematic Review

A small subset of people with nephrotic syndrome (NS) have genetically driven disease. However, the disease mechanisms for the remaining majority are unknown. Epigenetic marks are reversible but stable regulators of gene expression with utility as biomarkers and therapeutic targets. We aimed to identify and assess all published human studies of epigenetic mechanisms in NS. PubMed (MEDLINE) and Embase were searched for original research articles examining any epigenetic mechanism in samples collected from people with steroid resistant NS, steroid sensitive NS, focal segmental glomerulosclerosis or minimal change disease. Study quality was assessed by using the Joanna Briggs Institute critical appraisal tools. Forty-nine studies met our inclusion criteria. The majority of these examined micro-RNAs (n = 35, 71%). Study quality was low, with only 23 deemed higher quality, and most of these included fewer than 100 patients and failed to validate findings in a second cohort. However, there were some promising concordant results between the studies; higher levels of serum miR-191 and miR-30c, and urinary miR-23b-3p and miR-30a-5p were observed in NS compared to controls. We have identified that the epigenome, particularly DNA methylation and histone modifications, has been understudied in NS. Large clinical studies, which utilise the latest high-throughput technologies and analytical pipelines, should focus on addressing this critical gap in the literature.


Introduction
Nephrotic syndrome (NS) is a clinical diagnosis comprised of a triad of high urinary protein levels, low blood albumin levels and fluid retention. NS can lead to end-stage kidney failure and a lifelong need for renal replacement therapy (dialysis or transplantation). NS is labelled as primary, when it occurs in isolation, or secondary when it occurs as the consequence of systemic disease, infection, or medication use. Primary NS can affect both children and adults, with a reported worldwide incidence of 2-7/100,000 people [1]. However, the clinical classification system used to subgroup primary NS differs between children and adults; children are stratified based on their initial response to high dose steroid treatment, whereas adults are grouped based on their kidney histology. This review focuses on primary NS which has been labelled as steroid resistant (SRNS), steroid sensitive (SSNS), minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS).
A breakthrough in our understanding of NS came through the investigation of hereditary NS, which identified causative genetic variants affecting podocyte (a key renal filtration cell) function. Therefore, we now understand that roughly 30% of patients with SRNS have genetically driven disease [2]. The exact disease mechanisms for other patients with NS remain elusive but are generally thought to be mediated by a variety of different immune mechanisms. T lymphocytes are believed to have a key role as some NS patients exhibit

Protocol and Registration
The systematic review was designed using the Preferred Reporting Ideas for Systematic Review and Meta-analyses (PRISMA) systematic review checklist and was registered on PROSPERO, (ID: CRD42022311454, review protocol link: https://www.crd.york.ac.uk/ prospero/display_record.php?RecordID=311454 (accessed on 17 February 2022)).

Search Strategy-Eligibility Criteria, Information Sources and Search Terms
Original research articles written in English and published before 17 February 2022 were eligible for inclusion. Studies that examined any epigenetic mechanism in samples collected from humans with SRNS, SSNS, FSGS, or MCD were included. Studies which solely included patients with membranous nephropathy or secondary NS were excluded. Studies that included patients with chronic kidney disease of varied aetiology, in which NS, SRNS, SSNS, FSGS, or MCD were not explicitly stated or the results for these specific diseases were not reported separately from other disease groups, were also excluded. Conference abstracts were excluded. Our inclusion criteria did not include any age restrictions as we wished to capture both adults and children with NS.
Studies were identified from two databases: PubMed (MEDLINE) and Embase. The search was performed by using the terms listed in Figure 1 and was last conducted on 17 February 2022.

Study Selection and Data Extraction
Duplicate articles were removed from the literature search results. The titles and abstracts of the remaining articles were screened against the eligibility criteria by two independent authors. Any discrepancies between the authors were identified and discussed (with input from a third author if required). The remaining included articles proceeded to full-text screening, using the same eligibility criteria, by two independent authors. Data were extracted from the included studies by using a standardized data-extraction form created by the authors (Table 1). If the studies included work on both cell lines and patient samples, only data from the patient sample work were extracted. Only the results relating to SRNS, SSNS, FSGS, or MCD samples were extracted. If details of effect sizes were missing, the study was still included and available data extracted.

Study Selection and Data Extraction
Duplicate articles were removed from the literature search results. The titles and abstracts of the remaining articles were screened against the eligibility criteria by two independent authors. Any discrepancies between the authors were identified and discussed (with input from a third author if required). The remaining included articles proceeded to full-text screening, using the same eligibility criteria, by two independent authors. Data were extracted from the included studies by using a standardized data-extraction form created by the authors (Table 1). If the studies included work on both cell lines and patient samples, only data from the patient sample work were extracted. Only the results relating to SRNS, SSNS, FSGS, or MCD samples were extracted. If details of effect sizes were missing, the study was still included and available data extracted.

Data
Comments Study Design E.g., Case-control, cohort study etc.

Study population
Sample size Total number of participants and number with NS Diagnosis and control group E.g., SRNS v age matched controls Age E.g., 0-18 years only Epigenetic data Mechanism studied E.g., DNA methylation

Critical Appraisal
Study quality and risk of bias was assessed by using the Joanna Briggs Institute (JBI) critical appraisal tools [9]. The risk of bias in the studies was categorised based on the percentage of "yes" scores in the JBI checklist: less than 50% was considered high risk of bias, 50-69% was considered moderate risk, and 70% or greater was considered low risk of bias.
All included articles were summarised, however in-depth reporting of results was limited to higher quality studies, defined as those at low risk of bias (JBI of 70% or greater) and which included ≥20 people with FSGS, MCD, SRNS, or SRNS.

Results
The search identified 708 articles, 219 from PubMed (MEDLINE) and 489 from Embase. Duplicate records (n = 166) and articles that did not meet the inclusion criteria on title and abstract screening (n = 483) were removed, resulting in 59 articles which proceeded to full-text screening. A further 10 articles were removed on full-text screening as they did not meet the inclusion criteria or did not have full texts available. In total, 49 studies were included in the review ( Figure 2).
Forty-eight studies were case-control studies and one study had a repeated crosssectional design. Micro-RNAs were the most studied epigenetic mechanism (n = 35, 71%) reported. Only five studies investigated DNAm (10%), four long noncoding RNAs (8%), two histone modifications (4%), two small RNA (4%), and a single study examined a circular RNA (2%). Blood was the commonly investigated tissue, but kidney and urine were also quite common ( Figure 3). Twenty-four of the studies used samples that were collected exclusively from adults with NS and 13 used samples only from children. Seven studies included samples from both children and adults. Five studies did not explicitly state the participants' ages at sample collection or give any inclusion or exclusion criteria based on age. Only 23 of the 49 studies met our higher quality criteria (results summarised in Tables 2 and 3). The remaining low-quality studies are described in Supplementary Tables S1 and S2.

Higher Quality Studies-Micro-RNAs
The higher quality micro-RNA studies varied in their approach with some opting to investigate specific candidate micro-RNAs (n = 10) and others utilising array-based technology (n = 9), which captures thousands of micro-RNAs (Table 2). Despite the different methodologies, there were corroborating findings between the studies. By using a micro-RNA array, Luo et al. demonstrated higher serum levels of miR-191 in children with NS compared to healthy controls [10]. Bayomy et al. showed higher serum levels of a micro-RNA from the same family, miR-191a-5p, in children with NS compared to controls using a candidate approach [11]. In NS adults, Ramezani et al. used an array to show increasing levels of serum miR-30c from healthy controls to people with FSGS and MCD, with the highest levels demonstrated in MCD patients [12]. Hejazian et al. adopted a candidate micro-RNA approach and also found increased levels of serum miR-30c-5p in NS patients [13]. In another study from the same authors, which utilised the same approach and possibly included some of the same patients, increased levels of serum miR-30c were observed in people with FSGS compared to healthy controls [14]. Forty-eight studies were case-control studies and one study had a repeated crosssectional design. Micro-RNAs were the most studied epigenetic mechanism (n = 35, 71%) reported. Only five studies investigated DNAm (10%), four long noncoding RNAs (8%), two histone modifications (4%), two small RNA (4%), and a single study examined a circular RNA (2%). Blood was the commonly investigated tissue, but kidney and urine were also quite common ( Figure 3). Twenty-four of the studies used samples that were collected exclusively from adults with NS and 13 used samples only from children. Seven studies included samples from both children and adults. Five studies did not explicitly state the participants' ages at sample collection or give any inclusion or exclusion criteria based on age. Only 23 of the 49 studies met our higher quality criteria (results summarised in Tables  2 and 3). The remaining low-quality studies are described in Supplementary Tables S1 and S2.   Forty-eight studies were case-control studies and one study had a repeated crosssectional design. Micro-RNAs were the most studied epigenetic mechanism (n = 35, 71%) reported. Only five studies investigated DNAm (10%), four long noncoding RNAs (8%), two histone modifications (4%), two small RNA (4%), and a single study examined a circular RNA (2%). Blood was the commonly investigated tissue, but kidney and urine were also quite common ( Figure 3). Twenty-four of the studies used samples that were collected exclusively from adults with NS and 13 used samples only from children. Seven studies included samples from both children and adults. Five studies did not explicitly state the participants' ages at sample collection or give any inclusion or exclusion criteria based on age. Only 23 of the 49 studies met our higher quality criteria (results summarised in Tables  2 and 3). The remaining low-quality studies are described in Supplementary Tables S1 and S2.  Comparable results were also observed in urine micro-RNA studies. Both Feng et al. and Chen et al. found higher urinary exosomal levels of miR-23b-3p and miR-30a-5p in children with NS compared to controls followed by a decrease in miR-23b-3p when patients were treated with steroids and achieved remission [15,16]. Feng examined only a small number of micro-RNAs, whereas Chen examined the whole transcriptome. Chen et al. also validated these findings in a second independent cohort. In addition, increased urine and serum miR-30a-5p were demonstrated in paediatric NS patients by Luo et al.; these levels also declined after steroid treatment and NS remission [16]. Zhang et al. identified higher urinary miR-30a-5p in adults with active FSGS, compared to remission [17]. However, in this disease setting, only patients who had steroid-responsive FSGS demonstrated a decrease in urinary miR-30a-5p after treatment.

Higher Quality Studies-DNA Methylation
Two studies examined DNAm, and both opted to investigate specific candidate regions (NLRP3 promoter; SOCS3 and SOCS5 promoters) and demonstrated differences in methylation between SRNS and SSNS patients (Table 3) [18,19]. The promoter region of NLRP3 was examined as hypomethylation of this region is known to affect gene expression and cause steroid resistance in acute lymphoblastic leukaemia [20]. Indeed, in NS lower DNAm of this region was demonstrated in steroid resistant patients compared to those who were steroid sensitive [18]. The SOCS3 and SOCS5 promoters were investigated as previous work by the authors had demonstrated increased plasma levels of these proteins in SRNS compared to SSNS and healthy controls [21]. In this study, the authors identified lower DNAm in the promoter region of SOCS3 in SRNS compared to SSNS [19].

Higher Quality Studies-Small RNAs
Small RNAs were investigated by two studies (

Discussion
We have reported and summarized all published human studies of epigenetic mechanisms in NS. Overall, the epigenetics of NS is understudied, with only 23 high-quality studies published and 10 that attempted to replicate their findings in a second cohort of patients. Even in the higher-quality studies, the number of included patients were modest, with only seven studies including more than 100 patients. The research studies focused almost exclusively on micro-RNAs. Due to the high heterogeneity in methodology and the use of a diverse range of assays very few of the micro-RNA studies are truly comparable, allowing for only limited conclusions to be drawn. Despite this, concordant results were seen between a small number of the micro-RNA studies; serum miR-191, serum miR-30c, urinary miR-23b-3p, and urinary miR-30a-5p levels were observed to be increased in NS compared to healthy controls in multiple studies.
It is likely that epigenetic research in NS has been hampered by the fact that NS is a rare disease and so large numbers of patient samples are difficult to obtain. This will improve now that large national and multinational NS cohorts have been established, for example, the International Study of NS (International NephroS), the National Unified Renal Translational Research Enterprise (NURTuRE) and the NS Study Network (NEP-TUNE) [22,23]. Comparability between studies should also improve due to technological and methodological advances in epigenetic research. The combination of high-throughput array technology, greater standardisation of analytic pipelines and a better understanding of patient characteristics that may confound analyses, should lead to more consistent approaches between research teams and hopefully, more reproducible results. Nephrologists can be inspired by other medical specialties, such as oncology, which have been quicker to invest in epigenetic research and are now reaping the rewards with successful translation of the results into clinical practice.
There are many promising clinical applications for epigenetic data given that epigenetic mechanisms are known to respond to and sometimes play key roles in biological responses to the environment and disease processes. The flexibility and reversibility of epigenetic states suggest that, in some cases, epigenetic mechanisms may be therapeutic targets. In fact, a few drugs which act as broad reprogrammers of the epigenome have entered clinical practice, such as the histone deacetylase inhibitor panobinostat for treatment of multiple myeloma [24]. More recently, the discovery that CRISPR-cas9 can be used to perform locus-specific epigenome editing will likely lead to targeted epigenetic therapies in the not-too-distant future [25]. However, epigenetic variation is useful to medicine beyond causal roles in disease development and progression. Any variation that is merely associated with environmental or genetic risk factors or to disease processes may be used as a biomarker to estimate disease risk, diagnose disease, predict disease progression, or predict treatment response. For example, in the United States, hypermethylation in the promoter regions of BMP3, NDRG4, SEPT9, and VIM genes have been approved for colorectal cancer screening [8]. Similarly, the methylation status of MGMT is widely used in glioma patients as a predictive biomarker of response to alkylating chemotherapy agents and is included on National Comprehensive Cancer Network guidelines [26]. Each of these biomarkers was discovered by comparing DNA methylation between case and control tissues. None is known to play a role in disease.
For any molecular mechanism to be successfully translated into a clinical biomarker, it must be obtained from an easily accessible tissue, demonstrate low interlaboratory variation in measurements and be sufficiently stable. The studies identified in this review examined epigenetic mechanisms in blood, urine, renal tissue, or a combination of these. Obtaining samples from any of these tissues would be acceptable in clinical practice, although the less invasive options of blood and urine would be preferential. In general, interlaboratory reproducibility is improving, particularly with the use of DNAm microarrays and the sharing of analytical methods [27]. However, differences in micro-RNA isolation protocols persist and can lead to biased measurements hindering clinical utility [28]. Finally, a benefit of epigenetic mechanisms is their stability, for example, the half-life of micro-RNAs is roughly 10 times longer than that of messenger RNAs, and changes in DNAm can persist throughout adulthood [29,30]. Interestingly, synthetic micro-RNAs, which are being developed as epigenetic drugs, are less stable than their endogenous counterparts and can be rapidly degraded and cleared from circulation, which is a key limitation [31].

Conclusions
Overall, the epigenome is an attractive field of research and in certain disease settings, epigenetic research is beginning to alter clinical practice. However, NS research in this area is lagging behind, with a lack of high-quality epigenetic research. In particular, DNAm and histone modifications have been woefully understudied. Established large NS patient cohorts, alongside the technological and methodological advances in epigenetic research, should allow this gap in the literature to be addressed in the near future.         Table S1: Summary of the lower quality micro-RNA studies; Table S2: Summary of the lower quality DNA methylation and small RNA studies. References