Chronic kidney disease (CKD) is one of the most important risk factors for cardiovascular disease (CVD). Despite the kidney having no direct implications for lipoproteins metabolism, advanced CKD dyslipidemia is usually present in patients with CKD, and the frequent lipid and lipoprotein alterations occurring in these patients play a role of primary importance in the development of CVD. Although hypertriglyceridemia is the main disorder, a number of lipoprotein abnormalities occur in these patients. Different enzymes pathways and proteins involved in lipoprotein metabolism are impaired in CKD. In addition, treatment of uremia may modify the expression of lipoprotein pattern as well as determine acute changes. In renal transplantation recipients, the main lipid alteration is hypercholesterolemia, while hypertriglyceridemia is less pronounced. In this review we have analyzed lipid and lipoprotein disturbances in CKD and also their relationship with progression of renal disease. Hypolipidemic treatments may also change the natural history of CVD in CKD patients and may represent important strategies in the management of CKD patients. Chronic kidney disease (CKD) is one of the most important risk factors for cardiovascular disease (CVD) . SeveralAlthough the kidney has no direct implications for lipoproteins metabolism CRF dyslipidemia is usually present in patients with CRF . This meHypertriglyceridemia is a common feature in patients with CKD and this is due to an increased concentration of triglyceride-rich lipoproteins (TRL)  ,14,15. HAlthough LDL cholesterol is generally not elevated in CKD patients, a higher prevalence of small, dense LDL has been found . These pA recent study has analyzed the association between the change of lipoprotein and the kidney outcomes in patients with stage 3–5 CKD over 3.2 years of follow-up. It has been shown that stage 3 CKD subjects with increased and high variable LDL-C levels exhibit an increased risk of progression to advanced CKD stages to dialysis. No significant difference on this correlation was found in patients with CKD stage 4 or 5. These results suggest that an efficacious lipid-lowering treatment is crucial to improve clinical outcomes, especially in patients with CKD stage 3 .Patients with end-stage renal failure (ESRF) treated with different dialytic procedures  exhibit a different pattern of dyslipidemia. It was found that ESRF subjects on HD mainly present with hypertriglyceridemia, high apoB levels, low HDL-C and low apoA-I levels, while in patients on CAPD cholesterol serum levels also are increased . Lp(a) l2/day), low albuminemia levels, edema and hyperlipidemia. The latter is the result of different modifications of lipoprotein metabolism in terms of both qualitative and quantitative changes [Nephrotic syndrome is one of the most common manifestations of glomerular damage characterized by proteinuria , A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Haemodialysis: An Assessment of Survival and Cardiovascular Events (AURORA) and Study of Heart and Renal Protection (SHARP) ,96,97 s. While 42) aged ≥50 years who have pathological albuminuria  should be treated with a statin or statin plus ezetimibe combination [Moreover, lipid management in CKD subjects is based on recommendations from the major clinical practice guidelines as KDIGO and ACC/AHA, which suggest the use of statins rather than other lipid-lowering therapies, since their efficacy has been already tested in randomized controlled trials (RCTs) . Accordibination .2. So far, there are not randomized trials indicating the benefits of statins in patients with nephrotic syndrome. The effect of statin treatment on mortality in CKD subjects depends on the CKD stage. Patients with stages 2–4 CKD on statin therapy, do not exhibit any difference in terms of mortality compared to placebo. However it has been shown that statins can reduce the mortality in stage 5 CKD subjects [KDIGO recommends treatment of dyslipidemia on the basis of CHD risk, which depends maximally by age. However this guideline does not have an upper age limit for therapy recommendations contrary to ACC/AHA guidelines that suggest avoiding statin therapy for primary prevention in CKD patients older than 79. Moreover, KDIGO suggests a statin dose decrease in subjects with an eGFR < 60 mL/min/1.73 msubjects .Subgroups of FOURIER and ODYSSEY Outcomes trials have also demonstrated a similar benefit of PCSK9 inhibition independent of kidney function, despite subjects with severely reduced kidney function being excluded ,102,103,2. Furthermore, their benefits are dependent of the degree of cholesterol lowering and it seems that their efficacy decreases as CKD turns into more severe stages. Further studies are needed to prove the suitability of PCSK9 inhibitors in the treatment of CKD patients [There are limited data on PCSK9 inhibitors in CKD. The FOURIER trial evaluated the efficacy and safety of Evolocumab in CKD patients, and a recent analysis of 8 randomized trials on Alirocumab reported that these agents decrease LDL-C and reduce the CV risk among subjects with mild to moderate CKD spectrum. However these studies have some limitations that reflect the small number of patients with stage 3b and 4 CKD and the exclusion of patients with eGFR < 20 mL/min/1.73 mpatients .2. The only exception might be very high triglyceride levels  in which these drugs should be used reasonably by adjusting the dose according to kidney function [Other lipid-lowering agents are fibrates but to date, very little proofs recommend their use in subjects with CKD, especially in those with eGFR < 30 mL/min//1.73 mfunction .Bile acid sequestrants such as cholestyramine, colestipol and colesevelam are actually administered as second-line options in subjects with atherosclerotic CV disease, but there is only a little evidence for their use in CKD population. Moreover, since these agents might increase triglyceride levels, their utility in subjects with CKD or ESRF is very limited. To date, there is poor evidence to support the use of omega-3 fatty acids to decrease CV outcomes or ameliorate mortality in patients with CKD or ESRF. On the other hand, two placebo-controlled RCTs, REDUCE-IT  and STRENGTH , are evaluating the effect of high-dose (4 g) eicosapentaenoic acid on CV outcomes in subjects with hypertriglyceridaemia .Taken together, all these events contribute to the development of cardiovascular diseases  and, at the same time, to the progression of kidney disease . Indeed,Metabolism of lipids and lipoproteins is substantially altered in CKD and the frequent alterations occurring in these patients may promote atherogenesis, playing a role of primary importance in the development of CVD. Elevated plasma levels of apoC-III, apoA-I and the apoB-containing lipoproteins may modify lipoprotein metabolism, leading to the accumulation of atherogenic particles and a profile characterized by elevated triglycerides, Lp(a), dense LDL and low HDL cholesterol. Different enzymes pathways and proteins involved in lipoprotein metabolism are impaired in CKD, and these changes are mainly dependent of the different setting of kidney disease as well as the kidney transplant recipient state. These modifications may be controlled and, although there are limited data on CKD population, lipid-lowering therapies including statin or statin plus ezetimibe combination, PCSK9 inhibitors, omega 3 and fibrates  represent important strategies in the management of CKD patients and in the prevention of CV events.


Urine excretion of oxalate and citrate represent key determinants for the risk of calcium oxalate kidney stones, the most common type of kidney stone worldwide. Higher urine oxalate or lower urine citrate excretion will increase the risk for kidney stone recurrence. The molecular details underlying the regulation of urine oxalate and citrate excretion have become clearer over the past two decades. For oxalate, the anion transporter SLC26A6 plays a key role in oxalate homeostasis. SLC26A6 is expressed in the apical membrane of epithelial cells of the proximal tubule, pancreatic duct, stomach, and small intestine, and this transporter exchanges chloride for oxalate in brush border membrane vesicles from the proximal tubule and small intestine Aronson, . SLC26A6Xenopus laevis oocytes, SLC26A6 inhibited NaDC1, and NaDC1 reciprocally activated SLC26A6. But does a relationship exist between urine oxalate and citrate excretion in humans? For this to be a possibility, one must assume that this relationship between oxalate and citrate, operative in heterologous expression systems and transgenic animals, is recapitulated in humans of different ages, body weights, and genetic backgrounds. This is the question that Prochaska et al.  or endogenous  factors can independently affect urine oxalate and citrate excretion. The investigators attempt to control for some of these factors, but they do not report relevant factors that may affect this relationship such as age (for the non‐stone forming population), weight, serum chemistries, or kidney function. Obesity is associated with higher urine oxalate excretion (Amin et al., In conclusion, the simplicity of the original hypothesis by Prochaska et al. belies the complexity of the physiology underlying oxalate and citrate homeostasis in humans. The small intestine, liver, and proximal tubule all contribute to oxalate excretion, while the status of acid–base balance plays a significant factor in regulating citrate excretion. Without implementation of carefully controlled studies that account for age, sex, demographics, co‐morbid illness, and diet, it is unlikely that extrapolation of the interaction between SLC2646 and NaDC1 at the cellular level will lead to an accurate ab initio prediction of how the body integrates the control of oxalate and citrate excretion. Nonetheless, the investigators should be commended for testing whether a specific biological/cellular mechanism underlying urine citrate and oxalate handling exists in a human clinical population. Working from first principles, the investigators have taken the necessary first step.


Human athletic performance is a complex phenotype influenced by environmental and genetic factors, with most exercise-related traits being polygenic in nature. The aim of this article is to outline some of the challenge faced by sports genetics as this relatively new field moves forward. This review summarizes recent advances in sports science and discusses the impact of the genome, epigenome and other omics (such as proteomics and metabolomics) on athletic performance. The article also highlights the current status of gene doping and examines the possibility of applying genetic knowledge to predict athletes’ injury risk and to prevent the rare but alarming occurrence of sudden deaths during sporting events. Future research in large cohorts of athletes has the potential to detect new genetic variants and to confirm the previously identified DNA variants believed to explain the natural predisposition of some individuals to certain athletic abilities and health benefits. It is hoped that this article will be useful to sports scientists who seek a greater understanding of how genetics influences exercise science and how genomic and other multi-omics approaches might support performance analysis, coaching, personalizing nutrition, rehabilitation and sports medicine, as well as the potential to develop new rationale for future scientific investigation. The ever-advancing number and standard of sporting competitions has led to an increased demand to improve the existing methods used by scientists to understand athletic capability and to drive the innovation of new recommendations for athletes to improve both health and athletic performance. Sport and exercise scientists study the adaptation of athletes to training and competition using a variety of physiological, biochemical and biomedical indicators. In recent decades, exercise physiologists have begun to elucidate the cellular and molecular responses to exercise that lead to adaptation at the whole-body level. In recent years, an increasing number of post-genomic analysis methods have been employed in the search for new markers in sports that can be used to quantify changes in the physiological and functional state of the human body ,2. GenetIn the present review, we provide perspective on the current state of sports genomics and a range of potential applications for genomic data in elite sport. Here, we review the current understanding of genetics in modern sport, including several recent large-scale studies that have successfully identified and replicated the associations of genetic variants with athletic performance phenotypes. We subsequently explore recent studies that have focused on the potential of using genomic information to complement injury prevention and pre-participation screening. We will also address what impact the advancing fields of bioinformatics and omics approaches  are likely to have on the way we understand athletes’ physiology and whether information gained through these approaches could help to improve athletic performance.A comprehensive search of the Medline and PubMed databases was conducted. The last query was on 1 December 2021. Findings were limited to English articles. Articles published before 1990 were excluded, unless no newer information had been published on a topic or their inclusion was necessary for understanding. Due to the wide spectrum of this topic of article, several different search terms were used in the following layout: (sports genetics) OR (sports genomics) OR (exercise genomics) AND (XXX). All of the articles identified were evaluated by their title and/or abstract and included if considered appropriate. Additionally, references of the articles cited were searched for to find further publications not detected by the literature search.Interest in the genetic contribution to human sports performance can be traced back to the early 1970s, though the first major book on the topic, Genetics of Fitness and Physical Performance by Bouchard and colleagues, was not published until 1997 . AnotherSports genomics is the scientific discipline focusing on the organization and function of the genome in elite athletes and aims to develop molecular methods that may be used for sports medical practices, personalized exercise training, nutrition prescription and the prevention of exercise-related injury and/or disease ,10. ReceHuman physical performance traits (PPTs) and adaptations to exercise training both result from the interaction of multiple intrinsic and extrinsic factors, including those categorized as environmental, genetic, anatomical, physiological and psychological . Common In recent years, the potential to use genetic information to maximize athletic performance and prevent injuries has been proposed ,16,17,18ACE) gene, which is a known regulator of blood pressure, have been shown by multiple studies to be a determinant of endurance potential in athletes. Other examples of candidate genes that have received notable attention, and which have subsequently been associated with phenotypes underpinning sprint, power or endurance performance, include the skeletal muscle isoform of α-actinin 3 (ACTN3), adenosine monophosphate deaminase (AMPD1), muscle creatine kinase (CKM), insulin-like growth factor (IGF1) and the family of peroxisome proliferator-activated receptor (PPAR) genes. The associations of common genetic variants in these and other genes with a range of athletic performance phenotypes are comprehensively reviewed elsewhere [Considerable research has already been conducted regarding the roles of genetic and epigenetic factors in training adaptation and the determination of elite sports performance. Research investigating the genetic basis of PPTs was initiated by observational studies conducted in twins, families and pedigrees, evolving from genetic epidemiology research into the study of DNA sequence biomarkers ,4. The fnase AMPD, muscle lsewhere ,30,31,32At end of 2021, the total number of DNA polymorphisms associated with athlete status was 220, of which 97 markers have been found significant in at least two studies  . FurtherHFE rs1799945, MCT1 rs1049434, MYBPC3 rs1052373, NFIA-AS2 rs1572312, PPARA rs4253778, PPARGC1A rs8192678 and VEGFR2 rs1870377 for endurance; ACTN3 rs1815739, AMPD1 rs17602729, CPNE5 rs3213537, CKM rs8111989 and NOS3 rs2070744 for power; LRPPRC rs10186876, MMS22L rs9320823, PHACTR1 rs6905419 and PPARG rs1801282 for strength; and COL1A1 rs1800012, COL5A1 rs12722, COL12A1 rs970547, MMP1 rs1799750, MMP3 rs679620 and TIMP2 rs4789932 for soft-tissue injuries [injuries ,37,38.MSTN) gene lead to extreme growth of skeletal muscle, indicating that myostatin is a key regulator of muscle mass [MSTN mutations that lead to an inactive gene product have been shown to produce double skeletal muscle mass in mice, sheep and cattle. Such knowledge may be highly valuable in helping to explain the underlying causes of certain diseases and may subsequently aid the development of new treatment methods. Indeed, increased myostatin expression might contribute to the loss of muscle mass through normal aging, suggesting that exercise modalities known to reduce myostatin expression and/or pharmacological inhibition of myostatin, might represent suitable treatments designed to prevent the age-related loss of muscle mass in non-athletic cohorts [To date, studying the genetic profiles of elite athletes has revealed DNA sequence variations that are associated with a competitor’s predisposition to excel in strength, power, sprint or endurance sports, their intrinsic vulnerability to sports-related injuries and their individualized nutritional requirements . The majcle mass . Specifi cohorts . The canThe majority of sports genomics studies to date have adopted the candidate gene approach. This involves investigators selecting genes and variants they believe are related to a PPT of interest. Researchers first conduct case–control association studies to determine whether a genotype or allele is more prevalent in a cohort of athletes than a control group containing healthy non-athletes from the general population ,9. TypicThe most up-to-date GWAS platforms can screen more than one million DNA loci simultaneously in order to detect regions associated with traits of interest. One of the advantages of the GWAS approach is that it is unbiased with respect to genomic structure and previous knowledge of the trait (hypothesis-free), in contrast to candidate gene studies where a priori knowledge is used to identify candidate loci contributing to the trait of interest. In addition, some GWAS platforms enable the investigation of epigenetic modifications. To date, four GWAS have been performed in athletes ,42,43,44Despite the obvious advantages of the GWAS approach, an overarching limitation of these studies is that in some countries, and in certain sporting disciplines, there is a limited number of elite athlete cohorts (cohort homogeneity). This reflects another common constraint within exercise genomics, which is that research is often conducted using small sample sizes. Accordingly, major collaborative efforts are required to increase participant numbers and sufficiently advance the impact of exercise genomics research . In recoIn the field of sport performance research, genomics focuses on identifying genetic variants associated with PPTs, response to exercise or future prognosis of injury. DNA, RNA, protein and metabolite often have complementary roles in PPTs to jointly perform a particular biological function. High-throughput technologies enable omics studies  and provide researchers with a greater understanding of the flow of information, from the original process of PPTs biology  to the functional consequences. Multi-omics approaches integrate data obtained from different omics levels to understand their interrelation and combined impact on the biological mechanisms of physical performance. PPARGC1A, PPARD), accompanied by a concomitant overexpression of those genes [Epigenetics is a rapidly advancing field within molecular exercise physiology, referring to changes in gene function in the absence of changes in DNA sequence ,22. Epigse genes ,22,23.Proteomic and metabolomic research methods offer a quantitative measurement of metabolic profiles associated with exercise, with some researchers attempting to identify the distinct metabolic profiles of athletes from a range of different sports. Metabolomics studies characterize exercise-related adaptations that affect an abundance of small-molecule metabolites serving a wide range of biological functions, such as biomarkers of cardiometabolic fitness in skeletal muscle . “MolecuThe application of proteomics and metabolomics to sports research has already revealed a number of functional proteins and metabolic pathways that are altered by exercise in general or which demonstrate variable responses between different sporting disciplines. The majority of changes have been detected in cytoskeletal structure or within energy-related pathways. In addition, omics approaches have recently allowed scientists to uncover the transducers and profiles of secretomes , that is, the biologically active substances secreted from cells and tissues  via autocrine, paracrine and endocrine systems, as an emerging subdiscipline of proteomics to study exercise-regulated signals ,10. HoweA key objective for integrating omics technologies into sports science is the prospective ability to prescribe personalized nutrition and training programs to athletes. Understanding the genetic, epigenetic and epigenomics underpinnings of an athlete’s nutritional requirements may provide a valuable tool to further optimize his or her sporting performance . In addiCOL1A1, COL3A1, COL5A1 and COL12A1) and matrix metalloproteinase-encoding genes (MMP12 and MMP3) that are associated with injuries such as Achilles tendon pathology and anterior cruciate ligament rupture [Injury prevention is a priority for athletes, coaches and practitioners of sports medicine for whom the diagnosis, treatment and prevention of injuries constitute a large part of a daily practice. Exercise-induced injuries to the musculoskeletal system depend on race, age, body composition, physical activity/inactivity levels and their multifactorial interaction with other extrinsic and intrinsic factors. An individual’s genetic profile is a recognized intrinsic factor predisposing individuals to a higher risk of suffering a musculoskeletal injury and/or necessitating longer recovery after intense exercise . Recentl rupture ,15,16,49 rupture ,18.TTN, ABCC9, DMD, MYPN and SCN5A, amongst others, have been implicated in the causes of familial cardiomyopathy. Athletes in soccer, basketball, cross-country and long-distance running are most vulnerable to suffering SCD [In addition to the problem caused by sports injuries, there are a range of genetic conditions that may unknowingly increase the risk to an individual’s health when participating in exercise. The most severe examples include those that elevate the risk of sudden cardiac death (SCD). In this context, genetic data could potentially be applied during pre-participation screening for the identification of at-risk individuals and the prevention of SCD in sport. SCD refers to the unexpected natural death of an individual that is caused by cardiac arrest within a brief time interval  under conditions that would not normally be considered fatal, and it is the leading medical cause of death in young athletes during sport or exercise activity ,52,53. Hring SCD . Whilst ring SCD ,52, whicHigh-quality experimental data and accurate analysis continue to advance the understanding of the genetic and molecular basis of a range of human PPTs including sprint, strength and endurance performance. Here, we briefly describe a number of databases, as well as computational and statistical methods, that can be used when researching genetic markers related to PPTs.https://www.ebi.ac.uk/gwas/, accessed on 20 November 2021) to document genome-wide associations for PPTs and other traits of interest [Currently, several approaches are routinely employed to identify genetic variations affecting sport-related traits, including SNP array-based GWAS and next-generation sequencing (NGS) . GWAS uninterest . In addiinterest . Among t2 max, strength and/or specific anthropometric indicators of power output, associations with genetic markers are inferred using regression analysis or one-way analysis of variance (ANOVA) [There are two broad categories of computational and statistical methods in existence that are used to infer associations between genetic markers and PPTs: (i) methods for individual marker–trait association inference and (ii) methods utilizing polygenic scores to infer predispositions to enhanced physical fitness or sport-specific phenotypes. The first class of methods rely on simple statistical techniques that compare genotype and/or allele frequency distribution between cases and controls. For categorical traits, such as endurance versus sprint athlete status or power athlete status versus non-athlete control status, chi-square, Fisher’s exact or logistic regression tests are typically employed ,31,32,57 (ANOVA) ,59,60,61jAA = 2, SjAa = 1 and Sjaa = 0. Once scored, the sum of genotypes across an infinite number of variants can be calculated for each individual participant, meaning that possessing a greater number of trait-predisposing alleles from multiple variants will result in a higher TGS, indicating a greater predisposition to the trait of interest. For the total number of markers (N) included, the total genotype score is computed as: TGS = 100 × (Σj=1…NSj/2 × N). Subsequently, the discriminatory power of a TGS can be investigated in cohorts of athletes and/or controls in relation to a categorical or continuous trait. Where a statistically significant association with a trait can be established using individual markers in the first instance, a TGS can then be computed from that subset of markers, demonstrating statistically significant associations for each individual in order to quantify and score their polygenic predisposition to the trait of interest. Since the TGS concept was introduced, the approach has been adopted, and at times adapted, by several studies [SNAP25 rs362584 and ACTN3 rs1815739) has been described in gymnasts [In 2008, Williams and Folland  presente studies ,66. For  studies . Whilst  studies , suggestgymnasts .An additional problem arising from the relatively small samples of elite athlete populations is the limited ability to obtain strong evidence of genotype–phenotype associations. In such cases, published data of specific gene markers from multiple populations can be pooled in a meta-analysis to provide a stronger evidence based for genotype–phenotype associations ,69. In abFGF or NGF), this procedure could also be abused by athletic populations. However, understanding how gene therapy may be utilized for elicit purposes also allows experts to design new strategies to prevent doping in sport. For example, researchers have discovered that copy number variation in the UGT2B17 gene influences the efficacy of anti-doping methods and that tests designed to detect testosterone abuse may be less sensitive in athletes of some geographical ancestries where certain genotypes are more frequent [Advancing knowledge surrounding the genetic variants that predispose individuals to success in specific sporting domains also creates the risk that such information could be used for illegal doping. New technologies for the transfer of genetic material (DNA/RNA), known as a gene therapy, are actively being developed to treat severe disorders such as anemia, peripheral vascular diseases and muscular dystrophy ,71. Despfrequent ,71. In sEPO), vascular endothelial growth factor (VEGFA and VEGFD), insulin-like growth factor 1 (IGF-1), growth hormone (GH), phosphoenolpyruvate carboxykinase 1 (PCK1), myostatin (MSTN) and MSTN antagonists such as follistatin (FST) [MSTN, IGF, GH and rhGH (recombinant human GH) are the major determinants of successful performance in sprint/power sports, with EPO (and rEPO), VEGFA, HIF-1, PPARD and PCK1 associated with elite performance in endurance sports [Gene doping refers to the non-therapeutic use of gene therapy by healthy athletes to improve physical performance in sporting competition. Gene doping could have dangerous and even fatal outcomes, as the knowledge of gene therapy is still in its infancy . Throughin (FST) . These ge sports . In 2003e sports ,74. Accoe sports ,76. While sports ,71. The The current strategy to screen for gene doping involves the direct detection of cDNA sequences, and anti-doping laboratories must permanently incorporate new detection techniques in order to maintain a harmonized approach to detecting doping substances and methods on the worldwide scale .At present, the clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system is one of the most frequently used tools for genome editing. It enables the specific modification of any desired DNA sequence and surpasses all existing alternatives for gene editing . HoweverIn 2021, WADA released guidelines concerning gene doping detection methods   and seveExercise and sport-specific training induce the stimulation of many biological networks contributing to the complex yet coordinated response of the human body to perturbations in homeostasis and mechanical demand. Developments of omics technologies have created new possibilities in the study of the molecular mechanisms underlying athletic performance. Technological advances in genome-wide association studies and the profiling of gene expression have each contributed to our understanding of the genes, pathways and biological networks involved in the response to exercise, as well as the presence of variability in training responses among individuals. Sports genetics and genomics may one day help individuals to achieve superior outcomes in their general health and wellness. In athletic settings, genetic information has the potential to identify individuals with a natural predisposition to achieving success in specific athletic activities and may also be useful for athletes seeking to understand and make improvements in specific performance attributes where their genetic predisposition may be less favorable. In addition, there is scope for genetic information to assist in strategies aimed at reducing the risks of injury and to augment the process of rehabilitation once an athlete suffers an injury. Further collaborative research is needed to confirm the potential influence of genetic variants in athletes from different sporting backgrounds to identify more informative markers of athlete health and performance. The use of genetic testing in sports presents a number of possibilities for athletes, coaches and medical personnel to understand athletes’ susceptibility for specific pathological conditions including sports injuries, sudden death and cardiomyopathies, among others. With improved study designs and the increasing availability of advanced analytical tools and statistical approaches, the interaction of genetic variability with sport and exercise can be better understood and potentially utilized to improve human health, wellbeing and performance. Over the next decade, the application of genomics in sport is likely to progressively improve through the increasing availability of complete omics databases and high-throughput screening approaches for investigating human performance.


Pterygium is a common ocular surface condition frequently associated with irritative symptoms. The precise identity of its critical triggers as well as the hierarchical relationship between all the elements involved in the pathogenesis of this disease are not yet elucidated. Meta-analysis of gene expression studies represents a novel strategy capable of identifying key pathogenic mediators and therapeutic targets in complex diseases. Samples from nine patients were collected during surgery after photo documentation and clinical characterization of pterygia. Gene expression experiments were performed using Human Clariom D Assay gene chip. Differential gene expression analysis between active and atrophic pterygia was performed using limma package after adjusting variables by age. In addition, a meta-analysis was performed including recent gene expression studies available at the Gene Expression Omnibus public repository. Two databases including samples from adults with pterygium and controls fulfilled our inclusion criteria. Meta-analysis was performed using the Rank Production algorithm of the RankProd package. Gene set analysis was performed using ClueGO and the transcription factor regulatory network prediction was performed using appropriate bioinformatics tools. Finally, miRNA-mRNA regulatory network was reconstructed using up-regulated genes identified in the gene set analysis from the meta-analysis and their interacting miRNAs from the Brazilian cohort expression data. The meta-analysis identified 154 up-regulated and 58 down-regulated genes. A gene set analysis with the top up-regulated genes evidenced an overrepresentation of pathways associated with remodeling of extracellular matrix. Other pathways represented in the network included formation of cornified envelopes and unsaturated fatty acid metabolic processes. The miRNA-mRNA target prediction network, also reconstructed based on the set of up-regulated genes presented in the gene ontology and biological pathways network, showed that 17 target genes were negatively correlated with their interacting miRNAs from the Brazilian cohort expression data. Once again, the main identified cluster involved extracellular matrix remodeling mechanisms, while the second cluster involved formation of cornified envelope, establishment of skin barrier and unsaturated fatty acid metabolic process. Differential expression comparing active pterygium with atrophic pterygium using data generated from the Brazilian cohort identified differentially expressed genes between the two forms of presentation of this condition. Our results reveal differentially expressed genes not only in pterygium, but also in active pterygium when compared to the atrophic ones. New insights in relation to pterygium’s pathophysiology are suggested. In Brazil, the reported rate of pterygium in a city of the Southwest region was 8.12%3, while in indigenous populations of the Brazilian Amazon rainforest, the prevalence varied from 18.4 to 36.6%4. Several risk factors for pterygium have been listed, although the most frequent ones are exposure to ultraviolet radiation, chronic irritation and inflammation7. Surgery is considered for symptomatic and cosmetic improvement, but recurrence can happen in 0 to 88% of the cases, depending on the surgical technique employed8. The development of the pterygium has some similarities with tumor growth, such as fibrovascular proliferation, corneal invasion and high recurrence rate after surgical excision9. Among the mechanisms and structures involved, it is known that the proliferation and degeneration of collagen fibers, especially type I collagen, plays an important role in the development of the pterygium10. Several studies also demonstrate the role of anti-apoptotic mechanisms, modulation of extracellular matrix under ultraviolet radiation exposure, angiogenesis, inflammation and hereditary factors in the pathogenesis of pterygium13. However, the exact pathogenesis is not yet fully understood and the precise identity of the critical triggers as well as the hierarchical relationship between all the elements involved in the pathogenesis of this disease are yet to be described.Pterygium is a common disease of the ocular surface, consisting of a fibrovascular tissue that arises from the conjunctiva and extends to the cornea. It is related to irritative symptoms, poor aesthetics, astigmatism and, sometimes, worsening visual acuity, being a condition frequently associated with decreased quality of life. The prevalence of pterygium varies according to the region studied, being higher in tropical regions, where the prevalence is approximately 22%, and lower in countries outside these areas, where it is close to 2%15. Also, several other studies analyzed non-coding RNA expression, evidencing upregulated and downregulated miRNAs and lncRNAs that could be involved in pterygium pathogenesis25. Microarray-based studies generate large databases of raw gene expression that are deposited in data repositories for public reuse. In turn, meta-analysis of these data may enhance results and generate new biological insights regarding some conditions in comparison with individual studies, once it reduces study biases, increases statistical power and obtains a more accurate evaluation of differentially expressed genes (DEGs).In view of the controversies on the multiple mechanisms of pterygium formation, studies were carried out using high-throughput genomic technologies, such as microarrays, to attempt to elucidate mRNA expression patterns that could underlie specific molecular events of interest in its pathogenesis26. Consequently, the field of integrative analysis of high throughput gene expression datasets has grown exponentially, leading to the development of tools and guidance for conducting a meta-analysis of gene expression28. Meta-analysis of transcriptomic datasets deposited in public repositories represents an attractive strategy capable of identifying key pathogenic mediators and therapeutic targets in complex diseases30. By combining multiple study datasets, this strategy has the potential to increase the statistical power and to generate new biological insights that could not be accessed from original studies14. Moreover, several bioinformatics tools of curated biological databases33 have been recently developed, making even easier the interpretation of relevant differentially expressed (DE) genes sets identified from meta-analysis and the prediction of associated pathways and regulatory networks34. In this context, a well conducted meta-analysis has the potential to contribute to the generation of new hypothesis about the pathogenic mechanisms of complex diseases.Over the past decade, the availability of large public genomic datasets and robust bioinformatics tools enabled the development of new data mining approaches in biomedical researchThe aims of this study were to evaluate gene expression in pterygium samples, perform a meta-analysis with two databases available at the Gene Expression Omnibus public repository and then, correlate the findings through a miRNA-mRNA regulatory network.The present study was carried out with the approval of the Research Ethics Committee of the University of Campinas (UNICAMP) and was conducted in accordance with the tenets of the Declaration of Helsinki and current legislation on clinical research. Written informed consent was obtained from all subjects after explanation of the procedures and study requirements.Participants were selected by convenience, at the Ocular Surface outpatient clinic of the Hospital of University of Campinas. Participants that presented nasal pterygia, not previously operated, and wanted surgery were included. Individuals with conjunctival surface diseases such as previous chemical burns, pemphigoid and cicatricial diseases, or suspected ocular surface neoplasia were excluded.35.The pterygium was classified according to the fibrovascular tissue extension towards the cornea  and according to its biomicroscopic aspect . The latter classification is based on the classification proposed by Tan et al., in which the pterygia are divided according to tissue translucency. Grade T1 (atrophic) denotes a pterygium in which episcleral vessels underlying the body of the pterygium are unobscured and clearly distinguished. Grade T3 (active or fleshy) denotes a thick pterygium in which episcleral vessels underlying the body of the pterygium are totally obscured. All other pterygia that do not fall into these 2 categories are classified as Grade 2 (intermediate). An increased fleshiness or thickness of the fibrovascular component of the pterygium is believed to be associated with recurrence after excisionParticipants underwent pterygium excision followed by an upper bulbar conjunctival free autograft placed over the site of the original lesion. All pterygia specimens were nasally located. The entire pterygium tissue was collected, and the samples were immediately stored in RNAlater stabilization solution (Invitrogen) until RNA extraction.Tissue samples were transferred to liquid nitrogen and ground into powder. Total RNA extraction was performed using TRIzol Reagent (Invitrogen) and purified with RNeasy RNA extraction kit (Qiagen) according to the manufacturer's protocol. RNA concentration and purity were assessed through NanoDrop 2000 spectrophotometer (Thermo Scientific) whereas RNA integrity was measured in the Bioanalyzer 2100 System (Agilent Technologies). Samples with an RNA integrity number ≥ 7 were subjected to microarray analysis.Fifty nanograms of total RNA were used to generate ss-cDNA, which was hybridized on Human Clariom D Assay gene chip (Affymetrix). This array interrogates more than 540,000 human transcripts. Sample labeling, hybridization, washing and scanning were performed according to manufacturer’s protocols at Molecular Core Facility .36.The normalization of the transcriptomic data of 9 samples from our cohort was performed by SST-RMA summarization to generate gene level expression signals using Transcriptome Analysis Console  software after the evaluation of quality control metrics. Affymetrix probes were annotated to human genome hg38 version using TAC software and the Clariom D annotation file Clariom_D_Human.r1.na36.hg38.a1.transcript.csv. Probes mapped to more than one gene were removed and logarithm transformed expression data was exported for further analysisThis normalized expression data of the Brazilian cohort was later used for extracting the expression intensity of miRNAs and their respective targets to reconstruct the miRNA-mRNA regulatory network, that was based in data from the meta-analysis (described later).37 after adjusting variables by age. We also applied the Empirical Bayes framework to estimate the more precise expression of each gene to discriminate DE genes. DE genes were identified based on the adjusted p valor < 0.05 and filtered with the fold change values. To evaluate the discriminatory capacity of each DE gene, we computed the area under (AUC) the ROC curve using pROC package38.To identify the divergent gene expression pattern between active and atrophic pterygium, a differential gene expression analysis was performed using a linear model fitted in limma package39 implemented in the oligo package40 as previously described29. Briefly, RMA algorithm performs background subtraction which intends to minimize the effects of the noises inherent to microarray technology. Quantile normalization and median-polish steps are then applied to mitigate the effects of technical variables through the estimation of a common intensity distribution across samples. Probes are summarized into a single probe set corresponding to a single gene and expression is then transformed in log-scale. Probe sets were annotated using biomaRt packages41.Microarray raw data were pre-processed using the Robust Multichip Average (RMA) methodGene expression datasets from microarray studies including human patients that underwent pterygium excision were searched in the Gene Expression Omnibus (GEO) public repository, by December 2020. Search was conducted using the term “pterygium”. Datasets were only included if the studies analyze both affected patients and healthy tissues. All included datasets were from studies published in peer-reviewed journals. In addition, studies were excluded if samples were submitted to cell culture.42 using Rank product algorithm. A non-parametric method is applied to detect genes consistently ranked as DE by comparing patients to healthy controls in each dataset. Thus, this method overcomes the heterogeneity among multiple datasets by transforming the actual expression values into ranks42 and computes the p value and the false discovery rate (FDR) of each DE gene. The gene list was further filtered to include only genes that were up- or down-regulated in the same direction in both studies based on a false discovery rate (FDR) < 0.05 as previously described29. Heatmap visualization of the subset of 30 top DE genes was performed using the heatmap Bioconductor package43 after expression recalibration using a list of human housekeeping genes derived from the HRT Atlas database44.The meta-analysis was performed with RankProd package45. ClueGO is a plug-in of Cytoscape36, which predicts the functional gene ontology terms or biological pathways and organizes them in functionally grouped networks, highlighting the biological relationship between them. ClueGO’s fusion criterion was applied to reduce the redundancy of the terms that have similar associated gene sets. In order to filter the most important terms and pathways, we used two-sided hyper-geometric distribution tests and terms at a significant level Bonferroni adjusted p-value of ≤ 0.05.To facilitate the interpretation of the biological significance of the up-regulated genes identified by the meta-analysis, a functional gene set analysis (GSA) was performed using ClueGO34. Briefly, FAIME determines at a single sample level the cumulative quantitative effects of genes inside the differentiated Gene Ontology terms and biological processes. A FAIME score is then computed based on the gene expression pattern of each sample for the predicted terms or biological processes. Receiver operating characteristic (ROC) curve was computed to evaluate the discriminatory capacity of each of these enriched terms for a binary classification. In this analysis, the binary classification was pterygium vs control, across FAIME scores computed for each pathway. We computed the area under (AUC) the ROC curve using pROC R package38. Finally, we selected the enriched terms with AUC equal or more than 0.7 and network visualization was performed using cytoscape software36.To gain more insights into the biological processes associated with the down-regulated signature, these genes were used for an additional functional analysis based on the Functional Analysis of Individual Microarray/RNAseq Expression (FAIME) algorithm implemented in seq2pathway package33. To reconstruct the miRNA-mRNA regulatory network, the expression intensity of each miRNA and their respective targets was extracted from the normalized expression data of the Brazilian cohort of pterygium. Pairwise correlation was computed between miRNA and their targets. miRNAs that have a negative correlation  with at least one target were selected for the network reconstruction. Network visualization was performed in Cytoscape36 software considering miRNA and mRNA as source and target nodes respectively.The list of targeted genes included in this analysis was derived from the functional gene set analysis performed with the up-regulated genes identified by the meta-analysis. Briefly, we extracted the list of genes associated with the functional gene ontology terms and biological pathways predicted by ClueGO. The list of conserved miRNAs predicted to target these genes was identified in TargetScanHumanNine participants were included, being 6 males (aged 29–70 years old) and 3 females (aged 27–48 years old). The pterygia were classified as grade 1 in 1 case, grade 2 in 4 cases, grade 3 in 3 cases and grade 4 in 1 case. According to their biomicroscopic aspect, there were 2 atrophic and 7 active pterygia. Demographic information of the participants is presented in Table We evaluated the differential expression comparing active pterygium with atrophic pterygium using data generated from the Brazilian cohort. This analysis identified 219 up-regulated and 92 down-regulated genes. The pattern of gene expression generated with top 30 DE genes is presented as heatmap in Fig. Three studies  fulfilled the inclusion and exclusion criteria described in methods section of which two datasets (GSE51995 and GSE83627) were identified as duplicated datasets by PCA analysis , structural and adhesion molecules, including fibronectin (FN1), CEACAM5 (CEA), CD24, SPARC, MSMB and TFF146. In addition, Chen and colleagues performed RNA sequencing experiments on clinical pterygium tissues and normal conjunctival tissues and identified 200 upregulated DGEs and 139 downregulated DGEs. The upregulated genes were mainly associated with ECM and with cell adhesion and migration and included: FN1; ECM1, which encodes extracellular matrix protein 1; IQGAP2, associated with cell adhesion and cytoskeletal organization; GADD34, a growth cycle protein that is induced by cell stress; and CXCL12, an angiogenic chemokine50. Biological pathways overrepresented in the gene set analysis were highly related to remodeling of extracellular matrix, including its organization, degradation, associated proteoglicans and regulation of biomineral tissue development. It was evidenced not only in the network of up-regulated genes, but also in the correlation between this gene set with the miRNA expression obtained from the Brazilian cohort. Other pathways also represented in the network and in the correlation with miRNA expression included formation of cornified envelope, establishment of skin barrier and unsaturated fatty acid metabolic process, which are associated with wound healing response.A meta-analysis performed by Xu and colleagues, based on three datasets  available at the Gene Expression Omnibus public repository, disclosed upregulation of other genes in addition to the ones found in our meta-analysis, including FN1, a key molecule in the extracellular matrix, and PI3, involved in apoptosis, focal adhesion and extracellular matrix-receptor interactionAmongst the genes correlated with miRNA expression from the Brazilian cohort, GJA1 encodes a protein called connexin 43, a component of gap junctions, COL6A3 encodes one of the chains of type VI collagen, a component of connective tissues, and THBS4 and THBS2 encode for members of the thrombospondin family, which are adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. YAP1 is a gene known to play a role in the development and progression of multiple cancers as a transcriptional regulator of a signaling pathway. LOXL1 encodes a protein essential to the biogenesis of connective tissue, while LHFPL2 is a member of the superfamily of tetraspan transmembrane protein encoding genes.53. ALOX15B encodes a protein involved in the production of fatty acid hydroperoxides and FA2H, a protein that catalyzes the synthesis of a subset of sphingolipids. ABCC1 is involved in the transport across extra and intra-cellular membranes and UGCG, in the biosynthesis of glycosphingolipids, which are membrane components. PLA2G4A encodes a cytosolic phospholipase A2, an enzyme that catalyzes the hydrolysis of membrane phospholipids to release arachidonic acid. SCD encodes an enzyme involved in fatty acid biosynthesis, primarily the synthesis of oleic acid.Also, regarding the genes correlated with miRNA expression obtained from the Brazilian cohort, pathways involved with unsaturated fatty acid metabolic processes were found to be differentially expressed in pterygium. This supports other studies findings in which pterygium fibroblasts were found to have an increased activity of intracellular cholesterol metabolism and high expression of LDL receptors47, highlighting similarities between pterygium and neoplasia. Clinical evidence of tumoral behavior include fibrovascular proliferation, corneal invasion, and high recurrence rate after surgical excision. The reduced recurrence rate of pterygia with the application of mitomycin-C, 5-fluorouracil or cyclosporine during or after surgery supports this finding, as does the fact that the main risk factor for pterygium formation is UV light exposure, equivalently to the most common skin tumors54.Also reinforcing previous studies findings, increased cellular proliferation and reduced apoptosis appear to be involved in pterygium developmentFOS gene family consists of 4 members, which are FOS, FOSB, FOSL1 and FOSL2. The proteins encoded by these genes are leucine zipper proteins involved in regulation of cell proliferation, differentiation and transformation. In addition, these proteins play a role in cellular response to reactive oxygen species and in inflammatory response46. WIF1 consists of a tumor suppressor gene that is epigenetically silenced in various cancers. NR4A2 encodes a nuclear receptor of the steroid-thyroid hormone-retinoid receptor family and may be involved in pterygium once it is associated with cellular response to oxidative stress and regulation of apoptotic signaling pathway. DUSP1 encodes a protein that plays an important role in the cellular response to environmental stress and in the negative regulation of cellular proliferation. TNNI2 encodes a component of the troponin complex, present in corneal epithelium, acting as an inhibitor of angiogenesis and tumor suppressor. CYP26A1 encodes a member of the cytochrome P450 superfamily of enzymes, involved with regulation of retinoic acid receptor pathway and response to vitamin A. IGFBP3, one of the insulin-like growth factor binding proteins, is involved in apoptotic processes and regulation of cell population proliferation. IER2 is an immediate early gene involved in cell motility and response to fibroblast growth factor. Lastly, the protein encoded by BTG2 appears to have antiproliferative properties, being associated with DNA damage response and repair and having a function of negatively regulating the mitotic cell cycle.OLFM4 consists of an antiapoptotic factor that promotes tumor growth and also an extracellular matrix glycoprotein that facilitates cell adhesion. In pterygium’s pathogenesis, it could be associated an uncontrolled tissue growth. GJA1, a member of the connexin gene family, encodes the major protein of gap junctions and is related to cell signaling, apoptotic processes and chronic inflammatory processes. The TGM2, IGFBP3 and DUSP1) and stress-inducible transcription regulator genes  were down-regulated in pterygium, reinforcing the over-proliferative tendency in pterygium46. Downregulated genes in Chen and colleagues’ study, in its turn, included LCN1, a member of the lipocalin family, LTF, a component of the innate immune system, and SCGB2A150. Biological functions found to be down-regulated in pterygium in comparison with normal conjunctiva included regulation of cell differentiation and cellular component organization, oxidation–reduction processes and negative regulation of cell death. This reinforces the similarities between pterygium and tumor growth9, although differences still exist. Amongst the differences between the two conditions, the fact that extracellular matrix remodeling is the main mechanism found in pterygium’s pathogenesis, and not cellular proliferation, is one of them. The main histologic findings in pterygium’s specimens include squamous metaplasia, hyperplasia of goblet cells, underlying disrupted Bowman’s layer, stromal fibroblasts and vessels, altered extracellular matrix with accumulation of collagen and elastin fibers, and inflammatory infiltration55. As shown in previous studies, histopathology only rarely discloses neoplasia or moderate/severe atypia in excised pterygia57.In Tong and colleagues’ study, genes encoding for apoptosis (CXCL9 and PLA2G2D), angiogenesis (SERPINB5 and BM4), keratinization (DSG1), response to UV light (TYR) and negative regulation of apoptotic process (PIM1) are up regulated in relation to atrophic pterygium. On the other side, genes involved with negative regulation of transcription (CIART), response to osmotic stress (AQP9), chemotaxis (CXCL6), intracellular signal transduction (TREM1), extracellular matrix organization (COL6A3) and protein ubiquitination (NEURL4) are down regulated in relation to atrophic pterygium.Finally, we could determine differences in gene expression between atrophic and active pterygium, leading to the conclusion that distinct biological pathways may be differently combined in terms of up and down-regulation to result in certain phenotypic characteristics of pterygium. In active pterygium, genes involved with immune and inflammatory response (Limitations of the study included the Brazilian sample size, which was not large, in addition to an absence of healthy conjunctival samples to compare with pterygium samples. Consequently, these samples were not included in the analysis of up and down regulated genes of the main meta-analysis, which was based on already available datasets. Brazilian data was used for the reconstruction of the miRNA-mRNA regulatory network and for a transcriptomic analysis in which active and atrophic pterygia were compared. Even though the sample size of this analysis was limited, the top ranked genes made it possible to stratify active and atrophic pterygium in different clusters using unsupervised clustering method as shown in Fig. In conclusion, reinforcing previous studies, we identified crucial pathways in the pathophysiology of pterygium, with the main ones being related to extracellular matrix remodeling and dysregulated wound healing response. Furthermore, different pathways involved in the pathogenesis of pterygium were evidenced to be related to different phenotypic characteristics of this condition, which may also provide some clarity in our understanding of this condition’s pathophysiology. Thus, we believe this study enriches our understanding of molecular mechanisms involved with pterygium, providing insights that may contribute for the future development of new therapeutic targets for its management.


An organism’s diet is a major route of exposure to both beneficial nutrients and toxic environmental chemicals and natural products. The uptake of dietary xenobiotics in the intestine is prevented by transporters of the Solute Carrier (SLC) and ATP Binding Cassette (ABC) family. Several environmental chemicals and natural toxins have been identified to induce expression of these defense transporters in fish and aquatic invertebrates, indicating that they are substrates and can be eliminated. However, certain environmental chemicals, termed Transporter-Interfering Chemicals or TICs, have recently been shown to bind to and inhibit fish and mammalian P-glycoprotein (ABCB1), thereby sensitizing cells to toxic chemical accumulation. If and to what extent other xenobiotic defense or nutrient uptake transporters can also be inhibited by dietary TICs is still unknown. To date, most chemical-transporter interaction studies in aquatic organisms have focused on ABC-type transporters, while molecular interactions of xenobiotics with SLC-type transporters are poorly understood. In this perspective, we summarize current advances in the identification, localization, and functional analysis of protective MXR transporters and nutrient uptake systems in the digestive system of fish and aquatic invertebrates. We collate the existing literature data on chemically induced transporter gene expression and summarize the molecular interactions of xenobiotics with these transport systems. Our review emphasizes the need for standardized assays in a broader panel of commercially important fish and seafood species to better evaluate the effects of TIC and other xenobiotic interactions with physiological substrates and MXR transporters across the aquatic ecosystem and predict possible transfer to humans through consumption. Fish and other aquatic animals can be exposed to water-soluble environmental chemicals via uptake through the gills and hydrophobic xenobiotics through ingestion of contaminated food. Gills are commonly considered a part of the respiratory system of aquatic animals . In addiIn fish and mammals, most nutrients as well as water and ions in the diet are typically absorbed through the epithelial cells of the small and large intestines . TherebyYet, despite the similarities in intestinal macro- and micro-anatomy between terrestrial and aquatic organisms, little is known about the molecular composition and levels of both nutrient uptake and xenobiotic defense systems along the digestive tract of fish and other aquatic organisms. In addition, there is limited data available on the molecular interactions of aqueous and food-borne contaminants with these transport systems and how these interactions could affect nutrient homeostasis and toxic contaminant bioaccumulation.One of the key functions of the digestive system is to absorb nutrients from the diet that can be metabolized to provide energy for growth and development. At the same time, the intestinal tract serves as a major environmental barrier to prevent toxic xenobiotic uptake and accumulation. In the gills and intestine of fish and other aquatic organisms, several members of both the solute carrier (SLC) and ATP-Binding Cassette (ABC) family of transporters have been identified to participate in essential physiological functions such as nutrient uptake, ion flux, cell signaling processes, and toxic metabolite and xenobiotic efflux.Transepithelial transport of nutrients in the gastrointestinal tract is typically mediated by secondary active transporters of the solute carrier (SLC) family. In humans, SLCs represent the largest group of secondary active membrane transporters . SLCs arOncorhynchus mykiss) gills (Danio rerio) intestines (Oreochromis niloticus)  gills . Furthertestines . High inloticus) . In Zebrloticus) . Howeverloticus) . Intestiloticus) . Deficieloticus) .Members of both the ABC and SLC-type families have been shown to be part of a highly conserved defense mechanism against chemical insults, often referred to as multidrug-resistance (MDR) transporters . The mamIn the early 90s, a mechanism similar to MDR was reported in aquatic organisms and since then referred to as multixenobiotic-resistance or MXR . The MXRPleuronectes americanus using Southern blot techniques represented the first set of P-glycoprotein orthologs reported in lower vertebrates (The ABC-type transporter P-glycoprotein (aka MDR1 or ABCB1) is one the best characterized mammalian MDR/MXR transporters that shows high expression in biological barriers such as the liver, brain, kidney and the intestine . The idetebrates . Since ttebrates , carp  in the liver, brain, gonads, kidney, gills, and intestine of rainbow trout revealed similar expression patterns to mammalian tissues  and head (gills) of early larvae  and organic anion transporting polypeptide 2 (oatp2) genes were detected in yolk sac epithelium, while the mRNA levels of the organic anion transporting polypeptide 1d gene (oatp1d) were high in abdominal viscera  was shown to significantly decrease mRNA levels of the SLC15 peptide transporter gene family, but mRNA levels recovered upon refeeding (Fundulus heteroclitus), where short-term fasting increased, long-term fasting decreased, and re-feeding restored SLC15 mRNA levels  was shown to increase intestinal peptide transporter 1 (PepT1/SLC15A1) (Dicentrarchus labrax) diet was shown to increase PepT1 gene expression but not affect the expression of the monocarboxylate transporter SLC6A19  fed diets supplemented with saponins and pea protein were shown to have several metabolic systems greatly altered, including differential expression of several SLC proteins  . Similar SLC6A19 . Atlantiproteins . Studiesproteins .As a result of climate change and associated alterations in salinity, pH, and temperature of aquatic environments, the bioavailability and toxicity of environmental chemicals can increase . Recent Xenopus laevis oocytes  was shown to cause a decrease in copper absorption in rainbow trout gut sacs as well as a decrease in iron absorption by SLC11 proteins isolated from rainbow trout gills and expressed in  oocytes . The pH  oocytes . Zebrafi oocytes .Chionodraco hamatus) represents an extreme example, with structural changes to its PEPT1 protein facilitating in part its ability to function at very cold (−1.9°C) temperatures (Hypomesus transpacificus), the expression of the ion exchange transporter SLC8B1 was shown to have non-linear responses to increasing temperature, with decreased expression at 20°C and 25°C compared to the control (14°C) and re-elevated expression at 27°C , a linear increase in transport rate with increasing temperature has been demonstrated . The autMultixenobiotic Resistance transporters represent a critical line of defense in preventing xenobiotic chemicals from accumulating and harming an aquatic organism. Initial investigations identifying MXR transporters as molecular targets of xenobiotics focused on chemical exposure experiments and subsequent evaluation of transporter mRNA and/or protein expression levels . Therebyabcb1a and abcb1b, was significantly increased upon exposure to the antifungal agent clotrimazole (pgp (ABCB4) and abcc2 expression in zebrafish embryos , the abcb1 and abcc2 genes are induced in the liver upon exposure to cadmium ions  the expression levels of peptide uptake transporters SLC15a1a, SLC15a1b, SLC15a2, and SLC15a5 decreased after fasting (Ctenopharyngodon idella), the cotton derived phenol gossypol was shown to down-regulate several peptide and amino acid SLC transporters in the intestine, leading to intestinal damage and reduced growth  the ABCB1 homolog P-gp1 was shown to be inhibited by a series of pharmaceuticals, with sildenafil and simvastatin showing highest inhibition potency , moderately hydrophobic pesticides and PFAS compounds have been shown to inhibit gill tissue efflux of Rhodamine B, a known P-glycoprotein substrate (Mytilus coruscus), gill tissue pre-exposure to benzo(α)pyrene (BaP) reduced the inhibitory effect of ABCB1- and ABCC1-specific inhibitors reversin 205 and MK572 on Calcein-AM efflux pyrene led to increased deformities, impaired growth, and increased toxicant accumulation. For instance, in the Californian mussel  were exposed to combinations of the toxic MXR transporter substrates vinblastine and triclosan (TCS) or P-85 nanoparticles, the toxicity increased by a factor of 2-8 (Dreissena polymorpha) were exposed to a combination of vinblastine and model ABC transporter inhibitors, the toxicity increased super-additively  suggest that microcystin-LR may act as a substrate and competitive inhibitor of ABCC-like proteins in intestinal tissues  is capable of inhibiting ABCC-like transporters when absorbed by intestinal epithelial cells , ABCC2 activity was shown to be inhibited by the vasoconstrictor endothelin-1 via the ETB receptor and protein kinase C signaling . Transporter localization, gene expression levels and endogenous and xenobiotic substrate/inhibitor identification has mainly focused on two model organisms: zebrafish and rainbow trout. Furthermore, there have been only a few studies conducted on fish feed and dietary levels of contaminants and transporter-mediated uptake and bioaccumulation in commercial fish and other human-relevant aquaculture species. Finally, the extent to which other mutable environmental factors, such as salinity, temperature, and pH, may impair the levels and function of MXR and other nutrient transporters remains elusive. This perspective further highlights the urgent need to identify novel TICs, to determine their environmental levels in the natural diets and feeds of fish and other aquatic invertebrates, and to specify the transporter targets that regulate their uptake and disposition in aquatic organisms. Special emphasis should be placed on determining the interactions of TICs with protective MXR efflux transporters and essential nutrient uptake transporters lining the gills and intestinal epithelia to better predict toxic chemical accumulation and/or nutrient deprivation. The key strategies to mitigate toxicant and toxin accumulation in aquatic organisms include targeted chemical analysis of fish feed in aquaculture operations, a continuous biomonitoring of TIC levels in lower trophic level organisms that serve as prey or feed for commercial fish species and the development of regulatory guidelines that inform industrial chemical and agricultural pesticide management strategies to reduce or eliminate the use of environmentally persistent TIC compounds.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Since disease phenotypes associated with each individual target antigens can often blur the distinction between primary and secondary disease, we encourage the use of antigen-based classification of membranous nephropathy.The identification of the major target antigen phospholipase A2 receptor (PLA2R) in the majority of primary (idiopathic) cases of membranous nephropathy (MN) has been followed by the rapid identification of numerous minor antigens that appear to define phenotypically distinct forms of disease. This article serves to review all the known antigens that have been shown to localize to subepithelial deposits in MN, as well as the distinctive characteristics associated with each subtype of MN. We will also shed light on the novel proteomic approaches that have allowed identification of the most recent antigens. The paradigm of an antigen normally expressed on the podocyte cell surface leading to  The pathological hallmark of MN is the deposition of immunoglobulin G (IgG) and variable amounts of complement proteins within the subepithelial space. Immune complexes can form al space , 2. The Analogous to Moore’s Law which predicted an exponential increase in computing power as technology allowed more transistors per chip, MN research has recently been marked by an accelerated pace in the discovery and identification of antigens, more so than at any time since the first delineation of MN as a distinct clinicopathologic entity in 1957 . The resin situ   or if, instead, they formed in situ . Similarin situ , 7 or bein situ . This pa barrier , 10. Whi2 (LRP2) . Despite2 (LRP2) . Of note2 (LRP2) .Other animal models of MN had been developed, including those utilizing exogenous cationic bovine serum albumin (BSA) as an antigen that traversed the GBM and become planted in a subepithelial space. Mice injected with cationic BSA developed features of MN with proteinuria developing within two weeks . This moThe first evidence of an intrinsic glomerular antigen in human MN came through experiments of nature. Ronco and colleagues documented rare cases of antenatal MN occurring in newborns of mothers deficient in neutral endopeptidase (NEP), a known glomerular protein . These mIt was not until 2009 that a target antigen was identified in adult MN. Using patient serum to probe immunoblots of human glomerular proteins, Beck and Salant were able to find evidence of a common 185 kDa protein identified by the serum from a proportion of idiopathic MN cases. Mass spectrometric analysis of the gel band revealed this protein as the M-type (muscle derived) phospholipase A2 receptor (PLA2R) . PLA2R wDespite the identification of THSD7A and PLA2R, the specific target antigens in approximately 30% of primary MN cases remained unknown. There was a need to further characterize the antibody repertoire in MN for patients who were PLA2R and THSD7A-negative. However, for rare antigens, it was becoming increasingly difficult to identify common pattern of reactivity by Western blotting, confounded by a proportion of patients who are in immunologic remission at the time of serum sampling and therefore would not have these rare circulating autoantibodies. Therefore, new approaches were necessary that instead focused on the kidney biopsy as a tissue source to identify the antigen, under the assumption that the targeted antigen would be enriched in the subepithelial immune complexes relative to normal glomerular proteins , 22.vs. remission vs. relapse)  , 22, 23,Recent advances in proteomic technologies have enabled large-scale profiling of proteins in tissue and sera from patients, with the potential to better diagnose and classify autoimmune diseases such as MN . The powIn the field of MN, the use of sequential methods of LCM followed by MS has allowed the discovery of multiple new autoantigens including the exostosin 1/2 complex (EXT1/2) , neural This technique was subsequently followed by protein G immunoprecipitation (IP) from biopsy tissue, specifically focusing on immune complexes eluted from kidney tissue, followed by MS for identification of antigenic targets . FunctioAutoimmune profiling, based on serum reactivity with whole proteome arrays, has emerged as a promising methodology to complement the previously mentioned modalities –36. ThisAn overview of these methods is shown in e.g., PLA2R or THSD7A) has been considered to represent primary MN  or secondary disease. The presence of humoral autoreactivity against a known antigen  preceded clinical remission  and often presents with sub-nephrotic range proteinuria. PCDH7-associated MN was reported to account for 5.7-10.5% of PLA2R-, THSD7A-, EXT1/2-, SEMA3B-, and NELL1-negative MN cases . There ade novo membranous nephropathy in the setting of stem cell transplantation or kidney transplantation. It was discovered by LCMD, followed by mass spectrometry. Similar to the other protocadherin protein associated with MN, PCDH7, there was minimal complement deposition see by immunofluorescence  interacts with the anionic glomerular basement membrane and, with its corresponding antibodies, forms teinuria . ComplemMembranous lupus nephritis  is the most common secondary etiology of MN and disproportionately affects young females of African or Hispanic descent, as well as other minority populations. There are some histopathologic findings on kidney biopsy that increase suspicion for MLN over primary MN Figure 3While the antigenic targets involved in the majority of primary MN cases have been discovered, the majority of autoantigens in MLN are unknown. To date, three autoantigens have been described in membranous lupus nephritis, which together comprise approximately 1/3 of all MLN cases. These include the exostosin 1/exostosin 2 complex (EXT1/2) , neural The first biomarker to be described in MLN is the EXT1/EXT2 complex, identified by LCM-MS of kidney biopsies. The EXT1/EXT2 complex represents a hetero-oligomeric glycosyltransferase that requires both proteins for enzymatic function. It participates in GBM homeostasis through regulation of heparan sulfate . EXT1 muBoth the EXT1 and EXT2 components of the heterodimer are present within deposits in all cases and therefore staining for either component can be performed to identify positive cases. EXT1/EXT2-positive MN appears tightly associated with autoimmunity, with 70% of patients having a positive ANA, 35% having a clinical diagnosis of SLE and 12% of patients having mixed connective tissue disease at the time of biopsy . The freAlthough unclear at this time whether the EXT1/EXT2 complex is a putative autoantigen or a biomarker, the finding has prognostic significance. Two independent investigations indicated its value as a biomarker of favorable outcome with less progression of kidney disease in patients with MLN, when compared to EXT1/EXT2-negative MLN , 88. RemNCAM1 is an immunoglobulin superfamily cell surface glycoprotein that is expressed within the central nervous system, immune system, and within podocytes , 90. It Patients with NCAM1-associated MN were predominantly female (70%), with a mean age of 34 ± 12.1 years. Interestingly, 40% of NCAM1-associated MN patients with SLE had an increased frequency of neuropsychiatric manifestations at the time of biopsy (8/20 patients), a rate 4-5 times higher than reported in SLE overall . There are limited data to conclude whether NCAM1 may be a link between nephritis and neuropsychiatric disease in patients with SLE, but the question warrants further investigation to determine if such a connection exists. Manifestations of SLE vary in time and it could be helpful to determine if patients with NCAM1 autoantibodies are at future risk for neuropsychiatric disease and therefore might benefit from closer monitoring.+ T-cell specification to the Th1 lineage (The type III transforming growth factor-beta receptor (TGFBR3) was identified as a putative target in MLN by tissue-based proteomics utilizing LCM and tissue IP . TGFBR3  lineage . TGFBR3 TGFBR3 is positive within 5.5% of MLN biopsies and is not identified in primary MN cases . Patientin situ, an argument that is supported by the lack of mesangial or subendothelial deposits in some cases of MLN.Moreover, it conceivable that, despite the paradigm that immune complexes in lupus form extra-renally in the circulation, the pathogenesis of pure lupus nephritis may be more similar to that of primary MN with antibodies targeting intrinsic, induced, or planted podocyte antigens to form immune complexes In addition to SLE, other autoimmune diseases have been associated with MN, including sarcoidosis , 93, 94,Myeloperoxidase (MPO) is a target autoantigen associated with MN in the setting of ANCA-associated disease. A concurrent crescentic glomerulonephritis is frequent, but MN can occur in its absence. These cases are PLA2R-negative and MPO has been demonstrated in the epimembranous immune deposits in glomeruli , 108.Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is an autoimmune disease that can exhibit a membranous pattern on kidney biopsy and affects young women, often with positive antinuclear antibodies . In mostCNTN1 was recently identified as a target antigen in MN with chronic inflammatory demyelinating polyneuropathy (CIDP) and autoimmune myositis . It is aPerhaps relevant to these links to neuropsychiatric disease or autoimmune neuropathies, several MN autoantigens are proteins expressed in both neurons and podocytes. There are similarities in signaling between podocytes and neurons, both of which are specialized cells with arborized processes supported by robust cytoskeletal dynamics and an intercellular communication utilizing similar proteins expressed at synapses and foot processes, such as synaptopodin and dendrin , 114. OtAntigens targeted in MN can be intrinsic podocyte proteins, podocyte proteins that may be induced by certain stressors, extrinsic non-podocyte proteins that become planted in a subepithelial position, or those that precipitate from circulating immune complexes. The concept that target antigens in MN are often podocyte-expressed proteins is relatively recent. The antigenic component of rat proximal tubular brush border is megalin which, in a not-well-understood quirk of nature, is universally expressed by the podocyte in the rat, but not so in other species. PLA2R and THSD7A were identified as target antigens in MN before they were found to be podocyte proteins. Detailed scrutiny of the more-recently identified antigens may reveal that they are expressed by podocytes in some cases, including HTRA1, SEMA3B, NCAM1, and TGFBR3, but not in others, including EXT1/2, NELL1, PCDH7, and CNTN1. However, our knowledge of what is or is not expressed by normal human podocytes in the resting state is not comprehensive, and new studies consistently reveal novel podocyte proteins. There may also be age-related developmental changes in expression, or induced expression by disease or environmental factors. Such an induced expression may, for instance, explain the segmental pattern of NELL1-associated MN, since NELL1 does not seem to be normally expressed by the podocyte. In challenging the prevailing paradigm in understanding MN, the presence of antigens not inherently expressed in glomeruli, such as EXT1/2, NELL1, and PCDH7, may suggest more than one pathogenic pathway can lead to similar pathologic manifestations.Multiple medications are described to cause MN, comprising 6.6-14% of total MN in prior studies –124. DruSome infections have been identified in association with MN and in certain cases the microbial protein has been identified within immune complexes. It is unclear whether microbial proteins are true antigenic targets or are passively trapped within the subepithelial space. Hepatitis B virus (HBV) infection can induce MN with the hepatitis B e antigen (HBeAg) identified within subepithelial deposits. Proteinuria persists with HBV antigenemia and one-third develop progressive disease, despite treatment , 127. InSchistosoma mansoni by indirect immunofluorescence (Hepatitis C virus (HCV) infection has also been reported as a secondary cause of MN which can remit with antiviral therapy , 132. Merescence . Recentlrescence –146. TheOrientia tsutsugamushi is known to be antigenic and has been used in a vaccine developed against scrub typhus, demonstrating immunogenicity , a negative regulator of TLR signaling, was identified in a large genome wide association study for MN as a highly significant risk locus . IRF4 biAnother secondary etiology of MN is hematologic or solid organ malignancy, comprising approximately 10% of MN cases. MN is the leading cause of nephrotic syndrome in patients with malignancy and patients with MN have a three-fold increase in the incidence of cancer compared to the general population , 152. ThBecause both MN and malignancies tend to occur in older individuals, it can often be difficult to determine if the cancer is causal or coincidental . To clasThe mechanisms underlying malignancy-associated MN are largely unknown. It is possible that dysregulated expression of immunogenic proteins by the tumor incites autoimmunity with subsequent deposition of immune complexes within the subepithelial space. Genetic mutations within the tumor may alter the amino acid sequence to create neo-epitopes that are immunogenic. Alternatively, there could be molecular mimicry of a tumor antigen that shows sequence similarity to a podocyte protein .Increased expression within the tumor due to copy number increases, as has been seen with THSD7A due to polysomy of chromosome 7 , may alsThe categorization of MN as either primary or secondary was based on the absence or presence of a detectable underlying cause, such as systemic autoimmune disease, cancer, or infection, as discussed above. Subsequently, after the identification of PLA2R as target antigen in the majority of primary MN, PLA2R-negative cases elicited an extensive workup to identify a possible secondary etiology . RecentlAn overview of demographic, clinical, and histopathologic characteristics for the MN subtypes associated with each of these various antigens in MN is provided in However, as such an MS-based strategy for the typing of MN is currently impractical for routine clinical practice, we propose a staining algorithm for antigen subtyping based on clinical and pathologic features Figure 4Subepithelial immune complex formation is the hallmark of MN. Complement pathway activation has been considered as the paradigmatic mechanism leading to podocyte injury and failure of the glomerular filtration barrier, resulting in the nephrotic syndrome. However, other mechanisms inducing podocyte injury have been postulated and the primacy of complement as a pathomechanism in MN has been challenged. In the passive Heymann nephritis model, proteinuria depends on activation of the complement system with subsequent formation of the membrane attack complex (C5b-9) , 163. Otvia the classical pathway  air pollution, potentially implicating this environmental exposure as a risk factor for disease initiation . This haHLA-DQA1 locus has been associated with higher risk of developing PLA2R-associated MN  and whole exome sequencing (WES) have revealed highly significant risk alleles predisposing to MN. A single-nucleotide polymorphism (SNP) at the iated MN , more so  on the antigen to involve other region(s) of the same antigen (intramolecular spreading) or nearby or related antigens (intermolecular spreading) . EpitopeA better understanding of the evolution and repertoire involved in epitope spreading to any MN target antigen may help to guide prognosis, to understand the initial triggering events in disease pathogenesis, and to allow development of antigen-targeted therapies. While epitope spreading is important in the initiation and progression of MN and other autoimmune diseases –201 and i.e., antibody-mediated cytotoxicity due to one antigen resulting in exposure of the second protein to the immune system) is only speculative. The incidence of PLA2R and THSD7A dual positivity is approximately 1%, shown in a study where 2/258 cases of primary MN shown dual positivity by immunostaining and serologic testing  offers the ability to monitor immunologic disease activity and response to therapy in a non-invasive manner. As demonstrated with anti-PLA2R, autoantibody levels correlate with disease activity but with a lag time, decreasing and often disappearing prior to a full proteinuric response with treatment, or increasing after remission to signal oncoming relapse of disease. As serologic remission precedes clinical remission, it can be useful to adjust immunosuppressive therapy accordingly and can inform prognosis. Serologic diagnostic tests for PLA2R-associated MN  and THSDAs a proof of concept and extrapolating from the PLA2R experience, a combination of IFA and ELISA-based testing provided high specificity for disease. This has allowed providers in some situations to avoid the need to perform a kidney biopsy in patients who have with anti-PLA2R antibodies and rather to monitor them according to anti-PLA2R titers , 205. ThThe identification of new antigens in MN may also provide a means for clinical monitoring when circulating antibodies are detectable, yet there is a need for development of validated and reproducible immunoassays directed towards these antigens for use in the clinical setting. With such advances in serologic testing, the diagnosis and monitoring of the recently identified MN subtypes may be possible and more widely available. However, some subtypes of MN, including EXT1/2- and TGFBR3-associated disease, have yet to have autoantibodies identified, creating a challenge for development of these assays. Identifying autoantibodies directed against all distinct MN types could alter treatment paradigms for MN by adding to the list of treatment-specific biomarkers, identifying new therapeutic targets, and providing tools to forecast impending MN flares. This would allow for patients to be pre-emptively treated, perhaps without the need for biopsy, shorten exposure to toxic drugs by treating only to the point of immunologic remission and limiting permanent kidney damage.Treatment of MN has so far focused on non-specific immuno-suppressive therapy, similar to most other autoimmune diseases. Treatment for six months alternating cyclophosphamide with high-dose corticosteroids is still recommended for MN patients that are at very high risk of progression. This ‘gold standard’ in the treatment of MN of cyclic treatment with alkylating agents (cyclophosphamide or chlorambucil) and methyprednisolone, is described in the Ponticelli and ‘modified Ponticelli’ protocols  and to dversus addressing a stressed organelle such as endoplasmic reticulum, or disturbed cytoskeleton, or other methods. Ideally, specific depletion of the responsible clones may result in individualized medicine with improved efficacy and reduced off-target toxicities.Corticosteroids, cytotoxic medications, B-cell depletion, and calcineurin inhibitors lack specificity and are potently immunosuppressive. Unraveling the complexity of this disease might lead to more targeted therapy tailored to specific patient needs. Moreover, therapies targeting specific antigens through blockade with mimotopes or interference with synthesis can prove to be beneficial. This may be accomplished through targeting a certain antigen or targeting the epitope One challenge to clinically identifying and characterizing MN is the invasive nature of diagnosis by kidney biopsies. The current practice of collecting a renal biopsy is invasive and expensive, and it is impractical for physicians to closely monitor patients using serial biopsies. Future studies will build upon serum and tissue-based proteomic approaches to identify remaining specific biomarkers, potentially enabling development of non-invasive diagnostics and/or prognostic assays. Further characterization of temporal changes in autoantibodies and reactive epitope profiles throughout the different stages of MN is warranted.in vivo are not recapitulated in in vitro assays. There may be a mutation resulting in altered protein structure inducing antigenicity, which would not be re-capitulated through use of commercial recombinant proteins. Further work and novel approaches may yield autoantibody detection.Additionally, for some types of MN (EXT1/2 or TGFBR3), it is not yet known whether autoantibodies exist or are these proteins simply biomarkers of disease. The identification of immune complexes eluted from frozen kidney biopsy specimens by tissue IP suggests that a true antigen-antibody interaction exists. Therefore, why are we unable to detect autoantibodies in sera? Antibodies could have very low avidity binding, may circulate in multi-molecular immune complexes where epitopes are opsonized by complement and unavailable for binding, or post-translational modifications Further understanding of the etiopathogenesis of MN may elucidate further treatment targets. Currently, there is a lack of reliable preclinical models that recapitulate human pathology. To address this issue, future studies will likely introduce mouse models that might allow further investigation of the underlying molecular events of pathological development in this disease.in vitro studies of tolerogenic peptides suggested several mechanisms of tolerance induction, such as blocking of MHC class II presentation of the pathogenic peptides by using excess amount of the peptide mimetics or antagonism/partial agonism of TCR signaling and subsequent inhibition of T cell activation  technology will be leveraged to generate specific T cell clones that are able to recognize and destroy B cells expressing antigenic receptors with epitope specificity for target MN antigens , 214. Bativation , 218. ThIn summary, multiple advances have been made in MN over little more than a decade, with the discovery of PLA2R providing the ability to monitor disease. Within only the past three years, a plethora of autoantigens have been identified, reducing our knowledge gap for the protein targets of MN. This could open the door to precision classification through use of algorithmic and multiplex immunostaining, or alternatively, a mass spectrometry-based classification system. Knowledge of autoantigens also opens the door to develop assays for non-invasive monitoring through a serologic-based approach. Additionally, clinical associations of specific antigens with secondary manifestations  can guide the clinician to evaluate for underlying triggers of the disease. Our understanding of MN pathogenesis is incomplete and further studies will be informative to identify new potential treatment targets through precision medicine. The journey of membranous nephropathy has already reached a new avenue moving away from primary and secondary MN to an antigen-based classification system, enabling diagnosis and monitoring of all types of MN non-invasively, with novel treatments developed from the discoveries.All three authors  contributed equally to the writing of this article, and all authors are accountable for the content of the work.The preparation of this work was supported by institutional funding for the Glomerular Disease Center, Boston Medical Center (LB.) and by the Division of Nephrology & Hypertension, University of Utah Health (LA-R).LB reports being a co-inventor on the patent “Diagnostics for Membranous Nephropathy” and receives royalty income through his employer Boston University. LB has served on advisory boards on the topic of MN and other glomerular diseases for Visterra, Ionis, Alexion and Novartis, and receives royalties from UpToDate for topics related to MN.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.