Differential expression of microRNA miR-150-5p in IgA nephropathy as a potential mediator and marker of disease progression

Abstract

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases. Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases. Translational StatementA key unanswered question in IgA nephropathy is why some patients progress to kidney failure and others maintain lifelong normal kidney function. In this study, we have identified 4 microRNAs whose expression in the kidney at the time of diagnosis may help us understand the molecular pathways operating in high-risk patients and in the future could also be used as biomarkers to refine risk prediction in IgA nephropathy. These microRNAs are similarly dysregulated in other forms of progressive kidney disease and therefore may have a much broader utility in risk prediction and understanding the generic pathways and regulators of kidney fibrosis. Consistent with a role in generic kidney fibrosis, expression of these microRNAs significantly correlates with estimated glomerular filtration rate, proteinuria, and the T lesion of the Oxford classification, and in situ hybridization revealed staining patterns consistent with the known drivers of progression. A key unanswered question in IgA nephropathy is why some patients progress to kidney failure and others maintain lifelong normal kidney function. In this study, we have identified 4 microRNAs whose expression in the kidney at the time of diagnosis may help us understand the molecular pathways operating in high-risk patients and in the future could also be used as biomarkers to refine risk prediction in IgA nephropathy. These microRNAs are similarly dysregulated in other forms of progressive kidney disease and therefore may have a much broader utility in risk prediction and understanding the generic pathways and regulators of kidney fibrosis. Consistent with a role in generic kidney fibrosis, expression of these microRNAs significantly correlates with estimated glomerular filtration rate, proteinuria, and the T lesion of the Oxford classification, and in situ hybridization revealed staining patterns consistent with the known drivers of progression. Immunoglobulin A nephropathy (IgAN) is a mesangial proliferative glomerulonephritis characterized by the predominant deposition of IgA-containing immune complexes in the glomerular mesangium.1Wyatt R.J. Julian B.A. IgA nephropathy.N Engl J Med. 2013; 368: 2402-2414Crossref PubMed Scopus (614) Google Scholar It is unique among glomerular diseases in being defined by these immune deposits rather than by light microscopic appearances or clinical features. Intriguingly, the degree of histopathological injury is extremely variable and the tempo and severity of clinical presentation also vary widely, and both appear independent of the extent of immune complex deposition. Currently, the best predictors of future kidney function decline and end-stage kidney disease (ESKD) in IgAN are the extent of proteinuria and the coexistence of hypertension and low estimated glomerular filtration rate (eGFR) at presentation, alongside the specific histological features in the diagnostic kidney biopsy that comprise the Oxford classification.2Trimarchi H. Barratt J. Cattran D.C. et al.IgAN Classification Working Group of the International IgA Nephropathy Network and the Renal Pathology Society; Conference Participants. Oxford classification of IgA nephropathy 2016: an update from the IgA Nephropathy Classification Working Group.Kidney Int. 2017; 91: 1014-1021Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar Unraveling the mechanisms that switch on the inescapable progression to irreversible kidney scarring in those patients who progress to ESKD remains a major challenge. A clearer understanding of the drivers of inflammatory and fibrotic responses after mesangial IgA deposition is likely to identify novel therapeutic targets and potential biomarkers that may aid future risk stratification. It is becoming increasingly apparent that epigenetic modifications provide an additional tier of posttranscriptional regulation that functions to control normal and pathological processes.3Bechtel W. McGoohan S. Zeisberg E.M. et al.Methylation determines fibroblast activation and fibrogenesis.Nat Med. 2010; 16: 544-550Crossref PubMed Scopus (442) Google Scholar, 4Uhlenhaut N.H. Treier M. Transcriptional regulators in kidney disease: gatekeepers of renal homeostasis.Trends Genet. 2008; 24: 361-371Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 5Sun G. Reddy M.S.A. Yuan H. et al.Epigenetic histone methylation modulates fibrotic gene expression.J Soc Nephrol. 2010; 21: 2069-2080Crossref PubMed Scopus (171) Google Scholar, 6Meister G. Tusch T. Mechanisms of gene silencing by double stranded RNA.Nature. 2004; 431: 343-349Crossref PubMed Scopus (1863) Google Scholar Dysregulation of epigenetic factors including microRNAs (miRs)7Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (27032) Google Scholar, 8Soifer H.S. Rossi J.J. Sӕtrom P. MicroRNAs in disease and potential therapeutic applications.Mol Ther. 2007; 15: 2070-2079Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 9Stefani G. Slack F.J. Small non-coding RNAs in animal development.Nat Rev Mol Cell Biol. 2008; 9: 219-230Crossref PubMed Scopus (1077) Google Scholar may play a pivotal role in reprogramming cellular responses, including the extent of glomerular and interstitial inflammation and scarring and ultimately disease progression in IgAN.10Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis.J Am Soc Nephrol. 2010; 21: 212-222Crossref PubMed Scopus (635) Google Scholar These small noncoding RNAs have already been implicated in the control of inflammation (miR-155-5p, -146-5p, -150-5p, -181a-5p, and -223-5p),11Sonkoly E. Stahle M. Pivarcsi A. MicroRNAs in immunity: novel players in the regulation of normal immune function and inflammation.Semin Cancer Biol. 2008; 18: 131-140Crossref PubMed Scopus (420) Google Scholar whereas miR-192-5p, one of a number of miRs known to be enriched in the kidney compared with other organs,12Sun Y. Koo S. White N. et al.Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs.Nucleic Acids Res. 2004; 32: e188Crossref PubMed Scopus (273) Google Scholar has a complex and prominent role in diabetic nephropathy.13Jenkins R.H. Martin J. Phillips A.O. et al.Pleiotropy of microRNA-192 in the kidney.Biochem Soc Trans. 2012; 40: 762-767Crossref PubMed Scopus (27) Google Scholar A number of small discovery studies have reported miR expression in IgAN,14Dai Y. Sui W. Yan Q. et al.Microarray analysis of micro-ribonucleic acid expression in primary immunoglobulin A nephropathy.Saudi Med J. 2008; 29: 1388-1393PubMed Google Scholar, 15Tan K. Chen J. Li W. et al.Genome wide analysis of microRNAs expression profiling in patients with primary IgA nephropathy.Genome. 2013; 56: 161-169Crossref PubMed Scopus (35) Google Scholar, 16Wang G. Kwan B.C.H. Lai F.M.M. et al.Intrarenal expression of microRNAs in patients with IgA nephropathy.Lab Invest. 2009; 90: 98-103Crossref PubMed Scopus (92) Google Scholar, 17Wang N. Bu R. Duan Z. et al.Profiling and initial validation of urinary microRNAs as biomarkers in IgA nephropathy.Peer J. 2015; 3: e990Crossref PubMed Scopus (24) Google Scholar, 18Serino G. Sallustio F. Cox S.N. et al.Abnormal miR-148b expression promotes aberrant glycosylation of IgA1 in IgA nephropathy.J Am Soc Nephrol. 2012; 23: 814-824Crossref PubMed Scopus (132) Google Scholar but none have systematically investigated the expression of miRs in the kidney and related their expression to acknowledged risk factors for ESKD and actual future kidney function decline. The aim of the present study was to identify miRs likely to contribute to the future risk of ESKD in IgAN. Formalin-fixed paraffin-embedded and frozen kidney biopsy cores were obtained with written informed consent from the University of Leicester Hospitals NHS Trust kidney biopsy archive. Ethical approval for the study was obtained from the Northamptonshire, Leicestershire, and Rutland Ethics Committee (UHL 09873). Patients with biopsy-proven IgAN were classified as “nonprogressors” (IgANnp) if serum creatinine had changed by <10% for >10 years since diagnosis and “progressors” (IgANp) if serum creatinine had at least doubled or they had developed ESKD within 10 years of diagnosis. IgAN cases with rapidly progressive/crescentic glomerulonephritis were from the miR from patients with membranous nephropathy thin membrane nephropathy lupus nephritis and of kidney disease with at the time of biopsy were also miR were In was at the time of kidney biopsy and patients with IgAN and none The and clinical at the time of kidney biopsy for patients are in Full for the International IgAN Prediction are in and clinical at the time of kidney biopsy for discovery and validation cohorts kidney estimated glomerular filtration IgA nephropathy IgA nephropathy lupus membrane thin membrane are expressed as or as in a kidney estimated glomerular filtration IgA nephropathy IgA nephropathy lupus membrane thin membrane are expressed as or as is an that the prediction of the risk of progression on decline in proteinuria, and at the time of MEST-C R. H. et a in IgA Med. PubMed Scopus Google Scholar, P. validation of a Cox and Med 2013; PubMed Scopus Google Scholar, G. to in risk prediction to Med. PubMed Scopus Google Scholar A more is found in the frozen kidney biopsy cores were from the Leicester and cohorts for miR profiling by next generation was the generation sequencing was performed by a the are at miR validation was by miR by on from a independent and biopsy cores from the Leicester and samples were was performed with was used as miR to for In situ hybridization was performed on 4 paraffin-embedded biopsy as by the paraffin-embedded on were in and in of were with for at and with double for at were in of at with serum and with for at was with for at were with in of with to to and by light staining was performed on 4 paraffin-embedded biopsy were and at for were in normal serum for and with mouse or for at 4 were with and were and staining was on a a and were expressed as number of for miR by the number of by miRs whose were were were in expression were expressed as of gene expression time and the PubMed Scopus Google Scholar and an with for were expression were expressed as or were performed on and receiver operating were were performed curve as an PubMed Scopus Google Scholar a analysis of with was were used by for was at for were obtained biopsy miRs exhibited of of were upregulated and were between and and are correlated with the future risk of ESKD in patients with independent of primary diagnosis. we to identify those kidney miRs associated with the of proteinuria and in IgAN cases on the of biopsy to identify miRs likely to these key clinical miRs correlated with the of proteinuria in IgAN cases correlated with the of proteinuria in the cohorts by in miRs significantly correlated with in IgAN cases of and correlated with in the cohorts by in The Oxford classification histopathological features and independent of one and other clinical and of hypertension at are associated with a risk of future kidney function decline. therefore to identify those miRs that are associated with these features likely to be in the glomerular to IgA immune complex deposition in IgAN. on those miRs whose by by or by between and number of cases with was small to analysis to be and expressed at that these 4 Oxford scores in also correlated with both and in IgAN. classified of the IgAN cases as a future or a on the of a of after biopsy. us to identify miRs in the diagnostic biopsy that were associated with actual future kidney function decline and therefore likely to in IgAN and have the future potential to be used as biomarkers of progression risk in IgAN. we on those miRs whose by between and miRs were differentially expressed between the cohorts The and are in The for miRs for validation was as miR be differentially expressed in actual future progressors compared with miR be differentially expressed in compared with control and miR with at least high-risk risk or MEST-C score at and differentially expressed between and and between and and and and correlated with at least of the risk and have the and from other by exhibited expression between cohorts, was for validation in to and validation by in an independent of biopsy cores expression were correlated with and MEST-C -155-5p, and significantly correlated with the of proteinuria and -155-5p, -146b-5p, and significantly correlated with in IgAN the of miRs significantly associated with the and T whereas -155-5p, and also correlated with the of the miRs associated with the lesion However, after the of the T lesion was significantly associated with -155-5p, and In the validation cohorts, and also correlated significantly with the of proteinuria and also significantly correlated with of the revealed that 4 of the and significantly differentially expressed between future progressors and and were also differentially expressed between future progressors and and between future progressors and To the potential value of these 4 miRs to the future risk of were for miR and the validation miR-150-5p exhibited the best discrimination with an the curve of The for of the 4 miRs are in the best expression for predicting progression was a of and a of with a of statistical in discrimination with of the miRs was with and the from miR-150-5p with those from of the other miRs did not discrimination with to the the curve not of the 4 miRs are miR expression for and are as as the best to best with in a The of the 4 miRs are miR expression for and are as as the best to best with To these 4 miRs the we the prediction of the International IgAN Prediction or in with miR of -155-5p, and were to the prediction with the of the that miR was associated with an increased risk of the primary The for the for that was associated with a risk of the primary miR was to the with the increased compared with the prediction an in However, of the small number of none of the results statistical significance. The results were miRs were to the of miR significantly correlated with the risk of a decline in or progression to ESKD after biopsy prediction of the International IgAN Prediction with or to the or in with with to to to to to to to to to to to to to to to to to to to to to to to to to to a of were of The in the prediction with the with the prediction in a expression at biopsy correlated with the risk as by the International IgAN Prediction to to to to in a a of were of The in the prediction with the with the prediction The expression of -155-5p, -146b-5p, and a specific The expression of the 4 miRs in was from that in and whose were To the of -155-5p, -146b-5p, and we expression in lupus an immune disease to be associated with increased miR-150-5p H. P. et promotes renal fibrosis in lupus nephritis by Am Soc Nephrol. 2013; 24: PubMed Scopus Google Scholar and in a of biopsies from patients with established and low at the time of biopsy. miR expression in lupus nephritis was to that in and the of miR expression in with established renal was to that the miR we had identified was more associated with generic kidney fibrosis than with glomerular the of the identified 4 miR was the predominant increased expression of miR-150-5p and expression of miR-150-5p exhibited the best discrimination between and we used to identify the cellular and of miR-150-5p in the Consistent with a role in generic kidney fibrosis, the miR-150-5p expression was in kidneys in with infiltrates, in lymphoid in areas of and expression was also in areas of mesangial proliferation and miR-150-5p also to be expressed to in the in mesangial staining was also in To that miR-150-5p was likely from rather than kidney we the expression of miR-150-5p in kidney and Consistent with the the expression of miR-150-5p in was than that in of the primary kidney To were the likely of miR-150-5p in we performed staining for and and for miR-150-5p on kidney biopsy were in lymphoid of by T with the miR-150-5p in IgAN are T However, miR-150-5p and could contribute to and and areas in and To a functional role for miR-150-5p in the progression of kidney the targets of miR-150-5p were in M. N. et role of in microRNA Genet. 2007; PubMed Scopus Google Scholar G. N. et into functional analysis Acids Res. Scopus Google Scholar and M. A. et targets and Acids Res. 2008; PubMed Scopus Google Scholar To identify functions in the identified of a functional analysis was performed with the of and pathways as the for R. A. 3: a and molecular analysis for functional Acids Cell Res. 2012; 40: PubMed Scopus Google Scholar analysis potential of and pathways in potential A of pathways is in the have for and while was consistent with a role in kidney including kidney fibrosis. To refine the more specifically with in we the of miR-150-5p targets to those differentially expressed in glomerular and of patients with IgAN in a analysis with profiling of in and we E. G. a of mouse and 2015; PubMed Scopus Google Scholar for miR-150-5p gene targets that were differentially at least in the or the of patients who were of likely being compared with those defined as and between and Consistent with and a role in generic kidney fibrosis, miR-150-5p were dysregulated in the miR-150-5p were in the and were upregulated is the study to systematically and the miRNome of kidney from IgAN patients with the of a of by the expression with the clinical associated with clinical we have identified miRs and as potential of progression in could be used in the future as additional biomarkers for risk 4 miRs to the discrimination of that may play a role in the kidney to mesangial IgA immune complex deposition. -155-5p, and were associated with an increased risk of the primary whereas was associated with a However, of the small we did not have statistical to statistical this a much kidney biopsies from patients with these are consistent with independent studies that have reported an between miR-150-5p in the J. H. W. et microRNA of patients with IgA PubMed Scopus Google Scholar and in the G. et for the diagnosis of IgA Nephrol. PubMed Scopus Google Scholar and IgAN. In both the miR patients with IgAN from an with the risk of progression. the expression of miRs and in kidney apparent that the of miR expression was by the extent of kidney fibrosis and the degree of established kidney failure rather than the of the kidney is as that these miRs are likely to play an role in the progression of and could be used as biomarkers of the risk of progression in diseases other than The microRNA is in Scholar, G. G. et role of upregulated and in renal Med 2017; 16: PubMed Scopus Google Scholar, F. F. et promotes renal fibrosis in diabetic nephropathy by PubMed Scopus Google Scholar the 4 miRs the miR associated with the risk of progression was correlated with the of proteinuria, as as the T lesion of the Oxford miR-150-5p was also expressed in kidney biopsies from patients with IgAN who and exhibited discrimination between and in the revealed a of miR-150-5p expression consistent with the of progressive kidney disease with miR-150-5p in infiltrates, lymphoid that are found in areas of and is consistent with the between miR-150-5p expression and the T lesion of the Oxford The expression of miR-150-5p in lymphoid in IgAN kidneys is consistent with miR-150-5p as being expressed in the is in and and S. et a microRNA expressed in and T expressed A. 2007; PubMed Scopus Google Scholar A role of T miR-150-5p in kidney disease including is also consistent with a between the extent of fibrosis, and kidney function E. A. G. et of interstitial in J Full Text PDF PubMed Scopus Google Scholar, E. et role of interstitial in IgA a study with PubMed Scopus Google Scholar, P. Q. et T contribute to the progress of renal J Nephrol. 2012; PubMed Scopus Google Scholar, T a potential in renal Int. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, D.C. analysis in human Int. Full Text PDF PubMed Scopus Google Scholar, F. The role of in the progression of interstitial Google Scholar, T. R. et T for injury in PubMed Scopus Google Scholar In to a potential role of miR-150-5p in is that specific within the kidney are by miR-150-5p the of miR-150-5p from have been reported to Y. Chen et 2010; Full Text Full Text PDF PubMed Scopus Google Scholar and to J. Y. Y. et of from to promotes 2013; Full Text Full Text PDF PubMed Scopus Google Scholar in human while in miRs have been to S. et microRNAs and may contribute to 29: Full Text Full Text PDF PubMed Scopus Google Scholar analysis identified a number of potential within the and of and pathways known to be in kidney and associated with fibrosis. In were the of and are known to play key in the and of the by within the M. et in renal 2009; PubMed Google et of by mesangial with of and 2007; 23: PubMed Scopus Google Scholar In in lupus miR-150-5p has been to an increased expression of the of the of H. P. et promotes renal fibrosis in lupus nephritis by Am Soc Nephrol. 2013; 24: PubMed Scopus Google Scholar are a major to miR-150-5p expression in the kidney in they are not the also and this could contribute to disease progression. These are of as they play an functional role in the and of P. in kidney and 2013; PubMed Scopus Google Scholar they as for the of et of from glomerular Am Soc Nephrol. 2009; PubMed Scopus Google Scholar whereas they contribute to M. New insights into the of cellular PubMed Scopus Google Scholar and the deposition of the in E.M. et in the of in Am Soc Nephrol. PubMed Scopus Google Scholar To have been reported between IgAN progression and a role of in the and progression of inflammation in K. R. Renal inflammation in in Invest. PubMed Scopus Google Scholar The functional role of miR-150-5p in and the of miR-150-5p on kidney are the of within the performed and the and the and biopsy staining and the with the IgAN Prediction and performed performed receiver operating and the was by a from and the was by the Network by the and by an from the University of with

Topics & Concepts