Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Circulating Gremlin-1 is elevated in systemic sclerosis patients

Introduction: Systemic sclerosis is an autoimmune connective tissue disease in which there is activation of the immune system, vascular disease and fibrosis. Activation of quiescent fibroblasts to myofibroblasts is key to disease pathogenesis. Gremlin-1 is a bone morphogenetic protein antagonist which is important in development and we recently reported in skin fibrosis. The aim of this study was to determine the serum circulating levels of Gremlin-1 in early diffuse systemic sclerosis. Methods: Twenty-one early diffuse systemic sclerosis patients (less than 2 years from first non-Raynaud’s symptom) were included and age and sex-matched healthy controls. Serum was isolated from blood and measured with a specific enzyme-linked immunoassay for Gremlin-1. Clinical variables were also measured. Results: Significantly elevated Gremlin-1 was found in sera of early diffuse systemic sclerosis patients ( p < 0.001). In patients with interstitial lung disease, this compared to systemic sclerosis without evidence of interstitial lung disease, Gremlin-1 was significantly elevated ( p < 0.0007). A correlation was found between circulating Gremlin-1 and modified Rodnan Skin Score, albeit weak. Discussion: In early diffuse systemic sclerosis patients, elevated Gremlin-1 is found in serum. This is particularly prominent in systemic sclerosis–associated interstitial lung disease. This suggests that Gremlin-1 may be a biomarker for systemic sclerosis interstitial lung disease.

Evaluation of Coronary Artery Calcification and Gremlin-1 Serum Level Correlation in Patients with Chronic Ischemic Heart Disease

Background: Available evidences have shown that bone morphogenetic proteins (BMPs), particularly BMP 2 and BMP 4, are involved in vascular calcification. Gremlin 1 is one of the important endogenous inhibitors of BMPs. This extracellular antagonist of bone morphogenic proteins has a very complex and cysteine-rich chain and establishes non-colonial transmissions to the members of the family with varying degrees of dependence and prevents them from binding to the receptor, thereby inhibit their function. The main objective of this study was clinical evaluation of the correlation between Gremlin-1 serum concentration and Coronary Artery Calcification. Methods: Eighty-four patients with coronary artery disease from cardiology ward of Razavi Hospital, Mashhad, Iran, who completed the inclusion criteria, entered in the study between November 2015 and March 2016. CT-Angiography was performed to define coronary artery calcium score and Gremlin-1 serum concentrations were measured by an ELISA kit. Results: Eighty-one patients, with mean age of 57.19±10.18 years were included to the study. The mean serum level of Gremlin-1 was 10.92±8.46 pg/mL. There was a reverse significant correlation between Gremlin-1 serum concentration and Coronary Artery Calcification of Right coronary artery(RCA) (P<0.05), in contrast to total Coronary Artery Calcification score, left artery Descending (LAD), Left Main (LM) and Circumflex (CX). However, there was no evidence that age and sex risk factor, hypertension, diabetes mellitus, hyperlipidemia, positive family history, current smoking and high BMI to be associated with serum level of Gremlin-1. Conclusion: Based on the results, Gremlin-1 serum concentration may be a suitable biomarker for predication coronary artery calcification severity. However, more researches on larger population are necessary for its validation.

Bone Morphogenetic Protein Antagonist Gremlin-1 Increases Myofibroblast Transition in Dermal Fibroblasts: Implications for Systemic Sclerosis

ObjectiveSystemic Sclerosis is an autoimmune connective tissue disease which results in fibrosis of the skin and lungs. The disease is characterized by activation of myofibroblasts but what governs this is unknown. Gremlin-1 is a BMP antagonist that is developmentally regulated and we sought to investigate its role in Systemic Sclerosis.MethodsDermal fibroblasts were transfected with Grem1pcDNA3.1 expression vectors or empty vectors. Various markers of myofibroblasts were measured at the mRNA and protein levels. Scratch wound assays were also performed. Media Transfer experiments were performed to evaluate cytokine like effects. Various inhibitors of TGF-β signaling and MAPK signaling were used post-transfection. siRNA to Gremlin-1 in SSc dermal fibroblasts were performed to evaluate the role of Gremlin-1. Different cytokines were incubated with fibroblasts and Gremlin-1 measured. Bleomycin was used as model of fibrosis and immunohistochemistry performed.ResultsOverexpression of Gremlin-1 was achieved in primary dermal fibroblasts and lead to activation of quiescent cells to myofibroblasts indicated by collagen and α-Smooth muscle actin. Overexpression also led to functional effects. This was associated with increased TGF-β1 levels and SBE luciferase activity but not increased Thrombospondin-1 expression. Inhibition of Gremlin-1 overexpression cells with antibodies to TGF-β1 but not isotype controls led to reduced collagen and various TGF-β pathway chemical inhibitors also led to reduced collagen levels. In SSc cells siRNA mediated reduction of Gremlin-1 reduced collagen expression and CTGF gene and protein levels in these cells. IL-13 did not lead to elevated Gremlin-1 expression nor did IL-11. Gremlin-1 was elevated in an animal model of fibrosis compared to NaCl-treated mice.ConclusionGremlin-1 is a key regulator of myofibroblast transition leading to enhanced ECM deposition. Strategies that block Gremlin-1 maybe a possible therapeutic target in fibrotic diseases such as SSc.