Gremlin: A novel mediator of epithelial mesenchymal transition and fibrosis in chronic allograft nephropathy
ABSTRACT Chronic allograft nephropathy (CAN) is the most frequent cause of chronic dysfunction and late loss of renal allografts. Epithelial mesenchymal transition (EMT) has been identified as responsible for the presence of activated interstitial fibroblasts (myofibroblasts) and transforming growth factor beta (TGF-beta)/Smad is the key signaling mediator. It has been proposed that the bone morphogenetic protein 7 (BMP-7) antagonist, Gremlin, could participate in EMT, as a downstream mediator of TGF-beta.
We evaluated 33 renal allograft biopsies, 16 of which showed CAN, versus 17 controls. By in situ hybridization we studied the expression of TGF-beta and Gremlin mRNA. Gremlin, BMP-7, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) proteins were evaluated by immunohistochemistry and Smad3 activation by Southwestern. In cultured human tubuloepithelial cells (HK2 cell line), Gremlin induction by TGF-beta was studied by confocal microscopy.
Among renal biopsies of transplanted patients with CAN, we detected up-regulation of TGF-beta in colocalization with Gremlin (RNA and protein), mainly in areas of tubulointerstitial fibrosis. In the same tubules, we observed decreased expression of E-cadherin and induction of vimentin and alpha-SMA. BMP-7 was significantly decreased in the CAN biopsies. In addition, HK2 stimulated with TGF-beta (1 ng/mL) induced Gremlin production at 72 hours.
We postulated that Gremlin is a downstream mediator of TGF-beta, suggesting a role for Gremlin in EMT observed in CAN.
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ABSTRACT: A growing number of patients are recognized worldwide to have chronic kidney disease. Glomerular and interstitial fibrosis are hallmarks of renal progression. However, fibrosis of the kidney remains an unresolved challenge, and its molecular mechanisms are still not fully understood. Gremlin is an embryogenic gene that has been shown to play a key role in nephrogenesis, and its expression is generally low in the normal adult kidney. However, gremlin expression is elevated in many human renal diseases, including diabetic nephropathy, pauci-immune glomerulonephritis and chronic allograft nephropathy. Several studies have proposed that gremlin may be involved in renal damage by acting as a downstream mediator of TGF-β. To examine the in vivo role of gremlin in kidney pathophysiology, we generated seven viable transgenic mouse lines expressing human gremlin (GREM1) specifically in renal proximal tubular epithelial cells under the control of an androgen-regulated promoter. These lines demonstrated 1.2- to 200-fold increased GREM1 expression. GREM1 transgenic mice presented a normal phenotype and were without proteinuria and renal function involvement. In response to the acute renal damage cause by folic acid nephrotoxicity, tubule-specific GREM1 transgenic mice developed increased proteinuria after 7 and 14 days compared with wild-type treated mice. At 14 days tubular lesions, such as dilatation, epithelium flattening and hyaline casts, with interstitial cell infiltration and mild fibrosis were significantly more prominent in transgenic mice than wild-type mice. Tubular GREM1 overexpression was correlated with the renal upregulation of profibrotic factors, such as TGF-β and αSMA, and with increased numbers of monocytes/macrophages and lymphocytes compared to wild-type mice. Taken together, our results suggest that GREM1-overexpressing mice have an increased susceptibility to renal damage, supporting the involvement of gremlin in renal damage progression. This transgenic mouse model could be used as a new tool for enhancing the knowledge of renal disease progression.PLoS ONE 07/2014; 9(7):e101879. DOI:10.1371/journal.pone.0101879 · 3.53 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
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ABSTRACT: http://pubs.acs.org/doi/abs/10.1021/ab500060r Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of chronic open angle glaucoma. Elevated levels of TGFβ3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have been reported. The influence of TGFβ3 on the biochemical composition and biomechanics of ECM of human trabecular meshwork (HTM) cells was investigated. HTM cells from eye bank donor eyes were isolated, plated on aminosilane functionalized glass substrates and cultured in the presence or absence of 1 ng/mL TGFβ3 for 4 weeks. After incubation, samples were decellularized and decellularization was verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). Imaged by AFM, the surface features of the ECM from both sets of samples had a similar roughness/topography (as determined by RMS values) suggesting surface features of the ECM were similar in both cases; however, the ECM from the HTM cells treated with TGFβ3 was between 3- and 5-fold stiffer than that produced by the control HTM cells. Proteins present in the ECM were solubilized and analyzed using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Data indicate that multiple proteins previously reported to be altered in glaucoma were changed in the ECM as a result of the presence of TGFβ3, including inhibitors of the BMP and Wnt signaling pathways. Gremlin1and 4, SERPINE1 and 2, periostin, secreted frizzled related protein (SFRP) 1 and 4, and ANGPTL4 were among those proteins that were overexpressed in the ECM after TGFβ3 treatment.01/2015; 1(2). DOI:10.1021/ab500060r
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ABSTRACT: Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor- β (TGF- β ). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF- β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF- β , by a neutralizing antibody against active TGF- β , did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF- β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF- β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF- β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF- β .BioMed Research International 05/2014; 2014. DOI:10.1155/2014/802841 · 2.71 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.