Gremlin is a downstream profibrotic mediator of transforming growth factor-beta in cultured renal cells.

BACKGROUND/AIMS: Chronic kidney disease is characterized by accumulation of extracellular matrix in the tubulointerstitial area. Fibroblasts are the main matrix-producing cells. One source of activated fibroblasts is the epithelial mesenchymal transition (EMT). In cultured tubular epithelial cells, transforming growth factor-ß (TGF-ß1) induced Gremlin production associated with EMT phenotypic changes, and therefore Gremlin has been proposed as a downstream TGF-ß1 mediator. Gremlin is a developmental gene upregulated in chronic kidney diseases associated with matrix accumulation, but its direct role in the modulation of renal fibrosis and its relation with TGF-ß has not been investigated. METHODS: Murine renal fibroblasts and human tubular epithelial cells were studied. Renal fibrosis was determined by evaluation of key profibrotic factors, extracellular matrix proteins (ECM) and EMT markers by Western blot/confocal microscopy or real-time PCR. Endogenous Gremlin was targeted with small interfering RNA. RESULTS: In murine fibroblasts, stimulation with recombinant Gremlin upregulated profibrotic genes, such as TGF-ß1, and augmented the production of ECM proteins, including type I collagen. The blockade of endogenous Gremlin with small interfering RNA inhibited TGF-ß1-induced ECM upregulation. In tubular epithelial cells Gremlin also increased profibrotic genes and caused EMT changes: phenotypic modulation to myofibroblast-like morphology, loss of epithelial markers and in-duction of mesenchymal markers. Moreover, Gremlin gene silencing inhibited TGF-ß1-induced EMT changes. CONCLUSIONS: Gremlin directly activates profibrotic events in cul-tured renal fibroblasts and tubular epithelial cells. Moreover, endogenous Gremlin blockade inhibited TGF-ß-mediated matrix production and EMT, suggesting that Gremlin could be a novel therapeutic target for renal fibrosis.

CTGF promotes inflammatory cell infiltration of the renal interstitium by activating NF-kappaB.

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-kappaB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-kappaB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-gamma, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-kappaB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-kappaB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-kappaB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-kappaB pathway.

Angiotensin II activates the Smad pathway during epithelial mesenchymal transdifferentiation.

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor beta, TGFbeta) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-beta mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-beta upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-beta however, Smad-dependent transcription after 1 day was TGF-beta mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-beta-mediated processes; however, early changes were independent of endogenous TGF-beta. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-beta-independent processes, in vivo and in vitro, causing renal fibrosis.

TGF-beta signaling in vascular fibrosis.

Transforming growth factor-beta (TGF-beta) participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy and heart failure. TGF-beta exerts pleiotropic effects on cardiovascular cells, regulating cell growth, fibrosis and inflammation. TGF-beta has long been believed to be the most important extracellular matrix regulator. We review the complex mechanisms involved in TGF-beta-mediated vascular fibrosis that includes the Smad signaling pathway, activation of protein kinases and crosstalk between these pathways. TGF-beta blockade diminishes fibrosis in experimental models, however better antifibrotic targets are needed for an effective therapy in human fibrotic diseases. A good candidate is connective tissue growth factor (CTGF), a downstream mediator of TGF-beta-induced fibrosis. Among the different factors involved in vascular fibrosis, Angiotensin II (AngII) has special interest. AngII can activate the Smad pathway independent of TGF-beta and shares with TGF-beta many intracellular signals implicated in fibrosis. Blockers of AngII have demonstrated beneficial effects on many cardiovascular diseases and are now one of the best options to block TGF-beta fibrotic responses. A better knowledge of the intracellular signals of TGF-beta can provide novel therapeutic approaches for fibrotic diseases.

Gremlin activates the Smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells.

Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-ß (TGF-ß). 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-ß 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-ß, by a neutralizing antibody against active TGF-ß, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-ß independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-ß 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-ß 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-ß.

Inhibitory effect of interleukin-1beta on angiotensin II-induced connective tissue growth factor and type IV collagen production in cultured mesangial cells.

Connective tissue growth factor (CTGF) is overexpressed in kidney diseases associated with extracellular matrix accumulation. Angiotensin II (ANG II) participates in renal fibrosis by the upregulation of growth factors, including CTGF, and extracellular matrix proteins, such as type IV collagen. During renal injury, ANG II and the macrophage-produced cytokine interleukin-1beta (IL-1beta) may be present simultaneously in the glomerular environment. However, there are no studies about the interaction between ANG II and IL-1beta in renal fibrosis. For this reason, in cultured mesangial cells (MC), we investigated whether IL-1beta could regulate ANG II-mediated collagen accumulation and the mechanisms underlying this process. In MC, CTGF is a downstream mediator of type IV collagen production induced by ANG II. IL-1beta did not increase the production of CTGF and type IV collagen but significantly inhibited ANG II-induced CTGF and type IV collagen overexpression. Moreover, IL-1beta also inhibited type IV collagen upregulation caused by exogenous recombinant CTGF. Matrix metalloproteinase-9 (MMP-9) is the main enzyme involved in type IV collagen degradation. In MC, coincubation of IL-1beta and ANG II caused a synergistic increase in MMP-9 gene expression and activity, associated with type IV collagen inhibition. The described IL-1beta effects were dependent on activation of ERK/MAPK but independent p38-MAPK, JNK, phosphatidylinositol 3-kinase/Akt, and Rho-associated kinase pathways. In summary, these data indicate that IL-1beta inhibited ANG II-mediated type IV collagen production, via CTGF downregulation, and increased type IV collagen degradation, through MMP-9 upregulation. Our in vitro data show that the proinflammatory cytokine IL-1beta abrogates ANG II-induced CTGF production, describing antagonistic activities of proinflammatory cytokines on ANG II actions.

Expression of gremlin, a bone morphogenetic protein antagonist, in glomerular crescents of pauci-immune glomerulonephritis.

BACKGROUND: Recent evidence in vitro and in vivo suggests that gremlin, a bone morphogenetic protein antagonist, is participating in tubular epithelial mesenchymal transition (EMT) in diabetic nephropathy as a downstream mediator of TGF-beta. Since EMT also occurs in parietal epithelial glomerular cells (PECs) leading to crescent formation, we hypothesized that gremlin could participate in this process. With this aim we studied its expression in 30 renal biopsies of patients with pauci-immune crescentic nephritis. METHODS: Gremlin was detected by in situ hybridization (ISH) and immunohistochemistry (IMH) and TGF-beta by ISH and Smads by southwestern histochemistry (SWH). Phosphorylated Smad2, CTGF, BMP-7, PCNA, alpha-SMA, synaptopodin, CD-68, and phenotypic markers of PECs (cytokeratin, E-cadherin), were detected by IMH. In cultured human monocytes, gremlin and CTGF induction by TGF-beta was studied by western blot. RESULTS: We observed strong expression of gremlin mRNA and protein in cellular and fibrocellular crescents corresponding to proliferating PECs and monocytes, in co-localization with TGF-beta. A marked over-expression of gremlin was also observed in tubular and infiltrating interstitial cells, correlating with tubulointerstitial fibrosis (r=0.59; P<0.01). A nuclear Smad activation in the same tubular cells, that are expressing TGF-beta and gremlin, was detected. In human cultured monocytes, TGF-beta induced gremlin production while CTGF expression was not detected. CONCLUSION: We postulate that gremlin may play a role in the fibrous process in crescentic nephritis, both in glomerular crescentic and tubular epithelial cells. The co-localization of gremlin and TGF-beta expression found in glomeruli and tubular cells suggest that gremlin may be important in mediating some of the pathological effects of TGF-beta.

HMG-CoA reductase inhibitors decrease angiotensin II-induced vascular fibrosis: role of RhoA/ROCK and MAPK pathways.

3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) present beneficial effects in cardiovascular diseases. Angiotensin II (Ang II) contributes to cardiovascular damage through the production of profibrotic factors, such as connective tissue growth factor (CTGF). Our aim was to investigate whether HMG-CoA reductase inhibitors could modulate Ang II responses, evaluating CTGF expression and the mechanisms underlying this process. In cultured vascular smooth muscle cells (VSMCs) atorvastatin and simvastatin inhibited Ang II-induced CTGF production. The inhibitory effect of statins on CTGF upregulation was reversed by mevalonate and geranylgeranylpyrophosphate, suggesting that RhoA inhibition could be involved in this process. In VSMCs, statins inhibited Ang II-induced Rho membrane localization and activation. In these cells Ang II regulated CTGF via RhoA/Rho kinase activation, as shown by inhibition of Rho with C3 exoenzyme, RhoA dominant-negative overexpression, and Rho kinase inhibition. Furthermore, activation of p38MAPK and JNK, and redox process were also involved in Ang II-mediated CTGF upregulation, and were downregulated by statins. In rats infused with Ang II (100 ng/kg per minute) for 2 weeks, treatment with atorvastatin (5 mg/kg per day) diminished aortic CTGF and Rho activation without blood pressure modification. Rho kinase inhibition decreased CTGF upregulation in rat aorta, mimicking statin effect. CTGF is a vascular fibrosis mediator. Statins diminished extracellular matrix (ECM) overexpression caused by Ang II in vivo and in vitro. In summary, HMG-CoA reductase inhibitors inhibit several intracellular signaling systems activated by Ang II (RhoA/Rho kinase and MAPK pathways and redox process) involved in the regulation of CTGF. Our results may explain, at least in part, some beneficial effects of statins in cardiovascular diseases.

Renal and vascular hypertension-induced inflammation: role of angiotensin II.

PURPOSE OF REVIEW: We will focus on the recent findings concerning the inflammatory response in vascular and renal tissues caused by hypertension. RECENT FINDINGS: Angiotensin II is one of the main factors involved in hypertension-induced tissue damage. This peptide regulates the inflammatory process. Angiotensin II activates circulating cells, and participates in their adhesion to the activated endothelium and subsequent transmigration through the synthesis of adhesion molecules, chemokines and cytokines. Among the intracellular signals involved in angiotensin II-induced inflammation, the production of reactive oxygen species and the activation of nuclear factor-kappaB are the best known. SUMMARY: The pharmacological blockade of angiotensin II actions, by angiotensin-converting enzyme inhibitors or angiotensin receptor antagonists, results in beneficial organ protective effects, in addition to the effects of these agents on blood pressure control, that can be explained by the blockade of the angiotensin II-induced pro-inflammatory response. These data provide a rationale for the use of blockers of the renin-angiotensin system to prevent vascular and renal inflammation in patients with hypertension.