Mechanisms and targets of glomerular damage (2012)

&lt;b&gt;Introduction and Aims&lt;/b&gt;: Connective tissue growth factor (CTGF) has suggested as a risk marker for diabetic nephropathy and chronic kidney diseases (CKD) but its receptor and signalling are not defined. In the kidney, CTGF induces the recruitment of inflammatory cells via upregulation of Th1/Th2 cytokines and chemokines. In experimental models of renal damage, inhibition of endogenous CTGF, ameliorate disease progression. Some authors have suggested that CTGF could be an antifibrotic therapeutic target, but is important first to understand the renal effect and its molecular effector mechanisms. Epidermal growth factor receptor (EGFR) transactivation has been involved in CKD, but whether CTGF activates EGFR pathway is unknown. Our aim was to demonstrate that CTGF directly binds to EGFR and activates downstream mechanisms and cellular responses, showing that CTGF is a novel EGFR ligand &lt;b&gt;Methods&lt;/b&gt;: In Vivo studies were performed in adult male C57Bl/6 mice (9–12 weeks old, 20g). Systemic administration of CTGF was done with a single intraperitoneal injection of CTGF at the dose of 2.5 ng/g of body weight. To block EGFR activation, animals were treated with the EGFR kinase inhibitor Erlotinib (40mg/g of body weight) or its vehicle (control group) 24 hours before CTGF injection. In vitro experiments were done in human and mouse renal tubuloepithelial cells. Western blot, Inmunohistochemistry Chemical Cross-linking and coimmunoprecipitation assays, RT-PCR and inmunoassays (ELISA) were used for these experiments. &lt;b&gt;Results&lt;/b&gt;: In vivo, CTGF caused EGFR phosphorylation in the kidney mainly located in tubuloepithelial cells. Treatment with the EGFR kinase inhibitor Erlotinib inhibited CTGF-induced renal changes observed at 24 hours, including ERK activation (a downstream EGFR signalling), up-regulation of proinflammatory factors and interstitial inflammatory cell infiltration. In cultured tubular epithelial cells, CTGF led to CTGF-EGFR heterodimers formation, found by coimmunoprecipitation studies, and increase EGFR phosphorylation, suggesting that CTGF binds to EGFR and activates its signalling pathway. Matrix metalloproteases (MMPs) are not involved in CTGF-induced EGFR phosphorylation, as demonstrated by MMPs inhibitors (GM6001 and TAPI-2) and ADAM-17 gene silencing, by a siRNA approach. Some evidence suggest that Integrins could be CTGF receptors. CTGF-induced EGFR phosphorylation is regulated by integrins, as shown by incubation with RGD peptides, neutralizing antibody and RNA silencer against αV and β3 integrin. However, integrings are not involved in CTGF- EGFR complex formation. In tubular epithelial cells, CTGF induced ERK activation and overexpression of proinflammatory, that were inhibited by EGFR siRNA and EGFR kinase inhibitors. &lt;b&gt;CONCLUSIONS&lt;/b&gt;: Our results suggest that CTGF binds to EGFR and activates its signalling pathway leading to modulation of downstream mechanisms, such as ERK activation, and cellular responses, including renal inflammatory cell infiltration, and identify EGFR as a new therapeutic strategy for preventing progression of CKD.