Fibrotic processes in chronic kidney diseases are the leading cause of renal failure. Hepatocyte growth factor (HGF), effecting organ restructuring by its mitogenic, motogenic, morphogenic and anti-apoptotic activities, is one of the central mediators involved in tubular repair and regeneration after acute renal injury. In addition, HGF acts as an anti-inflammatory and anti-fibrotic factor antagonizing pro-fibrotic actions of transforming growth factor beta (TGF-beta). However, the molecular and cellular mechanisms underlying the anti-fibrotic function of HGF in chronic kidney disease are not well understood. Therefore, in the present study HGF signaling and HGF induced expression profiles were studied in renal interstitial fibroblasts that represent a central cell type in tubulointerstitial fibrosis due to their prominent production of extracellular matrix proteins. Furthermore, gene therapeutical HGF application using different serotypes of the adeno-associated viral vector (AAV), namely AAV2, AAV8 and AAV9, was tested in order to treat tubulointerstitial fibrosis in a COL4A3 knockout mouse model. Analyses of HGF signaling demonstrated that in agreement to signaling in epithelial cells HGF stimulation results in the activation of the Erk1/2 and the Akt pathway. However, the Stat3 signal transducer was not phosphorylated. Smad2/3 phosphorylation in response to Erk1/2 activation in HGF stimulated fibroblasts supports previous data showing the antagonistic interaction of the HGF- and TGF-beta-signaling. A comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridisation could further define the anti-fibrotic signals mediated by HGF. Functional cluster analyses and quantitative PCR assays indicated that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix roteins, various chemokines, and members of the CCN family. Interruption of the HGF signaling via the Akt pathway or support of the HGF signaling via the Erk1/2 pathway by RNA interference, using Akt-siRNA or Smad4-siRNA, proved that not only Erk1/2 activation but also Akt activation is responsible for anti-fibrotic signal transduction by HGF. These data clearly point out that the Akt signaling upon HGF stimulation acts as an auxiliary pathway in the anti-fibrotic function of HGF. In order to apply the anti-fibrotic effect of HGF to chronic kidney diseases, a gene therapeutical system was established, intended to reduce renal interstitial fibrosis by the use of HGF as transgene and the adeno-associated viral vector (AAV) as gene vehicle. COL4A3 knockout mice mimicking the human Alport syndrome served as model system for renal tubulointerstitial fibrosis. Different natural occurring AAV serotypes, namely AAV2, AAV8 and AAV9, were studied with regard to their capability to target renal epithelial cells compared to liver parenchyma. Furthermore, a mammalian promoter construct was generated that restricted transgene expression to the kidney and the liver for a combined endocrine and paracrine expression of HGF. Systemic application of AAV8 and AAV9 carrying HGF as transgene resulted in high serum levels of HGF in COL4A3 knockout mice, however, AAV9 achieved the highest HGF expression in both the liver and the kidney. HGF serum levels were associated with pronounced repression of fibrotic markers such as collagen1A1, PDGF-receptor-beta, and alpha-smooth-muscle actin. In addition, AAV mediated HGF expression resulted a remarkable reduction in the severity of fibrosis. In conclusion, HGF is a promising anti-fibrotic agent for the treatment of chronic kidney diseases. Additionally, this study established a proof-of-concept of AAV-based therapy as a promising vector platform to treat chronic kidney diseases
