In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1α1 (Col1A1), platelet-derived growth factor receptor-β (PDGFR-β), and α-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.