To determine whether previous renal injury accelerates the progression of glomerulosclerosis and interstitial fibrosis, we examined the effect of treating rats with angiotensin II after Habu venom injury. After initiating disease, we examined the origin of interstitial myofibroblasts by locating alpha-smooth muscle actin (alpha-SMA)-positive and Na+,K+-ATPase-positive cells relative to interstitial space, tubular epithelial cells, the tubular basement membrane (TBM), and vascular structures. Tubular epithelial-mesenchymal transition was also assessed by examining TBM integrity and by using Texas Red (TR)-dextran in intravital tracking experiments. The staining of alpha-SMA-positive myofibroblasts dramatically increased in peritubular interstitial spaces 48 hours after Habu venom plus angiotensin II, particularly in and around perivascular and periglomerular regions, while tubular epithelial cells were alpha-SMA-negative. Na+,K+-ATPase-positive and TR-dextran-labeled cells were restricted to the tubular epithelium and excluded from the interstitium. By 7 and 14 days, expanded interstitial space contained only alpha-SMA-positive myofibroblasts without TR-dextran endocytic particles. Epithelium of atrophic tubules containing TR-dextran remained confined by surrounding interstitium and myofibroblasts. These studies indicate that early expansion of alpha-SMA-positive cells in the interstitium and loss of tubular area occur via encroachment of interstitial myofibroblasts from perivascular into atrophic tubular spaces rather than via epithelial-mesenchymal transition and migration of tubular cells through the TBM into the interstitium.