Many factors are involved in the pathogenesis of diabetic nephropathy. A single gene abnormality may be prerequisite but insufficient to the disease to manifest. It is therefore only when a second or sometimes a third damage is associated that the consequences of pathogenic phenotypes become evident. We generated the triple transgenic mice overexpressing megsin (a novel glomerular-specific serpin), a receptor for advanced glycation end products (RAGE), and inducible nitric oxide synthase (iNOS). Compared with the single- or two-gene transgenic mice, the triple transgenic mice developed, at an early age (16 weeks), severe albuminuria and renal damage with all of the characteristics of human diabetic nephropathy (i.e., glomerular hypertrophy, diffuse mesangial expansion, inflammatory cell infiltration, and interstitial fibrosis). Interestingly, 30-40% of glomeruli exhibit nodule-like lesions. Oxidative and carbonyl stress makers (pentosidine, N(epsilon)-carboxymethyllysine, and 8-hydroxy-deoxyguanosine) were significantly higher in the triple transgenic mice. The iNOS transgenic mice have a diabetes phenotype, the renal consequences of which are moot, and the superimposition of RAGE leads to more conspicuous manifestations. By additional overexpression of megsin, a gene known to be involved in mesangial proliferation and expansion, these local consequences become dramatically manifest and approximate those observed in human pathology. This multiple hit approach is of interest in consideration of the sequential events during development of diabetic nephropathy.