It is recognized that oxidative stress plays a critical role in the development of salt-sensitive hypertension, especially in the kidney. The most abundant renal isoform is Nox4, but there is controversy regarding its distribution and function. There have been no studies of the role of Nox4 in a naturally occurring form of hypertension (Dahl salt-sensitive (SS) rat). We have therefore knocked out the Nox4 gene in the SS genetic background (SSNox4-/-) to determine the effect upon the development of salt-sensitive hypertension and renal kidney injury in the SS rat. The SSNox4-/- rat, developed using zinc finger nuclease (ZFN) techniques, exhibits an 8 base-pair frame-shift deletion within exon 7 resulting in a truncation of the Nox4 peptide by 195 amino acids with a molecular weight of 22 kD predicted (compared to the full length Nox4 peptide of 594 amino acids with a molecular weight of 68 kD). Western blot analysis confirmed the loss of the Nox4 band at ~65 kD in renal medullary tissue of SSNox4-/- rats vs. SS. The functional consequence of this deletion was substantial over a 21 day period following an increase in salt intake from 0.4 (LS) to 4.0% NaCl (HS). SSNox4-/- rats exhibited a change in MAP from 105±3 on LS to 130±2 mmHg (a change of 25 mmHg; n=10) on d21 of HS compared to a change from 107±2 mmHg in SS rats to 171±7 on d21 of HS (a change of 64 mmHg; n=10) as determined by telemetry. Albuminuria was virtually eliminated in SSNox4-/- rats who also exhibited a large reduction of tubular cast (2.4±0.3% positive strained region vs 12.6±1.3 in SS). Glomerular injury in both the cortex and medulla was significantly less in the SSNox4-/- than the SS. The % of glomeruli with an injury score of >2 (0-4 scale) was 43±1.5% (258 of 600 glomeruli) in the SSNox4-/- but 65±3.9% (390 of 600) in the SS. We conclude that Nox4 plays an important role in the development of this form of salt-sensitive hypertension and renal injury in SS rats.