Contrast-induced nephropathy (CIN) is a major complication of imaging in patients with chronic kidney disease (CKD). The publication of an academic RCT (n=83) reporting oral (N)-acetylcysteine (NAC) to reduce CIN led to >70 clinical trials, 23 systematic reviews, and two large RCTs showing no benefit. However, no mechanistic studies were conducted to determine how NAC might work; proposed mechanisms included renal artery vasodilatation and antioxidant boosting. We evaluated the proposed mechanisms of NAC action in participants with healthy and diseased kidneys. Four sub-studies were performed. Two randomised, double-blind, placebo-controlled, three-period cross-over studies (n=8) assessed the effect of oral and intravenous (IV) NAC in healthy kidneys in the presence/absence of iso-osmolar contrast (iodixanol). A third cross-over study in CKD3 patients (n=8) assessed the effect of oral and IV NAC without contrast. A three-arm randomised, double-blind, placebo-controlled parallel-group study, recruiting CKD3 patients (n=66) undergoing coronary-angiography, assessed the effect of oral and IV NAC in the presence of contrast. We recorded systemic (blood pressure, heart-rate) and renal (renal blood flow [RBF], glomerular filtration rate [GFR]) haemodynamics, and antioxidant status, plus biomarkers of renal injury in CKD3 patients undergoing angiography. Primary outcome for all studies was RBF over 8h after start of IV NAC/placebo. NAC at doses used in previous trials of renal prophylaxis was essentially undetectable in plasma after oral administration. In healthy volunteers, IV NAC, but not oral NAC, increased blood pressure (mean area-under-the-curve [AUC] arterial pressure [MAP]: mean difference 29 h.mmHg, p=0.019 vs. placebo), heart-rate (28 h.bpm, p<0.001), and RBF (714h.mL/min, 8.0% increase, p=0.006). Renal vasodilatation also occurred in the presence of contrast (RBF 917h.mL/min, 12% increase, p=0.005). In CKD3 patients without contrast, only a rise in heart-rate (34h.bpm, p=0.010) and RBF (288 h.mL/min, 6.0% increase, p=0.001) occurred with IV NAC, with no significant effect on blood pressure (MAP rise 26 h.mmHg, p=0.156). Oral NAC showed no effect. In CKD3 patients receiving contrast, IV NAC increased blood pressure (MAP rise 52 h.mmHg, p=0.008) but had no effect on RBF (151 h.mL/min, 3.0% increase, p=0.470), GFR (29h.mL/min/1.73m², p=0.122), or markers of renal injury. Neither IV nor oral NAC affected plasma antioxidant status. We found oral NAC to be poorly absorbed and have no reno-protective effects. Intravenous, not oral, NAC caused renal artery vasodilatation in healthy volunteers but offered no protection to CKD3 patients at risk of CIN. These findings emphasise the importance of mechanistic clinical studies before progressing to RCTs for novel interventions. Thousands were recruited to academic clinical trials without the necessary mechanistic studies being performed to confirm the approach had any chance of working.