Abstract Body: High dietary sodium intake is a major risk factor for hypertension and cardiovascular disease. We previously reported that the kidney microvasculature of rats fed a high sodium (HS) diet displays impaired nitric oxide (NO) signaling with increased reactive oxygen species (ROS). The disruption of NO signaling was attributable to a plasma-derived factor from HS-fed rats. However, the mechanisms by which a HS diet increases kidney microvascular ROS are unclear. We hypothesized that a HS diet leads to kidney microvascular dysfunction via mitochondrial-derived ROS. In male Sprague Dawley rats fed a normal sodium (NS; 0.49% NaCl) or HS (4% NaCl) diet for 2 weeks, we assessed afferent arteriolar (AA) function using in vitro blood-perfused juxtamedullary nephron preparations. Baseline AA diameter was similar between NS (13.8±0.3 µm) and HS rats (13.4±0.4 µm; p>0.05). Kidneys were treated ex vivo with increasing concentrations of L-NAME to assess the function of the NO pathway. Superfusion of L-NAME (10^-6–10^-3M) elicited concentration-dependent AA vasoconstriction in NS-fed rats (max vasoconstriction: 67±3% of control diameter, n=5), whereas AA vasoconstriction response from HS-fed rats was blunted (max vasoconstriction: 95±3% of control, n=6, p<0.05). Addition of mitoTEMPO (100 μM, mitochondria-targeted ROS scavenger) in the blood perfusate did not change the baseline AA diameter in HS-fed rats (p>0.05). Interestingly, mitoTEMPO restored the AA response to L-NAME in HS-fed rats (max vasoconstriction: 73±4% of control, n=6) similar to the response in NS-fed rats (p>0.05). To determine whether the implicated mitochondrial ROS are stimulated by a plasma-derived factor, we perfused kidneys from NS-fed rats with reconstituted blood from HS-fed rats (NS-kidney/HS-blood). AA’s of the NS-kidney/HS-blood group exhibited an attenuated L-NAME-induced vasoconstriction response similar to that of HS-fed rats (max vasoconstriction: 90±5% of control diameter, n=6; p>0.05). Here, we revealed that administration of mitoTEMPO in the perfusate of HS-fed rat blood prevented the attenuated L-NAME-induced vasoconstriction in NS-fed kidneys to an extent similar to the response of NS-fed rats (max vasoconstriction: 66±4% of control, n=5; p>0.05). The AA vasoconstriction to KCl (55 mM) was similar in all groups (p>0.05). These studies indicate that high dietary sodium intake drives kidney microvascular dysfunction through a plasma-mediated mitochondrial ROS mechanism.