No abstract

We previously characterized a H+ transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by NAD(P)H oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13BN) rats, and superoxide production was enhanced in low Na+ media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na+. We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H+ currents. In order to test this hypothesis, we developed HV1−/− null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1−/− rats (P<0.05), and more rapid recovery of intracellular pH in wild-type rats (ΔpHi 0.005U/sec vs. 0.002U/sec, p=0.046 respectively). Superoxide production was enhanced by low intracellular sodium (<10mM) in both thick limb and peritoneal macrophages only when HV1 was present. When fed a high salt diet, blood pressure, outer-medullary renal injury (tubular casts) and oxidative stress (4-Hydroxynonenal staining) were significantly reduced in HV1−/− rats compared to wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na+ is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.

BackgroundVascular congestion of the renal medulla—trapped red blood cells in the medullary microvasculature—is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined.MethodsIn this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion.ResultsWe found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla.ConclusionsBlockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla’s capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla’s susceptibility to ischemic injury.