Redox control of medullary function and blood pressure

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The overall goal of this project is to advance our understanding of the role that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play in determining renal function, especially that of the renal medulla, a region that is vulnerable to excess O2- production. We have previously established that changes in renal medullary blood flow play an important role in sodium homeostasis and the long-term regulation of arterial pressure and shown that nitric oxide (NO) production plays a key role in the regulation of blood flow to this region. We hypothesize that excess production of ROS in the renal medulla reduces NO bioavailability, lowers medullary blood flow, increases tubular sodium reabsorption and results in a salt-sensitive form of hypertension. This project, as all others in this PPG, utilizes Dahl salt-sensitive rats that are genetically defined (SS/Mcw) and a newly developed consomic control strain derived from the inbred SS/Mcw strain in which chromosome 13 from the salt-insensitive BN/Mcw strain has been introgressed into the genomic background of the SS/Mcw rat (SS.BN13 consomic strain). This consomic SS.BN13 strain is 98% identical to the SS/Mcw strain but is relatively salt-insensitive. Aim 1 will determine if the renal medulla of Dahl salt-sensitive rats (SS/Mcw) produces excess ROS (O2, H2O2, ONOO) that feeds back to uncouple nitric oxide syntheses enzymes (NOS) and reduce the redo ratio of tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) (i.e., a vicious cycle of O2. production). Newly developed fluorescent microdialysis and HPLC analytical techniques will enable comparison of ROS and NO pathways in salt-sensitive (SS/Mcw) and in salt-insensitive consomic SS.BN13 rats. Our preliminary data indicate that this control strain exhibits significantly lower levels of medullary ROS. Studies will also determine whether lowering of ROS in the medulla of SS/Mcw rats by chronic medullary infusion of antioxidants increases medullary blood flow and reduces salt-induced hypertension. Aim 2 will determine the effects of induced medullary elevations of ROS in salt-insensitive SS.BN13 consomic rats. Medullary blood flow, renal interstitial hydrostatic pressure (RIHP), arterial pressure (AP) and the pressure-natriuresis relationships will be determined in response to chronic elevations of O2-, H2O2, and ONOO-. The concept of NOS uncoupling will be examined by measurement of medullary tissue BH4/BH2 ratios determined by HPLC, and NO, O2 and H2O2 values obtained by microdialysis techniques. Aim 3 will determine the dynamic inter-relationships of O2 and NO within and between vascular and tubular segments of the renal medulla using thin, superfused medullary tissue stripes. NO responses to agents that stimulate and inhibit ROS will be compared in control SS.BN13 and salt-sensitive SS/Mcw rats. Novel fluorescent dyes, coupled with high speed capture of microscopic images will enable the measurement of intracellular NO independent from NO in the interstitial space thereby determining whether NO released from tubules could act as a paracrine substance to control medullary blood flow. Taken together, these studies will provide novel and clinically relevant data defining the role that ROS play in the regulation of renal medullary function and the development of salt-sensitive hypertension, information that would be expected to lead to new therapeutic modalities in the treatment of human hypertension.