The Role of Renal Medullary NAD(P)H Oxidase Subunit p67(phox) in the Development of Salt-sensitive Hypertension.

摘要

The first aim of this dissertation was to determine whether a strain difference exists between Dahl salt-sensitive (SS) and congenic SS.13BN26 in the p67phox sequence, mRNA and protein expression, and NADPH oxidase activity in the outer medulla. Rats used in these studies were fed on a 0.4% NaCl diet (low salt: LS) since weaning. At 6 weeks of age, a subgroup of these rats was switched to an 8% NaCl diet (high salt: HS). Outer medullary tissue was collected from rats on the LS diet and from rats on the HS diet 7 days after their diets were switched. The results indicated that the average mRNA levels of p67 phox were increased in both the SS rat and congenic SS.13BN26 after the diet was switched from LS to HS. The mRNA levels of p67phox were significantly higher in SS rats than in SS.13BN26 rats on both the LS diet and day 7 of the HS diet. The analysis of the promoter sequence revealed that SS rats had a 204 bp deletion and four single nucleotide polymorphisms (SNPs) compared to SS.13BN26 rats. The promoter activity of p67phox as assessed by luciferase fluorescence was significantly higher in the SS construct than in the SS.13BN26 construct. This result suggested that the genetic variances in the promoter region may contribute to higher mRNA expression of p67phox in the SS compared to SS.13 BN26 rats on both the LS and HS diet.;To pinpoint the source of elevated p67phox protein expression in the outer medulla, the second aim of this dissertation is to determine whether the medullary thick ascending limb (mTAL) of the SS rats exhibits higher levels of p67phox expression and NAD(P)H oxidase activity in response to a HS diet than that of congenic SS.13 BN26. Among different tubular segments, p67phox was previously identified to be expressed in the medullary thick ascending limb (mTAL), macula densa, and perhaps the outer medullary collecting ducts. The mTAL has been previously shown to produce greater amounts of O2 .- than the thin descending limb and outer medullary collecting duct. Moreover, increased luminal flow rate and/or [Na+] can stimulate the generation of O2.- in the mTAL. Increased levels of O2.- can enhance Na+ entry via the Na-K-2Cl cotransporter co-transporter and therefore increase Na+ reabsorption. We developed techniques to bulk dissect the mTAL with a purity of every preparation averaging between 90 to 95%. We then bulk dissected mTALs from SS and SS.13 BN26 strains on day 7 of the HS diet. We found that p67phox expression and NAD(P)H oxidase activity in the mTAL was significantly higher in SS rats compared to SS.13BN26.;In order to examine the functional relevance of p67 phox in salt-sensitive hypertension, the final aim of this dissertation sought to evaluate whether disruption of p67 phox in the SS rat would lead to a reduction of salt-sensitive hypertension and renal injury. We measured the mean arterial pressure (MAP) of the p67phox-/- rats and their WT littermates at 6 weeks of age when fed the LS and HS diet. Baseline MAP was not significantly different between WT and p67phox-/- rats. However, at the end of day 14 of the HS diet, the MAP of p67 phox-/- rats was 30 mmHg lower than their WT littermates. Moreover, NAD(P)H oxidase activity and total superoxide levels measured using outer medullary tissue homogenate on day 14 of the HS diet were also significantly reduced in p67phox-/- rats compared to WT littermates. (Abstract shortened by UMI.)