is known to be a key activator of NOX2-containing NAD(P)H oxidase in phagocytes (Maehara et al., 2009
; Noack et al., 1999
). However, the direct role of p67phox
in the development of salt-sensitive hypertension and renal injury has not been explored previously. The results of the present study using p67phox
null mutant rats provide strong evidence of an important role for this gene in salt-sensitive hypertension. Even in the face of very large increases of salt intake, suppression of p67phox
produced significant reductions of hypertension, oxidative stress, and renal injury.
p67phox was cloned and identified in the renal OM. Several genetic variances between SS and SS.13BN26 rats were found in the promoter region and contributed to higher promoter activity, perhaps explaining the higher p67phox mRNA expression in the OM of SS rats. It will now be important to identify the upstream transcription factors that differentially regulate p67phox expression through the genetic variances. The SS allele of the p67phox promoter contains a 204 bp deletion compared to the SS.13BN26 allele, yet the SS allele yielded higher promoter activity. This result suggests that there might be some transcriptional repressors binding to the deleted region. It will now be important to identify potential transcriptional factors that bind to this region and to validate the binding with Chromatin immunoprecipitation (ChIP) experiments.
In our current study, we also observed that p67phox
expression – not the expression of p22phox
or Rac1 – in the outer medullary region is associated with higher NAD(P)H oxidase activity and greater salt-sensitivity in SS rats. There is immunohistochemical evidence from kidneys of SHR rats suggesting that p67phox
is expressed in the thick ascending limb, macula densa, distal convoluted tubule, cortical collecting duct, and perhaps the outer and inner medullary collecting ducts (Chabrashvili et al., 2002
). We have confirmed these findings by immunostaining a single Sprague Dawley kidney. Additionally, we found no staining in the vasa recta of the outer medulla. Among different tubules in the outer medulla, the mTAL has been previously shown to produce greater amounts of O2.−
than the thin descending limb and outer medullary collecting duct (Li et al., 2002
). Moreover, increased luminal flow rate and/or [Na+
] can stimulate the generation of O2.−
in mTAL (Abe et al., 2006
). Increased levels of O2.−
can enhance Na+
entry via Na+
co-transport and therefore increase Na+
reabsorption (Juncos and Garvin, 2005
). It remains to be determined whether the mTAL of the SS rats exhibits higher levels of p67phox
expression and NAD(P)H oxidase activity in response to a HS diet than the mTAL of congenic SS.13BN26
A growing body of evidence suggests that T cells play an important role in the later phase of the development of hypertension (Harrison et al., 2011
). In SS rats following three weeks of a HS diet, significant infiltration of T cells was observed in the kidney (De Miguel et al., 2011
). These T cells were found to express NAD(P)H oxidase subunits – including p22phox
, and p67phox
– indicating that T cells contain the molecular machinery needed for O2.−
production. Administration of an immunosuppressive agent was found to reduce the number of infiltrated T cells in the kidney, reduce oxidative stress in the urine, and attenuate salt-sensitive hypertension and renal injuries in SS rats. Therefore, up-regulation of p67phox
in T cells of SS rats could also contribute to differences in salt-sensitivity between the two strains. The relative contribution of the mTAL and infiltrated T cells to oxidative stress in the outer medulla remains to be determined.
Finally, despite the crucial role of renal p67phox in the development of salt-sensitive hypertension, the role of this gene should still be examined in vascular systems and the central nervous system. Although SNPs in human p67phox have not been found to be associated with hypertension in genome-wide association studies, the present study suggests that stratification of the populations based on salt-sensitivity may uncover an association. However, even if mutations in this gene do not serve as a marker or predictor of salt sensitivity in human populations, our studies suggest that inhibitors targeting p67phox for the treatment of salt-sensitive hypertension should be explored.