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CARDIOVASCULAR

Early and Sustained Inhibition of Nuclear Factor-B Prevents Hypertension in Spontaneously Hypertensive Rats

The Renal Service, Hospital Universitario, Universidad del Zulia, Instituto de Investigaciones Biomédicas, Maracaibo, Venezuela (B.R.-I., A.F., V.V., Y.Q.); Department of Nephrology, Universidad Austral, Valdivia, Chile (S.M.); and Division of Nephrology and Hypertension, University of California, Irvine, California (N.D.V.)

Received April 15, 2005; accepted June 7, 2005.

	Abstract

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Compelling evidence has emerged pointing to the interaction of oxidative stress and renal interstitial inflammation and their mutual contribution to the pathogenesis of hypertension in experimental animals. Renal interstitial inflammation in spontaneously hypertensive rats (SHR) is accompanied by and largely due to activation of redox-sensitive, proinflammatory nuclear transcription factor-B (NF-B). Therefore, the present study was designed to test the hypothesis that long-term inhibition of NF-B, beginning early in the course of the disease, may attenuate renal interstitial inflammation and hypertension in SHR. To this end, we administered the reputed NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) (100 mg/kg daily intraperitoneally) to SHR from 7 to 25 weeks of age and compared the results with vehicle-treated SHR. Vehicle-treated and PDTC-treated Wistar Kyoto (WKY) rats served as controls. The untreated SHR exhibited a significant rise in arterial pressure; increased NF-B activation, elevated intercellular adhesion molecule (ICAM)-1 and in situ mRNA macrophage chemoattractant molecule-1 (MCP-1) expressions; and interstitial accumulation of lymphocytes, macrophages, and angiotensin-II-positive cells. PDTC administration prevented the rise in blood pressure, and normalized renal cortical NF-B activity as well as ICAM-1 and MCP-1 expressions. This was accompanied by a significant reduction in infiltration of immune cells, angiotensin II-expressing cells, and renal tissue malondialdehyde content to values that matched those found in the control WKY rats. Results suggest that NF-B-driven intrarenal inflammatory reactivity play a major role in the pathogenesis of hypertension in the SHR.

Because renal interstitial inflammation and oxidative stress promote sodium retention (Kitiyara et al., 2003; Rodríguez-Iturbe et al., 2004b) and could be either the cause or the consequence of hypertension (Wilcox and Welch, 2001), we recently investigated the evolution of interstitial inflammation and its relationship with the development of hypertension in the SHR. We found that activation of proinflammatory transcription factor NF-B and immune cell infiltration in the kidney of SHR occurred as early as 3 weeks of age, at which time blood pressure was still normal (Rodríguez-Iturbe et al., 2004a).

Because these events preceded the onset of hypertension, in the present study we investigated whether sustained suppression of NF-B activation (and thereby renal tubulointerstitial inflammation) beginning early in the course of the disease would prevent the development of hypertension in the SHR. For this purpose, we used pyrrolidine dithiocarbamate (PDTC), which is a known inhibitor of the activation of NF-B (Schreck et al., 1992; Liu et al., 1999).

Other investigators have previously used PDTC in hypertensive strains of rats. Müller et al. (2000) found that PDTC treatment improved end-organ damage in the double transgenic rats (dTGR) harboring human renin and angiotensin genes, and Hong et al. (1998) obtained a partial improvement in hypertension administering PDTC in the drinking water to SHR in studies designed to suppress inducible nitric-oxide synthase.

Because NF-B-induced intrarenal inflammation seems to be involved in the genesis and maintenance of hypertension in the SHR, we hypothesized that long-term administration of PDTC, in sufficient doses to suppress activation of this proinflammatory transcription factor, might prevent interstitial immune infiltration and hypertension in SHR. The results of this investigation support our hypothesis and demonstrate for the first time that early and sustained suppression of NF-B activation completely abrogates hypertension in the SHR

	Materials and Methods

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Animals and Experimental Groups. Studies were done in male SHR and control Wistar Kyoto (WKY) rats (Instituto Venezolano de Investigaciones Científicas, Los Teques, Venezuela) housed in institutional animal facilities with free access to standard (Purina) rat feed and water. The Animal Care Committee of the Centro de Medicina y Cirugía Experimental, Universidad del Zulia, approved the study.

Preliminary studies demonstrated that normalization of NF-B activation (reduction of p65 NF-B renal content to levels similar to those in WKY rats) could be achieved with the daily administration of 100 mg of PDTC per kilogram of body weight intraperitoneally. Lesser doses given parenterally and the administration of this compound in the drinking water were less reliably associated with a reduction of p65 NF-B renal content to normal levels. The appropriate interval of time for the administration of PDTC by the intraperitoneal route was determined in a separate group of SHR that were sacrificed 3, 12, and 24 h after the intraperitoneal administration of 100 mg/kg PDTC. The lowest p65 NF-B contents in renal tissue were consistently obtained 3 h after the PDTC dose, returning to baseline after 24 h. Therefore, the PDTC was administered daily.

Five SHR and five WKY rats, 7 weeks of age, were sacrificed and kidneys were harvested for immunohistochemical studies and determination of NF-B (see below). The rest of the SHR were randomly assigned to receive PDTC or vehicle from 7 weeks to 25 weeks of age, and the following experimental groups were studied. 1) The SHR.PDTC group (n = 15) consisted of rats that received PDTC (Sigma-Aldrich, St. Louis, MO) administered intraperitoneally at a dose of 100 mg/kg daily, diluted in a total volume of 0.5 ml of saline. 2) The SHR group (n = 15) received 0.5 ml of saline intraperitoneally daily. 3) The WKY.PDTC group (n = 10) received PDTC in similar doses and by a similar route as the SHR.PDTC group and were used to rule out potential blood pressure-lowering effects of the PDTC. 4) The WKY group (n = 15). Received no treatment and were used as controls.

Five SHR and WKY rats of each experimental group were sacrificed after 5 weeks of treatment (12 weeks old), and the rest of the rats were sacrificed at the end of the experiment, after 18 weeks of treatment (25 weeks old).

Experimental Protocol. Blood pressure was determined in 7-week-old SHR and Wistar Kyoto rats before sacrifice by direct intraaortic monitoring as described previously (Rodríguez-Iturbe et al., 2004a) and in older rats by tail-cuff plethysmography (Quiroz et al., 2001; Rodríguez-Iturbe et al., 2001; Alvarez et al., 2002). Blood pressure and body weight were determined every 2 weeks. Serum creatinine (AutoAnalyzer technology) and 24-h urinary protein excretion (sulfosalicylic acid methodology) were evaluated at 7 (baseline), 12, and 25 weeks of age. Histology and immunohistology and renal cortical content of p65 NF-B were evaluated in kidneys harvested in rats euthanized at 7, 12, and 25 weeks of age. Adhesion molecules, MCP-1 mRNA expression in the kidney (in situ hybridization), and malondialdehyde (MDA) renal content were studied in kidneys harvested at the end of the experiment.

At the time of sacrifice the rats were euthanized under general anesthesia (diazepam /ketamine) and after perfusion with cold (4°C) saline solution the kidneys were harvested. One kidney was used for determination of NF-B or MDA, and the contralateral kidney was divided in two parts: one part was snap frozen and kept at –70°C, and the other part was fixed methyl Carnoy and embedded in Paraffin for immunohistochemical studies.

NF-B. Kidneys harvested from SHR and WKY rats 7 weeks of age (n = 5 each) and kidneys harvested from each experimental group after 5 weeks (n = 5 each) and after 18 weeks (SHR group, n = 4; SHR.PDTC group, n = 5; WKY group, n = 5) were used for NF-B determination. Renal cortex was immediately separated, minced, weighed, and collected in a prechilled container as detailed previously (Rodríguez-Iturbe et al., 2004a). Nuclear extracts were obtained using commercially available nuclear extraction kits (Active Motif, Carlsbad, CA) following the manufacturer's recommendations. Determination of the p65 DNA binding subunit of NF-B was done by an enzyme-linked immunosorbent assay-based assay (Renard et al., 2001) with commercially available kits (TransAm NF-B p65; Active Motif). The evaluation of the renal p65 NF-B content was done in kidneys of rats sacrificed 3 h after the administration of the PDTC compound, for reasons indicated above. As described previously (Rodríguez-Iturbe et al., 2004a), lower detection limits were 3 ng/ml extract. Results are expressed in nanograms of p65 NF-B subunit per gram of tissue.

In Situ Hybridization for MCP-1. In situ hybridization was performed in 5-µm sections of five kidneys from each experimental group harvested at the end of the experiment. Tissues fixed in methyl Carnoy and embedded in Paraffin that were mounted on 3-aminopropyltriethoxysilane (Sigma Chemical Co.)-coated slides treated with diethylpyrocarbonate (DEPC; Sigma Chemical Co.). Sections were dewaxed with xylene and rehydrated through a series of decreasing ethanol solutions and then treated with 2x SSC at 60°C for 10 min and washed with DEPC-treated water. Tissue sections were then digested with proteinase K (5 µg/ml in 0.05 M Tris, pH 7.6) for 60 min at 37°C. After washing with DEPC-treated water, sections were incubated with a prehybridization solution (Dako mRNA in situ hybridization solution; DakoCytomation California Inc., Carpinteria, CA) for 60 min at 37°C. The hybridization reaction was performed overnight at 60°C with 100 µl of digoxigenin-labeled antisense probe for rat MCP-1 (200 ng/ml) (Molecular Research Reagents, Inc., Tetralink International, licensed by Roche Diagnostics, Mannheim, Germany) in a humidified chamber. Then, slides were washed twice with 4x SSC 30% formamide, 2x SSC 30% formamide, and 0.2x SSC 30% formamide at 37°C for 5 min and then with 1x Tris-buffered saline containing 2% Tween 20 at room temperature for 15 min. After blocking the reaction for 15 min (blocking solution 100 mM Tris-HCl, 150 mM NaCl, 2% bovine serum albumin, and 0.3% Triton X-100), the slides were incubated overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin F(ab)₂ antibody (Roche Diagnostics) (dilution 1:100). Detection was done with alkaline phosphatase conjugate (DakoCytomation California Inc.) for 30 min at room temperature, washed for 5 min with 1x Tris-buffered saline, and using nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate as the enzyme substrate for 120 min at 37°C in the dark. Tissues were then dehydrated in an ethanol series and mounted in Permount (FSP15-100; Fisher Scientific Co., Pittsburgh, PA). The specificity of the reaction was confirmed by running the test without a probe and with a negative control (plasmid DNA).

Histology and Immunohistology. Paraffin-embedded kidney biopsies were stained with periodic acid-Schiff, hematoxylin and eosins, and trichromic stains for light microscopy studies. Severity of glomerulosclerosis was evaluated using an index score originally proposed by Raij et al. (1984) and used by us in previous communications (Quiroz et al., 2001; Rodríguez-Iturbe et al., 2001; Alvarez et al., 2002). In brief, glomeruli were graded from 0 to +4 with grade 0, normal; grade 1, <25%; grade 2, 25 to 50%; grade 3, 50 to 75%, and grade 4, sclerosis occupying >75% of the glomerular tuft. The glomerulosclerosis score was obtained as follows: [(1 x n glomeruli with +1) + (2 x n glomeruli with +2) + (3 x n glomeruli with +3) + (4 x n glomeruli with +4)] x 100/total number of glomeruli examined. All glomeruli suitable for analysis were examined in each biopsy (range 38–68).

Tubulointerstitial damage was scored using a 0 to 5 scale depending on the extent of areas with tubular (dilatation, disruption of basement membrane, and cell defacement) and interstitial (cellular infiltration, widening, and sclerosis) damage. Extent of damage was evaluated by computer image analysis in successive fields at 20x magnification of all the cortical and juxtamedullary regions of each biopsy and graded as follows: 0, no changes; grade 1, <10%; grade 2, 10 to 25%; grade 3, 25 to 50%; grade 4, 50 to 75%; and grade 5, 75 to 100%, as described in previous investigations (Quiroz et al., 2001; Rodríguez-Iturbe et al., 2001; Alvarez et al., 2002).

Avidin-biotin-peroxidase methodology was used to study to identify lymphocytes (CD5-positive cells), macrophages (ED1-positive cells), and angiotensin II-positive cells and the expression of intercellular adhesion molecule (ICAM)-1. Details of the immunostaining techniques in our laboratories have been described previously (Parra et al., 2003). Positive cells detected with the immune stainings were defined separately in the glomeruli (positive cells per glomerular cross-section) and in tubulointerstitial areas (positive cells per square millimeter). ICAM expression was evaluated by computer-assisted image analysis.

All histological and immunohistological studies were done in a blinded manner. Computer-assisted image analyses were done in an Olympus BX51 system microscope and DP70 microscope digital camera, with Sigma Pro (Leesburgh, VA) image analysis software.

MDA Determination. Renal content of MDA was analyzed in four kidneys harvested at the end of the experiment in each experimental group by the method of Ohkawa et al. (1979) in supernatants of kidney slices placed in a cold mixture of 100 mM KCl and 0.003 M EDTA, homogenized and centrifuged at 600g. Results are expressed as nanomoles of MDA per milligram of kidney homogenate protein. Specific details of all these methods in our laboratories have been reported previously (Nava et al., 2000).

Antisera. Anti-CD5 and anti-ED1 monoclonal antibodies (Biosource, Camarillo, CA) were used to identify lymphocytes and macrophages, respectively. Rabbit anti-human angiotensin II antiserum with cross-reactivity to rat angiotensin II (Peninsula Laboratories, Belmont, CA) was used to identify angiotensin II-positive cells as detailed in previous communications (Quiroz et al., 2001; Rodríguez-Iturbe et al., 2001, 2002; Alvarez et al., 2002). Specificity of the staining was tested by preincubating the antibody with human angiotensin II (Rodríguez-Iturbe et al., 2004a). Anti ICAM-1 (CD54) monoclonal antibodies were purchased from Seikagaku Corp. (Tokyo, Japan). Secondary biotin-conjugated affinity-pure antibodies with minimal reactivity to rat serum proteins were purchased from Accurate Chemical & Scientific Co. (Westbury, NY). Nonrelevant antibodies were used for negative control studies.

Statistical Methods. Multigroup analysis of variance (ANOVA) was used to compare results from different groups, and Tukey post tests were applied if differences were p < 0.05. Increments in blood pressure from baseline values in the same rats were studied by repeated measurements ANOVA. Two-tailed p values <0.05 were considered significant. All data are presented as mean ± S.D.

	Results

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General Effects of PDTC Administration, Body Weight, and Renal Function. The administration of PDTC intraperitoneally was associated with a short period (15–20 s) of agitation in the rats that moved rapidly in the cage; no such effects were noted with the injection of vehicle. After this immediate reaction, there were no apparent ill effects, and the rats behaved and fed normally.

At the beginning of the experiment, the rats had similar body weight (grams) (WKY group, 138 ± 12.2; SHR group, 136 ± 13.2; SHR.PDTC group, 134 ± 11.4), similar plasma creatinine (milligrams per deciliter) (WKY group, 0.18 ± 0.04; SHR group, 0.19 ± 0.06; SHR.PDTC group, 0.19 ± 0.05) and similar urinary protein excretion rates (milligrams per 24 h) (WKY group, 0.8 ± 1.6; SHR group, 1.3 ± 1.8; SHR.PDTC group, 0.5 ± 1.2). The findings at the end of the experiment are shown in Table 1. The mean urinary protein excretion at the end of the study period was significantly higher in the untreated SHR than in the PDTC-treated SHR group (Table 1).

Blood Pressure. Data are shown in Fig. 1. As expected, the untreated SHR group exhibited a progressive increment in blood pressure; in contrast, the PDTC-treated SHR group maintained a normal blood pressure throughout the study period, comparable with the values in the WKY group. As shown in Fig. 1, the administration of PDTC to WKY rats did not modify the blood pressure (baseline, 129 ± 12 mm Hg and after PDTC treatment, 130 ± 12).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Serial BP measurements of blood pressure in SHR (, uninterrupted lines), SHR treated with PDTC (, uninterrupted lines), WKY control rats (, interrupted lines), and WKY rats treated with PDTC (, interrupted lines). a, p < 0.05 versus baseline, p < 0.01 versus the rest. *, p < 0.05 versus the rest; ***, p < 0.001 versus the rest.

Light Microscopy. Renal structure was largely preserved in all experimental groups. Renal histology was essentially normal in renal biopsies taken at 7 weeks of age (beginning of the experiment) and was not significantly different in the biopsies taken at 12 weeks of age. Glomerulosclerosis scores at the end of the experiment were mildly increased in the SHR group (Table 2). Tubulointerstitial damage scores were mildly increased (p < 0.05) in the SHR at 25 weeks (2.1 ± 0.74) from baseline scores (0.9 ± 0.69). However, there were no significant differences between the SHR group, the SHR.PDTC group (1.4 ± 0.83), and the WKY group (1.1 ± 0.24) (Table 2).

Suppression of NF-B Activation. The p65 NF-B content in the nuclear extracts of the renal cortex was increased at 7 weeks of age in the SHR group and rose further in the untreated SHR at 12 and 25 weeks of age. In contrast, values obtained in the PDTC-treated SHR at 12 and 25 weeks of age were similar to those found in the WKY control rats (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Renal cortical content of p65 NFB is increased in the SHR and increases as hypertension becomes severe. Administration of PDTC inhibits NF-B activation and p65 NFB levels are maintained at levels found in the WKY control rats throughout the experiment. *, p < 0.05 versus WKY control group. a, p < 0.05 versus the rest; b, p < 0.01 versus the rest.

Adhesion Molecules. ICAM-1 expression in glomeruli and tubulointerstitial regions are summarized in Fig. 3, A and B, respectively. The expression of ICAM-1 was increased in the untreated SHR. PDTC treatment suppressed it to levels that were similar to those found in WKY rats. In the untreated SHR, ICAM-1 was overexpressed in most glomeruli and tubulointerstitial areas. Occasionally, glomeruli showing overexpression of ICAM-1 were surrounded by tubulointerstitial areas with negative ICAM-1 staining. Representative microphotographs are shown in Fig. 3, C to E.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Glomerular (A) and tubulointerstitial (B) ICAM-1 is overexpressed in the SHR and reduced to normal levels with PDTC treatment (**, p < 0.01). C and D, SHR 25 weeks of age, demonstrating that positive ICAM-1 staining can be found in both glomeruli and tubulointerstitium as well as in glomeruli surrounded of negative tubulointerstitial areas. E, reduction in ICAM expression in both glomerular and tubulointerstitial areas resulting from PDTC treatment (immunoperoxidase staining). Original magnification, 200x).

Immune Cell Infiltration. Glomerular infiltration of lymphocytes and macrophages was scarce and not significantly different in the experimental and control groups. In contrast, increased numbers of immune cells in tubulointerstitium were present already at 7 weeks of age in the SHR. At this time, there were 21.4 ± 3.1 CD5-positive cells/mm², and 37.1 ± 4.9 ED1-positive cells/mm² in the SHR group, whereas the WKY group had 11.3 ± 2.5 CD5-positive cells/mm² and 4.6 ± 2.1 ED1-positive cells/mm² (p < 0.001 versus each one of the SHR values). The accumulation of lymphocytes and macrophages in tubulointerstitial areas further increased at the end of the experiment in the untreated SHR. Administration of PDTC prevented the accumulation of immune cells in the treated SHR (Table 2). Representative microphotographs are shown in Fig. 4, A and B.

View larger version (123K): [in this window] [in a new window]   Fig. 4. Infiltration of macrophages (ED1-positive cells) in the SHR (A) is reduced by PDTC treatment (B). Angiotensin II-positive cells in tubulointerstitial areas in the SHR (C) is reduced by PDTC treatment (D) (immunoperoxidase staining). Original magnification, 400x.

Angiotensin II-Positive Cells. Angiotensin II-positive cells were increased in the renal tubulointerstitial areas of the SHR group and were reduced by PDTC treatment (Table 2). Representative microphotographies are shown in Fig. 4, C and D. Double-staining studies (data not shown) revealed that 15 to 20% of the infiltrating macrophages as well as tubular epithelial cells, were angiotensin II-positive, as reported in previous work (Quiroz et al., 2001; Rodríquez-Iturbe et al., 2001).

MCP-1 in Situ Hybridization. MCP-1 mRNA was increased in the SHR and reduced to normal levels by PDTC treatment (Fig. 5A). Representative microphotographs are shown in Fig. 5, B and C.

View larger version (41K): [in this window] [in a new window]   Fig. 5. In situ hybridization showing increased MCP-1 mRNA in the renal tissues of the SHR (B) and reduction resulting from PDTC treatment (C). *, p < 0.05; **, p < 0.01.

MDA Renal Content. Renal MDA content was increased in the SHR and reduced to normal values by PDTC treatment (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Renal MDA abundance is increased in the SHR and reduced by PDTC treatment to levels similar to those in the WKY control rats. **, p < 0.01.

	Discussion

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The central and novel finding of this study is the observation that arterial hypertension and inflammatory immune infiltration of the kidneys in the SHR rat can be completely abrogated by suppression of NF-B activation to the levels found in control WKY rats. These findings are in concert and complement previous work from our group showing that reduction of the immune cell accumulation ameliorates the hypertension of the SHR (Rodríguez-Iturbe et al., 2002).

PDTC effects included a reduction in the expression of ICAM-1 and MCP-1 mRNA. Because transcriptional stimulation of cell adhesion molecule ICAM-1 and MCP-1 induction is regulated by NF-B in endothelial cells (Martin et al., 1997), smooth muscle cells (Landry et al., 1997), renal tubular cells (Viedt et al., 2002), and mesangial cells (Rovin et al., 1995), their down-regulation is an expected result of PDTC-driven suppression of this transcription factor (Wang et al., 1999).

The possibility of reducing the vascular inflammation and cardiac hypertrophy in the dTGR rats by PDTC was explored by Müller et al. (2000). These double transgenic rats, harboring both human renin and angiotensin genes, are severely hypertensive, develop renal and cardiac damage, and die at a young age. In their study (Müller et al., 2000), the administration of 200 mg/kg/day of PDTC subcutaneously for 3 weeks resulted in improved survival, amelioration of the angiotensin II-induced inflammatory damage, and a 16 to 17% reduction in the mean systolic blood pressure levels. PDTC administration was also used by Hong et al. (2000) to investigate the prohypertensive role of the inducible nitric-oxide synthase in the SHR. For this purpose, they used 10 mg/kg/day of PDTC administered in the drinking water and found that this dose reduced the systolic blood pressure from a mean value of 195 to 160 mm Hg at 16 weeks of age. These elegant studies, therefore, demonstrated a partial effect on the systolic blood pressure by PDTC administration. However, it remained undefined whether a complete suppression of NF-B activation starting at an early age and maintained for an extended period would result in prolonged suppression of the immune cell infiltration and prevention of hypertension in SHR. This issue is important because it would offer additional and compelling evidence of the pivotal role of the renal inflammatory reactivity in the pathogenesis of hypertension. The quantitative evaluation of the suppression of NF-B activation in serial studies was made possible by work of Renard et al. (2001) who described an enzyme-linked immunosorbent assay-based sensitive test for the p65 and p50 subunits. We determined p65 content because, in contrast with p50, it contains transcription activation domain (Ping et al., 1999; Sun and Andersson, 2002).

The PDTC-induced reduction of the blood pressure cannot be attributed to direct effects of the drug because, in agreement with similar observations by others (Hong et al., 2000), the blood pressure was unmodified by this compound in the WKY rats (WKY.PDTC group; Fig. 1). One obvious effect of PDTC treatment was the reduction of intrarenal inflammation and oxidative stress. As reported previously (Rodríguez-Iturbe et al., 2004a), the accumulation of immune cells in tubulointerstitial areas begins at an early age and increases with advancing age in association with the severity of hypertension.

Because the suppression of interstitial inflammation and oxidative stress improves hypertension, the effects of PDTC could be the result of inhibition of NF-B. However, several other consequences of PDTC administration could contribute to reducing the blood pressure in the SHR. The modest reduction in blood pressure observed in the studies of Müller et al. (2000) was attributed to the amelioration of renal failure in the dTGR strain; however, this factor was unlikely to play a significant role in our studies because the renal function in the SHR and SHR.PDTC groups, at least as judged by serum creatinine determinations, was similar. Hong et al. (2000) focused their experiments on the potential role of excess nitric oxide (NO) production by inducible nitric-oxide synthase (iNOS) in the pathogenesis of hypertension in the SHR. Excessive NO would result in peroxynitrite anion formation, protein tyrosine nitration, hydroxyl radicals generation and thereby oxidative/nitrosative stress and hypertension (Klahr, 1998; Espey et al., 2002; Modlinger et al., 2004). It should be noted that the reduction in NO bioavailability in the SHR is, in part, due to angiotensin II-mediated increase in superoxide production and impaired superoxide scavenger activity (Adler and Huang, 2004). Nonetheless, iNOS expression is significantly increased in the SHR (Chou et al., 1998; Vaziri et al., 1998) and its down-regulation by PDTC, which is a known to inhibit iNOS induction (Liu et al., 1997; Hong et al., 1998) could have contributed to the prevention of hypertension in our PDTC-treated SHR

The well known antioxidant properties of PDTC could also be involved in the antihypertensive effects of this compound because increased ROS production is characteristic of the SHR and is, in part, responsible for the associated hypertension (Zalba et al., 2001; Zhan et al., 2004). In fact, the suppression of NF-B activity by PDTC may be mediated by a direct inhibition of its intranuclear translocation (Ziegler-Heitbrook et al., 1993) as well as by antioxidant properties of this compound (Schreck et al., 1992). It should be noted, however, that in specific biological systems the PDTC can be pro-oxidant and proapoptotic (Galter et al., 1994; Nobel et al., 1995). Although important, all these PDTC-related effects cannot be separated from its suppression of the proinflammatory transcriptional activity of the NF-B.

Furthermore, interstitial inflammation and oxidative stress mutually support one another as pathogenic conditions in the SHR (Rodríguez-Iturbe et al., 2003). As recently reviewed (Modlinger et al., 2004; Rodríguez-Iturbe et al., 2004), the unchecked generation of reactive oxygen species and intrarenal angiotensin II, favor sodium retention by reduction in filtered sodium, increased sodium reabsorption, and impaired pressure diuresis, which culminate in a new equilibrium maintained by elevated blood pressure. These effects are further amplified by a high salt intake that increases NAD(P)H oxidase abundance and superoxide generation (Kitiyara et al., 2003). The interrelation between oxidative stress and interstitial inflammation is underlined by the present study, which demonstrates that long-term inhibition of NF-B improves both conditions and prevents the development of hypertension in SHR. Several pathophysiological conditions, such as unchecked oxidative stress and inflammatory cytokines, may trigger NF-B activation in the SHR; however, the role of these factors was not defined in the present studies.

In conclusion, the present study confirms the early increase in NF-B activation in the SHR and shows that long-term inhibition of this proinflammatory transcription factor can result in prevention of hypertension in this model. Taken in context with previous investigations, the present work provides compelling evidence for the role of intrarenal inflammation in the pathogenesis of hypertension.

	Footnotes

 
Financial support for this study was provided by a grant from the Asociación de Amigos del Riñón (Maracaibo) and Fondo Nacional de Ciencia y Tecnología Grant S1-2001001097 (Venezuela). A.F. and V.V. are recipients of grants from the Asociación de Amigo del Riñon, Maracaibo, Venezuela.

doi:10.1124/jpet.105.088062.

ABBREVIATIONS: SHR, spontaneously hypertensive rats; NF-B, nuclear transcription factor-B; PDTC, pyrrolidine dithiocarbamate; dTGR, double transgenic rat; WKY, Wistar Kyoto; MCP-1, macrophage chemoattractant molecule-1; MDA, malondialdehyde; DEPC, diethyl pyrocarbonate; SSC, standard saline citrate; ICAM, intercellular adhesion molecule; NO, nitric oxide; iNOS, inducible nitric-oxide synthase.

Address correspondence to: Dr. Bernardo Rodríguez-Iturbe, Servicio de Nefrología, Hospital Universitario de Maracaibo, Ave Goajira s/n, Maracaibo, Estado Zulia, Venezuela. E-mail: bernardori{at}telcel.net.ve

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