Original Report: Laboratory Investigation

Effects of Dietary Salt on Intrarenal Angiotensin System, NAD(P)H Oxidase, COX-2, MCP-1 and PAI-1 Expressions and NF-B Activity in Salt-Sensitive and -Resistant Rat Kidneys
G. Chandramohan^{a, b}, Y. Bai^c, K. Norris^{a, b}, B. Rodriguez-Iturbe^{d, e}, N.D. Vaziri<sup>c

a</sup>Charles R. Drew University of Medicine and Science and
^bKing Drew Medical Center, Los Angeles, Calif., and
^cDivision of Nephrology and Hypertension, University of California, Irvine, Calif., USA;
^dRenal Service, Hospital Universitario, Universidad del Zulia, and
^eCentro de Investigaciones Biomédicas, IVIC-Zuli, Maracaibo, Venezuela

Address of Corresponding Author

Am J Nephrol 2008;28:158-167 (DOI: 10.1159/000110021)

 Outline

• Key Words
• Abstract
• Introduction
• Methods
• Animals
• Measurement of Arterial Pressure
• Plasma Renin Activity Assay
• Tissue Preparation and Western Blot Analyses
• Immunohistological Tests
• Statistical Analysis
• Results
• General Data
• Plasma Renin Activity
• AT₁ Receptor and Ang II Data
• NAD(P)H Oxidase and Antioxidant Enzymes
• Phospho-IB, COX, MCP-1 and PAI-1 Data
• Renal Interstitial T Cell and Macrophage Infiltration
• Discussion
• Acknowledgement
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Hypertension
• Oxidative stress
• Inflammation
• Renal injury
• Dietary sodium
• Renin-angiotensin system
• Cardiovascular disease

  Abstract

Background: Chronic consumption of a high-salt diet causes hypertension (HTN) and renal injury in Dahl salt-sensitive (SSR) but not salt-resistant rats (SRR). These events are, in part, mediated by oxidative stress and inflammation in the kidney and vascular tissues. Activation of the angiotensin II type 1 (AT₁) receptor plays an important role in the pathogenesis of oxidative stress and inflammation in many hypertensive disorders. However, the systemic renin-angiotensin system (RAS) is typically suppressed in salt-sensitive HTN. This study was designed to test the hypothesis that differential response to a high-salt diet in SSR versus SRR may be related to upregulation of tissue RAS and pathways involved in inflammation and reactive oxygen species (ROS) production. Methods and Results: SSR and SRR were studied 3 weeks after consumption of high- (8%) or low-salt (0.07%) diets. The SSR consuming a low-salt diet exhibited significant increases in AT₁ receptor, cyclooxygenase (COX) 2, plasminogen activator inhibitor (PAI) and phospho-IB in the kidney as compared to those found in SRR. The high-salt diet resulted in severe HTN and proteinuria (in SSR but not SRR) and marked elevations of renal tissue monocyte chemoattractant protein 1, p22^phox, NADPH oxidase subunit 4, angiotensin-II-positive cell count, infiltrating T cells and macrophages and further increases in AT₁ receptor, COX-2, PAI-1 and phospho-IB in the SSR group. The high-salt diet significantly lowered plasma renin activity (PRA) in SRR but not in the SSR. COX-1 abundance was similar on the low-salt diet and rose equally with the high-salt diet in both groups. Among subgroups of animals fed the low-salt diet, kidney glutathione peroxidase (GPX) abundance was significantly lower in the SSR than SRR. The high-salt diet raised GPX and mitochondrial superoxide dismutase (SOD) abundance in the SRR kidneys but failed to do so in SSR. Cu/Zn-SOD abundance was similar in the subgroups of SSR and SRR fed the low-salt diet. The high-salt diet resulted in downregulation of Cu/Zn-SOD in SSR but not SRR. Conclusions: Salt sensitivity in the SSR is associated with upregulations of the intrarenal angiotensin system, ROS-generating and proinflammatory/profibrotic proteins and an inability to raise antioxidant enzymes and maximally suppress PRA in response to high salt intake. These events can contribute to renal injury with high salt intake in SSR.

Copyright © 2007 S. Karger AG, Basel

 Introduction

Consumption of a high-salt diet results in severe hypertension (HTN) in Dahl salt-sensitive (SSR) but not salt-resistant rats (SRR). Development of HTN with a high-salt diet is accompanied by early onset of significant renal injury and dysfunction in the SSR [1,2,3]. The associated renal injury and dysfunction in SSR consuming a high-salt diet is accompanied by and, in part, mediated by oxidative stress and inflammation [2,3,4,5]. The prototypical phagocytic and tissue-specific isotypes of NAD(P)H oxidase are the main source of reactive oxygen species (ROS) in the kidney and cardiovascular tissues [6,7,8,9]. Oxidative stress and excess production of ROS in the hypertensive SSR kidney and vascular tissues have been shown to be attenuated by inhibitors of NAD(P)H oxidase [10, 11]. These observations suggest that activation and/or upregulation of NAD(P)H oxidase may be a cause of excess ROS production in the hypertensive SSR kidney and vascular tissues.

Inflammation, oxidative stress and activation/upregulation of NAD(P)H oxidase in the kidney and cardiovascular tissues in hypertensive disorders are frequently mediated by angiotensin II (Ang II). These effects are mediated by activation of the Ang II type 1 (AT₁) receptor which is largely responsible for most actions of Ang II including those leading to vasoconstriction, sodium reabsorption, inflammation and oxidative stress [7, 8,12,13,14,15,16,17,18]. It is of note that development of severe HTN, endothelial dysfunction, left ventricular hypertrophy and nephropathy with high-salt intake is accompanied by marked suppression of the circulating renin-angiotensin system (RAS) in SSR. Accordingly, it may appear counterintuitive to implicate the RAS in the pathogenesis of renal and cardiovascular diseases in SSR which is a prototypical model of low-renin HTN. It should be noted, however, that the kidney expresses all components of RAS and that renal RAS can operate independently of its circulating counterpart [19,20,21,22]. In fact, marked reductions of plasma renin activity (PRA) and angiotensinogen concentration are accompanied by paradoxical elevations of renal tissue and urinary excretion of angiotensinogen in SSR fed a high-salt diet [23, 24]. Since most hemodynamic and nonhemodynamic actions of the RAS in adult tissues are mediated by AT₁ receptor, we sought to explore the effects of strain, dietary salt intake and interactions thereof on Ang-II-producing cells and protein expression of AT₁ receptor as well as those of the major mediators of oxidative stress and inflammation in the SSR kidney. Accordingly, protein expressions of AT₁ receptor, NAD(P)H oxidase subunits, phospho-IB, cyclooxygenase (COX) 1, COX-2, monocyte chemoattractant protein (MCP) 1 and plasminogen activator inhibitor (PAI) 1 as well as numbers of Ang-II-positive and inflammatory cells were compared in the kidneys of SSR and SRR given low- (0.07%) or high-salt (8% NaCl) diets for 3 weeks.

 Methods

 Animals

Experiments were performed using 20-week-old inbred lines of male Dahl SR/JrHsd (SRR) and Dahl SS/JrHsd (SSR) rats maintained as inbred colonies (Harlan, Indianapolis, Ind., USA). The Charles Drew University Animal Care and Use Committee approved all experimental protocols. The rats were maintained on tap water and regular diet ad libitum until switched to low-salt (0.07%) or high-salt (8%) diets. Six rats were used in each of the 4 subgroups. The diets were continued for 3 weeks. Blood pressure and body weight were measured twice weekly. Twenty-four-hour urine collections were obtained using metabolic cages twice before and after initiation of the assigned diets. At the conclusion of the study period, under general anesthesia (using 0.3% of isoflurane inhalation), the animals were sacrificed by decapitation. Blood samples were collected, and kidneys were immediately harvested. A piece of the kidney was fixed in 10% formalin for immunohistological examinations, and the remainder was snap-frozen in liquid nitrogen and stored at -80°C until processed. Serum creatinine was determined by a DT-II chemistry analyzer (Ortho Clinical Diagnostics Inc., Raritan, N.J., USA). Urine protein and creatinine concentrations were analyzed by Randil Chemistry Laboratory (Michigan, Pa., USA).

 Measurement of Arterial Pressure

Collection of blood pressure data was begun after a week of conditioning. Conscious rats were placed in a restrainer on a warming pad and allowed to rest insidethe cage for 15 min before blood pressure measurements.Rat tails were placed inside a tail cuff, and the cuff was inflatedand released several times to allow the animal to be conditionedto the procedure. Three consecutiveblood pressure readings were taken by a rat tail blood pressure monitor attachedto a student oscillograph (Harvard Apparatus) and averagedfor presentation.

 Plasma Renin Activity Assay

PRA was determined by measuring the amount of Ang I produced from endogenous substrate following incubation at 37°C. To this end, the plasma Ang I concentration in each specimen was measured prior to and after incubation at 37°C for 1 h. The Ang I concentration was quantified by radioimmunoassay using ¹²⁵I-Ang-I and anti-Ang-I antibody. PRA was determined by subtracting preexisting Ang I from that found after incubation and expressed as nanograms Ang I generated per milliliter of plasma per hour. The sensitivity of the assay is 0.1 ng/ml/h. The interassay variation is less than 12%.

 Tissue Preparation and Western Blot Analyses

Kidney cortex was separated and homogenized in 10 mM HEPES buffer, pH 7.4, containing 320 mM sucrose, 1 mM EDTA, 1 mM DTT, 10 mg/ml leupeptin, 2 mg/ml aprotinin and 1 µM phenylmethylsulfonyl fluoride at 0-4°C. A Polytron tissue mixing and blending device was used to blend the tissue into a smooth homogenate. Homogenates were centrifuged at 12,000 g for 10 min at 4°C to precipitate tissue debris. The supernatant was used to perform the Western analyses. Total protein concentration was determined with the use of a Bio-Rad kit (Bio-Rad Laboratories, Hercules, Calif., USA).

Protein abundance of AT₁ receptor, COX-1, COX-2, MCP-1, PAI-1 and NADPH oxidase subunits (NOX-4, gp91^phox, 67^phox, 47^phox and p22^phox) were measured by Western blot analysis as described in our earlier studies [17, 25]. AT₁ receptor and NOX-4 antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif., USA). Specificity of the AT₁ receptor antibody has been verified by the supplier with the use of several positive control preparations including NIH/3T3 and C3H/10T1/2 cell lysates and loss of detectable AT₁ receptor protein with the siRNA technique. The lack of cross-reactivity of this antibody with AT₂ receptor has been confirmed in earlier studies [26]. Polyclonal antibodies against gp91^phox and 67^phox were purchased from Upstate Inc., Lake Placid, N.Y., USA. Antibody against p22^phox was generously provided by Dr. A.J. Jesaitis, Montana State University. Antibody to p47^phox was purchased from BD Biosciences (San Diego, Calif., USA). Antibodies against cytosolic (Cu/Zn-SOD), mitochondrial (MnSOD) superoxide dismutase isoforms and glutathione peroxidase (GPX) were purchased from Calbiochem Inc. (San Diego, Calif., USA). Polyclonal antibodies against COX-1 and COX-2 were purchased from Cayman Chemical, Ann Arbor, Mich., USA. Anti-MCP-1 antibody was purchased from Abcam Inc. (Cambridge, Mass., USA), and anti-PAI-1 antibody was purchased from BD Biosciences. The polyclonal rabbit antibody against phospho-IB was purchased from Cell Signaling Technology Inc. (Denver, Colo., USA). Secondary peroxidase-conjugated immunopure goat anti-rabbit IgG (H + L) antibodies were purchased from Pierce Biotechnology (Rockford, Ill., USA) and diluted in 5% nonfat milk at 1:10,000. Actin monoclonal antibody was purchased from Sigma Chemical Inc. (St. Louis, Mo., USA). Western blot blue staining (Perkin Elmer, Boston, Mass., USA) was used to verify the uniformity of protein load and transfer efficiency across the test samples. Experiments failing this test were discarded. Optical densities of protein bands were determined by a laser densitometer (Molecular Dynamics, Sunnyvale, Calif., USA) and expressed as arbitrary units.

 Immunohistological Tests

Lymphocytes (CD5-positive cells) and macrophages (ED1-positive cells) were identified using the avidin-biotin-peroxidase methodology, as described before [27]. Immune cell infiltration was evaluated in a blinded fashion and expressed as the number of positive cells per square millimeter. Lymphocytes and macrophages were identified with monoclonal anti-CD5 and anti-ED1 antibodies (Biosource Inc., Camarillo, Calif., USA), respectively. Rabbit anti-human Ang II antiserum with cross-reactivity to rat Ang II (Peninsula Laboratories Inc., San Carlos, Calif., USA) was used to identify Ang-II-positive cells; specificity of the staining was tested by preincubating the antibody with human Ang II, as described in a previous communication [27].Secondary biotin-conjugated affinity-pure antibodies with minimal reactivity to rat serum proteins were purchased from Accurate Chemical and Scientific Inc., Westbury, N.Y., USA. Nonrelevant antibodies were used for negative control studies.

 Statistical Analysis

The statistical analyses were performed using the SPSS 13.0 program. Data for continuous variables are presented as means ± SEM. The paired t test was employed to compare the initial and follow-up measures of blood pressure within each group. One-way ANOVA was employed to compare the outcome variables among low-salt SSR, high-salt SRR, low-salt SSR and high-salt SSR groups. A univariate general linear model was used to uncover the primary and interaction effects of two categorical independent variables (strain and diet) on outcome variables. The general linear model allows detecting both main effect and interaction effects of the dependent variables. A 'main effect' is the direct effect of an independent variable on the dependent variable. An 'interaction effect' is the joint effect of independent variables (strain and diet) on the dependent variables. p values equal to or less than 0.05 were considered significant.

 Results

 General Data

Data are shown in table 1. The subgroups of SRR and SSR receiving the low-salt diet experienced normal growth as evidenced by significant weight gain during the study period. Consumption of the high-salt diet prevented the growth in the SRR and actually led to a significant weight loss in the SSR. Consumption of the high-salt diet resulted in a marked rise in arterial pressure in the SSR but had no significant effect on arterial pressure in the SRR. Creatinine clearance was significantly lower in SSR compared to SRR consuming the low-salt diet. Consumption of the high-salt diet resulted in a significant increase in creatinine clearance in SRR but not in the SSR. Urinary protein excretion was significantly higher in the SSR than in the SRR on the low-salt diet. Consumption of the high-salt diet resulted in a significant rise in urinary protein excretion in SSR but not in SRR.

Table 1. The body weight, blood pressure, creatinine clearance and urinary protein excretion of SRR and SSR with high- and low-salt diets

 Plasma Renin Activity

Consumption of the high-salt diet resulted in an approximately sixfold reduction in the mean PRA in the SRR group (3.87 ± 0.5 vs. 0.56 ± 0.1 ng/h, p < 0.05). PRA in the SSR consuming the low-salt diet (4.34 ± 0.7 ng/h) was not significantly different from that found in the corresponding SRR group and did not fall significantly in response to the high-salt diet (2.9 ± 1.1 ng/h).

 AT₁ Receptor and Ang II Data

Data are shown in figures 1 and 2. The number of Ang-II-positive cells in the renal tissue was similar in SRR and SSR groups consuming the low-salt diet. Consumption of the high-salt diet significantly increased the tissue Ang-II-positive cell count in the SSR but not in the SRR kidney. Compared to the SRR, the SSR exhibited a significant increase in renal tissue AT₁ receptor protein abundance on the low-salt diet. Consumption of the high-salt diet did not significantly alter the AT₁ receptor abundance in the SRR but significantly raised it in the SSR.

Fig. 1.a, b Representative photomicrographs depicting Ang-II-positive cells in the kidney tissues of Dahl SRR and SSR maintained on a high-salt diet for 3 weeks. Arrows point to Ang-II-positive cells. c Group data depicting Ang-II-positive cell count in the kidneys of SRR and SSR maintained on low-salt (LS) or high-salt (HS) diets for 3 weeks. n = 6 animals in each group; * p < 0.01 versus other groups.

Fig. 2. Representative Western blot and group data depicting AT1 receptor and -actin abundance in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05, SSR versus SRR; b p < 0.05, SSR-HS versus all other groups.

 NAD(P)H Oxidase and Antioxidant Enzymes

Data are shown in figures 3 and 4. Protein abundances of NOX-4, gp91^phox, p22^phox, p47^phox and p67^phox subunits of NAD(P)H oxidase were similar in the renal tissues of the SSR and SRR consuming the low-salt diet (gp91^phox, p47^phox and p67^phox, data not shown). Consumption of the high-salt diet resulted in a significant upregulation of p22^phox in the SSR group and of NOX-4 in both SRR and SSR groups. No significant change was observed in gp91^phox, p47^phox or p67^phox with the high-salt diet in either group (data not shown). Cu/Zn-SOD abundance in the kidney was similar in the SSR and SRR groups consuming the low-salt diet. Consumption of the high-salt diet resulted in a significant decline in renal tissue Cu/Zn-SOD abundance in the SSR. However, the high-salt diet did not alter kidney tissue Cu/Zn-SOD in the SRR group. Renal tissue MnSOD abundance was similar among the groups on the low-salt diet. Consumption of the high-salt diet resulted in a significant upregulation of MnSOD in the SRR but failed to do so in the SSR. GPX abundance in the kidney was significantly lower in the SSR than in the SRR group receiving the low-salt diet. Consumption of the high-salt diet significantly raised renal tissue GPX abundance in the SRR but failed to do so in the SSR.

Fig. 3. Representative Western blot and group data depicting p22phox (a), NOX-4 (b) and -actin abundance in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05, SSR-HS versus all other groups; b p < 0.05, HS versus LS diet.

Fig. 4. Representative Western blot and group data depicting Cu/Zn-SOD (a), MnSOD (b), GPX (c) and -actin abundance in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05 versus all other groups; b p < 0.05, SSR versus SRR.

 Phospho-IB, COX, MCP-1 and PAI-1 Data

Data are shown in figures 5 and 6. Phospho-IB abundance was significantly greater in the kidneys of SSR compared to those of SRR consuming the low-salt diet. The high-salt diet led to a significant rise in kidney phospho-IB abundance in both groups. However, kidney phospho-IB abundance in animals consuming the high-salt diet was markedly greater in the SSR than the SRR group. Compared to the SRR, the SSR exhibited a significant upregulation of COX-2 in the kidney on the low-salt diet. Consumption of the high-salt diet led to a further rise in renal tissue COX-2 expression in SSR but not in SRR. COX-1 abundance in the kidney was similar in the SSR and SRR groups given the low-salt diet and rose significantly in both groups in response to high-salt intake. Kidney tissue MCP-1 abundance was not significantly different among the SRR and SSR groups consuming the low-salt diet. Consumption of the high-salt diet resulted in a significant rise in MCP-1 abundance in both groups but more so in the SSR than in the SRR. No significant difference was found in kidney PAI-1 abundance between the two groups consuming the low-salt diet. Consumption of the high-salt diet resulted in a marked upregulation of kidney PAI-1 abundance in the SSR. However, the high-salt diet had no effect on the kidney PAI-1 abundance in the SRR.

Fig. 5. Representative Western blot and group data depicting phospho-IB and -actin abundance in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05, HS versus LS diet; b p < 0.05, SSR-LS versus SRR; c p < 0.05, SSR-HS versus all other groups.

Fig. 6. Representative Western blot and group data depicting COX-1 (b), COX-2 (a), PAI-1 (d), MCP-1 (c) and -actin abundance in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05, HS versus LS diet; b p < 0.05, SSR versus SRR; c p < 0.05, SSR-HS versus all other groups.

 Renal Interstitial T Cell and Macrophage Infiltration

Data are depicted in figure 7. No significant difference was found in the numbers of T cells (CD5-positive cells) or macrophages (ED1-positive cells) in the renal tissue among the SSR and SRR groups fed a low-salt diet. Consumption of the high-salt diet led to a significant increase in interstitial T cell infiltration in both SSR and SRR groups but more so in the former than in the latter group. The high-salt diet significantly increased interstitial macrophage infiltration in the SSR group but not in the SRR group.

Fig. 7. Bar graphs depicting the numbers of infiltrating T cells (CD5-positive, a) and macrophages (ED1-positive, b) in the kidney cortex of Dahl SRR and SSR fed a low- (LS) or high-salt (HS) diet for 3 weeks. n = 6 animals in each group; a p < 0.05, HS versus LS diet; b p < 0.05, SSR-HS versus all other groups.

 Discussion

As expected, consumption of the high-salt diet resulted in a marked exacerbation of HTN and proteinuria in SSR but did not alter arterial pressure or urinary protein excretion in SRR. Renal tissue AT₁ receptor was markedly elevated in the SSR on the low-salt diet and rose further with the high-salt diet. Elevation of renal tissue AT₁ receptor was accompanied by a significant rise in the Ang-II-positive cells in the kidneys of SSR consuming the high-salt diet. These observations, together with the previously reported increase in renal tissue angiotensinogen in SSR maintained on a high-salt diet [23, 24], provide compelling evidence for upregulation of the intrarenal RAS in this model.

Upregulation of the intrarenal RAS in SSR was compounded by an impaired ability to suppress PRA in response to the high-salt diet. In an earlier study, Campbell et al. [28] found a significant reduction of plasma renin and prorenin concentrations following consumption of a high-salt (4%) diet for 2 weeks in 7-week-old Brookhaven SSR but not SRR. The changes in plasma renin/prorenin values in their animals paralleled those seen in renin mRNA and immunodetectable protein in the kidney. In contrast, consumption of the high-salt diet for 3 weeks failed to cause a significant suppression of PRA and paradoxically increased renal cortical tissue AT₁ receptor and Ang-II-positive cells in our 20-week-old SSR. The reason for apparent disparities in the results of the two studies is uncertain. However, it may be due to differences in the age and strain of rats used, duration of salt loading, presence of renal damage and the nature of parameters measured (renin, prorenin concentrations/expression vs. renin activity, AT₁ receptor and Ang-II-positive cells in plasma and kidney, respectively).

The elevation of the intrarenal RAS appears to be primarily involved in the pathogenesis of the associated nephropathy as opposed to the circulating RAS which is intimately involved in the regulation of sodium balance, vascular resistance and arterial pressure. This viewpoint is supported by the studies of Hayakawa et al. [1], who showed that blockade of the RAS reversed endothelial dysfunction, attenuated proteinuria and reduced renal injury without reducing blood pressure in the SSR fed a high-salt diet. As noted earlier, AT₁ receptor mediates most hemodynamic and nonhemodynamic actions of Ang II [7, 8, 12,14,15,16, 29, 30]. For instance, AT₁ receptor activation results in increased ROS production and oxidative stress by activating and/or upregulating NAD(P)H oxidase in the kidney and cardiovascular tissues [7, 8, 15]. In addition, AT₁ receptor activation promotes inflammation by stimulating production of proinflammatory and profibrotic cytokines, and chemokines as well as expression of adhesion molecules, events which can lead to leukocyte infiltration, fibrosis and tissue injury [31,32,33,34].

The SSR consuming the high-salt diet exhibited a marked increase in renal tissue NOX-4 and p22^phox subunits of NAD(P)H oxidase which is the main source of ROS in the kidney and cardiovascular tissues. This was accompanied by significant downregulation of Cu/Zn-SOD which is responsible for conversion of superoxide to hydrogen peroxide in the cytoplasm (O₂^-· + O₂^-· + ₂H^{+ SOD} H₂O₂ + O₂). The reduction of Cu/Zn-SOD was compounded by a significant downregulation of GPX in the salt-sensitive animals regardless of their dietary salt intake. GPX is the key enzyme for reduction of lipoperoxides and conversion of hydrogen peroxide to water using reduced glutathione as substrate (H₂O₂ + 2GSH ^GPX ₂H₂O + GS-SG). The observed upregulation of the ROS-producing enzyme NAD(P)H oxidase and impaired antioxidant defense system can work in concert to promote oxidative stress and tissue injury in the kidneys of SSR consuming a high-salt diet. The link between activation of angiotensin system and upregulation of ROS-generating pathways in the kidney observed here is consistent with the findings of Zhou et al. [31], who showed increased superoxide production in the aorta of SSR maintained on a high-salt diet.

AT₁ receptor activation promotes production of proinflammatory/profibrosis cytokines and chemokines which, in turn, cause inflammation, fibrosis and tissue damage [32,33,34,35,36]. The SSR consuming the high-salt diet exhibited a significant increase in the renal tissue abundance of MCP-1 which is a potent proinflammatory chemokine. This phenomenon can contribute to renal injury and dysfunction by promoting inflammation and leukocyte infiltration as seen in our SSR fed a high-salt diet. Similarly, PAI-1 expression was significantly increased in the kidneys of SSR consuming the high-salt diet. This can, in turn, contribute to glomerulosclerosis and interstitial fibrosis by inhibiting matrix metalloproteinases. Upregulations of MCP-1, PAI-1, p22^phox and NOX-4, and interstitial T cell/macrophage infiltration were accompanied by an increased abundance of phospho-IB in the renal cortex, denoting activation of redox-sensitive nuclear transcription factor B (NF-B) in SSR fed the high-salt diet. NF-B is the general transcription factor for a variety of proinflammatory cytokines and chemokines, and as such its activation can account for the upregulations of MCP-1 and PAI-1 in the kidneys of SSR fed a high-salt diet. Ang II has been shown to promote NF-B activation in renal and vascular cells [18, 32]. Conversely, activation of NF-B stimulates gene expression of angiotensinogen [37]. Thus, activations of the tissue angiotensin system and NF-B appear to be involved in a vicious cycle that contributes to renal injury and inflammation.

Byproducts of the main enzymes of the arachidonic acid metabolism play an important part in the pathogenesis of inflammation, oxidative stress as well as dysregulations of renal and systemic hemodynamics [38,39,40,41,42]. For instance, COX-2 has been shown to promote ROS production, inflammation and hemodynamic alterations in the kidney and other tissues [38,39,40, 43, 44]. The SSR consuming the high-salt diet exhibited a significant upregulation of renal tissue COX-2 abundance. The observed upregulation of renal tissue COX-2 expression can contribute to renal injury and inflammation in SSR consuming a high-salt diet. This viewpoint is supported by studies of Hermann et al. [45], who found amelioration of endothelial dysfunction, tubulointerstitial inflammation, glomerulosclerosis and proteinuria with administration of a selective COX-2 inhibitor in SSR fed a high-salt diet.

Although consumption of the high-salt diet resulted in mild, but significant, upregulations of renal tissue NOX-4, COX-2, COX-1, MCP-1 and NF-B activity, it did not raise arterial pressure or urinary protein excretion in SRR. It should be noted that high dietary salt intake was associated with a robust expression of antioxidant enzymes in the kidneys of SSR. In contrast, the opposite was observed in the SSR kidneys. The ability to raise the antioxidant defense system and to suppress PRA in SRR and the lack thereof in SSR may, in part, account for their differential response to high salt intake. This is because oxidative stress and inflammation are inseparably linked and together they play a major role in the pathogenesis of HTN and renal and cardiovascular diseases [29].

In conclusion, salt sensitivity in the SSR is associated with upregulations of the intrarenal RAS, increased abundance of ROS-generating and proinflammatory/profibrosis proteins and an inability to raise antioxidant enzymes in response to a high salt intake. These events may contribute to the pathogenesis of salt-induced HTN and renal injury in salt-sensitive animals and perhaps humans. Further studies are needed utilizing AT₁ receptor blockade in order to investigate causal relationships between the upregulation of AT₁ receptor and oxidative and inflammatory processes in this model.

 Acknowledgement

This study was funded by an NIH-Center for Research Resources grant (5-U54-RR019234).

  References

1.

Hayakawa H, Coffee K, Raij L: Endothelial dysfunction and cardiorenal injury in experimental salt-sensitive hypertension: effects of antihypertensive therapy. Circulation 1997;96:2407-2413.

2.

Meng S, Roberts LJ 2nd, Cason GW, Curry TS, Manning RD Jr: Superoxide dismutase and oxidative stress in Dahl salt-sensitive and -resistant rats. Am J Physiol Regul Integr Comp Physiol 2002;283:R732-R738.

3.

Tian N, Thrasher KD, Gundy PD, Hughson MD, Manning RD Jr: Antioxidant treatment prevents renal damage and dysfunction and reduces arterial pressure in salt-sensitive hypertension. Hypertension 2005;45:934-939.

4.

Kushiro T, Fujita H, Hisaki R, Asai T, Ichiyama I, Kitahara Y, Koike M, Sugiura H, Saito F, Otsuka Y, Kanmatsuse K: Oxidative stress in the Dahl salt-sensitive hypertensive rat. Clin Exp Hypertens 2005;27:9-15.

5.

Makino A, Skelton MM, Zou AP, Roman RJ, Cowley AW Jr: Increased renal medullary oxidative stress produces hypertension. Hypertension 2002;39:667-672.

6.

Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox CS: Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension 2002;39:269-274.

7.

Griendling KK, Sorescu D, Ushio-Fukai M: NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 2000;86:494-501.

8.

Taniyama Y, Griendling KK: Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension 2003;42:1075-1081.

9.

Griendling KK: Novel NAD(P)H oxidases in the cardiovascular system. Heart 2004;90:491-493.

10.

Taylor NE, Glocka P, Liang M, Cowley AW Jr: NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats. Hypertension 2006;47:692-698.

11.

Zhuo JL, Carretero OA, Li XC: Effects of AT₁ receptor-mediated endocytosis of extracellular Ang II on activation of nuclear factor-kappa B in proximal tubule cells. Ann NY Acad Sci 2006;1091:336-345.

12.

Anders HJ, Ninichuk V, Schlondorff D: Progression of kidney disease: blocking leukocyte recruitment with chemokine receptor CCR1 antagonists. Kidney Int 2006;69:29-32.

13.

Egido J: Vasoactive hormones and renal sclerosis. Kidney Int 1996;49:578-597.

14.

Hoffmann S, Podlich D, Hahnel B, Kriz W, Gretz N: Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats. J Am Soc Nephrol 2004;15:1475-1487.

15.

Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG: Role of p47^phox in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 2002;40:511-515.

16.

Timmermans PB, Benfield P, Chiu AT, Herblin WF, Wong PC, Smith RD: Angiotensin II receptors and functional correlates. Am J Hypertens1992;5:2215-2355.

17.

Xu ZG, Lanting L, Vaziri ND, Li Z, Sepassi L, Rodriguez-Iturbe B, Natarajan R: Upregulation of angiotensin II type 1 receptor, inflammatory mediators, and enzymes of arachidonate metabolism in obese Zucker rat kidney. Circulation 2005;111:1962-1969.

18.

Zhou MS, Hernandez Schulman I, Pagano PJ, Jaimes EA, Raij L: Reduced NAD(P)H oxidase in low renin hypertension: link among angiotensin II, atherogenesis, and blood pressure. Hypertension 2006;47:81-86.

19.

Ichihara A, Kobori H, Nishiyama A, Navar LG: Renal renin-angiotensin system. Contrib Nephrol 2004;143:117-130.

20.

Navar LG: The intrarenal renin-angiotensin system in hypertension. Kidney Int 2004;65:1522-1532.

21.

Navar LG, Harrison-Bernard LM, Nishiyama A, Kobori H: Regulation of intrarenal angiotensin II in hypertension. Hypertension 2002;39:316-322.

22.

Navar LG, Nishiyama A: Why are angiotensin concentrations so high in the kidney? Curr Opin Nephrol Hypertens 2004;13:107-115.

23.

Kobori H, Nishiyama A: Effects of tempol on renal angiotensinogen production in Dahl salt-sensitive rats. Biochem Biophys Res Commun 2004;315:746-750.

24.

Kobori H, Harrison-Bernard LM, Navar LG: Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension. Hypertension 2001;37:1329-1335.

25.

Zhan CD, Sindhu RK, Vaziri ND: Up-regulation of kidney NAD(P)H oxidase and calcineurin in SHR: reversal by lifelong antioxidant supplementation. Kidney Int 2004;65:219-227.

26.

Thomas MA, Fleissner G, Stohr M, Hauptfleisch S, Lemmer B: Localization of components of the renin-angiotensin system in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats. part B. Brain Res 2004;1008:224-235.

27.

Rodriguez-Iturbe B, Quiroz Y, Ferrebuz A, Parra G, Vaziri ND: Evolution of renal interstitial inflammation and NF-kappaB activation in spontaneously hypertensive rats. Am J Nephrol 2004;24:587-594.

28.

Campbell WG Jr, Gahnem F, Catanzaro DF, James GD, Camargo MJ, Laragh JH, Sealey JE. Plasma and renal prorenin/renin, renin mRNA, and blood pressure in Dahl salt-sensitive and salt-resistant rats. Hypertension 1996;27:1121-1133.

29.

Vaziri ND, Dicus M, Ho N, Sindhu R: Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency. Kidney Int 2003;63:179-185.

30.

Vaziri ND, Rodriguez-Iturbe B: Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2006;2:582-593.

31.

Zhou MS, Adam AG, Jaimes EA, Raij L: In salt-sensitive hypertension, increased superoxide production is linked to functional upregulation of angiotensin II. Hypertension 2003;42:945-951.

32.

Douillette A, Bibeau-Poirier A, Gravel SP, Clement JF, Chenard V, Moreau P, Servant MJ: The proinflammatory actions of angiotensin II are dependent on p65 phosphorylation by the IkappaB kinase complex. J Biol Chem 2006;281:13275-13284.

33.

Nahmod KA, Vermeulen ME, Raiden S, Salamone G, Gamberale R, Fernandez-Calotti P, Alvarez A, Nahmod V, Giordano M, Geffner JR. Control of dendritic cell differentiation by angiotensin II. Faseb J 2003;17:491-493.

34.

Ruiz-Ortega M, Lorenzo O, Ruperez M, Konig S, Wittig B, Egido J: Angiotensin II activates nuclear transcription factor kappaB through AT₁ and AT₂ in vascular smooth muscle cells: molecular mechanisms. Circ Res 2000;86:1266-1272.

35.

Wolf G, Neilson EG: Angiotensin II as a renal growth factor. J Am Soc Nephrol 1993;3:1531-1540.

36.

Chow F, Ozols E, Nikolic-Paterson DJ, Atkins RC, Tesch GH: Macrophages in mouse type 2 diabetic nephropathy: correlation with diabetic state and progressive renal injury. Kidney Int 2004;65:116-128.

37.

Brasier AR, Li J: Mechanisms for inducible control of angiotensinogen gene transcription. Hypertension 1996;27:465-475.

38.

Cheng HF, Harris RC: Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms: role of p38-mediated pathways. J Biol Chem 2002;277:45638-45643.

39.

Goncalves AR, Fujihara CK, Mattar AL, Malheiros DM, Noronha Ide L, de Nucci G, Zatz R: Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory. Am J Physiol Renal Physiol 2004;286:F945-F954.

40.

Kramer BK, Kammerl MC, Komhoff M: Renal cyclooxygenase-2 (COX-2): physiological, pathophysiological, and clinical implications. Kidney Blood Press Res 2004;27:43-62.

41.

Ota T, Takamura T, Ando H, Nohara E, Yamashita H, Kobayashi K: Preventive effect of cerivastatin on diabetic nephropathy through suppression of glomerular macrophage recruitment in a rat model. Diabetologia 2003:46:843-851.

42.

Reddy MA, Adler SG. Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R: Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells. Am J Physiol Renal Physiol 2002;283:F985-F994.

43.

Kiritoshi S, Nishikawa T, Sonoda K, Kukidome D, Senokuchi T, Matsuo T, Matsumura T, Tokunaga H, Brownlee M, Araki E: Reactive oxygen species from mitochondria induce cyclooxygenase-2 gene expression in human mesangial cells: potential role in diabetic nephropathy. Diabetes 2003:52:2570-2577.

44.

Komers R, Lindsley JN, Oyama TT, Schutzer WE, Reed JF, Mader S, Anderson S: Effects of cyclooxygenase-2 (COX-2) inhibition on plasma and renal renin in diabetes. J Lab Clin Med 2002;140:351-357.

45.

Hermann, M, Shaw S, Kiss E, Camici G, Buhler N, Chenevard R, Luscher TF, Grone HJ, Ruschitzka F: Selective COX-2 inhibitors and renal injury in salt-sensitive hypertension. Hypertension 2005;45:193-197.

46.

Vaziri ND, Bai Y, Ni Z, Quiroz Y, Rodriguez-Iturbe B: Intra-renal angiotensin II/AT₁ receptor, oxidative stress, inflammation and progressive injury in renal mass reduction. J Pharmacol Exp Ther 2007;323:85-93.

  Author Contacts

N.D. Vaziri, MD
Division of Nephrology and Hypertension, UCI Medical Center
101 The City Drive, Bldg 53, Rm 125, Rt 81
Orange, CA 92868 (USA)
Tel. +1 714 456 5142, Fax +1 714 456 6034, E-Mail ndvaziri@uci.edu

  Article Information

Received: June 22, 2007
Accepted: September 4, 2007
Published online: October 19, 2007
Number of Print Pages : 10
Number of Figures : 7, Number of Tables : 1, Number of References : 46

  Publication Details

American Journal of Nephrology

Vol. 28, No. 1, Year 2008 (Cover Date: November 2007)

Journal Editor: Bakris, G. (Chicago, Ill.)
ISSN: 0250-8095 (print), 1421-9670 (Online)

For additional information: http://www.karger.com/AJN

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.