Lombardi et al have recently reported that pressor doses of angiotensin II (Ang II) administered for two weeks resulted in the subsequent development of salt-sensitive hypertension (SSHTN) in the rat1. This investigation established an experimental model that permits the analysis of the sequential structural changes in the kidney that are acutely induced by Ang II as well as with the development of persistent SSHTN. The structural changes present in the kidney consist of focal vascular and tubulointerstitial damage with areas of tubular atrophy and a reduction in peritubular capillaries, while in contrast, the glomeruli were minimally abnormal. These findings supported the postulate that impairment of pressure-natriuresis response caused by tubulointerstitial injury could play a pivotal role in the development of hypertension in a variety of clinical conditions2.
Nevertheless, histologic findings in the studies of Lombardi et al as well as in a variety of other experimental and clinical conditions associated with SSHTN include infiltration, at times prominent, of mononuclear cells in tubulointerstitial areas of the kidney1. Examples of mononuclear cell infiltration in other experimental hypertension models include the "vicious cycle" of two-kidney one-clip Goldblatt hypertension3,4, aging5, cyclosporine nephropathy6, and catecholamine infusion7. An influx of immunocompetent cells may be induced by chemokines and leukocyte adhesion proteins expressed by injured renal cells [reviewed in 8]. Infiltrating cells could then produce cytokines in situ, which could modify local vascular reactivity [reviewed in 9] and promote inflammation and scarring [reviewed in 10]. Therefore, the mononuclear cellular infiltrate could be critically involved in modifications of the pressure-natriuresis and sodium reabsorption mechanisms and, thereby, in the pathogenesis of SSHTN.
The present studies were designed to evaluate the role, if any, of infiltrating immunocompetent cells that occur in the kidney during the angiotensin exposure, on the subsequent development of hypertension. For this purpose, we treated rats with the immunosuppressive drug mycophenolate mofetil (MMF) during the angiotensin infusion phase of this experimental model.
Mycophenolate mofetil is a widely used drug for the treatment of transplant recipients. It reversibly inhibits the enzyme inosine monophosphate dehydrogenase that regulates de novo purine synthesis, and consequently is a selective lymphocyte immunosuppressor11. Recently, MMF has been shown to diminish the expression of intracellular adhesion molecules and the infiltration of immune cells in the remnant kidney model12 and in the atherosclerotic lesions in the rabbit aorta13. Furthermore, MMF treatment has antiproliferative effects in mesangial cells14,15 and tubulointerstitial cells16, and reduces collagen deposition16. Since the drug has relatively minor side effects17 and in contrast to cyclosporine A and Tacrolimus is not nephrotoxic, it appeared well suited for the purposes of this work. Our results indicate that treatment with MMF prevents the development of SSHTN in the Ang II model. This effect was associated with a reduction of tubulointerstitial lymphocyte infiltration and Ang II-producing cells, a reduction in histologic damage and a reduction in superoxide producing cells and urinary lipid peroxidation products.
METHODS
Experimental design
The experimental protocol included three successive phases over an eight-week period: the angiotensin infusion phase (2 weeks of angiotensin administration), washout phase (5 to 7 days following angiotensin), and a high-salt diet phase (from the end of the washout phase until the 8th week).
Male Sprague-Dawley rats (Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela) weighing 290 to 340 g were divided in three groups that received Ang II infusion and MMF (Ang II + MMF group) or vehicle (Ang II group) or were sham operated (control group). The rats of the Ang II + MMF group (N = 15) and the Ang II group (N = 15) received 435 ng 
kg min-1 of Ang II (Sigma Chemical Co. St. Louis, MO, USA) dissolved in Ringer's lactate, for two weeks by osmotic minipumps (Alzet model 2002; Alza Corporation, Palo Alto, CA, USA) that were surgically placed subcutaneously and removed at the end of two weeks, as described elsewhere18.
In addition to angiotensin, the Ang II + MMF group received MMF (CellCept; Roche Laboratories, Montclair, NJ, USA) by gastric gavage, in doses of 30 mg kg day-1, during the angiotensin infusion phase and the washout phase of the experiment. As described in previous work, the MMF was suspended in 500 
L of water by vigorous agitation immediately before administration because the drug is insoluble in water12. The Ang II group received 500 L water daily by gastric gavage during the angiotensin and washout phases. The control group (N = 9) consisted of rats in which the same surgical procedure was performed (including both the implantation and removal of the minipumps), but minipumps were not inserted.
All of the animals (Ang II group, Ang II + MMF group, and control group) had a similar dietary protocol, consisting of standard rat chow with normal sodium content (Ratarina Protinal Company, Valencia, Venezuela) during the initial three weeks of the study (angiotensin and washout periods) and a high-sodium (4% NaCl) diet (Harlan Teklad, Madison, WI, USA) from weeks 4 to 8.
Serum creatinine and urinary protein were determined by autoanalyzer methodology (Express Plus; Ciba Corning, Essex, UK). Urinary malondialdehyde (MDA) was determined in 24-hour urine samples obtained at the end of the angiotensin phase and at the end of the experiment. Rats from the Ang II and Ang II + MMF groups were sacrificed under ether anesthesia by aortic desanguination after removal of the kidneys for histologic studies at two time periods: at the end of the angiotensin administration and at the end of the experiment at the eighth week.
Blood pressure measurements
Systolic and mean arterial blood pressures were determined in conscious restrained rats by tail-cuff plethysmography (IITC; Life Scientific Instruments, Woodland Hills, CA, USA). Before the experiments were begun, rats were conditioned three to four times to the procedure. Once the experiments were started, blood pressures were measured weekly. In each session, the blood pressure recorded was the mean of three separate measurements for each rat.
Urinary MDA determinations
Thiobarbituric reactive substances were determined by the method of Ohkawa, Ohishi, and Yagi19 as described previously12. Briefly, MDA was measured in 24-hour urine samples centrifuged at 2000 
g and processed the same day. A sample of 400 L was added to a reaction mixture consisting of 200 L of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), 1.5 mL of 0.5% thiobarbituric acid, and 400 L of bidistilled water. The mixture was heated at 95°C in a water bath for 60 minutes, and 1 mL of water and 5 mL of n-butanol-pyridine were added; the mixture was agitated and centrifuged at 2000 g for 15 minutes. The absorbance of the upper organic layer was read at 532 nm (Shimatzu Spectrophotometer model UV21100S, Kyoto, Japan). Malonaldehyde bis-dimethyl acetal was used as the external standard.
Histologic studies
Coronal kidney sections were fixed in Methyl Carnoy and embedded in Paraplast Plus (Monoject, Sherwood Medical Scientific Division, St. Louis, MO, USA). The remainder of the tissue was included in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC, USA), frozen in dry ice and acetone, and stored at -70°C.
Paraffin-embedded sections 4 m thick were stained with periodic acid-Schiff reagent (PAS) and hematoxylin and eosin (HE). Tubulointerstitial injury was evaluated in the entire cortex and corticomedullary areas. As previously described6,7, tubulointerstitial injury was defined by any of the following findings: tubular dilation, atrophy or sloughing, cellular infiltration (identified by HE staining), interstitial widening, or basement membrane thickening. The severity of the injury was expressed as a semiquantitative score based on the extension of the finding: 0 = no changes present; grade 1 = <10%; grade 2 = 10 to 25%; grade 3 = 25 to 50%; grade 4 = 50 to 75%; and grade 5 = 75 to 100%6.
Computer-assisted image analysis software was used to study osteopontin (OPN; Optimas, version 6.2; Media Cybernetics, Silver Springs, MD, USA) and fibronectin expression (Sigma Scan Pro 5.0; SPSS Inc., Chicago, IL, USA) in cortical tubulointerstitial areas. The digitized images of OPN were acquired using a Leica DMRB microscope fitted with a microimage i308 low-light video camera with a 1/2-inch HyperHAD sensor and a Flashpoint video framegrabber board (Bartels and Stout, Bellevue, WA, USA) as described before1. The fibronectin studies were done on images acquired from a Zeiss Axioscope with a Kodak DC 120 digital megapixel camera. Results are given as the percentage of positive area for each biopsy (exclusive of glomeruli).
Cellular infiltration and proliferation (positive cells with a given staining/mm2) were counted by two different observers using an ocular fitted with a grid in at least 20 microscopic fields (40 magnification) in cortical and corticomedullay areas, and the mean value of the counts was used in each biopsy. Glomerular cell counts were expressed as positive cells per glomerular cross section (gcs).
Histologic evaluation (individual cell counts and computer-assisted image analysis) was done without previous knowledge of the source of the tissue.
Histologic technique and reagents
Indirect immunoperoxidase methodology was used to identify OPN, macrophages, and proliferating cells. Staining with goat anti-OPN (OP 199; gift from C. Giachelli, University of Washington, Seattle, WA, USA) and ED-1 (Harlan Bioproducts, Indianapolis, IN, USA), was described in previous work1. Proliferating cell nuclear antigen was identified with the appropriate antibody [monoclonal antibody (mAb) anti-PCNA clone PC10; concentration 50 g/mL; Zymed Laboratories, Inc. San Francisco, CA, USA] as described in a previous study20.
Superoxide production in renal cells was studied in 8 m cryostat sections by the cytochemical method of Briggs et al21. Slides were incubated for 60 minutes at 37°C in the following solution: 50 mL 0.05 mol/L Tris-HCl buffer, 1 mL diaminobenzidine (DAB) stock solution (5 g DAB/132 mL Tris buffer 0.05 mol/L, pH 7.6), 250 L 8% NiCl, 32.5 L 10% NaN3, and 50 L 0.5 mol/L MnCl2. Sections were fixed with 10% formalin and counterstained with 1% methyl green.
Indirect immunofluorescence was used to identify T lymphocytes (mAb anti-rat CD5, clone MRCOX19; concentration 50 g/mL; Biosource International, Camarillo, CA, USA), interleukin-2 (IL-2) receptor (mAb anti-CD25; concentration 100 g/mL; Accurate Chemical and Scientific Corporation, Westbury, NY, USA), Ang II-producing cells [rabbit anti-Ang II (human) IgG, dilution 1:20; Peninsula Labs. Inc., CA, USA], and fibronectin (mAb rabbit anti-rat fibronectin; dilution 1:50; CalBiochem Laboratories, La Jolla, CA, USA) as described previously19. The secondary antibody for staining CD5 and IL-2 receptor was FITC-conjugated affinity-pure F(ab')2 fragment rat anti-mouse IgG with minimal cross-reactivity with rat protein (Accurate Chemical and Scientific Corporation). The secondary antibody for fibronectin staining was donkey anti-rabbit FITC (concentration 20 g/mL; Accurate Chemical and Scientific Corporation).
Double staining of Ang II-positive cells was done as described before22. Briefly, tissue sections were first incubated with either anti-CD5 or anti-ED-1 mAb at the concentration described earlier and then with rhodamine-conjugated (TRIC) affinity-pure F(ab')2 rat anti-mouse IgG with minimal cross reactivity with rat proteins (Accurate Chemical and Scientific Co.). Afterward, tissues were incubated overnight with the anti-Ang II antibody (discussed previously) and finally, with the secondary fluorescein conjugated (DTAF) affinity-pure donkey anti-rabbit IgG antibody (dilution 1:50; Accurate Chemical and Scientific Co.).
Immunoperoxidase staining was used to localize angiotensin receptors using rabbit or goat anti-rat angiotensin II type 1 and 2 (AT1 and AT2) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously23. Briefly, AT1 receptors were studied in kidney sections incubated with the corresponding antibody (dilution 1:30) and then with a secondary antibody conjugated with a peroxidase-labeled polymer (Envision System; Dako A/S, Carpinteria, CA, USA). Finally, the slides were incubated with 3,3'-diaminobenzidine (DAB; Sigma). AT2 localization was done by the avidin-biotin methodology. Biotinylated mouse anti-goat IgG (Amersham, Arlington Heights, IL, USA) was used as a secondary antibody, and positive sites were also evidenced with DAB. Control slides were treated with nonrelevant antisera. Sections were counterstained with Mayer's hematoxylin (Sigma Chemical Co.). Double immunostaining was also performed to determine whether lymphocytes were expressing Ang II receptors. For this purpose, mAb mouse anti-rat CD5 and affinity-pure rabbit anti-Ang II type 1 receptor (anti-AT1) and anti-Ang II type 2 receptor (anti-AT2) antisera (Alpha Diagnostics, San Antonio, TX, USA) were used as primary antibodies. Secondary antibodies were rhodamine-conjugated rat anti-mouse IgG and fluorescein-conjugated (DTAF) donkey anti-rabbit IgG.
All histologic and immunohistochemical microphotography are collected in two plates (Figures 9 and 10; Discussion section).
Figure 9.
Structural changes, proliferative activity, and lymphocyte infiltration. (A) Structural changes (PAS staining) found at the end of angiotensin infusion (2 weeks) in the Ang II group present focal damage, including tubular dilation and sloughing (arrows) and areas of interstitial sclerotic widening (arrowheads). (B) A biopsy section from a rat from the Ang II + MMF group appears essentially normal. Proliferative activity as shown by PCNA-positive cells (anti-PCNA staining, avidin-biotin-peroxidase technique) at the end of angiotensin infusion is more pronounced in the Ang II group (C) than in the Ang II + MMF group (D); PCNA-positive cells are infiltrating cells, while the resident glomerular and tubular cells are negative. T-lymphocyte infiltration (staining with anti-CD5 mAb) in the Ang II group (E) is reduced by MMF treatment (F).
Full figure and legend (92K)Figure 10.
Lymphocyte activation, superoxide-producing cells, Ang II-positive cells, and AT1 receptor expression. Cells expressing IL-2 receptor demonstrated by indirect immunofluorescence (staining with anti-CD25) in the tubulointerstitium of a rat untreated with MMF (A). Superoxide-producing cells (arrows) shown with staining for intracellular superoxide in the Ang II group (B). Ang II-positive cells are increased in the renal interstitium of rats from the untreated Ang II group (C) in relationship to the MMF-treated rats (D). Double staining demonstrates two lymphocytes (E; TRIC staining, mAb anti-CD5) that also stain positive for Ang II (FITC staining, rabbit anti-Ang II IgG) indicated by arrows in (F). Staining for AT1 receptor is shown in (G).
Full figure and legend (3K)Statistical analysis
Statistical analyses were done with the help of a commercial statistical package (Instat GraphPad®, San Diego, CA, USA). Comparisons between groups were done by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-tests or by unpaired t tests using Welch's correction when the differences in SD were significant. Serial changes were evaluated with repeated-measures ANOVA and Tukey-Kramer post-tests. Two-tailed P values <0.05 were considered significant. Results are given as mean 
SD.
RESULTS
Effects of MMF treatment on blood pressure, weight, and renal function
The effects of MMF on the systolic blood pressure are shown in Figure 1. During the administration of Ang II, the blood pressure increased in both experimental groups. In the first week, the blood pressure increment was more pronounced in the Ang II group (P < 0.01), but both groups had a similar blood pressure elevation at the end of two weeks. In the washout period, the blood pressure returned to baseline levels in both groups, and after the fourth week, corresponding to one week in the high-salt diet, the blood pressure progressively rose in the Ang II group. In contrast, the blood pressure in the Ang II + MMF group remained at the same baseline-washout levels, well within the 95% CI of the control group Figure 1.
Figure 1.
Angiotensin (Ang II) infusion induces salt-dependent hypertension in untreated rats (
) but not in mycophenolate mofetil (MMF)-treated rats (
). Dotted lines correspond to the 95% CI in control (sham-operated) rats. Data are mean SD. *P < 0.05 and **P < 0.01 vs. the values at the fourth week (end of the wash-out period after angiotensin infusion). During angiotensin infusion and washout periods, rats received a normal Na diet.
As shown in Figure 2, the rats from both experimental groups lost weight during the Ang II infusions (P < 0.001), and the difference in weight loss between the Ang II and the Ang II + MMF groups was not statistically significant. During the washout period, the body weight started to increase in both experimental groups, and this tendency continued throughout the high-salt diet period Figure 2.
Figure 2.
Weight changes during the experiment. The values in untreated rats () and the values in MMF-treated rats () are not statistically different at any time interval. The weight in both groups of experimental rats diminished during the angiotensin infusion (period a: P < 0.001 vs. each previous week) and increased afterward (period b: P < 0.001 vs. each previous week after week 2), without reaching the 95% CI of the weight in control rats. Data are mean SD.
Baseline serum creatinine was similar in the experimental groups (Ang II group, 0.33 0.08; Ang II + MMF, 0.33 0.05 mg/dL). At the end of angiotensin infusion, the serum creatinine had doubled in the Ang II and the Ang II + MMF groups and then returned to control levels in both experimental groups Figure 3.
Figure 3.
Serum creatinine levels (mean SD) increased during angiotensin infusion and returned to normal during the period of high-salt diet. The Ang II group () and the Ang II + MMF group () are not statistically different. Dotted lines indicate the 95% CI of the control rats.
Baseline levels of proteinuria (mg/24 hours) were 2.2 1.4 in the Ang II group, 2.2 0.7 in the Ang II + MMF group, and 2.6 1.0 in the control group. At the end of eight weeks, the values in the Ang II group (5.7 1.5) and in Ang II + MMF group (4.7 1.6) were higher (P < 0.05) than the values in the control rats (2.3 1.1) but were still within normal limits.
Histologic findings
Tubulointerstitial damage was evaluated after two weeks of Ang II. At this time, the PAS score (0 to 5) was 2.9 0.7 in the Ang II group and 1.8 1.2 in the Ang II + MMF group (P < 0.05). Representative areas of the histologic damage are shown in Figure 9a and b.
Cellular proliferation and infiltration
At the end of angiotensin infusion, proliferating cell nuclear antigen (PCNA)-positive cells were four times more numerous in the Ang II group than in the Ang II + MMF group (P < 0.001). Six weeks after stopping the angiotensin infusion (end of the high-salt diet period), the proliferative activity had returned to almost normal (control) levels in both groups Figure 4. PCNA staining appeared to label infiltrating cells, while fibroblasts, resident glomerular cells, and tubular cells were negative Figure 9c, d.
Figure 4.
Tubulointerstitial proliferative activity (PCNA-positive cells) is increased at the end of angiotensin infusion. The values in the Ang II group () are significantly higher than in the Ang II + MMF group (
). Controls (C) are shown as open columns. Data are mean SD. ***P < 0.001.
The macrophage infiltration (positive ED-1 cells/mm2 in cortical tubulointerstitial areas) was more intense at the end of the Ang II infusion than at the end of the experiment in both experimental groups. ED-1–positive macrophages were less numerous in the MMF-treated rats at the end of Ang II infusion (Ang II group, 41 34 vs. Ang II + MMF group, 28 14) and at the end of the SSHTN period (Ang II group, 17 10 vs. Ang II + MMF group = 10 5), but the difference observed between groups was not statistically significant. In contrast, the infiltration of T lymphocytes (CD5-positive cells/mm2 in the cortical tubulointerstitium) was more prominent (P < 0.05) in the Ang II group than in the Ang II + MMF group at the end of the Ang II phase (2 weeks). At the end of the high-salt period (8th week), the lymphocyte infiltration had diminished, but the Ang II group continued to have a more intense infiltration than the Ang II + MMF group (P < 0.05). These findings are shown in Figure 5 and photomicrographs in Figure 9e and f.
Figure 5.
Infiltrating lymphocytes resulting from angiotensin infusion (Ang II group; ) are suppressed by MMF treatment (Ang II + MMF group;). Control group (C) is shown as open column. Data are mean SD; *P < 0.05.
As shown in Figure 6, infiltrating T cells in the cortical tubulointerstitial areas expressing IL-2 receptor were approximately 18 times more numerous in the Ang II group than in the Ang II + MMF group at the end of the angiotensin phase. The findings were essentially the same at the end of the salt-sensitive hypertensive phase (8 weeks; Figure 6). A representative example is shown in Figure 10a. The number of cells expressing IL-2 receptor was essentially unchanged at two weeks and at eight weeks in the corresponding experimental groups Figure 6.
Figure 6.
Activated T cells expressing interleukin-2 (IL-2) receptor are suppressed by MMF treatment. Symbols are: () Ang II group; () Ang II + MMF group; () controls. Data are mean SD; **P < 0.01.
Superoxide staining
At the end of the angiotensin phase, the number of superoxide-positive cells in glomeruli (positive cells/gcs) was not statistically different in the Ang II (0.42 0.22) and in the Ang II + MMF groups (0.24 0.21). However, within the cortical tubulointerstitial areas (excluding glomeruli), the group treated with MMF had significantly fewer cells staining for intracellular superoxide (Ang II group, 94 23 cells/mm2 vs. Ang II + MMF group, 24 18 cells/mm2, P < 0.05). A representative example is shown in Figure 10b.
Angiotensin II-producing cells
Intraglomerular angiotensin positive cells were scarce at two weeks (Ang II group, 0.23 0.11 cells/gcs, Ang II + MMF group, 0.14 0.07 cells/gcs) and at the end of the SSHTN phase (Ang II group, 0.21 0.13 cells/gcs; Ang II + MMF group, 0.20 0.21). There were essentially no angiotensin-positive cells in the interstitium of sham-operated rats. In contrast, tubulointerstitial cells showing intense staining for Ang II were found in both experimental groups. As shown in Figures 7 and 10c and D, MMF treatment was associated with a reduction of angiotensin-positive interstitial cells (cells/mm2) at the end of Ang II infusion (Ang II group, 45 6 vs. Ang II + MMF group, 21 3; P < 0.001) and at the end of the salt-sensitive period at the eighth week (Ang II group, 65 31 vs. Ang II + MMF group, 20 21; P < 005).
Figure 7.
Angiotensin-positive cells in tubulointerstitial areas were reduced by MMF treatment. Abbreviations are: () Ang II group; () Ang II + MMF group. There were no interstitial angiotensin-positive cells in the control (C) group. Data are mean SD; *P < 0.05; ***P < 0.0001.
Double-staining methodology demonstrated that 9 to 10% of the intraglomerular lymphocytes stained positive for Ang II at two and eight weeks. Intraglomerular ED1+ cells very seldom were positive for Ang II (<4%). In contrast, at two weeks in the tubulointerstitial areas, 41 4.5% of the CD5+ cells in the MMF-untreated group and 40 5.4% of the CD5+ cells in the MMF-treated group stained positive for Ang II. A similar proportion of infiltrating lymphocytes produced Ang II at eight weeks (41 4.6% of CD5+ cells in the MMF-untreated group and 42 6.3% of CD5+ cells in the MMF-treated group). Figure 10e and f demonstrate the double immunostaining. In tubulointerstitium, 8 to 10% of infiltrating macrophages stained positive for Ang II at two and eight weeks in the MMF-treated and untreated groups.
Angiotensin receptors
Angiotensin II type 2 receptors were scarce but present in vascular locations and rarely found in proximal tubular cells. Rats in both experimental groups and sham-operated rats had similar AT2-positive cells in this location (mean values in all groups, <3 positive cells/100 proximal tubular cells). AT1 receptors were found in glomeruli, proximal tubules Figure 10g, and the renal vasculature. The number of AT1-positive cells in glomeruli (AT1 + cells/gcs) was comparable in the Ang II group (4.9 5.6) and in the Ang II + MMF group (3.3 2.9). Similarly, the number of proximal tubular cells expressing AT1 receptors (AT1 + cells/100 tubular cells) was equivalent at two and eight weeks (sham, 18.8 3.4; Ang II group at 2 weeks, 19.3 17.8; Ang II + MMF group at 2 weeks, 17.4 8.4; Ang II group at 8 weeks, 19.3 18.6; Ang II + MMF group at 8 weeks, 17.5 7.1).
Mononuclear cells were negative for the AT1 and AT2 Ang II receptors by immunostaining, and similarly, by double immunostaining, no Ang II receptors could be identified in CD5-positive lymphocytes.
Osteopontin and fibronectin expression
At the end of angiotensin infusion, OPN expression (percentage of cortex) was comparable in the Ang II group (0.89 0.4) and in the Ang II + MMF group (0.85 0.7). Fibronectin was expressed in 16.0 5.1% of cortex in the Ang II group and in 11.7 6.9% in the Ang II + MMF group (P = NS).
Urinary malondialdehyde excretion
The urinary excretion of MDA increased significantly (P < 0.001) with the angiotensin infusion. Basal levels of urinary MDA excretion were 102 28.1 and 98 26.3 nmol/24 hours for the Ang II group and Ang II + MMF groups, respectively. As shown in Figure 8, at the end of the angiotensin phase (2 weeks), the values of urinary MDA were two to three times higher than the basal levels. At this time, the difference between the group treated with MMF and the untreated group was significant (Ang II group, 351 56.8; Ang II + MMF group, 257 53.7 nmol/24 hours, P < 0.01). Lipid peroxidation products in the urine returned to basal levels at the end of the high-salt period (8 weeks; Figure 8).
Figure 8.
Malondialdehyde (MDA) urinary excretion. Urinary MDA excretion is increased after angiotensin infusion in both experimental groups in relationship to basal (pre-angiotensin) values, but MMF treatment reduced the urinary lipid peroxidation products. Symbols are: () Ang II group; () Ang II + MMF group. Data are mean SD; **P < 0.01.
DISCUSSION
Johnson and Schreiner have theorized that salt-sensitive essential hypertension could be the result of microvascular and tubulointerstitial damage that would interfere with the pressure-natriuresis physiologic mechanism2. In support of this hypothesis, they have presented evidence derived from experimental1,3,4,5,6,7, as well as clinical conditions24,25,26,27,28 in which focal or diffuse interstitial disease results in hypertension when the salt content of the diet is increased. Since the histologic appearance of all of the experimental models and clinical conditions mentioned previously in this article include infiltration of mononuclear cells, we further hypothesized that these cells could play a determinant role in the interstitial injury and, thereby, in the pathogenesis of SSHTN.
We decided to evaluate the effect of MMF treatment in the model of salt-sensitive hypertension induced by Ang II infusion1 because this drug has a variety of potentially beneficial actions in this model. Not only is MMF a selective lymphocyte immunosuppressive agent11, but it also inhibits proliferation of resident renal cells14,15 and collagen deposition16. Furthermore, MMF inhibits the production of lymphocyte and macrophage-derived transforming growth factor-
, tumor necrosis factor-
, interferon-
in specific experimental models29,30 and reduces T helper cell-1 (Th1)31 and Th232 cytokine responses. The dose, route of administration, and preparation of the drug prior to use were similar to our previously reported experiments12,13.
Mycophenolate mofetil or vehicle was administered during the period of Ang II infusion and during the washout phase. MMF was not administered during the high-salt diet phase to eliminate the possibility of potential direct effects of MMF on blood pressure. Hypertension was initially less pronounced in the Ang II + MMF group than in the Ang II group, but after two weeks of Ang II infusion, hypertension was similar in both groups, and during the washout period, the blood pressure returned to normal in both groups Figure 1.
The major finding in this study was that MMF treatment effectively prevented the development of hypertension in the subsequent phase when rats were placed on a high-salt diet. Whereas the Ang II group developed progressive hypertension (systolic blood pressure of 170 27 mm Hg at the 8th week; Figure 1), the MMF-treated group maintained the blood pressure within the 95% CI of the control group Figure 1.
Several mechanisms may be responsible for the protection offered by MMF from post-Ang II SSHTN. First, there is a reduction in the tubulointerstitial damage (PAS score) induced by Ang II infusion Figure 9a, b. Surprisingly, OPN expression, a marker of Ang II-induced tubular injury33, was not reduced by MMF treatment. However, this may reflect the fact that OPN is stimulated by ischemia34, and MMF may not block the acute vasoconstrictive and hemodynamic effects of Ang II. The reduction in tubulointerstitial damage may be a consequence of suppression of the local inflammatory reaction fueled by the infiltration of immunocompetent cells. As expected11,12,13, MMF treatment was associated with a reduction in infiltration of lymphocytes (Figures 5 and 9e, f), lymphocyte activation (IL-2 receptor expression; Figures 6 and 10a), and to a lesser extent, macrophages. A second mechanism is suggested by the novel observation that Ang II is expressed in infiltrating mononuclear cells located in the cortical and juxtamedullary tubulointerstitial areas and that the number of these Ang II-positive cells is significantly reduced by MMF treatment.
These findings are not totally unexpected, since increased renal production of Ang II35,36 and angiotensin-converting enzyme expression37 have been reported in angiotensin infusion experiments, and several investigations have shown that mononuclear cells may express angiotensin-converting enzyme and produce Ang II38,39,40,41,42,43,44,45,46,47. In fact, our double-staining studies demonstrate that approximately 40% of the lymphocytes and 10% of the macrophages in tubulointerstitium stain positive for Ang II at two and eight weeks. Since the proportion of Ang II-positive lymphocytes and macrophages did not change with MMF treatment, it is likely that the decrease in the number of Ang II-positive tubulointerstitial cells obtained with MMF is related to the reduction of the immune infiltrate. Suppression of renal Ang II is likely associated with reduction in the inflammatory reactivity and oxidative stress48,49,50 as well as with improvement in the physiologic mechanisms for sodium excretion, including pressure natriuresis51.
The observed distribution of Ang II receptors in the kidney is in agreement with the description of others38. We found no changes in the expression of Ang II receptors in the experimental groups with respect to control (sham) values. Previous studies on the expression of AT1 receptors in the kidney after Ang II infusions have produced conflicting results37,52,53,54,55. Since AT1 receptors can be expressed by mononuclear cells46,47,56, we performed double-staining experiments that failed to show their presence in the infiltrating lymphocytes. The possibility that in Ang II-positive lymphocytes, antibody recognition of type 1 receptors would be prevented by ligand (Ang II) fixation is considered unlikely because antibodies to type 1 receptors are made to a 10 amino acid peptide sequence near the N-terminus of rat Ang II, which is located at a site different from the ligand binding site (personal communication; A. Masarrat, Director of Alpha Diagnostic International, San Antonio, TX, USA).
An additional potential mechanism for the protective effects of MMF on the post-Ang II salt-sensitive hypertension is the reduction in oxidative stress. A role for oxidative stress in renal injury [reviewed in 57] as well as in hypertension58,59 has been reported. In angiotensin-mediated hypertension, increased vascular superoxide production results from activation of the NADH/NADPH oxidase system50. Whether resulting from its action as lymphocyte immunosuppressant or as a consequence of a decrease in local Ang II, MMF reduced the number of superoxide-producing cells Figure 10b and the amount of MDA production Figure 8.
One concern of the MMF treatment is the potential development of gastrointestinal side-effects. Diarrhea and lack of food intake could result in loss of weight and volume contraction that would minimize the hypertensive response to a high-salt diet. In our rats, there was no diarrhea, and the changes in weight were similar in the groups with and without MMF treatment, consisting of weight loss during Ang II infusion and subsequent weight gain during the high-salt period Figure 2. We did not measure food consumption; nevertheless, the weight loss during Ang II infusion was probably due to both an increased metabolic rate and relative anorexia resulting from severe hypertension and the subcutaneous Ang II minipumps. During the period of high-salt diet, when the rats were not receiving angiotensin or MMF, both groups gained weight in an almost similar fashion without reaching the 95% CI of the body weight in the control group Figure 2. Despite maintaining a similar weight at every week in the high-salt period, hypertension developed in the Ang II group, while the Ang II + MMF group remained normotensive Figure 1.
Since infiltration of mononuclear cells in the kidney is a feature present in experimental1,6,7,60,61 and genetic models62 of hypertension as well as in essential hypertension in humans27, it is attractive to consider a pathogenetic role for immunocompetent cells in SSHTN. Such a role could be the development of a chronic low-grade inflammatory process in the tubulointerstitial areas with increased local generation of Ang II, oxidants, renal vasoconstriction, and impairment of the physiologic mechanisms for sodium excretion. Nevertheless, other nonimmune mechanisms may also be participating in the beneficial effects of MMF, including a reduction in proliferative activity of resident renal cells14,15,16 and suppression of angiotensin-driven cytokines63. Further studies are needed to clarify these issues.
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Acknowledgments
Financial support for these studies was provided by the Asociación de Amigos del Riñón, Maracaibo, Venezuela; U.S. Public Health Service Grants DK-43422, DK-52121, and DK-47659; and Ministerio de Educación (97/0085), Fondo de Investigaciones Sanitarias (99/0425), Spain.
