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Nephrology Research and Training Center, Comprehensive Cancer Center, and Cell Adhesion and Matrix Research Center, Division of Nephrology, Departments of Medicine and of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham
Department of Veterans Affairs Medical Center, Birmingham, Alabama
ABSTRACT
The Dahl/Rapp salt-sensitive (S) rat is a model of salt-sensitive hypertension and hypertensive renal disease. This study explored the role of vascular remodeling in the development of renal failure in S rats. Groups of S and Sprague-Dawley rats were given 0.3 and 8.0% NaCl diets for up to 21 days and evidence of smooth muscle proliferation identified using immunohistochemistry that showed nuclear accumulation of proliferating cell nuclear antigen and 5-bromo-2'-deoxy-uridine. Compared with the other three groups, S rats on 8.0% NaCl diet showed increased nuclear labeling of cells of the aorta and arteries and arterioles of the kidney by the end of the first week of study. Progressive luminal narrowing of the interlobular arteries and preglomerular arterioles occurred in S rats over the 3 wk on the 8.0% NaCl diet. Accumulation of pimonidazole adducts and nuclear accumulation of hypoxia-inducible factor-1 (HIF-1) were used as markers of tissue hypoxia. By the end of the second week of study, pimonidazole levels increased in S rats on 8.0% NaCl diet and deposition was apparent in tubular cells in the cortex and medulla. At the completion of the experiment, HIF-1 levels were increased in nuclear extracts from the cortex and medulla of S rats on this diet, compared with the other three groups of rats. The data demonstrated a disorder of the vascular remodeling process with proliferation of vascular smooth muscle cells temporally followed by development of tissue hypoxia in the hypertensive nephropathy of S rats on 8.0% NaCl diet.
salt-sensitive hypertension; kidney disease; hypoxia; hypoxia-inducible factor-1; pimonidazole
RENAL FAILURE FROM HYPERTENSION is the second most common cause of end-stage kidney disease in the United States (25). However, hypertension is a very common medical problem, occurring in as many as 43 million individuals (3), so only about 1 in 2,500 hypertensive patients develops clinically important end-stage kidney failure from hypertension. While several interpretations are possible, this low frequency of end-organ renal damage suggests a potential genetic predisposition to this complication.
The Dahl/Rapp salt-sensitive (S) rat is an inbred strain that serves as an excellent model of salt-sensitive hypertension. These animals also have a striking predisposition to develop progressive renal failure; within 3 wk following the development of hypertension, S rats uniformly demonstrated a severe reduction in glomerular filtration rate (6). Renal morphological abnormalities consisted of mesangial expansion and tubular atrophy with tubular epithelial cell dropout from apoptosis (22, 31, 36), but the striking changes were observed in the vessels. Small arteries and arterioles, which were indistinct in the kidneys of the Dahl/Rapp salt-resistant (R) rats and S rats treated to maintain normal blood pressures, were prominent in the kidneys of untreated, hypertensive S rats (6). Morphometric analysis demonstrated an increase in wall thickness of the interlobular arteries and preglomerular arterioles, whereas qualitative light microscopic and ultrastructural analyses suggested that the vascular remodeling that occurred in these hypertensive rats included excess matrix deposition and increased numbers of smooth muscle cells in the vessel wall (6).
The intent of the present study was to characterize further the vascular remodeling process in S rats, focusing particularly on vascular smooth muscle proliferation. To determine if this vigorous remodeling contributed to the progressive renal failure in S rats, markers of tissue hypoxia were also examined over a 3-wk time frame.
MATERIALS AND METHODS
Animal preparation. The Institutional Animal Care and Use Committee at the University of Alabama at Birmingham approved the project. Studies were conducted using 86 male Sprague-Dawley (SD) rats and 86 Dahl/Rapp salt-sensitive (S) rats. The rats were obtained from Harlan Sprague Dawley (Indianapolis, IN) and were 28 days of age at the start of study. The protocol that was followed has been standardized in our laboratory (3234). The rats were housed under standard conditions and given formulated diets (AIN-76A, Dyets, Bethlehem, PA) that contained 0.3 or 8.0% NaCl. These diets were prepared specifically to be identical in protein composition and differed only in NaCl and sucrose content. The rats were studied at baseline and days 7, 14, and 21 of the study. Systolic blood pressures were determined in awake animals by tail-cuff sphygmomanometry (4). The rats were anesthetized by intraperitoneal injection of pentobarbital sodium injection (Abbott Laboratories, North Chicago, IL), 50 mg/kg body wt, and the kidneys were perfused in situ through the aorta for 2 min with 0.9% heparinized saline. Both kidneys and aorta were harvested and either placed in 4% paraformaldehyde or the cortex and medulla were dissected for protein analysis, as described below.
Proliferating cell nuclear antigen staining and analysis. Proliferating cell nuclear antigen (PCNA) is a nuclear protein that is found in dividing cells and participates integrally in DNA replication (28). The appearance of PCNA provides supportive evidence of cell proliferation. Paraffin-embedded sections were deparaffinized by immersion twice into xylene for 5 min each, followed by immersion twice for 3 min each in 100% ethanol and then 95% ethanol. Slides were rinsed for 30 s using deionized water and then immersed twice in deionized water for 5 min. Slides were covered in 1% SDS in Tris-buffered saline (TBS), which contained 100 mM Tris?HCl, pH 7.4, 138 mM NaCl, and 27 mM KCl, for 5 min at room temperature, then rinsed in TBS. The slides were immersed in 0.1% H2O2 for 10 min at room temperature and then incubated for 1 h at room temperature in 50 μM Tris?HCl, pH 7.2, containing 10% goat serum and a mouse monoclonal antibody directed against PCNA (Dako, Carpinteria, CA), 1:1,600 dilution. Slides were rinsed with PBS and covered with 10% rat serum containing a peroxidase-labeled polymer conjugated to goat anti-mouse IgG (Dako Envision System, Dako) for 1 h at room temperature. Color was developed using 3,3'-diaminobenzidine (DAB) for 5 min. Cells were counterstained using hematoxylin and the slides were mounted in standard fashion. As a negative control, the primary antibody was omitted from the reaction.
To quantify cellular proliferation, cells with nuclear staining for PCNA were counted manually in whole aortic cross-section and averaged for each of the four groups (n = 4 rats in each group) of rats at days 7 and 21 of the experiment. A total of 16 SD and 16 S rats were examined in this portion of the study. In the kidney, the entire trichrome-stained cortex was scanned for interlobular arteries and preglomerular arterioles that were completely cross-sectioned. Arteries and arterioles were identified by their anatomic location and branching pattern. Because of tapering of the interlobular artery, sections of interlobular artery located in the outer third of the cortex were excluded from analysis. Digitized photomicrographs obtained at the same magnification were projected on a monitor and the luminal diameter and outer diameter measured directly to calculate luminal area, wall area and the wall-to-lumen ratio (WTL). Nuclei present in the media were counted manually. An average of 5.9 interlobular arteries and 6.5 preglomerular arterioles were examined in each kidney. The mean diameters of the interlobular arteries and preglomerular arterioles were 49.8 ± 0.6 and 24.5 ± 0.2 μm, respectively.
BrdU labeling and analysis. Thirty-two SD and 32 S rats were used in this study. To evaluate DNA synthesis in vascular cells, 5-Bromo-2'-deoxy-uridine (BrdU; Roche Molecular Biochemicals, Indianapolis, IN), 100 mg/kg body wt, was administered intraperitoneally 18 h before examination. The dose was similar to that published by other investigators (26). Kidney and aortic tissue were fixed in 4% paraformaldehyde and then transferred to 70% ethanol until paraffin embedding. The presence of BrdU in 5-μm sections was detected using an anti-BrdU antibody and a kit (BrdU Labeling and Detection Kit II, Roche Diagnostics, Indianapolis, IN). Briefly, antigen retrieval was performed by immersing the sections in 10 mM citrate buffer, pH 6.0, and warmed in a microwave oven at 700 W for 10 min. The slides were left in the hot citrate buffer for an additional 30 min and then were rinsed for 5 min in 50 mM TBS, pH 7.6. BrdU detection proceeded following the directions supplied by the manufacturer. The number of BrdU-labeled nuclei in a cross section of aorta was counted and averaged for each of the four groups (n = 4 rats in each group) of rats at days 7 and 21 of the experiment.
In some experiments, double immunohistochemical staining was performed to demonstrate colocalization of cytoplasmic staining for smooth muscle -actin with nuclear labeling by BrdU. Deparaffinized tissue sections were dehydrated according to routine procedure. Monoclonal mouse anti-smooth muscle -actin (DakoCytomation, Carpinteria, CA), 1:200 dilution, was used initially as the primary antibody, followed by biotinylated secondary antibody and then peroxidase-conjugated streptavidin (LSAB2 Kit, DakoCytomation) and DAB, using the protocol provided by the manufacturer. Negative controls used mouse IgG2a (DakoCytomation). Following a 3-min incubation in the double-stain blocking solution, antigen retrieval proceeded as described in the preceding paragraph. The samples were then incubated in anti-BrdU monoclonal antibody (BrdU Labeling and Detection Kit II), 1:10 dilution, followed by alkaline phosphatase-conjugated anti-mouse IgG (EnVision Doublestain System, DakoCytomation), 1:10 dilution, for 30 min at 37°C and development using Fast Red Chromogen Solution (DakoCytomation).
Protein-bound pimonidazole adduct analysis using immunohistochemistry and ELISA. Nitroimidazole compounds are activated at low-oxygen concentrations and form adducts with thiol groups of proteins (18); under hypoxic conditions, the rate of formation of adducts increases and can be quantified in a variety of tissues including the kidney (1, 2, 9, 19, 23, 30). At the initiation and on days 7, 14, and 21 of the study, SD and S rats received pimonidazole hydrochloride (Hypoxyprobe-1, Chemicon International, Temecula, CA), 120 mg/kg body wt ip, 2 h before death. Paraffin-embedded 5-μm kidney sections were deparaffinized, hydrated, and covered in 3% H2O2 for 5 min. Antigen retrieval consisted of incubation in 0.01% pronase at 40°C for 40 min. Slides were then washed with 0.2% Brij in PBS for 2 min at 0°C and then incubated in a blocking solution (DakoCytomation) for 5 min at room temperature. A mouse monoclonal IgG1 (Hypoxyprobe-1MAb1, Hypoxyprobe-1 Kit, Chemicon International), 1:100 dilution, was applied to each section for 40 min at room temperature. The sections were incubated for 10 min with a biotin-conjugated F(ab')2, 1:200 dilution. After being washed with 0.02% Brij in PBS buffer, the samples were incubated with peroxidase-conjugated streptavidin (DakoCytomation), followed by DAB (DakoCytomation). The sections were counterstained with hematoxylin and analyzed in standard fashion.
For ELISA, 200 mg of kidney were homogenized and suspended in 10 volumes of PBS containing 0.05% Tween (PBS-Tween) solution. Protein concentration of the homogenates was determined using bicinchoninic acid reagent (Micro BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). The homogenates were diluted 1:1 with PBS-Tween containing 1 mg/ml of proteinase K, 20 U/mg protein, and the incubated 37°C overnight in a shaking water bath. PMSF, 200 μM, was added and the homogenates were heated for 10 min at 95°C to completely inactivate the protease. The samples were centrifuged for 10 min at 9,300 g and the supernatant was used for the ELISA. To quantify pimonidazole-protein adducts, a competitive ELISA method that was described by Raleigh (2, 19) and successfully applied to kidney tissue (23, 30, 37) was followed. Hypoxyprobe-1 antigen and rabbit polyclonal anti-Hypoxyprobe primary antisera were generously provided by Dr. James A. Raleigh in the Department of Radiation Oncology at the University of North Carolina at Chapel Hill.
Determination of nuclear levels of hypoxia-inducible factor-1 in the cortex and medulla. The protocol was similar to that published by Zou et al. (38). Briefly, dissected kidney cortical and medullary tissues from 8 S and 8 SD rats on the two diets for 21 days (n = 4 in each group) were minced and washed with PBS, then homogenized in ice-cold hypotonic buffer, which contained 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10% Nonidet P-40. The suspension was centrifuged at 10,000 g for 5 min at 4°C. The pellets were collected and incubated for 15 min in an ice-cold extraction buffer, which contained 5 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 26% glycerol. The nuclear extract was obtained by centrifugation at 32,140 g for 30 min. The nuclear extract was snap-frozen in liquid nitrogen and stored at 20°C until use. Protein concentrations were determined using bicinchoninic acid reagent (Micro BCA Protein Assay Reagent Kit, Pierce). Sixty-five micrograms of total protein were separated 8% SDS-PAGE and then transferred onto nitrocellulose membrane. After being blocked using 5% nonfat milk (Bio-Rad, Hercules, CA) in TBST (10 mM Tris?HCl, pH 8.0, 200 mM NaCl, 0.05% Tween 20) overnight at 4°C, the membranes were incubated for 4 h at room temperature with a rabbit polyclonal IgG antibody for hypoxia-inducible factor-1 (HIF-1; sc-10790, Santa Cruz Biotechnology, Santa Cruz, CA), 1:200 dilution in the blocking solution. After being washed with TBST, the blots were incubated 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), 1:10,000 dilution in blocking solution, and the bound antibody was identified and quantified using chemiluminescence (SuperSignal Chemiluminescent reagents, Pierce) and densitometry.
Statistical analysis. All data were presented as means ± SE. Statistical differences were determined using one-way analysis of variance with standard post hoc testing (Statview, version 5.0, SAS Institute, Cary, NC) for parametric data. Logarithmic transformation of the wall area, lumen area, and WTL permitted normal distribution of the data. Because the PCNA and BrdU analyses generated nonparametric data, the Kruskal-Wallis test was used to determine significance. A P value of <0.05 assigned significance.
RESULTS
Although no significant changes in kidney morphology were observed in SD rats on either diet and S rats maintained on the 0.3% NaCl diet over the 21 days of the study, S rats on the 8.0% NaCl diet demonstrated marked thickening of the arteries and arterioles of the kidney by day 21 (Fig. 1A). Associated mesangial expansion and tubular atrophy with occasional cast formation were also present at that time. These findings reproduced those obtained from this laboratory previously (6, 22). Mean systolic blood pressures increased progressively in S rats on the 8.0% NaCl diet (109 ± 2 mmHg at day 0, 135 ± 2 mmHg at day 7, 132 ± 4 mmHg at day 14, 141 ± 8 mmHg at day 21). Morphometric analyses did not demonstrate differences in mean wall areas and luminal areas of interlobular arteries and preglomerular arterioles of SD and S rats at the start of the experiment (Table 1). At 1 wk on the 8.0% NaCl diet, the mean luminal areas of both interlobular arteries and preglomerular arterioles decreased and WTL ratios increased specifically in S rats and persisted through the duration of the experiment. At 3 wk on the 8.0% NaCl diet, mean wall thickness of interlobular arteries and preglomerular arterioles of S rats was greater than the other three groups of rats. A progressive decrease in luminal area and increase in the number of nuclei in the media of the interlobular arteries and preglomerular arterioles of S rats on 8.0% NaCl were apparent.
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Immunohistochemical detection of PCNA identified nuclear labeling of smooth muscle cells throughout the arterial tree of S rats maintained on the high-salt diet and were especially prominent in the small arteries and arterioles of the kidney by day 21 of the study (Fig. 1, BE). Nuclear labeling of cells of the tubular epithelium was also striking in S rats and was similar to that reported previously (36). To confirm these findings, BrdU labeling experiments were then performed. Incorporation of BrdU into nuclei of cells of the vascular tree of S rats on the 8.0% NaCl diet was observed by day 7 and progressed over the course of the study (Fig. 2). Double immunohistochemical staining experiments showed nuclear localization of BrdU in cells that also showed staining for cytoplasmic smooth muscle -actin (Fig. 2, D and E). Quantification of nuclear PCNA and BrdU labeling of aortic cross-sections demonstrated greater (P < 0.05) numbers of labeled nuclei in aortas from S rats on the 8.0% NaCl diet, compared with the other three groups at days 7 and 21 of study (Fig. 3).
To determine if the blood supply to the kidney was compromised as a result of the vascular remodeling process, tissue hypoxia was determined using pimonidazole, which forms adducts with thiol groups at low-oxygen concentrations (18). While medullary localization of these adducts was shown in every specimen examined, staining of the tubules in the cortex was increased specifically in S rats on the 8.0% NaCl diet (Fig. 4). Protein-bound pimonidazole adducts in kidneys were quantified using an ELISA (Fig. 5). By day 14 of study, tissue pimonidazole levels were greater (P < 0.05) in S kidneys on the 8.0% NaCl diet, compared with the other three groups in the study.
As an additional indicator of tissue hypoxia, nuclear accumulation of HIF-1 in cortical and medullary tissues from rats on the diets for 21 days was quantified using western analysis (Fig. 6). A single band that migrated to the same position as that described by Zou and associates (38) was identified. The mean relative intensity of HIF-1 in nuclei from medullary tissue of S rats on 8.0% NaCl (10,145 ± 825/mm2) was greater (P < 0.05) than mean intensities of bands from S rats on 0.3% NaCl (8,277 ± 277/mm2) and SD rats on 8.0% NaCl (3,997 ± 251/mm2) and 0.3% NaCl (2,814 ± 155/mm2). Using this technique, nuclear accumulation of HIF-1 in the cortex was not quantifiable in SD rats on either diet. Mean levels of HIF-1 in the cortex of S rats on 8.0% NaCl (56,797 ± 2,144/mm2) was greater (P < 0.05) than mean levels observed in S rats on 0.3% NaCl (42,279 ± 4,192/mm2).
DISCUSSION
The Dahl/Rapp salt-sensitive (S) rat is a widely studied genetic model of salt-sensitive hypertension. Hypertensive S rats also rapidly and consistently develop end-organ kidney damage and have been used as a model of progressive renal injury. An interesting study by Cowley and associates (7) suggested that different genes confer salt-sensitive hypertension and the renal injury. A consomic strain in which chromosome 13 from the Brown-Norway strain was introgressed into the S genome demonstrated significantly less vascular reactivity and indices of tubulointerstitial damage compared with S rats on the same diet. Under the conditions of the present study, progressive increases in wall thickness and associated luminal narrowing of interlobular arteries and preglomerular arterioles occurred in S rats during the course of hypertension related to the 8.0% NaCl diet. In addition, two different methods (PCNA detection and BrdU incorporation) were used to demonstrate a role for hyperplasia of vascular smooth muscle in the exuberant vascular remodeling that emerged in the renal resistance vessels of S rats in the response to hypertension. Remodeling, however, promoted tissue hypoxia in the renal cortex and medulla. Dietary salt alone did not stimulate vascular smooth muscle proliferation, since the data did not differ between SD rats on either the low-salt or high-salt diet. The combined findings supported a primary role for dysregulated growth of vascular smooth muscle in the etiopathogenesis of hypertensive nephropathy in the Dahl/Rapp rat.
Tissue hypoxia was demonstrated using two different methods. The first method used pimonidazole, which has been shown to form adducts with thiol groups under hypoxic conditions (18) and has been used to quantify tissue hypoxia in the kidney (23, 30, 37). As shown by other investigators (30, 37), medullary deposition of pimonidazole was routinely demonstrated in all the groups under study. Compared with the other three groups, however, after two wk on the high-salt diet, pimonidazole adducts in the kidney of S rats increased, particularly in the tubular epithelium of the kidney cortex. The findings correlated with a previous study that showed the reduction of the glomerular filtration rate in S rats was present by this time point; hypertension developed within the first week in S rats on 8.0% NaCl diet, but glomerular filtration rate was preserved until the end of the second week of study (6). Pimonidazole deposition in the renal cortex was therefore considered suggestive of tissue hypoxia.
An important sensor of tissue levels of oxygen is HIF, which is a heterodimeric transcription factor that is composed of an -subunit bound to HIF-1; the biology of this system has been recently reviewed (21, 29). Hypoxia increases the cellular abundance of HIF-1 protein (10, 14, 27); the mechanism relates to oxygen-dependent proline hydroxylation of HIF-1 that in turn modulates the binding of an E3 ubiquitin ligase that contains the von Hippel-Lindau tumor suppressor protein (pVHL) (11, 12). Stabilization of HIF-1, which contains the transactivation domains, permits nuclear import by HIF-1, which possesses the nuclear transporter signal, and subsequent transcriptional activation of hypoxia-regulated genes (21, 29). HIF-1 is abundantly expressed in rodent kidney particularly in tubular epithelial cells and is regulated by changes in oxygen tension (20, 38). Because of the relative hypoxic conditions of the medulla, expression of HIF-1 is observed in normal rats (38). In the present study, using extracts of medullary tissue from SD rats on both diets, nuclear localization of HIF-1 was observed. Increased amounts of HIF-1 were seen in nuclear extracts from both the cortex and medulla of S rats on 8.0% NaCl diet for three wk, confirming a significant decrease in oxygen tension in these tissues.
A recent study by Khan and associates (15) demonstrated the appearance of tissue hypoxia with an associated increase in apoptosis of tubular epithelial cells in a murine model of progressive renal failure, providing support for the link between hypoxia and apoptosis in the kidney. ATP-depleted Madin-Darby canine kidney cells exhibit apoptosis that is dependent upon upregulation and activation of Fas and Fas ligand (FasL) (8). A major mechanism of renal failure in S rats is tubular epithelial cell apoptosis, related to activation of Fas/FasL pathway and the intrinsic pathway involving mitochondrial release of cytochrome c (22, 31, 36); both pathways can be activated by hypoxia. While albuminuria and expansion of the glomerular mesangium are part of the features of hypertensive nephropathy in S rats (6), the present findings supported an important role for vascular smooth muscle hyperplasia with the subsequent development of tissue hypoxia as the proximate cause of progressive renal failure in this genetic model of hypertension and hypertensive renal disease. The underlying mechanism may be related to endothelial dysfunction, which is observed in S rats even prior to development of hypertension and may be related to an intrinsic disturbance of growth mechanisms of vascular smooth muscle cells, or to altered production of vasoactive agents (46, 35). The absence of augmented production of NO in response to an increase in dietary salt intake (46) may also unmask intrinsic renal vasoconstriction, as suggested to occur in other models of salt-sensitive hypertension (13). The imbalance between vasoconstrictor and vasodilator influences and subsequent vascular remodeling all contribute to the impaired myogenic responses of the renal resistance vessels of S rats (24). Progressive tissue hypoxia can also explain the paradoxical activation of the intrarenal renin-angiotensin axis in S rats on 8.0% NaCl diet (16) and facilitate the imbalance between vasoconstrictors and vasodilators. S rats demonstrate a progressive increase in blood pressure as renal function deteriorates (6); the increase in intrarenal angiotensin II promotes capillary rarefaction (17), which also contributes to salt-sensitive hypertension and tissue hypoxia. Thus a positive feedback loop is generated with the final result of end-stage kidney failure in this genetic model of salt-sensitive hypertension.
GRANTS
National Institutes of Health Grant R01-DK-46199 and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs supported this work.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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