Th1 inflammatory response with altered expression of profibrotic and vasoactive mediators in AT1A and AT1B double-knockout mice
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《美国生理学杂志》
Division of Nephrology, Department of Medicine, University of Florida, Gainesville, Florida
Department of Medicine, Duke University, and Durham Veterans Affairs Medical Centers, Durham, North Carolina
Department of Nephrology, Ignacio Chavez, Mexico City, Mexico
Nephrology Service, Hospital Universitario, Maracaibo, Venezuela
ABSTRACT
AT1 double receptor (AT1A and AT1B) knockout mice have lower blood pressure, impaired growth, and develop early renal microvascular disease and tubulointerstitial injury. We hypothesized that there would be an increased expression of vasoactive, profibrotic, and inflammatory mediators expressed in the kidneys of AT1 double-knockout mice. We examined the renal expression of various mediator systems in control (n = 6) vs. double-knockout mice (n = 6) at 3–5 mo of age by real-time PCR, immunohistochemistry, and Western blot analysis. AT1 double-knockout mice show activation of Th1-dependent pathways (with increased expression of IFN-, IL-2 mRNA) with increased expression of both monocyte (MCP-1 mRNA) and T cell (RANTES mRNA) chemokines, infiltration of CD4+ and CD11b+ cells, increased fibrosis-associated mediators (CTGF, TGF- and TNF- mRNA) and extracellular matrix (collagens I and III mRNA and protein) deposition compared with controls (P < 0.05 for all markers). These changes were associated with increased mRNA expression of endothelin (ET)-1 and ET-A receptor (P < 0.05), cyclooxygenase (COX)-2/TXA2 synthase (P < 0.05), NADPH oxidase (p40-phox, p67-phox, P < 0.05) and iNOS and nNOS (P < 0.05). COX-2 and nNOS protein were also increased in the kidneys of AT1 double-knockout mice by Western blot analysis (P < 0.05). Although renin and angiotensinogen mRNA expression were increased in the knockout mice, AT2 receptor mRNA expression was not significantly different from wild-type mice. In conclusion, the absence of the AT1 receptor is associated with marked renal alterations in vasoactive, profibrotic, and immune mediators with an inflammatory pattern favoring a Th1 phenotype.
endothelin; cyclooxygenase-2; NADPH oxidase
THE RENIN-ANGIOTENSIN SYSTEM (RAS) modulates a diverse set of physiological processes including development, blood pressure, renal function, and inflammation. In the kidney, all of the components of the RAS [renin, angiotensinogen, angiotensin-converting enzyme (ACE), ANG II, and ANG II type 1 and 2 receptors (AT1 and AT2)] are synthesized locally (2).
In humans, there is only a single AT1 receptor type, whereas in rodents, two subtypes of the AT1 receptor have been identified (AT1A and AT1B). To date, AT1 receptors have been shown to mediate most of the physiological actions of ANG II and this subtype is predominant in the control of ANG II-induced vascular functions (15). The AT1 receptor mediates most of the deleterious effects of ANG II, such as vasoconstriction, endothelial damage, and cell growth. AT1 receptor blockade appears to offer both active and passive therapeutic benefits (30). However, the absence of a functional RAS has been associated with development of microvascular disease and tubulointerstitial inflammation. This is observed in mice genetically lacking renin (33), angiotensinogen (11), ACE (12), and AT1 receptors (19). Renal microvascular disease has also been reported in marmosets or Wistar-Kyoto rats immunized against renin (16, 17) and in neonatal spontaneously hypertensive or Wistar rats treated with AT1 receptor inhibitors or ACEI (3, 5, 20, 29).
Characterization of the inflammatory changes in AT1A and AT1B double-knockout mice has not been previously described. We hypothesized that the inflammatory changes would be associated with marked alterations in vasoactive and profibrotic mediators. We further hypothesized that the inflammatory response might involve both T cells and macrophages.
MATERIALS AND METHODS
Animals. Kidneys harvested from double homozygous Agtr1a –/– Agtr1b –/– mice were provided by the Thomas M. Coffman Laboratories (Duke University and Durham VA Medical Centers). These mice were produced as previously described (19). Because of poor survival of animals with combined AT1A/AT1B receptor deficiencies, animals for experiments were generated by selective breeding on mixed backgrounds of 129/SvEv and C57BL/6. Wild-type mice of similar mixed backgrounds were used as controls. All experiments were performed using male mice between 3–5 mo of age. All mice were bred and maintained in the American Association for Accreditation of Laboratory Animal Care-accredited animal facility of the Durham Veterans Affairs Medical Center under National Institutes of Health guidelines. Animals were anesthetized with isoflurane, blood was collected by a heart stick under the xiphoid using a 1-ml syringe and a 25-gauge needle, and kidney tissue samples were collected for RNA, protein, and immunohistochemical analysis.
Renal pathological and histological studies. Renal tissues were fixed in MethylCarnoy's fixative, and 3-μm paraffin sections were stained with periodic acid-Schiff (PAS) and hematoxylin. Immunohistochemistry was performed using the following affinity-purified primary antibodies: rabbit polyclonal antibodies against nNOS (Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal antibodies against cyclooxygenase (COX)-2 (Santa Cruz Biotechnology), rat polyclonal antibodies against CD11b (BD Pharmingen, San Diego, CA), rat polyclonal antibodies against CD4 (BD Pharmingen), goat polyclonal antibodies against collagen (Col) I and Col III (Southern Bioteck, Birmingham, AL), monoclonal antibody against -SMA (1A4, Sigma, St. Louis, MO). The following secondary antibodies (Rockland) were used in this study: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit anti-goat IgG. For immunohistochemistry, renal tissues were fixed in 4% (wt/vol) buffered paraformaldehyde. Cryosections (4 μm) were stained as previously described (13). Sections were preincubated with 10% fetal calf serum and 10% normal sheep serum for 20 min and then incubated with first antibody as described above overnight at 4°C. Sections were then washed in PBS, inactivated with endogenous peroxidase in 0.3% H2O2 in methanol, labeled with second antibody as described above followed by mouse peroxidase anti-peroxidase, and developed with DAB substrate kit (Vector Laboratories, Burlingame, CA) to produce a brown color, or developed with DCIP/NBT substrate kit (Vector Laboratories) to produce a blue color.
For quantification of immunohistochemistry staining, PAS-stained sections were imaged using a Axioplan 2 imaging microscope (Zeiss), CR5 digitized color camera, image analyzed using Zeiss Auto image software (Axiovision 4.1), and a 133-MHz Pentium computer (96 Mb RAM). For studies of vascular and glomerular morphology, cross sections of all the arteriole were traced and examined morphometrically. Both inner and outer areas (μm2) were measured for each arteriole, and outer area-inner area (designated as arterial wall thickness) was calculated and compared among wild-type and AT1 receptor knockout mice.
For studies of glomerular morphology, both glomerular tuft and whole glomerular areas (μm2) were measured, and whole glomerular area/glomerular tuft (designated as glomerular tuft/whole glomerular ratio) was calculated and compared among wild-type and AT1 receptor knockout mice. Single image frames (700 x 550 μm) were captured at x200 magnification, and 20 frames/sample were used to count the number of CD4 and CD11b-positive cells [expressed as cell number/(700 x 550 μm)], and for quantification of Col I, Col III and -SMA [positive area /(700 x 550 μm)].
RNA isolation, reverse transcription, and real-time PCR. Total RNA was isolated from total kidney tissue using the SV Total RNA Isolation kit (Promega, Madison, WI) according to the manufacturer's protocol. The RNA was eluted with 50 μl of RNase-free water. All RNA was quantified by spectrophotometer and the optical density (OD) 260/280 nm ratios were determined. Reverse transcription was performed in a one-step protocol using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocols. Reactions were incubated at 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, and cooled at 4°C in a Thermocycler (Eppendorf, Hamburg, Germany). Primers (Table 1) were designed by Genetool software (BioTools, Alberta, Canada), and oligonucleotides were synthesized by Sigma Genosys. Real-time PCR analyses were performed using the Opticon PCR machine (MJ Research, Waltham, MA). The SYBR Green master mix kit (Bio-Rad) was used for all reactions with real-time PCR. Briefly, PCR was performed as follows: 94°C for 2 min followed by 40 cycles of denaturation, annealing, and extension at 94°C for 15 s, 64°C for 30 s, 72°C for 45 s, respectively, and final extension at 72°C for 10 min. PCR reaction for each sample was done in duplicate for all the product and for the GAPDH control. Ratios for each product/GAPDH mRNA were calculated for each sample and expressed as means ± SD. The data presented are expressed as the fold-increase or fold-decrease in mRNA.
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Western blot analysis. Whole kidney samples were mixed with SDS-polyacrylamide gel electrophoresis sample buffer (Cell Signaling), boiled for 5 min, and electrophoresed on a 7.5% SDS polyacrylamide gel (Bio-Rad). Proteins were transferred onto Immobilon-NC Transfer Membrane (Millipore) with a Bio-Rad Transblot cell at 0.5 A overnight. The membrane was blocked in TBS (Bio-Rad) containing 0.05% Tween 20 for 2 h and then incubated overnight with rabbit anti-mouse nNOS antibody or goat anti-mouse COX-2 antibody (Santa Cruz Biotechnology). After being washed, the membrane was incubated with a 1/5,000 dilution of fluorescence-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG in TBS containing 5% skim milk powder and 0.05% Tween 20 for 1 h. Blots were developed using Odyssey Infrared Imager (Odyssey) and analyzed by densitometry using Odyssey Image Software (Version 1.2).
Serum uric acid measurement. Serum uric acid concentration was determined by a carbonate phosphotungstate method and uric acid standard (Sigma) (6).
Statistical analysis. All data are presented as means ± SD. Differences in the various parameters between groups were evaluated by single-factor ANOVA. Significance was defined as P < 0.05.
RESULTS
Renal vascular disease and fibrosis in AT1 receptor knockout mice. The routine light microscopy study of the kidney was normal in the wild-type group. In contrast, the kidneys in the AT1 receptor knockout mice demonstrated focal areas of interstitial fibrosis with mononuclear cell infiltration (Fig. 1). Immunohistochemistry demonstrated expression of Col I and Col III in the fibrotic areas (Fig. 2). An increase in Col I and III mRNA was also observed in whole kidney RNA extracts (Fig. 2). Preglomerular arteriolar vessels were thickened, resulting in a threefold increased medial area compared with wild-type controls (Fig. 3). The increase in medial wall thickness was associated with dramatic increases in -SMA expression (Fig. 4). In addition, AT1 receptor knockout mice displayed an increase in -SMA-positive cells in glomeruli (reflecting activated mesangial cells) and in areas of periglomerular and interstitial fibrosis (reflecting myofibroblasts; Fig. 4). Glomeruli displayed evidence of ischemia, with wrinkling of the basement membrane and collapse of glomerular tufts (Fig. 3). The ratio of glomerular tuft to total glomerular area (demarcated by Bowman's space) was significantly lower in the AT1 receptor knockout mice, reflecting the higher frequency of glomerular collapse in these mice (Fig. 3).
T cell and monocyte infiltration in AT1 receptor knockout mice: association with a Th1 phenotype and oxidative stress. Infiltration of both monocytes (CD11b-positive cells) and CD4-positive T cells were present in the cortex of AT1 receptor knockout mice, especially in areas of interstitial fibrosis, compared with the wild-type mice (Fig. 1). Real-time PCR on the whole kidneys demonstrated marked expression of proinflammatory cytokines and mediators of oxidative stress (Fig. 5). An increase in chemotactic factors for both monocytes (monocyte chemotactic factor 1, or MCP-1) and T cells (RANTES) was observed. There was also a marked increase in the mRNA for enzymes involved in oxidative stress, including p40-phox, p67-phox, and gp91-phox (the phagocyte NADPH oxidase) and the xanthine oxidase (XO)/xanthine dehydrogenase (XDH) systems (Fig. 5). The increase in XO expression was associated with higher serum uric acid levels, although this did not reach statistical significance (1.1 ± 0.3 vs. 1.6 ± 0.5 mg/dl, P = 0.06). Interestingly, there was a relative downregulation of NOX4, which is the major catalytic component of an endothelial NAD(P)H oxidase. Consistent with the inflammatory and profibrotic response, we observed an increase in TNF- mRNA and TGF- mRNA, respectively, in the AT1 receptor knockout mice (Fig. 5).
Given the infiltration of T cells in the kidneys of AT1 receptor knockout mice, we performed real-time PCR to determine whether the phenotype was consistent with a Th1 or Th2 response (Fig. 5). AT1 receptor knockout mice demonstrated a Th1 phenotypic pattern in their kidneys, as manifested by increased mRNA expression of IFN- and IL-2 (Th1 cytokines), whereas IL-4 and IL-10 (Th2 cytokine) mRNA levels were not changed.
AT1 receptor knockout mice have evidence for activation of vasoactive pathways in their kidneys. Consistent with previous studies, AT1 receptor knockout mice had an increase in renin and angiotensinogen mRNA in their kidneys compared with wild-type mice (Fig. 6). Interestingly, neither ACE nor AT2 receptor mRNA expression was altered (Fig. 6). There was also evidence for activation of the endothelin system, with upregulation of ET-1 and ET-A receptor mRNA but not ET-B receptor mRNA, consistent with activation of the endothelin vasoconstriction. AT1 receptor knockout mice also demonstrated increased expression of nNOS protein (Fig. 7) and COX-2 (Fig. 8), consistent with previous studies suggesting activation of these pathways involved in tubuloglomerular feedback (10). Inducible NOS, which is present in tubules and inflammatory cells and has been reported to be increased in conditions associated with renal inflammation and injury (4), was also increased. Interestingly, endothelial NOS (eNOS), an important vasodilatory enzyme, was not altered (Fig. 6).
DISCUSSION
ANG II, the main effector of the RAS, plays a central role in the hemodynamic and nonhemodynamic mechanisms of chronic renal disease and is currently the main target of interventions aimed to prevent the onset of chronic nephropathies and their progression to end-stage renal disease (24). ANG II is known as a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of ANG II with AT1 and AT2 receptors. AT1 receptor activation leads to cell growth, vascular contraction, inflammatory responses, and salt and water retention (27). AT1 receptor blockade appears to offer both active and passive therapeutic benefits in humans, but interestingly, the absence of AT1 receptor has been associated with development of microvascular disease and tubulointerstitial inflammation in kidney, the same phenomenon observed in experimental and human salt-sensitive hypertension (9, 23, 32). In these latter models, a key role for T cells (bearing a Th1 phenotype), oxidative stress, and renal vasoconstriction has been reported (22). We thus hypothesized that there would be an alteration in vasoactive and profibrotic mediators and that the inflammatory response would include both T cells and macrophages, with the former expressing a Th1 phenotype.
We first confirmed previous studies that demonstrated that AT1 receptor knockout mice develop premature vascular disease and interstitial fibrosis (19, 28). The vascular disease was marked and was associated with an increase in -SMA-positive cells and by the marked increase in arteriolar medial area and medial:lumen ratios (Figs. 1 and 3). The vascular disease was also associated with glomerular collapse with shrinkage of the glomerular tuft, suggesting glomerular ischemia. Focal area of fibrosis was also present, as demonstrated by the increase in Col I and III staining, and an increase in TGF- mRNA by quantitative real-time PCR.
The first new finding was the characterization of the inflammatory cells and cytokines in the AT1 receptor knockout mice. Specifically, we found that the renal tissue had elevated levels of both monocyte (MCP-1) and T cell (RANTES) cytokine mRNA expression in association with an increase in monocytes and CD4-positive T cells. The T cells were likely of a Th1 phenotype, as the kidneys also have a marked induction of mRNA expression of Th1 cytokines (IFN- and IL-2) but not Th2 (IL-4 and IL-10) cytokines. There was also evidence for the increased expression of monocyte/macrophage inflammatory cytokines (TNF- mRNA). The local inflammatory response was associated with upregulation of oxidative enzymes, including the NADPH oxidase system and the XO enzymes. Thus the renal injury in AT1 receptor knockout is associated with a T cell and monocyte response with local activation of inflammatory mediators and oxidative enzyme systems.
The second new finding was the observation that there was an induction of cytokines and vasoactive mediators. An upregulation of proximal pathways involved in the RAS, such as renin and angiotensinogen, was expected. However, we also found an increased expression of the renal endothelin system (including ET-1 and the ET-A receptor) consistent with a vasoconstrictive response. Although eNOS expression was not modulated in AT1 receptor-deficient state, nNOS and COX-2 mRNA and protein were increased in our AT1 receptor knockout mice consistent with a vasodilatory response. An increase in the nNOS and COX-2 pathways has been previously reported in rodents in which the RAS is blocked, and this is thought to represent a consequence of interrupting the negative feedback of ANG II on the nNOS-COX-2 pathways involved in tubuloglomerular feedback (1, 10, 31). Moreover, the afferent arteriolar diameter might be directly regulated by NO derived from nNOS in the macula densa (26), and COX-2 participates in tubular flow-dependent afferent arteriolar tone via interation with nNOS (7).
An interesting observation in the current study was the finding that both the NADPH oxidase isoform P40-phox and P67-phox mRNA were increased in the AT1 receptor knockout mice, whereas a crucial flavin-containing catalytic subunit of NADPH oxidase, Nox4, was reduced. Unlike other subunits, neither Nox1 nor Nox4 is present in leukocytes but is highly expressed in vascular cells and upregulated in vascular remodeling, such as that found in hypertension and atherosclerosis (8). This suggests that the upregulation of NADPH oxidase in the kidney of AT1 receptor knockout mice may largely represent activation by infiltrating monocytes and macrophages, which have been shown to produce oxidants in other models of salt-sensitive hypertension (14, 24, 25, 34). One might posit that NADPH oxidases should also be induced in the preglomerular vessels, as activation of these oxidases has been shown to play a critical role in vascular remodeling (18). However, most studies suggest that the induction of NADPH oxidases in vascular cells is dependent on ANG II (21), raising the possibility that other mechanisms may be required when the RAS is completely blocked.
In conclusion, while ANG II has been definitively shown to have a role in mediating inflammation and fibrosis in the kidney, in this study we demonstrate that a complete absence of AT1 receptor is also associated with the development of preglomerular vascular disease and interstitial inflammation. Characterization suggests the induction of chemokines driving a Th1 and monocytic/macrophage response coupled with oxidative stress, an alteration in vasoactive mediators, and fibrosis.
GRANTS
This study was supported by National Institutes of Health Grants DK-52121, HL-68607, and George O'Brien Center Grant DK-64233 (to R. J. Johnson). This study was also supported by the Gatorade Fund, University of Florida.
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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Department of Medicine, Duke University, and Durham Veterans Affairs Medical Centers, Durham, North Carolina
Department of Nephrology, Ignacio Chavez, Mexico City, Mexico
Nephrology Service, Hospital Universitario, Maracaibo, Venezuela
ABSTRACT
AT1 double receptor (AT1A and AT1B) knockout mice have lower blood pressure, impaired growth, and develop early renal microvascular disease and tubulointerstitial injury. We hypothesized that there would be an increased expression of vasoactive, profibrotic, and inflammatory mediators expressed in the kidneys of AT1 double-knockout mice. We examined the renal expression of various mediator systems in control (n = 6) vs. double-knockout mice (n = 6) at 3–5 mo of age by real-time PCR, immunohistochemistry, and Western blot analysis. AT1 double-knockout mice show activation of Th1-dependent pathways (with increased expression of IFN-, IL-2 mRNA) with increased expression of both monocyte (MCP-1 mRNA) and T cell (RANTES mRNA) chemokines, infiltration of CD4+ and CD11b+ cells, increased fibrosis-associated mediators (CTGF, TGF- and TNF- mRNA) and extracellular matrix (collagens I and III mRNA and protein) deposition compared with controls (P < 0.05 for all markers). These changes were associated with increased mRNA expression of endothelin (ET)-1 and ET-A receptor (P < 0.05), cyclooxygenase (COX)-2/TXA2 synthase (P < 0.05), NADPH oxidase (p40-phox, p67-phox, P < 0.05) and iNOS and nNOS (P < 0.05). COX-2 and nNOS protein were also increased in the kidneys of AT1 double-knockout mice by Western blot analysis (P < 0.05). Although renin and angiotensinogen mRNA expression were increased in the knockout mice, AT2 receptor mRNA expression was not significantly different from wild-type mice. In conclusion, the absence of the AT1 receptor is associated with marked renal alterations in vasoactive, profibrotic, and immune mediators with an inflammatory pattern favoring a Th1 phenotype.
endothelin; cyclooxygenase-2; NADPH oxidase
THE RENIN-ANGIOTENSIN SYSTEM (RAS) modulates a diverse set of physiological processes including development, blood pressure, renal function, and inflammation. In the kidney, all of the components of the RAS [renin, angiotensinogen, angiotensin-converting enzyme (ACE), ANG II, and ANG II type 1 and 2 receptors (AT1 and AT2)] are synthesized locally (2).
In humans, there is only a single AT1 receptor type, whereas in rodents, two subtypes of the AT1 receptor have been identified (AT1A and AT1B). To date, AT1 receptors have been shown to mediate most of the physiological actions of ANG II and this subtype is predominant in the control of ANG II-induced vascular functions (15). The AT1 receptor mediates most of the deleterious effects of ANG II, such as vasoconstriction, endothelial damage, and cell growth. AT1 receptor blockade appears to offer both active and passive therapeutic benefits (30). However, the absence of a functional RAS has been associated with development of microvascular disease and tubulointerstitial inflammation. This is observed in mice genetically lacking renin (33), angiotensinogen (11), ACE (12), and AT1 receptors (19). Renal microvascular disease has also been reported in marmosets or Wistar-Kyoto rats immunized against renin (16, 17) and in neonatal spontaneously hypertensive or Wistar rats treated with AT1 receptor inhibitors or ACEI (3, 5, 20, 29).
Characterization of the inflammatory changes in AT1A and AT1B double-knockout mice has not been previously described. We hypothesized that the inflammatory changes would be associated with marked alterations in vasoactive and profibrotic mediators. We further hypothesized that the inflammatory response might involve both T cells and macrophages.
MATERIALS AND METHODS
Animals. Kidneys harvested from double homozygous Agtr1a –/– Agtr1b –/– mice were provided by the Thomas M. Coffman Laboratories (Duke University and Durham VA Medical Centers). These mice were produced as previously described (19). Because of poor survival of animals with combined AT1A/AT1B receptor deficiencies, animals for experiments were generated by selective breeding on mixed backgrounds of 129/SvEv and C57BL/6. Wild-type mice of similar mixed backgrounds were used as controls. All experiments were performed using male mice between 3–5 mo of age. All mice were bred and maintained in the American Association for Accreditation of Laboratory Animal Care-accredited animal facility of the Durham Veterans Affairs Medical Center under National Institutes of Health guidelines. Animals were anesthetized with isoflurane, blood was collected by a heart stick under the xiphoid using a 1-ml syringe and a 25-gauge needle, and kidney tissue samples were collected for RNA, protein, and immunohistochemical analysis.
Renal pathological and histological studies. Renal tissues were fixed in MethylCarnoy's fixative, and 3-μm paraffin sections were stained with periodic acid-Schiff (PAS) and hematoxylin. Immunohistochemistry was performed using the following affinity-purified primary antibodies: rabbit polyclonal antibodies against nNOS (Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal antibodies against cyclooxygenase (COX)-2 (Santa Cruz Biotechnology), rat polyclonal antibodies against CD11b (BD Pharmingen, San Diego, CA), rat polyclonal antibodies against CD4 (BD Pharmingen), goat polyclonal antibodies against collagen (Col) I and Col III (Southern Bioteck, Birmingham, AL), monoclonal antibody against -SMA (1A4, Sigma, St. Louis, MO). The following secondary antibodies (Rockland) were used in this study: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit anti-goat IgG. For immunohistochemistry, renal tissues were fixed in 4% (wt/vol) buffered paraformaldehyde. Cryosections (4 μm) were stained as previously described (13). Sections were preincubated with 10% fetal calf serum and 10% normal sheep serum for 20 min and then incubated with first antibody as described above overnight at 4°C. Sections were then washed in PBS, inactivated with endogenous peroxidase in 0.3% H2O2 in methanol, labeled with second antibody as described above followed by mouse peroxidase anti-peroxidase, and developed with DAB substrate kit (Vector Laboratories, Burlingame, CA) to produce a brown color, or developed with DCIP/NBT substrate kit (Vector Laboratories) to produce a blue color.
For quantification of immunohistochemistry staining, PAS-stained sections were imaged using a Axioplan 2 imaging microscope (Zeiss), CR5 digitized color camera, image analyzed using Zeiss Auto image software (Axiovision 4.1), and a 133-MHz Pentium computer (96 Mb RAM). For studies of vascular and glomerular morphology, cross sections of all the arteriole were traced and examined morphometrically. Both inner and outer areas (μm2) were measured for each arteriole, and outer area-inner area (designated as arterial wall thickness) was calculated and compared among wild-type and AT1 receptor knockout mice.
For studies of glomerular morphology, both glomerular tuft and whole glomerular areas (μm2) were measured, and whole glomerular area/glomerular tuft (designated as glomerular tuft/whole glomerular ratio) was calculated and compared among wild-type and AT1 receptor knockout mice. Single image frames (700 x 550 μm) were captured at x200 magnification, and 20 frames/sample were used to count the number of CD4 and CD11b-positive cells [expressed as cell number/(700 x 550 μm)], and for quantification of Col I, Col III and -SMA [positive area /(700 x 550 μm)].
RNA isolation, reverse transcription, and real-time PCR. Total RNA was isolated from total kidney tissue using the SV Total RNA Isolation kit (Promega, Madison, WI) according to the manufacturer's protocol. The RNA was eluted with 50 μl of RNase-free water. All RNA was quantified by spectrophotometer and the optical density (OD) 260/280 nm ratios were determined. Reverse transcription was performed in a one-step protocol using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's protocols. Reactions were incubated at 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, and cooled at 4°C in a Thermocycler (Eppendorf, Hamburg, Germany). Primers (Table 1) were designed by Genetool software (BioTools, Alberta, Canada), and oligonucleotides were synthesized by Sigma Genosys. Real-time PCR analyses were performed using the Opticon PCR machine (MJ Research, Waltham, MA). The SYBR Green master mix kit (Bio-Rad) was used for all reactions with real-time PCR. Briefly, PCR was performed as follows: 94°C for 2 min followed by 40 cycles of denaturation, annealing, and extension at 94°C for 15 s, 64°C for 30 s, 72°C for 45 s, respectively, and final extension at 72°C for 10 min. PCR reaction for each sample was done in duplicate for all the product and for the GAPDH control. Ratios for each product/GAPDH mRNA were calculated for each sample and expressed as means ± SD. The data presented are expressed as the fold-increase or fold-decrease in mRNA.
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Western blot analysis. Whole kidney samples were mixed with SDS-polyacrylamide gel electrophoresis sample buffer (Cell Signaling), boiled for 5 min, and electrophoresed on a 7.5% SDS polyacrylamide gel (Bio-Rad). Proteins were transferred onto Immobilon-NC Transfer Membrane (Millipore) with a Bio-Rad Transblot cell at 0.5 A overnight. The membrane was blocked in TBS (Bio-Rad) containing 0.05% Tween 20 for 2 h and then incubated overnight with rabbit anti-mouse nNOS antibody or goat anti-mouse COX-2 antibody (Santa Cruz Biotechnology). After being washed, the membrane was incubated with a 1/5,000 dilution of fluorescence-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG in TBS containing 5% skim milk powder and 0.05% Tween 20 for 1 h. Blots were developed using Odyssey Infrared Imager (Odyssey) and analyzed by densitometry using Odyssey Image Software (Version 1.2).
Serum uric acid measurement. Serum uric acid concentration was determined by a carbonate phosphotungstate method and uric acid standard (Sigma) (6).
Statistical analysis. All data are presented as means ± SD. Differences in the various parameters between groups were evaluated by single-factor ANOVA. Significance was defined as P < 0.05.
RESULTS
Renal vascular disease and fibrosis in AT1 receptor knockout mice. The routine light microscopy study of the kidney was normal in the wild-type group. In contrast, the kidneys in the AT1 receptor knockout mice demonstrated focal areas of interstitial fibrosis with mononuclear cell infiltration (Fig. 1). Immunohistochemistry demonstrated expression of Col I and Col III in the fibrotic areas (Fig. 2). An increase in Col I and III mRNA was also observed in whole kidney RNA extracts (Fig. 2). Preglomerular arteriolar vessels were thickened, resulting in a threefold increased medial area compared with wild-type controls (Fig. 3). The increase in medial wall thickness was associated with dramatic increases in -SMA expression (Fig. 4). In addition, AT1 receptor knockout mice displayed an increase in -SMA-positive cells in glomeruli (reflecting activated mesangial cells) and in areas of periglomerular and interstitial fibrosis (reflecting myofibroblasts; Fig. 4). Glomeruli displayed evidence of ischemia, with wrinkling of the basement membrane and collapse of glomerular tufts (Fig. 3). The ratio of glomerular tuft to total glomerular area (demarcated by Bowman's space) was significantly lower in the AT1 receptor knockout mice, reflecting the higher frequency of glomerular collapse in these mice (Fig. 3).
T cell and monocyte infiltration in AT1 receptor knockout mice: association with a Th1 phenotype and oxidative stress. Infiltration of both monocytes (CD11b-positive cells) and CD4-positive T cells were present in the cortex of AT1 receptor knockout mice, especially in areas of interstitial fibrosis, compared with the wild-type mice (Fig. 1). Real-time PCR on the whole kidneys demonstrated marked expression of proinflammatory cytokines and mediators of oxidative stress (Fig. 5). An increase in chemotactic factors for both monocytes (monocyte chemotactic factor 1, or MCP-1) and T cells (RANTES) was observed. There was also a marked increase in the mRNA for enzymes involved in oxidative stress, including p40-phox, p67-phox, and gp91-phox (the phagocyte NADPH oxidase) and the xanthine oxidase (XO)/xanthine dehydrogenase (XDH) systems (Fig. 5). The increase in XO expression was associated with higher serum uric acid levels, although this did not reach statistical significance (1.1 ± 0.3 vs. 1.6 ± 0.5 mg/dl, P = 0.06). Interestingly, there was a relative downregulation of NOX4, which is the major catalytic component of an endothelial NAD(P)H oxidase. Consistent with the inflammatory and profibrotic response, we observed an increase in TNF- mRNA and TGF- mRNA, respectively, in the AT1 receptor knockout mice (Fig. 5).
Given the infiltration of T cells in the kidneys of AT1 receptor knockout mice, we performed real-time PCR to determine whether the phenotype was consistent with a Th1 or Th2 response (Fig. 5). AT1 receptor knockout mice demonstrated a Th1 phenotypic pattern in their kidneys, as manifested by increased mRNA expression of IFN- and IL-2 (Th1 cytokines), whereas IL-4 and IL-10 (Th2 cytokine) mRNA levels were not changed.
AT1 receptor knockout mice have evidence for activation of vasoactive pathways in their kidneys. Consistent with previous studies, AT1 receptor knockout mice had an increase in renin and angiotensinogen mRNA in their kidneys compared with wild-type mice (Fig. 6). Interestingly, neither ACE nor AT2 receptor mRNA expression was altered (Fig. 6). There was also evidence for activation of the endothelin system, with upregulation of ET-1 and ET-A receptor mRNA but not ET-B receptor mRNA, consistent with activation of the endothelin vasoconstriction. AT1 receptor knockout mice also demonstrated increased expression of nNOS protein (Fig. 7) and COX-2 (Fig. 8), consistent with previous studies suggesting activation of these pathways involved in tubuloglomerular feedback (10). Inducible NOS, which is present in tubules and inflammatory cells and has been reported to be increased in conditions associated with renal inflammation and injury (4), was also increased. Interestingly, endothelial NOS (eNOS), an important vasodilatory enzyme, was not altered (Fig. 6).
DISCUSSION
ANG II, the main effector of the RAS, plays a central role in the hemodynamic and nonhemodynamic mechanisms of chronic renal disease and is currently the main target of interventions aimed to prevent the onset of chronic nephropathies and their progression to end-stage renal disease (24). ANG II is known as a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of ANG II with AT1 and AT2 receptors. AT1 receptor activation leads to cell growth, vascular contraction, inflammatory responses, and salt and water retention (27). AT1 receptor blockade appears to offer both active and passive therapeutic benefits in humans, but interestingly, the absence of AT1 receptor has been associated with development of microvascular disease and tubulointerstitial inflammation in kidney, the same phenomenon observed in experimental and human salt-sensitive hypertension (9, 23, 32). In these latter models, a key role for T cells (bearing a Th1 phenotype), oxidative stress, and renal vasoconstriction has been reported (22). We thus hypothesized that there would be an alteration in vasoactive and profibrotic mediators and that the inflammatory response would include both T cells and macrophages, with the former expressing a Th1 phenotype.
We first confirmed previous studies that demonstrated that AT1 receptor knockout mice develop premature vascular disease and interstitial fibrosis (19, 28). The vascular disease was marked and was associated with an increase in -SMA-positive cells and by the marked increase in arteriolar medial area and medial:lumen ratios (Figs. 1 and 3). The vascular disease was also associated with glomerular collapse with shrinkage of the glomerular tuft, suggesting glomerular ischemia. Focal area of fibrosis was also present, as demonstrated by the increase in Col I and III staining, and an increase in TGF- mRNA by quantitative real-time PCR.
The first new finding was the characterization of the inflammatory cells and cytokines in the AT1 receptor knockout mice. Specifically, we found that the renal tissue had elevated levels of both monocyte (MCP-1) and T cell (RANTES) cytokine mRNA expression in association with an increase in monocytes and CD4-positive T cells. The T cells were likely of a Th1 phenotype, as the kidneys also have a marked induction of mRNA expression of Th1 cytokines (IFN- and IL-2) but not Th2 (IL-4 and IL-10) cytokines. There was also evidence for the increased expression of monocyte/macrophage inflammatory cytokines (TNF- mRNA). The local inflammatory response was associated with upregulation of oxidative enzymes, including the NADPH oxidase system and the XO enzymes. Thus the renal injury in AT1 receptor knockout is associated with a T cell and monocyte response with local activation of inflammatory mediators and oxidative enzyme systems.
The second new finding was the observation that there was an induction of cytokines and vasoactive mediators. An upregulation of proximal pathways involved in the RAS, such as renin and angiotensinogen, was expected. However, we also found an increased expression of the renal endothelin system (including ET-1 and the ET-A receptor) consistent with a vasoconstrictive response. Although eNOS expression was not modulated in AT1 receptor-deficient state, nNOS and COX-2 mRNA and protein were increased in our AT1 receptor knockout mice consistent with a vasodilatory response. An increase in the nNOS and COX-2 pathways has been previously reported in rodents in which the RAS is blocked, and this is thought to represent a consequence of interrupting the negative feedback of ANG II on the nNOS-COX-2 pathways involved in tubuloglomerular feedback (1, 10, 31). Moreover, the afferent arteriolar diameter might be directly regulated by NO derived from nNOS in the macula densa (26), and COX-2 participates in tubular flow-dependent afferent arteriolar tone via interation with nNOS (7).
An interesting observation in the current study was the finding that both the NADPH oxidase isoform P40-phox and P67-phox mRNA were increased in the AT1 receptor knockout mice, whereas a crucial flavin-containing catalytic subunit of NADPH oxidase, Nox4, was reduced. Unlike other subunits, neither Nox1 nor Nox4 is present in leukocytes but is highly expressed in vascular cells and upregulated in vascular remodeling, such as that found in hypertension and atherosclerosis (8). This suggests that the upregulation of NADPH oxidase in the kidney of AT1 receptor knockout mice may largely represent activation by infiltrating monocytes and macrophages, which have been shown to produce oxidants in other models of salt-sensitive hypertension (14, 24, 25, 34). One might posit that NADPH oxidases should also be induced in the preglomerular vessels, as activation of these oxidases has been shown to play a critical role in vascular remodeling (18). However, most studies suggest that the induction of NADPH oxidases in vascular cells is dependent on ANG II (21), raising the possibility that other mechanisms may be required when the RAS is completely blocked.
In conclusion, while ANG II has been definitively shown to have a role in mediating inflammation and fibrosis in the kidney, in this study we demonstrate that a complete absence of AT1 receptor is also associated with the development of preglomerular vascular disease and interstitial inflammation. Characterization suggests the induction of chemokines driving a Th1 and monocytic/macrophage response coupled with oxidative stress, an alteration in vasoactive mediators, and fibrosis.
GRANTS
This study was supported by National Institutes of Health Grants DK-52121, HL-68607, and George O'Brien Center Grant DK-64233 (to R. J. Johnson). This study was also supported by the Gatorade Fund, University of Florida.
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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