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J Am Soc Nephrol 15:1514-1529, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Angiotensin II, via AT1 and AT2 Receptors and NF-{kappa}B Pathway, Regulates the Inflammatory Response in Unilateral Ureteral Obstruction

Vanesa Esteban*, Oscar Lorenzo*, Mónica Rupérez*, Yusuke Suzuki{dagger}, Sergio Mezzano{ddagger}, Julia Blanco§, Mathias Kretzler||, Takeshi Sugaya{dagger}, Jesús Egido* and Marta Ruiz-Ortega*

*Vascular and Renal Research Laboratory, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain; {dagger}Division of Nephrology, Juntendo University, Japan; {ddagger}Division of Nephrology, School of Medicine, Universidad Austral, Valdivia, Chile; §Hospital Clínico de San Carlos, Madrid, Spain; and ||Nephrology Center, University of Munich, Munich, Germany

Correspondence to Dr. Marta Ruiz-Ortega, Vascular and Renal Research Laboratory, Fundación Jiménez Díaz, Avda Reyes Católicos, 2, 28040 Madrid, Spain. Phone: 34-91-550-48-00, ext 3168; Fax: 34-91-5442636; E-mail: mruizo{at}fjd.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Inflammatory cell infiltration plays a key role in the onset and progression of renal injury. The NF-{kappa}B participates in the inflammatory response, regulating many proinflammatory genes. Angiotensin II (Ang II), via AT1 and AT2 receptors, activates NF-{kappa}B. Although the contribution of Ang II to kidney damage progression is already established, the receptor subtype involved in the inflammatory cell recruitment is not clear. For investigating this issue, the unilateral ureteral obstruction (UUO) model was used in mice, blocking Ang II production/receptors and NF-{kappa}B pathway. Two days after UUO, obstructed kidneys of wild-type mice presented a marked interstitial inflammatory cell infiltration and increased NF-{kappa}B activity. Treatment with AT1 or AT2 antagonists partially decreased NF-{kappa}B activation, whereas only the AT2 blockade diminished monocyte infiltration. Obstructed kidneys of AT1-knockout mice showed interstitial monocyte infiltration and NF-{kappa}B activation; both processes were abolished by an AT2 antagonist, suggesting AT2/NF-{kappa}B involvement in monocyte recruitment. In wild-type mice, only angiotensin-converting enzyme inhibition or combined therapy with AT1 plus AT2 antagonists blocked monocyte infiltration, NF-{kappa}B activation, and upregulation of NF-{kappa}B–related proinflammatory genes. Therefore, AT1 and AT2 blockade is necessary to arrest completely the inflammatory process. Treatment with two different NF-{kappa}B inhibitors, pirrolidin-dithiocarbamate and parthenolide, diminished monocyte infiltration and gene overexpression. These data show that Ang II, via AT1 and AT2 receptors and NF-{kappa}B pathway, participates in the regulation of renal monocyte recruitment and may provide a rationale to investigate further the role of AT2 in human kidney diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the main features of renal damage is mononuclear cells infiltration (1). The renin-angiotensin system (RAS) is involved in the pathophysiology of several kidney diseases, but there are still some important unresolved questions. Angiotensin II (Ang II) regulates cell proliferation, apoptosis, fibrosis, and, as recent works have demonstrated, the inflammatory response (2,3). Two subclasses of Ang II receptors (AT1 and AT2) have been described (4). AT1 participates in BP control and exerts growth-promoting and fibrotic effects, whereas AT2 is involved in cell growth inhibition and diuresis/natriuresis (2–4). The AT2 is overexpressed in pathologic processes involving tissue remodeling or inflammation, including renal damage (4,5). In relation to the inflammatory process, AT1 regulates several proinflammatory genes, including cytokines (interleukin-6 [IL-6]), chemokines (monocyte chemoattractant protein 1 [MCP-1]), and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1]) (2), but others, as the chemokine RANTES, are regulated by AT2 (6). Some evidence suggests that AT2 participates in the inflammatory response in renal and vascular tissues (2,6,7). NF-{kappa}B is a transcription factor that regulates many genes involved in immune and inflammatory responses. In kidney damage, increased tissue NF-{kappa}B activity and upregulation of proinflammatory parameters have been described, and those processes were diminished by pharmacologic blockade of RAS (8–11). In vivo and in vitro studies have shown that Ang II activates NF-{kappa}B in the kidney, via both AT1 and AT2 receptors (2,12,13). Studies with AT2 agonists, cells from AT1 knockout mice, and cells that express only AT2 have also demonstrated the implication of AT2/NF-{kappa}B pathway (14–16). The aim of our work was to investigate the role of Ang II receptors and the NF-{kappa}B pathway in the regulation of inflammatory cell infiltration in renal damage.

We used the unilateral ureteral obstruction (UUO) model of renal injury in mice, characterized by interstitial inflammatory cell infiltration, NF-{kappa}B activation, oxidative stress, apoptosis, and fibrosis (10,11,17–25). Early investigations in the rat model have demonstrated that Ang II contributes to renal damage after UUO (10,11,17,18). Studies using specific antagonists or knockout mice for AT1 or AT2 receptors have demonstrated that both receptors modulate kidney disease progression in this model (11,19–23). These studies have clearly shown the role of AT1 in renal fibrosis (19,21). However, the receptor subtype involved in the regulation of the inflammatory process remains to be elucidated. In the UUO model, AT1 antagonists did not diminish inflammatory cell infiltration and VCAM-1 expression (20), but both AT1 and AT2 antagonists diminished NF-{kappa}B activation (11). Studies using AT1 or AT2 knockout mice or AT2 antagonists have not investigated the inflammatory response in a systematic and complete way (21,23), leading to important open questions. AT1 antagonists are currently used to treat hypertension and renal and cardiovascular diseases. AT1 blockade causes an increase in plasma Ang II concentrations that could selectively bind to AT2, exerting potential undefined effects. In UUO-induced renal damage, we have investigated the role of Ang II/NF-{kappa}B pathway in the inflammatory process. We have studied the receptor subtype involved in this process and the effect of simultaneous blockade of both AT1 and AT2 receptors. The elucidation of the receptor involved in Ang II–induced inflammatory cell infiltration may have important clinical consequences.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Design
Animals used were male wild-type (WT; C57BL/6) and AT1a receptor knockout (AT1 –/–) mice, with the same genetic background. The AT1 knockout mice were generated by a homologous recombination method (26) (Tanabe Seiyaku Corp., Osaka, Japan) and have been previously used to study the role of AT1 receptors in several diseases (27,28). The UUO model was performed under pentobarbital-induced anesthesia; the left ureter was ligated with silk (4/0) at two locations and cut between ligatures to prevent urinary tract infection (obstructed kidney). For determining the role of Ang II receptors, animals were treated with the AT1 antagonist Losartan (MSD, Spain; 10 mg/kg per d; drinking water) or the AT2 antagonist PD123319 (Sigma, St. Louis, MO; 30 mg/kg per d, subcutaneously, osmotic minipumps) or a combination of both antagonists, at doses that block each receptor (12,13). The role of Ang II was evaluated by the ACE inhibitor quinapril (30 mg/kg per d) (28). NF-{kappa}B inhibitors used were pirrolidin-dithiocarbamate (PDTC; 15 mg/kg per d) or parthenolide (3.5 mg/kg per day). Those inhibitors have previously demonstrated beneficial effect on renal damage (29,30). Treatments were started 1 d before UUO and continued for 2 d. After the period of study, animals were killed under anesthesia, and both kidneys (contralateral and obstructed) were removed and processed for histologic studies and frozen in liquid nitrogen for RNA and transcription factor activity evaluations. In all of the experiments, control animals (WT and AT1 knockout) of the same age were also evaluated.

Morphology, Inflammatory Cell Infiltration, and Immunohistochemistry
Morphology was evaluated by Masson’s staining and light microscopy. Inflammatory cell infiltration was determined by monoclonal antibodies against F4/80 antigen (Serotec, Oxford, UK), present in murine monocytes/macrophages. Briefly, paraffin-embedded sections were rehydrated, and endogenous peroxidase and unspecific binding were blocked. Then, sections were incubated with primary antibodies and revealed by standard methods. In each experiment, negative controls without the primary antibody or with an unrelated antibody were done. Morphology was scored by semiquantitative determination as previously reported (12) and graded as follows: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, marked staining; and 4+ severe staining. The whole interstitium was examined from each animal, separately evaluating proximal, distal, and collecting ducts. Tubular damage was defined as epithelium flattening, lumen increase, vacuolization, desquamations, necrosis, and loss of brush border in proximal tubules. Glomerular damage was defined as increase in mesangial matrix expansion and interstitial damage such as the presence of fibrosis. Although by Masson’s staining the presence of infiltrating cells can be observed, the inflammatory cells were quantified only by immunohistochemistry with specific antibodies. In WT animals, contralateral kidneys did not present renal lesions during the period of study. There were no changes in the morphology of contralateral kidneys of untreated WT mice, although in AT1 knockout, some contralateral kidneys presented slight tubular damage (shown in Results). We have also evaluated the effect of the different treatments, analyzing contralateral kidneys. At the doses used, none of the compounds caused renal damage (not shown).

Immunohistochemical quantification of infiltrating cells was evaluated by image analysis using a KZ 300 imaging system 3.0 (Zeiss, Munchen-Hallbergmoos, Germany). Briefly, the percentage of the stained area was calculated as the ratio of suitable binary thresholded image and the total field area. For each sample, the mean staining area was obtained by analysis of 20 different fields (x40). The staining score is expressed as F4/80-positive cells/mm2. The immunohistochemistry experiments were performed in two to four kidney sections from each experimental animal that were stained and analyzed to obtain a mean score for each of them. In all cases, evaluations were performed by two independent observers in a blinded manner, and the mean score value was then calculated for each mouse.

Evaluation of Ang Receptors (AT1 and AT2)
Total RNA was obtained from half kidney by homogenization and isolated with Trizol (Invitrogen, San Diego, CA). Reverse transcription–PCR (RT-PCR) was used to study gene expression of AT2 receptors because by Northern blot, their gene expressions were undetectable, maybe because of low mRNA levels in the kidney (control experiments; not shown). Specific primers and PCR conditions are shown in Table 1. In all RT-PCR experiments, several cycles were analyzed to determine linearity of the reaction; only data of the cycle described in Table 1 were used for calculations. Control experiments were done with RNA samples but without AMV reverse transcriptase. The PCR products were analyzed on 6% polyacrylamide gels, dried, and exposed to X-OMAT AS films (Eastman Kodak, Rochester, NY). The autoradiographs were scanned using the GS-800 Calibrated Densitometer (Quantity One; BioRad, Spain). Data were normalized against those of the corresponding glyceraldehyde-3 phosphate dehydrogenase (GAPDH).


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  		Table 1. RT-PCR conditions and primers used for RT-PCRa
 
In a set of control experiments, we showed that AT1a mRNA was expressed in the kidney of WT mice and in cultured renal cells but absent in cells from AT1 knockout mice. In the kidney and cultured renal cells, AT1b was not detected in either WT or knockout mice (not shown), as others and we have previously described (14,26–28). AT2 receptor protein levels were evaluated by immunohistochemistry in paraffin-embedded tissue sections using a specific goat anti-AT2 antibody (sc-9040; Santa Cruz, Santa Cruz, CA; tested by Western blot (5,15)) and with secondary anti-IgG biotinylated-conjugated antibody.

Proinflammatory Gene Expression
Proinflammatory gene expression was analyzed by RT-PCR or real-time PCR. RT-PCR was done as described above. Primers and conditions used are shown in Table 1. For calculating the data of gene expression, in each animal the mRNA data (densitometric arbitrary units) obtained from densitometry of PCR products from obstructed kidney were compared with mRNA data of contralateral kidney to obtain the fold increase. First, each datum was corrected by GAPDH gene levels. For comparing different experiments, ratio gene/GAPDH from control WT mice was arbitrarily equal to 1. In addition, all samples were analyzed in duplicate to obtain the mRNA value of each sample. Results were calculated as fold increase over control and expressed as means ± SEM of n animals of each group. To validate some of the results of semiquantitative RT-PCR, we used real-time PCR. First, we compared the gene expression of MCP-1 and RANTES by the two methods, obtaining similar results. In brief, 2 µg of RNA underwent random primed reverse transcription using a modified Maloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies, Gaithersburg, MD) for 1 h at 42°C. Real-time RT-PCR was performed on a TaqMan ABI 7700 Sequence Detection System using heat-activated TaqDNA polymerase (Amplitaq Gold). After an initial hold of 2 min at 50°C and 10 min at 95°C, the samples were cycled 40 times at 95°C for 15 s and 60°C for 60 s. For all quantitative cDNA analysis, the {Delta}Ct technique was applied (32). GAPDH and 18S rRNA served as housekeeping genes and were amplified in parallel with the genes of interest. The expression of each target gene was normalized to both different housekeeping transcripts. Sequences with the following GeneBank accession numbers served for the design of the predeveloped TaqMan assay reagents or primer and probes: PDAR used for MCP-1, M1968; RANTES, M77747; TNF{alpha}, M11731. Target gene, forward and reverse primers, and probes were designed using Primer Express 1.5 software (Applied Biosystems, Foster City, CA) and searched against the public databases to confirm unique amplification products. All primers, probes, and reagents were obtained from Applied Biosystems. All measurements were performed in duplicate. Controls consisting of ddH2O were negative in all runs.

Determination of Renal NF-{kappa}B Activity
NF-{kappa}B activity was evaluated by binding of 80 µg of protein extracts with NF-{kappa}B consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) labeled [{gamma}-32P]ATP; the complexes formed were analyzed by electrophoretic mobility shift assay (EMSA), and autoradiographs were obtained after exposure of dried gels. Protein extraction was done as described previously (12). Frozen kidney pieces were pulverized in a metallic chamber and resuspended in a cold extraction buffer (20 mmol/L HEPES-NaOH [pH 7.6], 20% [vol/vol] glycerol, 0.35 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.5 mmol/L PMSF). The homogenate was shaken vigorously for 30 min, and the insoluble materials were precipitated by centrifugation at 40,000 x g for 30 min at 4°C. Negative controls without cellular extracts and competition assays with a 100-fold excess of unlabeled NF-{kappa}B, mutant NF-{kappa}B, and AP-1 (unrelated) oligonucleotides were performed to establish the specificity of the reaction (not shown). When competition assays were done, the unlabeled probe was added to this buffer 10 min before the addition of the labeled probe. The results of EMSA experiments were analyzed using a Densitometer. Data of renal NF-{kappa}B activity of each animal were obtained from the relation of densitometric arbitrary units between obstructed/contralateral kidney and expressed as fold of increase. For comparing different groups and for diminishing differences between experiments performed on different days (interassays), each experiment included a control sample (without obstruction or treatment) and an untreated animal (both obstructed and contralateral kidneys) as reference. Data were normalized by the mean NF-{kappa}B activity of contralateral kidneys of WT mice, and then means ± SEM of all groups studied were calculated.

Southwestern histochemistry was used for localization of cells that contained NF-{kappa}B active complexes (12). In brief, renal sections were rehydrated, fixed with 0.5% paraformaldehyde, treated with 5 mmol/L levamisole for 30 min, digested with 0.5% pepsine in 1 N HCl for 30 min at 37°C, and then treated with 0.1 mg/ml DNAsa for 20 min. Samples were then incubated overnight at 37°C with NF-{kappa}B oligonucleotide digoxigenin labeled (100 pmol/ml) and revealed by standard methods. Results were scored by semiquantitative determination and graded as follows: 1+, mild staining; 2+, moderate staining; 3+, marked staining; and 4+ severe staining. Identification of different cell types was based on topographical criteria commonly used by our group (12). The mean number of positive cells per glomerular cross-section was determined by evaluating 10 to 15 glomeruli. The whole interstitium was examined from each animal. Evaluations were performed by two independent observers in a blinded manner, and the mean value was then calculated for each kidney.

Statistical Analyses
Significance was established by the GraphPAD Instat using t test (GraphPAD Software, San Diego, CA), Mann-Whitney test (nonparametric t test), and ANOVA nonparametric (Kruskal-Wallis test), and differences were considered significant at P < 0.05. To compare the effect of the different treatments, we always compare each genotype separately.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evolution of the Disease in WT and AT1 Knockout Mice after UUO
Our main aim was to investigate the Ang II receptor subtype involved in renal inflammatory cell infiltration during kidney damage. Therefore, we compared time-dependent evolution of inflammatory cell infiltration, fibrosis, tubular damage, and apoptosis between WT and AT1 knockout mice after UUO.

In WT mice at day 2 after UUO, obstructed kidneys showed only interstitial inflammatory cell infiltration and slight tubular dilation. At day 4, obstructed kidneys also presented marked tubular damage, focal interstitial fibrosis, and slight glomerular injury. At day 7, besides infiltrating cells, important renal lesions were observed, including atrophic tubules, marked interstitial fibrosis, and some glomerular sclerosis (Figure 1Go). In AT1 knockout mice with UUO, at day 2 only inflammatory cells and some tubular damage was shown, and at day 4 slight tubular dilation and focal fibrosis was added. These data indicate that disease evolution in AT1 knockout mice was slower than in WT. Importantly, at day 7, both WT and AT1 knockout mice showed comparable renal injury (Figure 1, A and BGo), indicating AT1-independent processes involved in renal damage progression. This results is in agreement with previously published data in AT1 knockout mice. At day 5, AT1 knockout mice had less fibrosis than WT, but at day 10, there were no differences between genotypes (21).



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  		Figure 1, A and B. Evolution of morphologic lesions in obstructed kidneys of wild-type (WT) and AT1 knockout mice. (A) Renal lesions evaluated by a semiquantitative score described in Materials and Methods (glomerular sclerosis, tubular atrophy, and interstitial fibrosis). {square}, data of WT mice; {blacksquare}, data of AT1 knockout (AT1 –/–) mice. Data are shown as means ± SEM of 6 to 12 animals per group. *P < 0.05 versus contralateral of each genotype, respectively. (B) Masson staining of a representative animal of each group. Magnification, x200.

 


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  		Figure 1 C. Inflammatory cell infiltration. Obstructed kidneys of WT or AT1 knockout mice present inflammatory cell infiltration at days 2, 4, and 7 as shown by immunostaining with anti-F4/80 (positive for monocytes/macrophages). As control of the technique, a sample was incubated without primary antibody. The picture of AT1 –/– control represents an AT1 knockout mice without obstruction/treatment. Figures show a representative animal of 6 to 12 studied in each group. Magnifications: x200; WT 7 days and AT1 –/– 7 days, x400.

 
To evaluate better the inflammatory response in this model, we examined inflammatory cell infiltration by immunohistochemistry with a specific anti-F4/80 antibody that recognizes murine monocytes/macrophages (Figure 1CGo). In control samples and in noninjured kidneys of both genotypes, only a few cells were positive for F4/80. Two days after UUO, obstructed kidneys of WT mice presented a high number of positive interstitial F4/80 cells focally distributed mainly at the juxtamedullary level (Figure 1Go). In obstructed kidneys of AT1 knockout mice, an elevated number of monocytes/macrophages was also observed, but the mean staining score was lower than in WT (Figure 2). However, at day 7, there were no significant differences in the number of monocytes/macrophages between genotypes. We examined the expression of extracellular matrix proteins (type I and III collagens) and of apoptosis-related proteins (proapoptotic, Fas-L; antiapoptotic, Bcl-2) by immunohistochemistry. Two days after UUO, there were no significant changes in the staining of collagen or Fas-L between contralateral and obstructed kidneys of both genotypes (data not shown). After 7 d, increased expression of collagen and Fas-L was found, as described previously (24), showing no differences between WT and AT1 knockout mice (not shown). These data clearly indicate that 2 d after UUO, the renal lesions are mainly interstitial infiltration of monocytes/macrophages, whereas fibrosis, tubular damage, and apoptosis occur several days after. Therefore, this time point is adequate to investigate the effect of Ang receptor blockade and the NF-{kappa}B pathway contribution in the inflammatory response.



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  		Figure 2. (A) Effect of angiotensin II (Ang II) receptor antagonists and angiotensin-converting enzyme (ACE) inhibition on renal lesion in WT and AT1 knockout mice with unilateral ureteral obstruction (UUO). Animals were treated daily with the AT1 antagonist losartan, the AT2 PD123319, both antagonists together, or the ACE inhibitor quinapril. All treatments were started 1 d before unilateral obstruction, and animals were studied 2 d after obstruction. The figure shows a semiquantitative score of renal lesions. (B) Inflammatory cell infiltration of a representative animal of each group that was stained with an antibody against anti-F4/80 (monocytes/macrophages). (C) Computer analysis of monocytes/macrophages score. Results are expressed as F4/80-positive cells/mm2. In A and C, {square}, data of WT mice; {blacksquare}, data of AT1 knockout mice. Data are shown as means ± SEM, n = 6 to 8 animals for each group. *P < 0.05 versus contralateral kidney; #P < 0.05 versus untreated-obstructed. P value is calculated comparing animals of the same genotype. Magnification: x400.

 
Effect of Ang II System Blockade on Renal Damage Progression Two Days after UUO
In obstructed kidneys of WT mice, compared with untreated ones, treatment with the ACE inhibitor quinapril markedly diminished tubular lesions and inflammatory cell infiltration, demonstrating the implication of Ang II in renal damage progression after UUO (Figure 2), as previously demonstrated in the rat model (10). In obstructed kidneys of WT mice, the AT1 antagonist losartan ameliorated tubular damage and interstitial fibrosis, whereas the AT2 antagonist PD123319 had no effect (Figure 2A). Previous studies have demonstrated that AT1 receptors are involved in fibrosis in this model (19), as well as in the induction of profibrotic factors and extracellular matrix accumulation in Ang II–treated cultured renal cells (3). These data confirm that AT1 receptors regulate fibrotic-related processes. In contrast, losartan only partially but not significantly decreased the number of inflammatory cells in the obstructed kidney (Figure 2, B and C).

In obstructed kidneys of AT1 knockout mice, the ACE inhibitor quinapril ameliorated renal lesions and restored the number of inflammatory cells to normal levels, clearly indicating that Ang II contributes to renal damage in UUO in the absence of AT1 receptors (Figure 2). In AT1 knockout mice, we also evaluated the effect of losartan. This AT1 antagonist blocks both AT1a and AT1b receptors. Obstructed kidneys from losartan-treated AT1 knockout mice presented similar renal lesions and number of infiltrating cells as those of untreated AT1 knockout mice.

In WT mice, the AT2 antagonist PD123319 markedly diminished inflammatory cell infiltration in the obstructed kidneys. These data suggest that AT2 receptor regulates renal inflammatory cell infiltration in UUO. It is interesting that in AT1 knockout mice, the AT2 antagonist diminished renal lesions and the number of monocytes/macrophages, suggesting that the progression of renal damage in AT1 knockout mice could be due to the presence of inflammatory cells.

Studies in the UUO model using the AT2 knockout mice or AT2 blockade have shown an exaggerated fibrosis (22,23). In our model, we did not observe additional fibrosis with the AT2 antagonist maybe because of the time point of our study.

Simultaneous Blockade of Both AT1 and AT2 Receptors Ameliorates Renal Damage and Interstitial Inflammatory Cell Infiltration
We assessed the effect of combined therapy with the AT1 and AT2 antagonists to investigate what happens when both receptors are blocked. The simultaneous blockade of both AT1 and AT2 receptors normalized UUO-induced renal lesions and monocyte infiltration in both genotypes (Figure 2), showing that blockade of both AT1 and AT2 receptors abolished inflammatory cell infiltration after UUO.

AT2 Receptors are Upregulated in UUO
AT2 gene expression was analyzed by RT-PCR. In control kidneys, AT2 mRNA expression was detected as a band corresponding to the predicted size. In obstructed kidneys of WT and AT1 knockout mice, AT2 mRNA levels were increased (Figure 3A). Protein levels of AT2 in the kidney were evaluated by immunohistochemistry (Figure 3B). Renal sections of control mice showed positive AT2 immunostaining in the tubuloepithelial cells, whereas no staining was observed in glomeruli. In obstructed kidneys of both WT and AT1 knockout mice, an increased staining for AT2 was observed in tubular cells, but there was no induction of AT2 expression in glomeruli (Figure 3).



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  		Figure 3. AT2 expression is upregulated by UUO. (A) Analysis of AT2 mRNA expression by reverse transcription–PCR. In both genotypes, AT2 mRNA expression was increased in obstructed kidneys compared with contralateral ones. (B) In control WT mice, a weak staining for AT2 in tubular cells was observed. Unilateral obstruction upregulated tubular AT2 expression. Similar results were found in AT1 knockout mice showing no differences between genotypes. Magnification: x200.

 
Both AT1 and AT2 Receptors Participate in NF-{kappa}B Activation after Ureteral Obstruction
Previous studies in the rat UUO model have shown elevated NF-{kappa}B activity in obstructed kidneys that were diminished by AT1 or AT2 antagonists (11); however, studies in AT1 knockout mice showed only a slight NF-{kappa}B activation in obstructed kidneys (21). We further investigated the role of Ang II receptors in renal NF-{kappa}B activation after UUO.

Two days after UUO, obstructed kidneys of WT mice showed increased NF-{kappa}B activity compared with contralateral ones (Figure 4). Obstructed kidneys of AT1 knockout mice at day 2 also presented increased NF-{kappa}B activity, suggesting an AT1-independent NF-{kappa}B activation in UUO. However, the increase in renal NF-{kappa}B activity in AT1 knockout was lower than in WT (Figure 4), showing that some NF-{kappa}B activation could be AT1 mediated. By Southwestern histochemistry, we identified cells with activated NF-{kappa}B complexes (Figure 4B). Contralateral kidneys of both genotypes showed no nuclear staining for NF-{kappa}B. In obstructed kidneys of WT mice, there was an increase in positive nuclear staining, with activated NF-{kappa}B complexes located mainly in the glomeruli (mesangial, endothelial, and epithelial cells) and tubulointerstitial areas (tubuloepithelial and infiltrating cells). In AT1 knockout mice, activated NF-{kappa}B complexes were located in some glomerular, tubular, and infiltrating cells, clearly showing an AT1-independent NF-{kappa}B activation. This activation can be mediated directly by Ang II via AT2 receptors or by other factors (induced by Ang II via AT2 or independent of Ang II, as mechanical stress).



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  		Figure 4. UUO increased renal NF-{kappa}B activity in WT and AT1 knockout mice. (A) Representative electrophoretic mobility shift assay (EMSA) experiment that shows NF-{kappa}B activity of contralateral and obstructed kidneys of three different animals of each genotype (left) and data of densitometric analysis expressed as fold increase versus contralateral as means ± SEM of 8 to 12 animals of each group analyzed in duplicate (right). *P < 0.05 versus contralateral of each genotype. (B) Localization of activated NF-{kappa}B complexes by Southwestern histochemistry. Obstructed kidneys of WT presented many cells that contained activated NF-{kappa}B complexes, in glomeruli (marked by arrow heads) and in tubulointerstitial cells (*) and infiltrating cells (arrows). In obstructed kidneys of AT1 knockout, there were also some positive glomerular, tubuloepithelial, and infiltrating cells.

 
In WT mice, the AT1 antagonist partially diminished NF-{kappa}B activation in the obstructed kidney (~60% inhibition versus untreated obstructed WT; P < 0.05; EMSA; Figure 5, A and BGo), showing that AT1 is involved in NF-{kappa}B activation in UUO. AT2 blockade also partially diminished NF-{kappa}B activation (~55%; Figure 5, A and BGo). Each antagonist diminished the presence of activated NF-{kappa}B complexes in the same cell population (Southwestern histochemistry; Figure 5, C and D Go). Only when both AT1 and AT2 receptors were blocked was renal NF-{kappa}B activity in obstructed kidneys diminished to control levels (84% inhibition; Figure 5, A and BGo). These data clearly show that the NF-{kappa}B pathway is activated by both AT1 and AT2 receptors.



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  		Figure 5, A and B. Effect of Ang II receptor antagonists and ACE inhibition on renal NF-{kappa}B activity in WT and AT1 knockout UUO mice. Animals were treated daily with the AT1 antagonist losartan, the AT2 PD123319, both antagonists together, or the ACE inhibitor quinapril. Representative EMSA of three independent experiments done that shows one animal of each group. (B) Data of densitometric analysis expressed as fold increase versus contralateral as means ± SEM of six to eight animals of each group. {square}, data of WT mice; {blacksquare}, data of AT1 knockout mice. *P < 0.05 versus contralateral; #P < 0.05 versus untreated obstructed. Each P value is calculated versus each genotype.

 


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  		Figure 5, C and D. Semiquantitative score of NF-{kappa}B staining of Southwestern histochemistry. Results, scored as described in Materials and Methods, are expressed as mean ± SEM of six to eight animals of each group. (D) Localization of activated NF-{kappa}B complexes by Southwestern histochemistry. In obstructed kidneys of WT mice, AT1 antagonist diminished the number of positive cells for NF-{kappa}B–positive staining in glomeruli (marked by arrowheads) and tubulointerstitial cells (*). It is interesting that some infiltrating cells (arrows) presented activated NF-{kappa}B complexes. Treatment with AT2 antagonist also diminishes the number of positive glomeruli and tubulointerstitial cells in both genotypes.

 
In AT1 knockout obstructed kidneys, NF-{kappa}B was activated in some glomerular and tubular cells (Figure 5CGo). The AT2 blockade restored tissue NF-{kappa}B activity to control levels (Figure 5Go). These data demonstrate that AT2/NF-{kappa}B activation plays a key role in UUO. We also determined the role of endogenous Ang II production in NF-{kappa}B activation. The use of an ACE inhibitor quinapril diminished renal NF-{kappa}B activity in all renal structures, in both obstructed WT and AT1 knockout mice (~85% in each genotype; Figure 5, A and B Go), showing that Ang II participates in the NF-{kappa}B activation in the UUO model. In control experiments, we also observed that other transcription factors (AP-1 and NF-AT) are regulated only via AT1 receptors (not shown).

Role of the NF-{kappa}B Pathway in the Progression of Renal Injury in the UUO Model
For evaluating the contribution of the NF-{kappa}B pathway in the progression of renal injury in this model, some animals were treated with two different NF-{kappa}B inhibitors: PDTC (which also possesses antioxidant properties (8,29)) and parthenolide (a sesquiterpene lactone extracted from common medicinal Asteracae plants used in folk medicine because of their anti-inflammatory activity, which acts as a potent NF-{kappa}B inhibitor (30,33)). Both inhibitors diminished the presence of monocytes/macrophages (Figure 6) and renal lesions (not shown) in WT and AT1 knockout mice. These results show that the NF-{kappa}B pathway is involved in the recruitment of inflammatory cells during renal injury. PDTC and parthenolide blocked NF-{kappa}B activation, with a similar effect in both genotypes (Figure 6), showing that, at the doses used, these compounds inhibit renal NF-{kappa}B activity and indicating that there were no differences in the NF-{kappa}B pathway between WT and AT1 knockout mice.



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  		Figure 6. The blockade of the NF-{kappa}B pathway diminished renal inflammatory cell infiltration in the UUO model. Animals were treated daily with the NF-{kappa}B inhibitors pirrolidin-dithiocarbamate (PDTC; 15 mg/kg per d) or parthenolide (3.5 mg/kg per day). Treatments were started 1 d before unilateral obstruction, and animals were studied 2 d after obstruction. (A) Monocytes/macrophages infiltration in obstructed kidneys of a representative animal of each group (left) and the score (right) as F4/80-positive cells/mm2. (B) Both treatments effectively inhibited renal NF-{kappa}B activation in obstructed kidneys, as shown by Southwestern histochemistry (right) or by EMSA (left). Data are expressed as means ± SEM of four to six animals for each group. *P < 0.05 versus control; #P < 0.05 versus untreated obstructed. Magnification: x400.

 
Overexpression of NF-{kappa}B–Related Genes during UUO Was Diminished Only by ACE Inhibition or Combined Blockade of AT1 and AT2 Receptors
In obstructed kidneys of WT mice, mRNA expression of proinflammatory cytokines (TNF-{alpha}, IL-6), chemokines (MCP-1, RANTES) and growth factors (connective tissue growth factor [CTGF] and prepro–endothelin-1 [prepro-ET-1]) was significantly upregulated (real-time PCR and RT-PCR data; Figure 7). These genes are also upregulated in cultured renal cells stimulated with Ang II (2).



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  		Figure 7. Gene expression of NF-{kappa}B–related genes in WT and AT1 knockout mice after 2 d of UUO. (A) Several genes regulated by NF-{kappa}B, such as TNF-{alpha}, RANTES, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) are significantly upregulated in obstructed kidneys (P < 0.05 versus contralateral) and only ACE inhibition, combined blockade of AT1 and AT2 antagonists, or NF-{kappa}B inhibitors abolished that gene overexpression (P < 0.05 versus untreated obstructed). In AT1 knockout mice, gene overexpression was lower than in WT. (B) Gene expression of growth-related genes (CTGF, prepro–endothelin-1 [prepro-ET-1]), cytokines (TNF-{alpha}), and chemokines (MCP-1) in WT mice. In Materials and Methods, technique and explanation of data analysis are described.

 
NF-{kappa}B regulates the gene expression of some Ang II–induced genes, such as TNF-{alpha}, IL-6, MCP-1, and RANTES (9). In WT mice, treatment with the ACE inhibitor or combined treatment with AT1 and AT2 antagonists significantly diminished gene overexpression of these proinflammatory genes (P < 0.05 versus untreated obstructed; Figure 7A). These data demonstrate the contribution of Ang II in the pathogenesis of tissue injury in UUO and clearly show that the blockade of both receptors is necessary to stop the upregulation of proinflammatory genes.

To investigate this point further, we focused on MCP-1 gene regulation, the main chemokine involved in monocyte recruitment. In WT mice, AT1 antagonists partially diminished MCP-1 gene upregulation, whereas the AT2 antagonist caused a slight decrease and only combined blockade of both receptors significantly diminished it (P < 0.05 versus untreated obstructed; Figure 7B). In AT1 knockout mice, MCP-1 was elevated (P < 0.05 versus contralateral) and AT2 antagonist decreased it to control levels (Figure 7A). These data clearly show that the blockade of both AT1 and AT2 receptors is necessary to inhibit MCP-1 overexpression. Another important gene in UUO renal damage is TNF-{alpha} (34). In both WT and AT1 knockout obstructed kidneys, TNF-{alpha} and RANTES were upregulated (P < 0.05 versus contralateral) and diminished to control levels by AT2 antagonist (Figure 7). It is interesting that in WT mice, the diminution caused by ACE inhibition in MCP-1 and TNF-{alpha} genes is higher than the combination of AT1 and AT2 antagonists (Figure 7B). In this sense, at days 4 and 7 after UUO, those genes were upregulated, showing no differences between WT and AT1 knockout mice (data not shown). These data suggest that those genes may also be regulated by other receptors (e.g., AT4) or independent of Ang II.

In WT mice, mRNA upregulation of CTGF and prepro–ET-1 was diminished only by AT1 (P < 0.05 versus untreated obstructed) but not AT2, antagonist (Figure 7B) and was not increased in obstructed kidneys of AT1 knockout mice (data not shown). These data clearly demonstrate that these genes are specifically regulated by AT1 receptors in UUO. We have observed that treatment with the two NF-{kappa}B inhibitors PDTC and parthenolide blocked the overexpression of proinflammatory genes in obstructed kidneys (P < 0.05 versus untreated obstructed; Figure 7A), showing that NF-{kappa}B pathway is involved in gene upregulation during UUO.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mononuclear cell infiltration of glomeruli and interstitium occurs in most progressive renal diseases and plays a crucial role in the outcome of irreversible structural changes (1). We have observed that 2 d after UUO, there is a focal interstitial macrophage infiltration, elevated NF-{kappa}B activity, and upregulation of proinflammatory genes. Ang II contributes to tissue damage progression in renal diseases, including UUO, although some important questions are not resolved yet. In this work, we have demonstrated that Ang II, through the regulation of the NF-{kappa}B pathway, contributes to the inflammatory response in UUO. In WT mice, only ACE inhibition or combined therapy with AT1 plus AT2 antagonists blocked renal monocyte infiltration, NF-{kappa}B activation, and upregulation of NF-{kappa}B–related proinflammatory genes. These data suggest that blockade of Ang II generation or both AT1 and AT2 receptors is necessary to block completely the NF-{kappa}B pathway and to stop the inflammatory process.

NF-{kappa}B activation has been described in kidney diseases (8). In UUO, we have observed that two different NF-{kappa}B inhibitors, PDTC and parthenolide, which act to block renal NF-{kappa}B activation, diminished the inflammatory cell infiltration and downregulated gene expression of several proinflammatory factors, in both WT and AT1 knockout mice. Previous studies have demonstrated that PDTC ameliorated end-organ damage and diminished tissue NF-{kappa}B activity (29). Parthenolide has also shown beneficial effect in experimental renal injury and in reperfusion-induced myocardial damage (30,35). These effects are due to IKK/NF-{kappa}B pathway inhibition, because parthenolide inhibited IKK activity, enhanced stability of I{kappa}B{alpha}, and blocked nuclear translocation of NF-{kappa}B (30,33,35). Our data clearly demonstrate that renal NF-{kappa}B activation blockade, by NF-{kappa}B inhibitors, ACE inhibition, or combined therapy with AT1 plus AT2 antagonists, abolished the inflammatory process in the kidney, supporting the importance of the regulation of this transcription factor during renal damage.

Ang II activates NF-{kappa}B; however, depending on the cell type, different receptors are involved (2). In glomerular mesangial and vascular smooth muscle cells, NF-{kappa}B activation occurs via both AT1 and AT2 receptors (14,15), whereas in tubuloepithelial cells, it occurs only through AT1 (12) and in glomerular endothelial cells via AT2 (16). In UUO, we have observed that in WT, treatment with AT1 or AT2 antagonists partially decreased NF-{kappa}B activation, as described in rats (11). Obstructed kidneys from AT1 knockout mice presented high NF-{kappa}B activity that was diminished to control levels by the AT2 antagonist, showing the involvement of the AT2/NF-{kappa}B pathway in the UUO model. One important question is the biologic meaning of AT2/NF-{kappa}B activation. In WT mice with UUO, ACE inhibition and AT2 but not AT1 blockade reduced inflammatory cell recruitment. In UUO rats, only ACE inhibitors but not AT1 antagonists diminished monocytes/macrophages infiltration and VCAM-1 expression (20). Moreover, inflammatory cells were observed in obstructed kidneys of AT1 knockout mice, which were diminished by both AT2 antagonist and ACE inhibitor. Other data also support the hypothesis that AT2/NF-{kappa}B activation is involved in the recruitment of inflammatory cells in the kidney. In antithymocyte serum-induced nephritis, AT1 blockers reduced inflammatory infiltration only by ~50% (36). In Ang II–infused rats, only the AT2 but not AT1 antagonists diminished glomerular and interstitial mononuclear infiltration, NF-{kappa}B activation in inflammatory cells, and RANTES overexpression (6,12). We have observed similar responses when NF-{kappa}B or AT2 was blocked, such as decreased inflammatory cell infiltration and downregulation of gene expression of TNF-{alpha} and RANTES. In several models of renal and vascular damage characterized by an inflammatory response, an AT2 overexpression has been described (2,5), similar to that found by us in the UUO model. It is interesting that Ma et al. (23) observed that at day 5 after UUO, AT2 knockout mice present less infiltrating cells in obstructed kidneys than WT, although there was no significant statistical difference. The study of infiltrating cells at day 2 and the effect of AT1 antagonists or ACE inhibitors were not approached in that paper, leaving some unresolved questions, such as the role of Ang II (and AT1) in the inflammatory response in AT2 knockout mice, that could explain the exacerbated fibrosis noted in these animals. Studies of vascular lesions in AT2 knockout mice have demonstrated that AT2 mediates the effect of inflammation on vascular smooth muscle cell proliferation (7). Inflammatory mediators, including IL-1{beta}, insulin, interferon regulatory factors, and Ang II, upregulate AT2 expression (7,37). All of these data further strengthen the role of AT2 in the inflammatory process in renal and vascular diseases. Because experimental manipulations that reduced the number of interstitial macrophages ameliorate tissue damage, our data, showing that AT2 blockade diminished infiltration, provide information that could have important clinical implications. Some authors have suggested that the beneficial effects of AT1 blockade are due not only to the stop of AT1-mediated processes (fibrosis/proliferation) but also to the binding of free Ang II to AT2 receptors, which can elicit some other beneficial effects (vasodilation, antiproliferative or antifibrotic response). In WT mice, MCP-1 gene upregulation was only partially diminished by AT1 antagonists, but the simultaneous blockade of both receptors abolished it. These data clearly show that both AT1 and AT2 blockade is needed to reduce MCP-1 overexpression and the amount of infiltrating monocytes in obstructed kidney. This challenges the above-described hypothesis, at least regarding the regulation of inflammatory cell infiltration after UUO.

Studies with specific antagonist and Ang II receptor knockout mice have demonstrated the importance of AT1 in renal fibrosis (3). In cultured renal cells, Ang II via AT1 regulates the expression of profibrotic growth factors and matrix production (3,38). We have observed that in UUO, the AT1 antagonist ameliorates tubular atrophy and fibrosis as described previously (19), associated with downregulation of profibrotic factors (CTGF and ET-1). However, our studies in UUO show that in the absence of AT1, renal damage still progressed. At day 4, obstructed kidneys of AT1 knockout mice presented similar gene upregulation and inflammatory cell infiltration as WT, and after 7 d, the same renal injury. These lesions could be attributed to the migration of mononuclear cells into the interstitium, where they mature to macrophages and activate renal cells through the release of a wide range of growth factors, contributing to the perpetuation of kidney damage. Other potential non–AT1/AT2-related mechanisms could also participate in this process. Studies of acute pressure overload or mechanical stretch in the absence of AT1 showed that Ang II may upregulate genes by non-AT1 processes (34,39). Studies with double knockout of TNF-{alpha} receptors (TNFR1/TNFR2) and treatment with an ACE inhibitor have demonstrated that blocking each system individually leads to partial blunting of fibrosis, whereas blocking both Ang II and TNF-{alpha} systems further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy (40), showing an interrelation between these systems. It is interesting that in WT mice, the ACE inhibitor–induced reduction of TNF-{alpha} and MCP-1 gene expression is higher than that achieved by combined AT1 and AT2 blockade. These data suggest that these genes may also be regulated by other receptors (e.g., AT4) or independent of Ang II. We recently observed that in vascular smooth muscle cells, Ang IV, by AT4 receptors, increases MCP-1 via NF-{kappa}B activation (41). AT1 blockade leads to more free ligand (Ang II) that can be degraded to other metabolites, such as Ang III and Ang IV, which may cause adverse effects such as NF-{kappa}B activation (2,14), although there is little evidence of these mechanisms in vivo during renal damage.

Clinical data comparing ACE inhibitor and AT1 receptor antagonist therapy in renal disease are limited to short-term studies, which indicate that AT1 receptor antagonists have equivalent effects to ACE inhibitors on the major determinants of renal disease progression, specifically BP and proteinuria (42). However, there are no data of renal inflammatory parameters. Our results show that only combined therapy with AT1 plus AT2 antagonists blocked renal monocyte infiltration, NF-{kappa}B activation, and upregulation of NF-{kappa}B–related proinflammatory genes, showing that the blockade of both AT1 and AT2 receptors is necessary to stop completely the inflammatory process. Important is that our results suggest that AT2, via the NF-{kappa}B pathway, could play a key role in the inflammatory cell recruitment and therefore contribute to the progression of the disease. These results provide a rationale to investigate further the role of the AT2/NF-{kappa}B pathway in the inflammatory response in kidney diseases, with potential clinical consequences in the treatment of severe human nephritis.31


   Acknowledgments
 
This work wad supported by grants from Fondo de Investigación Sanitaria (PI020513), Comunidad Autónoma de Madrid (08.4/0018/2001), Sociedad Española de Nefrología, Fundación Renal Iñigo Alvarez de Toledo, European Project (QLG1-CT-2002-01215), and Fondecyt 1040163, República de Chile. V.E., O.L., and M.R. are fellows of FIS.

We thank Dr. Anna Henger for help with real-time PCR. We thank L. Gulliksen for secretarial assistance and Dr. Julio Osende for careful reading of the manuscript. We also thank Prof. Yasuhiko Tomino (Juntendo University, Japan) for support to Y.S.


   Footnotes
 
V.E. and O.L. contributed equally to the work.


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 Results
 Discussion
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Received for publication June 21, 2002. Accepted for publication February 23, 2004.




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