1Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of São Paulo, 01246-903 São Paulo; and 2Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, 13081-970 Campinas, Brazil
Submitted 1 July 2003 ; accepted in final form 12 December 2003
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ABSTRACT |
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prostaglandin-endoperoxide synthase; angiotensin II; anti-inflammatory agents; kidney failure; chronic; inflammation
The beneficial effect of RAS suppressors was initially attributed to amelioration of the glomerular hemodynamic dysfunction associated with progressive nephropathies. However, recent observations suggest that the nonhemodynamic effects of RAS suppressors may be equally important, given the strong proinflammatory and profibrotic effects of ANG II (35). A substantial fraction of this proinflammatory ANG II may originate in the renal parenchyma, rather than in renal vessels or in the systemic circulation (44). Increased intrarenal production of ANG II was described in various models of renal fibrosis (12, 30, 34). A preliminary report has suggested that, in the renal ablation (Nx) model, ANG II is expressed in renal interstitial cells, paralleling the severity of renal injury (28).
Both the hemodynamic and proinflammatory effects of ANG II are mediated by AT-1 receptors (AT1R) (35), extensively expressed in renal tissue. In the normal rat kidney, AT1R are predominantly expressed in tubular cells and vessels (15). Recent data obtained with the Nx model have suggested that AT1R expression is shifted from the glomerular to the tubulointerstitial compartment 4 wk after ablation (6). However, the renal distribution of AT1R in this model and its temporal evolution have not been established.
Cyclooxygenase (COX) derivatives may play an important role in the pathogenesis of progressive nephropathies, comparable to their role in arthritis. Increased renal expression of isoforms 1 and 2 of COX has been reported in nephropathies of immunological and nonimmunological origin, such as systemic lupus erythematosus (43), glomerulosclerosis in Fawn-Hooded hypertensive rats (50), Heymann nephritis (40), and renal ablation (9, 49). In a recent study by this laboratory, increased renal expression of COX-2, which correlated significantly with the extent of renal damage, was shown in Nx rats (9). In addition, chronic use of COX inhibitors greatly attenuated renal injury in the Nx model (9, 10, 48).
In view of the complexity and the large number of events leading to progressive renal fibrosis, the interruption of two or more pathogenic pathways by a combination of drugs with different mechanisms of action is likely to be more effective than the respective monotherapies. Three independent studies showed that the combination of a RAS suppressor with an immunosuppressive agent exerted a much stronger protective effect on Nx rats than either drug alone (11, 13, 33).
In the present study, we evaluated in Nx rats the renal distribution of both the AT1R and the COX-2 isoform, as well as the variation of their renal expression with time. In addition, we estimated the amount of ANG II present in glomerular arterioles and in the cortical interstitium. To evaluate the role of these mediators in progression, Nx rats were treated with nitroflurbiprofen (NOF), a nonsteroidal anti-inflammatory drug (NSAID) with low gastrointestinal toxicity, or losartan potassium (Los), an AT1R blocker. In an attempt to obtain more effective renal protection, a group of Nx rats receiving a combined NOF/Los treatment was studied as well.
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METHODS |
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Experimental groups. Thirty days after Nx, the tail-cuff pressure (TCP) was measured by an indirect method (11). The animals were then placed in metabolic cages for determination of daily urinary albumin excretion rate (UalbV). Animals that at this time had failed to develop hypertension (defined as TCP 140 mmHg) or albuminuria (UalbV
50 mg/day) were excluded from the study. The kidneys of 12 Nx rats were then perfusion-fixed with Duboscq-Brazil solution (0.45% picric acid in a mixture of ethanol, formaldehyde, and acetic acid) after a brief saline washout and prepared for light microscopic and immunohistochemical analysis as described below. This group, designated Nxpre, was used to evaluate the extent of renal injury at 30 days after Nx and served to assess the therapeutic efficacy of treatments started thereafter. The remaining Nx rats were then followed for an additional 3 mo (up to 4 mo after nephrectomy) after having been distributed among four experimental groups: NxV (n = 12), Nx rats receiving inert vehicle; NxNOF (n = 14), Nx rats receiving NOF (Nicox, Sophia Antipolis), 7.5 mg/kg dissolved in a mixture of DMSO 5% in olive oil and given by gavage twice daily; NxLos (n = 14), Nx rats receiving Los (Merck Sharp & Dohme, Rahway, NJ) dissolved in the drinking water at 20 mg/dl, corresponding to a daily ingestion of
50 mg/kg; and NxLos/NOF (n = 13), rats given simultaneous NOF and Los treatments. The concentration of Los in the drinking water was adjusted to compensate for variations in daily water intake to ensure that both NxLos and NxLos/NOF groups received a constant dosage of 50 mg·kg-1·day-1. A group of S rats (n = 12) was also followed. To mimic the situation usually found in clinical practice, all drug treatments were started only 30 days after renal ablation, when the process of progressive renal injury associated with this model is already in motion. The distribution of rats into the four Nx groups was performed in such a way as to ensure that both mean TCP and albumin excretion rates were similar among groups. The mean initial blood pressure values (in mmHg) and albuminuria values (in mg/day) are shown in Table 1. None of these differences was statistically significant.
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Experimental protocol. To evaluate the efficiency of NOF as a COX blocker, the daily urinary excretion of thromboxane B2 (TxB2), a derivative of thromboxane A2 (TxA2), was measured by radioimmunoassay (NEN, Boston, MA) 60 days after renal ablation (30 days of treatment). Because, after renal mass reduction, most of the urinary prostanoids is believed to originate from renal synthesis (26), we also estimated the thromboxane production per nephron by factoring urinary TxB2 by the total number of nephrons, assumed to be 6x104 in S and 104 in Nx.
All groups were followed until 120 days after surgery (90 days of treatment), with monthly determination of TCP and UalbV (11). At the end of the study, rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a blood sample was collected from the abdominal aorta to assess serum creatinine (Screat). The renal tissue was then perfusion-fixed with Duboscq-Brazil solution after saline washout and prepared for morphological analysis as described below.
Histological analysis. Sections 4-µm thick were stained by periodic acid-Schiff reaction or Masson trichrome. All morphometric evaluations were performed in a blinded fashion by a single observer. The extent of glomerular sclerosis (GS) was evaluated by attributing a score to each glomerulus according to the apparent extent of the tuft area affected by the sclerotic injury, as described previously (11). At least 100 glomeruli were evaluated for each rat. A GS index (GSI) was calculated for each rat as the weighted average of all individual glomerular scores thus obtained (11). To evaluate the extent of renal interstitial expansion, the fraction of renal cortex occupied by interstitial tissue staining positively for extracellular matrix components (%INT) was quantitatively evaluated in Masson-stained sections by a point-counting technique in 25 consecutive microscopic fields, at a final magnification of x100 under a 176-point grid (11).
Immunohistochemical analysis. All immunohistochemical studies were performed in 4-µm-thick, paraffin-embedded renal sections. Sections were mounted on glass slides coated with 2% gelatin, deparaffinized, and rehydrated using standard techniques. Sections were then exposed to microwave irradiation in citrate buffer to enhance antigen retrieval and preincubated with 5% normal rabbit (for ED-1) or horse (for COX-2, AT1, and ANG II) serum in Tris-buffered saline to prevent nonspecific protein binding. Incubation with the primary antibody was always carried out overnight at 4°C in a humidified chamber. Negative control experiments for all antigens were performed by omitting incubation with the primary antibody.
For macrophage detection, a monoclonal mouse anti-rat ED-1 antibody (Serotec, Oxford, UK) was used. After being washed, sections were incubated with rabbit anti-mouse Ig (Dako, Glostrup, Denmark), then with an alkaline phosphatase anti-alkaline phosphatase (Dako) complex. Finally, sections were developed with a fast-red dye solution, counterstained with Mayer's hemalaum, and covered with Kaiser's glycerin-gelatin (Merck, Darmstadt, Germany).
COX-2-, ANG II-, and AT1R-positive cells were detected by an indirect streptavidin-biotin alkaline phosphatase technique. The primary (monoclonal mouse anti-rat) antibody for COX-2 was purchased from Transduction Laboratories (Lexington, KY). For ANG II detection, a monoclonal rabbit anti-human ANG II (Peninsula Lab, San Carlos, CA) was used, whereas AT1R was detected with a monoclonal rabbit anti-rat AT1R antibody (RDI, Flanders, NJ). Sections were preincubated with avidin and biotin solutions to block nonspecific binding of these compounds (Blocking Kit, Vector Labs, Burlingame, CA). After being washed, the sections were incubated at room temperature with rat-adsorbed biotinylated anti-mouse or anti-rabbit IgG (Vector Labs) for 45 min, then with a streptavidin-biotin-alkaline phosphatase complex (Dako) for an additional 30 min. Sections were finally incubated with a freshly prepared substrate, consisting of naphthol AS-MX-phosphate and developed as described above.
The extent of renal infiltration by macrophages, ANG II-positive cells, and COX-2-positive cells was evaluated in a blinded manner at x250 magnification and expressed as cells per square millimeter. For each section, 25 microscopic fields, each corresponding to an area of 0.06 mm2, were examined. Because interstitial AT1R in Nx rats was often so densely expressed as to preclude the individualization of positively stained cells, AT1R expression had to be estimated by a point-counting technique similar to the one employed to determine %INT. This technique allowed us to assess the distribution of AT1R among several compartments of the renal cortex (glomeruli, vessels, tubules, and interstitium). The glomerular expression of COX-2 was evaluated by counting positively stained cells in a total of 100 glomeruli/rat, and the results were expressed as cells/100 glomeruli. The fraction of stained macula densa regions was also estimated. The expression of COX-2 in arteries/arterioles was evaluated by counting the number of stained cells in a total of 50 cortical vessels and given as cells/50 vascular profiles.
Statistical analysis. One-way ANOVA with paired comparisons according to the Newman-Keuls formulation was used in this study (9). The Spearman correlation coefficient was used to evaluate the existence of significant linear correlation between parameters obtained in individual rats. Because GSI and albumin excretion rates behaved as continuous variables with nonnormal distribution, an approximately Gaussian distribution was obtained in all groups by performing log transformation of these data before statistical analysis. For similar reasons, parameters expressed as proportions underwent arcsine transformation before analysis (9). P values < 0.05 were considered significant.
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RESULTS |
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As expected, UalbV (Table 1) was markedly increased at 30 days of surgery (Nxpre group), reaching 104.3 ± 7.1 mg/day (P < 0.05 vs. S). Albuminuria was aggravated at 120 days after Nx (178.5 ± 43.1 mg/day, P < 0.05 vs. S and P > 0.05 vs. Nxpre). Monotherapy with LOS numerically decreased UalbV relative to untreated Nx (112.9 ± 14.7 mg/day, P > 0.05 vs. NxV) and prevented further increases in albuminuria, which remained at levels similar to those observed in the Nxpre group. Monotherapy with NOF significantly reduced albuminuria relative to pretreatment values (72.6 ± 11.2 mg/day, P < 0.05 vs. NxV, NxLos, and Nxpre). Combined Los+NOF treatment exerted a much more efficient antiproteinuric action than any of the monotherapies, reversing albuminuria to 26.2 ± 3.5 mg/day (P < 0.05 vs. NxV, NxNOF, NxLos, and Nxpre).
As expected, Screat levels were markedly increased by renal mass reduction. None of the monotherapies promoted a significant decline in Screat relative to either NxV or Nxpre. In the Los+NOF group, Screat was significantly reduced compared with either NxV or Nxpre (1.0 ± 0.1 vs. 1.2 ± 0.1 in Nxpre, P < 0.05 and 1.4 ± 0.2 mg/day in NxV, P < 0.05).
As shown in Table 2, urinary excretion of TxB2, measured at 30 days of treatment, was slightly increased in NxV compared with S (20.6 ± 1.7 vs. 17.8 ± 1.9 ng/day in S, P > 0.05). Calculated TxB2 excretion per nephron was increased seven-fold in Nx rats 60 days after surgery (2.1 ± 0.2 vs. 0.3 ± 0.1 pg·nephron-1·day-1 in S, P < 0.05). As expected, total urinary TxB2 excretion was markedly depressed compared with S and NxV in NxNOF and NxLos/NOF rats (7.0 ± 1.0 and 8.6 ± 0.8 ng/day, respectively, P < 0.05 vs. S and NxV). TxB2 excretion in the NxNOF and NxLos/NOF groups appeared even more depressed when factored by the estimated number of nephrons, reaching values close to those verified in S (0.7 ± 0.1 and 0.9 ± 0.1 ng·nephron-1·day-1, respectively, P < 0.05 vs. NxV and P > 0.05 vs. S). Monotherapy with Los resulted in a slight but statistically significant reduction in the calculated TxB2 excretion per nephron relative to Nx (1.7 ± 0.2 ng·nephron-1·day-1, P < 0.05 vs. NxV).
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Glomerular segmental sclerotic lesions were evident 30 days after surgery (Nxpre group), the GSI reaching values almost 20-fold higher than in S (Table 3). Ninety days later (120 days after surgery), considerable progression of glomerular injury had occurred. In NxV rats, the GSI attained values almost 10-fold as high as in Nxpre and 200-fold higher than in S. Treatment with any of the monotherapies was associated with a less pronounced increment of the GSI, although the respective differences relative to the NxV group were not statistically significant. Combined Los/NOF treatment arrested the progression of glomerular injury, keeping the GSI at levels close to those verified in the Nxpre group (43 ± 11 vs. 19 ± 4 in Nxpre, P > 0.05). There was a significant correlation between GSI and TCP (r = 0.74, P < 0.01). Interstitial expansion was also a prominent component of renal injury after Nx (Table 3), %INT reaching values more than threefold higher than in Nxpre 120 days after Nx. Unlike NOF and Los monotherapies, combined Los/NOF treatment significantly attenuated the progression of interstitial expansion (4.8 ± 0.5 vs. 7.8 ± 0.7 in NxV, P < 0.05).
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Immunohistochemistry. Thirty days after Nx (Nxpre group), macrophage infiltration in the renal tissue (Table 3), assessed by the density of ED-1-positive cells, more than quadrupled compared with S values (131 ± 12 vs. 29 ± 3 cells/mm2 in S, P < 0.05). This was aggravated 120 days after surgery, macrophage density reaching values of 178 ± 23 cells/mm2 in the NxV group. None of the monotherapies had any significant influence on macrophage infiltration. However, the combined Los/NOF treatment reduced the renal macrophage density to levels significantly lower than in the Nxpre group (81 ± 6 cells/mm2 vs. Nxpre, P < 0.05). A significant linear correlation (r = 0.67, P < 0.001) was observed between the macrophage density and the GSI.
Figure 2A shows a typical staining pattern for ANG II in an afferent arteriole obtained from an S rat (the efferent arteriole, not stained, is shown as well). In Fig. 2B, ANG II-positive cells unrelated to vascular tissue are shown in the renal cortical interstitium of an Nx rat 120 days after ablation. Most of these ANG II-positive cells appeared in association with inflamed areas. Figure 3 shows the intensity of both modalities of ANG II expression in graphic form. Afferent arteriolar ANG II expression was deeply depressed in untreated Nx (0.04 ± 0.02 positively stained arterioles/mm2 in Nxpre and 0.03 ± 0.02 in NxV vs. 1.55 ± 0.16 in S, P < 0.05) and was not influenced by any of the treatments. By contrast, the density of ANG II-positive cells in the interstitium was already markedly increased at 30 days after ablation (Nxpre group) compared with S. These values were tripled 120 days after Nx (NxV group). The increasing interstitial ANG II expression was completely arrested and kept at pretreatment levels by the combined Los/NOF treatment. None of the monotherapies had any significant effect on interstitial ANG II expression.
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AT1R expression patterns are shown in Figs. 4 and 5. In the S group, AT1R expression was almost entirely confined to the tubular compartment and only sparsely expressed at glomeruli, vessels, and interstitium. Renal mass reduction increased only numerically the total renal expression of AT1R but promoted a drastic change in its intrarenal distribution. In the Nxpre group (30 days after Nx), total renal AT1R expression was clearly shifted to the interstitial area. This pattern became even more marked 120 days after ablation (NxV group), when AT1R expression at the tubules declined to very low levels (Fig. 5). None of the treatments promoted any significant change in the intensity or distribution of AT1R expression in Nx rats.
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As described previously (9, 49), COX-2 was constitutively expressed in cells of the macula densa region of S rats (Fig. 6A). Only rare COX-2-positive cells were found in glomeruli, vessels, or interstitium of intact kidneys. In accordance with previous observations of this laboratory (9), renal mass reduction numerically increased the frequency of macula densa staining positively for COX-2 (Table 4). Monotherapy with NOF or Los further augmented the expression of COX-2 at the macula densa, although only with the latter was this change statistically significant (Table 4). Combined Los/NOF treatment nearly doubled COX-2 expression at the macula densa (Table 4).
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As previously reported by this laboratory (9), there was a dramatic elevation in the density of COX-2-positive cells at glomeruli (Fig. 6B), vessels (Fig. 6C), and interstitium (Fig. 6D) 30 days after renal mass reduction, which was exacerbated after 120 days (Table 4). Monotherapies had no effect on the frequency of glomerular, vascular, or interstitial COX-2-positive cells, whereas combined Los/NOF treatment reduced vascular and glomerular COX-2 expression to values indistinguishable from those seen in S (Table 4). There was a strong positive correlation (r = 0.83, P < 0.001) between the glomerular density of COX-2-positive cells and the GSI.
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DISCUSSION |
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As described previously, intrarenal ANG II distribution was profoundly changed after renal mass reduction (28). Thirty days after ablation there was a marked decrease in ANG II expression in afferent arterioles, whereas large amounts of ANG II-positive cells appeared in the inflamed interstitial areas. This intrarenal shift of ANG II-positive cells was intensified 120 days after Nx, which may have contributed substantially to aggravate renal injury. These findings support and extend previous observations made in Nx and chronic nitric oxide (NO) inhibition models, which showed the presence of ANG II-positive cells in inflamed areas of the renal interstitium (28, 34). The pattern of intrarenal ANG II distribution suggests completely distinct roles for ANG II located in each of these "compartments." It is possible that "vascular" ANG II, the expression of which is reduced after renal ablation, is primarily linked to regulation of sodium balance. According to this view, vascular ANG II would be depressed by the extracellular fluid volume expansion known to occur in the Nx model (17). By contrast, "interstitial" ANG II seems to be insensitive to volume expansion and, given its association with interstitial expansion and cellular infiltration, it appears to be linked to renal inflammation. The origin of this interstitial ANG II was not addressed in the present study. ANG II may have been produced locally, because tubular epithelial cells, macrophages, and myofibroblasts all possess the appropriate biochemical machinery (12, 29, 39). Alternatively, ANG II may have originated outside the renal interstitium and undergone internalization by local cells after binding to AT1R (44, 57). Local ANG II may have had a proinflammatory and profibrotic effect at the renal interstitium. ANG II stimulates inflammatory cells such as lymphocytes (25) and activates nuclear factor-B in monocytes (36). In addition, ANG II stimulates the release of MCP-1 by vascular smooth muscle cells (36). In renal tissue, ANG II stimulates the proliferation of mesangial cells, glomerular endothelial cells (53), and myofibroblasts (55), as well as the secretion of chemokines and growth factors such as RANTES (52), PDGF (8), and MCP-1 (36). In addition, ANG II strongly stimulates collagen synthesis and renal fibrosis by activating TGF-
(54) and the MAPK/ERK pathway (41). Blockade of interstitial ANG II is a possible explanation for the well-known beneficial effect of RAS suppressors in progressive nephropathies, also observed in the NxLos and NxLos/NOF groups.
In agreement with previous observations (15), renal expression of AT1R in S rats appeared mostly in tubular cells, and to a lesser extent, at the interstitial area, whereas weaker expression was seen in vessels and glomeruli. This pattern was completely disrupted 30 days after Nx, when dense AT1R expression could be demonstrated in interstitial cells, far exceeding in intensity the expression of AT1R in tubules. The exact meaning of this finding and the cell types involved are uncertain. Several inflammatory cells known to infiltrate the renal interstitium in the Nx model have the potential to express AT1R, such as lymphocytes (25) and macrophages (29). In addition, AT1R may be expressed by myofibroblasts originating from tubular cell transdifferentiation (27). This hypothesis is particularly attractive because it helps to explain the progressive shift in AT1R expression, from tubules to the interstitial area, observed in Nx rats, and also because tubular cells already express AT1R under normal conditions. The simultaneous presence at the interstitial area of large amounts of ANG II and of the AT1R may accelerate the progression of the nephropathy by a positive-feedback mechanism. Consistent with this view is the aggravation of the renal structural injury at 120 days of Nx, which was paralleled by the intensity of the inflammatory infiltration and of the interstitial expression of ANG II.
The potential role of prostanoids in the pathogenesis of progressive nephropathies has long been acknowledged. The stimulation of podocytes by complement fractions can increase the local synthesis of prostanoids (40). Similarly, nonimmune mechanisms such as mesangial stretching can augment the expression of COX and enhance the production of its derivatives (1). Accordingly, studies of the Nx model showed increased urinary excretion of prostanoids per nephron (26).
Increased production of prostanoids can enhance inflammation and, therefore, accelerate renal injury. Prostanoids derived from COX-2 are thought to modulate proliferation and activation of T lymphocytes (20). Dendritic cells, described in the remnant kidney (33), constitutively express COX-2 and utilize prostanoids as an autocrine stimulus for cytokine secretion and for their own proliferation (51). In addition to its well-known vasoconstrictor effect (24), TxA2 stimulates the expression of adhesion molecules and of MCP-1 in endothelial cells (21), promotes the proliferation of mesangial cells (4), and enhances platelet aggregation and extracellular matrix production (5, 24).
Previous studies have shown that the renal cortical expression of COX-2 increases after renal ablation, whereas the expression of COX-1 remains unchanged (18, 49). We showed recently that a large fraction of the excess COX-2 expressed in remnant kidneys localizes in glomeruli, vessels, and the interstitium, especially in areas of injury and inflammation (9). The present study confirms these observations, reinforcing the concept that COX-2 can exert a dual role in this model: at the MD, COX-2 appears to exert a physiological effect, possibly related to sodium homeostasis. At "anomalous" locations such as glomeruli and vessels, COX-2 and its products would mediate inflammation and structural injury. The consistent presence of COX-2 in damaged areas, and the strong correlation between the intensity of its expression and parameters of renal injury, strengthens the notion that COX-2-derived prostanoids play an important pathogenic role in this model. The mechanisms by which COX-2 may have been induced in these areas are obscure. COX-2 may have been activated by ANG II anomalously produced in the interstitium (56), by stretching of mesangial cells resulting from glomerular hypertension (1) and/or by the action of other mediators such as TNF-
and interleukin-1
(7, 31). Once synthesized, prostanoids can further activate COX-2, thereby contributing to amplify and perpetuate the inflammatory process (42). The protective effect of chronic treatment with either COX-2-specific inhibitors (9, 48) or NOF (10) lends further support to the notion that prostanoids play a fundamental role in the pathogenesis of progressive renal injury in the Nx model.
Consistent with previous observations, Los monotherapy lowered blood pressure by 40 mmHg 1 mo after treatment was started, although TCP returned to pretreatment levels at the end of the study. In addition, Los limited UalbV, GSI, interstitial damage, macrophage infiltration, and ANG II-positive cell infiltration (11). However, protection conferred by losartan monotherapy was only partial, because progression of renal inflammation and of renal structural injury was not arrested in the NxLos group. There are at least three possible reasons for the limited efficacy of Los treatment in this study. First, rats failing to attain blood pressures higher than 140 mmHg or albuminuria in excess of 50 mg/dl 30 days after nephrectomy were excluded from the study to ensure that the attending nephropathy had a progressive nature. Second, unlike in most previous studies of this model, treatments were started only 30 days after nephrectomy, when renal injury was already established. Third, rats were followed up to 4 mo after renal mass reduction, whereas in most other studies of this model observations were ended at 2 mo or less. In the face of the unusual severity of renal injury, the relative resistance to Los treatment was not unexpected. At any instance, these findings agree with previous experimental observations, obtained in this laboratory and elsewhere (11, 19), as well as in large clinical trials (3), all of which indicate that, once set in motion, progressive nephropathies can be attenuated, but not detained, by isolated treatment with AT1R blockers or ACE inhibitors. As a whole, these observations suggest that events antecedent to the initiation of treatment may be of crucial importance in the pathogenesis of renal injury associated with this model. Additionally, the relative inefficiency of Los monotherapy in the present study may reflect the presence of ANG II-independent inflammatory events, as well as the recrudescence of arterial hypertension after the first few weeks of treatment. Finally, it is conceivable that the "conventional" dose of Los employed in the present study, although high enough to exert full vascular effect, was insufficient to effectively block the enormous amount of AT1R already present at the renal tissue when treatment was started.
NOF is a nonsteroidal anti-inflammatory compound with low gastrointestinal toxicity, presumably due to its NO-releasing properties (10, 47). It appears unlikely that NO released by NOF has a direct therapeutic effect because the NOF molecule is rapidly degraded in the intestinal lumen, releasing flurbiprofen (10). However, we cannot exclude the possibility that nitroso proteins, which have a much longer half-life than NO itself, propagate a possible protective effect of NO released into the intestinal lumen (37). Flurbiprofen is a potent inhibitor of both COX isoforms (45). In the present study, this property was demonstrated by the marked reduction in urinary TxB2 excretion obtained in the NxNOF and NxLOS/NOF groups. Monotherapy with NOF reduced proteinuria and attenuated the progression of glomerular injury and the intensity of local macrophage infiltration as effectively as monotherapy with Los, even though NOF exerted no effect on blood pressure. However, as in the case of Los treatment, progression of renal injury was not arrested. Moreover, no protective effect was observed regarding interstitial expansion or glomerular and vascular COX-2 expression. In addition, the effects of NOF were much more modest than those obtained when treatment was begun immediately after surgery (10), once again underlining the pathogenic importance of early events in this model.
Treatment of Nx rats with the Los/NOF combination promoted a significant regression of hypertension, albuminuria, and inflammatory signs such as macrophage infiltration and tissue COX-2 expression, whereas the parameters of structural tissue injury remained stable, or were strongly attenuated, compared with pretreatment levels. The protection achieved with combined treatment was much greater than that obtained with either drug alone. On the basis of the present study, one cannot exclude the hypothesis that the success of combined treatment was due to a particularly effective hemodynamic action, although previous observations from this laboratory (10) indicated that NOF had no significant effect on glomerular hemodynamics. Because treatment with NOF alone had no effect on blood pressure, it seems unlikely that the hemodynamic effect of LOS was directly intensified by its association with NOF. Therefore, the efficacy of combined treatment was likely due to the simultaneous blockade of the hemodynamic and proinflammatory actions of ANG II and COX derivatives and by abrogation of the complex interplay between hypertension and inflammation (34). The present findings support previous observations of the Nx model, which similarly indicated the superiority of the combination of a RAS suppressor with an anti-inflammatory agent (11, 13, 33). It is noteworthy that combined treatment afforded partial regression of the nephropathy associated with Nx even though it was started 1 mo after surgery, when renal injury was already established. This observation suggests that both continued stimulation of AT1 receptors and production of prostanoids continue to play an important pathogenic role even during the late phases of the process, necessitating vigorous and persistent treatment to prevent further renal deterioration.
Despite the promising response of Nx rats to combined treatment, it must be noted that NSAIDs are potentially nephrotoxic drugs, especially in the setting of chronic renal insufficiency. NOF promoted no glomerular filtration rate decline in these and in previous studies (10). However, there have been numerous anecdotal reports of acute renal failure, hyperkalemia, hypertension, and edema attributed to NSAIDs (14, 16), although few studies have directly assessed the objective risk associated with the clinical use of these compounds (38, 46). It is possible that simultaneous administration of a RAS suppressor, by limiting or suppressing the vascular effect of ANG II, reduces the risk of excessive renal vasoconstriction in the presence of a COX inhibitor (32). Nevertheless, any future systematic use of COX inhibitors in chronic nephropathies, alone or in combination, will necessitate the development of careful clinical studies to assess the safety of these compounds in this particular group of patients.
In summary, combined treatment with Los/NOF partially reversed the nephropathy and renal inflammation associated with the Nx model, showing much more effective protection than with either drug alone. Clinical studies are needed to establish whether this scheme may eventually become a new weapon in the limited arsenal currently available to attenuate or prevent human progressive nephropathies.
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ACKNOWLEDGMENTS |
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Preliminary results of this study were presented at the American Society of Nephrology/International Society of Nephrology World Congress of Nephrology, San Francisco, CA, October 1017, 2001, and published in abstract form (J Am Soc Nephrol 12: 814A, 2001).
GRANTS
This work was supported by Grants 95/47102 and 98/095694 from the State of São Paulo Foundation for Research Support. During these studies, R. Zatz was the recipient of a Research Award (326.429/81) from the Brazilian Council of Scientific and Technologic Development.
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FOOTNOTES |
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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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REFERENCES |
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