Chronic inhibition of nuclear factor- B attenuates renal injury in the 5/6 renal ablation model

【摘要】  Recent studies indicated that the nuclear transcription factor, NF- B, activates a number of proinflammatory genes in subjects with progressive nephropathies. We investigated whether NF- B inhibition limits progressive renal injury in the 5/6 renal ablation model (Nx). Adult male Munich-Wistar rats were subdivided in four groups: S ( n = 16), subjected to sham operation; S+PDTC ( n = 18), sham-operated rats receiving the NF- B inhibitor pyrrolidine-dithiocarbamate (PDTC; 60 mg·kg -1 ·day -1 ) in drinking water; Nx ( n = 16), Nx rats receiving vehicle only; and Nx+PDTC ( n = 19), Nx rats given PDTC as above. Thirty days after renal ablation, Nx rats exhibited systemic and glomerular hypertension. Only the former was attenuated by PDTC treatment. Sixty days after renal ablation, Nx rats exhibited marked hypertension, albuminuria and creatinine retention, as well as glomerulosclerosis and cortical interstitial expansion/inflammation. Immunohistochemical analysis of Nx rats showed renal interstitial infiltration by macrophages and by cells staining positively for ANG II and its receptor, AT 1. Glomerular and interstitial cells expressing the p65 subunit of the NF- B system were also found. PDTC treatment attenuated renal injury and inflammation, as well as the density of cells staining positively for the p65 subunit. Activation of the NF- B system plays an important role in the pathogenesis of renal injury in the Nx model. Inhibition of this system may represent a new strategy to prevent the progression of chronic kidney disease.

【关键词】  chronic kidney disease inflammation glomerulosclerosis angiotensin II

THE PATHOGENESIS OF CHRONIC kidney disease (CKD) involves a complex interaction between hemodynamic abnormalities, represented mainly by systemic and glomerular hypertension, and inflammatory events, which include infiltration of glomeruli and interstitium by macrophages, lymphocytes, fibroblasts, and myofibroblasts, as well as exaggerated production of extracellular matrix ( 9, 10, 30 ). The interaction between all these phenomena to promote CKD is heavily dependent on the production of cytokines, chemokines, and growth factors, as well as on the simultaneous activation of a number of intracellular signaling cascades, which in turn govern the production of new inflammatory mediators, thus amplifying the initial inflammatory stimulus ( 12, 40 ).

The NF- B complex is one of the most important proinflammatory intracellular signaling systems, consisting of a heterodimer (subunits p50 and p65) attached to an inhibiting subunit (I- B). Activation of this complex requires phosphorylation and degradation of the I- B subunit and migration of the p50/p65 heterodimer to the nucleus, where it promotes further synthesis of inflammatory molecules ( 16 ). Phosphorylation of the I- B subunit can follow exposure to infectious agents or oxidative stress, as well as activation of superficial receptors by common inflammatory mediators, such as TNF- and IL-1 ( 16 ). In recent years, evidence has accumulated that locally produced ANG II can act as an inflammatory mediator, promoting, mainly through the AT 1 receptor (AT 1 R), the activation of the NF- B system, among a host of other cellular events ( 27 ).

Activation of the NF- B complex has been demonstrated in association with a number of renal diseases in which chronic inflammation plays a prominent role ( 11, 33 ). Accordingly, inhibition of the NF- B system limited or abolished the associated inflammatory response ( 16, 26 ). Pyrrolidine dithiocarbamate (PDTC) has been widely employed as an inhibitor of the NF- B system ( 5, 38 ). The exact mechanisms by which PDTC exerts this effect are presently unclear. PDTC may directly impede the degradation of the I- B subunit. In addition, PDTC may act through its anti-oxidant properties, inhibiting the well-known stimulant effect of oxidative stress on the NF- B system ( 34 ).

In the 5/6 renal ablation (Nx), a commonly used model of CKD, severe and progressive renal injury has been attributed to hemodynamic abnormalities, especially glomerular hypertension, as well as to inflammatory events such as macrophage infiltration, excessive production of extracellular matrix, and local activation of the renin-angiotensin system ( 9, 12 ). In these animals, increased activity of the NF- B system was reported especially at the interstitial area, whereas amelioration of proteinuria and renal structural damage was associated with decreased activity of the NF- B system ( 6, 13 ). However, the possible renoprotective effect of maneuvers intended to inhibit the NF- B system has not been examined in the Nx model.

In the present study, we sought to determine whether treatment of Nx rats with PDTC would afford renal protection comparable to that obtained in other experimental models. We also investigated whether such protection would be linked to a hemodynamic effect, to an effect on the NF- B system, and therefore on the associated inflammatory process, or to a combination of both effects.

METHODS

We used in this study 69 adult male Munich-Wistar rats obtained from a local colony, weighing 240-260 g. All rats received food (22% protein) and tap water ad libitum. Surgical reduction of renal mass (Nx) was performed under anesthesia with pentobarbital sodium, 50 mg/kg ip, through removal of the right kidney and infarction of approximately two-thirds of the left one. Sham-operated rats (S) underwent anesthesia, ventral laparotomy, and manipulation of the renal pedicles without removal of renal mass.

Experimental groups. Immediately after surgery, rats were randomly assigned to four experimental groups: S ( n = 16), sham-operated rats; S+PDTC ( n = 18), sham-operated rats receiving PDTC, 60 mg·kg -1 ·day -1; Nx ( n = 16), rats subjected to 5/6 renal ablation (Nx) and treated with vehicle only; Nx+PDTC ( n = 19), Nx rats receiving PDTC, 60 mg·kg -1 ·day -1. PDTC was dissolved in drinking water and untreated rats received vehicle only. All experimental procedures were conducted in conformity with international and institutional guidelines and approved by the local Ethics Committee (CAPPesq No. 159/06).

Renal hemodynamic studies. Thirty days after renal ablation, rats from groups S ( n = 7), S+PDTC ( n = 7), Nx ( n = 7), and Nx+PDTC ( n = 7) were anesthetized with 100 mg/kg ip Inactin and prepared for whole kidney and glomerular hemodynamic studies. The glomerular hydraulic pressure (P GC ) was determined through direct micropuncture of superficial glomeruli. The details of the experimental procedures and analytic techniques utilized in these studies are given elsewhere ( 10 ).

Long-term studies. A separate cohort consisting of 9 S rats, 11 rats from group S+PDTC, 9 rats from group Nx, and 12 rats from group Nx+PDTC was followed up to 60 days of treatment. At 30 and 60 days after ablation, the tail-cuff pressure (TCP) and 24-h urinary albumin excretion (U alb V) were determined. The rats were anesthetized with 50 mg/kg ip pentobarbital sodium, and blood was collected from the abdominal aorta for determination of serum creatinine concentration (S creat ). The kidneys were then perfusion-fixed as described previously ( 10 ). After fixation, the renal tissue was weighed and two midcoronal sections were postfixed in buffered 4% formaldehyde solution. The material was then embedded in paraffin by conventional sequential techniques for assessment of glomerular and interstitial injury, as well as for immunohistochemical detection of the macrophage-specific antigen, ED-1, ANG II, AT 1 R, and the p65 subunit of the NF- B complex.

Histomorphometry. Sections 3- to 4-µm thick were stained by the periodic acid Schiff and Masson trichrome methods to assess the extent of glomerulosclerosis (GS) and interstitial expansion, respectively. A single observer (DMACM), blinded to the groups, performed all morphometric procedures. The severity of GS was assessed by attributing to each glomerulus a score that reflected the proportion of the tuft area that was taken by the sclerotic lesion, as described previously ( 10 ). A GS index (GSI) was derived for each rat as the weighted average of the individual glomerular scores, multiplied by 100. At least 120 glomeruli were examined for each rat. To evaluate the magnitude of renal interstitial expansion, the fraction of renal cortex taken by interstitial tissue was quantitatively evaluated in Masson-stained sections by a point counting technique ( 18 ) in 25 consecutive microscopic fields.

Immunohistochemical techniques. To detect ANG II, AT 1 R, the ED-1 antigen, and the NF- B p65 subunit, 4-µm-thick paraffin-embedded sections were mounted on glass slides coated with 2% gelatin and deparaffinized by conventional sequential techniques. Sections were then subjected to microwave irradiation in citrate buffer to enhance antigen retrieval. Negative control experiments for all antigens were performed by omitting incubation with the primary antibody.

For immunohistochemical detection of macrophages, a monoclonal mouse anti-rat ED-1 antibody (Serotec, Oxford, UK) was used. Sections were preincubated with normal rabbit serum (Vector Laboratories, Burlingame, CA) and then incubated overnight with the primary antibody at 4°C in a humidified chamber. After being washed, sections were incubated with rabbit anti-mouse immunoglobulins (Dako). To complete the sandwich technique, incubation with a soluble complex of alkaline phosphatase anti-alkaline phosphatase (APAAP; Dako) was performed. The last two steps were repeated to enhance the intensity of the reaction product. Finally, the slides were developed with a fast-red dye solution and counterstained.

Specific monoclonal primary antibodies were purchased from Peninsula Laboratories (San Carlos, CA; for ANG II), RDI (Flanders, NJ; for AT 1 R), and Santa Cruz Biotechnology (Santa Cruz, CA; for the p65 subunit). Sections were preincubated with avidin and biotin solutions to block nonspecific binding of these compounds and then preincubated with normal horse serum (Vector Laboratories) to reduce nonspecific staining. Detection of the p65 subunit required additional previous incubation with a hydrogen peroxide-methanol solution to block endogenous peroxidase activity. The incubation with the respective primary antibodies was carried out overnight at room temperature. Sections were then incubated with rat-adsorbed biotinylated anti-mouse IgG (Vector Laboratories) for 45 min at room temperature, followed by incubation, for 30 min at room temperature, with a streptavidin-biotin-alkaline phosphatase complex (Dako; for ANG II and AT 1 R), or a streptavidin-biotin peroxidase complex (Dako) for the p65 subunit. Sections were thereafter incubated with a freshly prepared substrate, consisting of naphthol AS-MX-phosphate (Sigma, St. Louis, MO) and fast red dye (Sigma) or diaminobenzidine solution (Dako), counterstained with Mayer's hemalaum (Merck, Darmstadt, Germany) and covered with Kaiser's glycerin-gelatin (Merck). Further details of the immunohistochemical procedures followed in this study are given in detail elsewhere ( 10, 12 ).

Quantitative analysis of ED-1-, ANG II-, and p65-positive cells was performed by cell counting in a blinded fashion by a single observer (DMACM) under x 250 magnification and expressed as cells per millimeter squared. For each section, 25 microscopic fields, each corresponding to an area of 0.06 mm 2, were examined. For AT 1 R, a point-counting technique was utilized ( 12 ) under the same magnification and the same blinding of the observer as for the other molecules sought in this study.

Analytic methods. Urinary albumin excretion rate was determined by radial immunodiffusion ( 22 ). Total plasma protein concentration was determined by refractometry. Serum creatinine concentration was assessed by a colorimetric method.

Statistics. One-way ANOVA with post hoc pairwise comparisons according to the Bonferroni method was employed in this study ( 39 ). P levels of 0.05 or less were significant. Since GSI and albumin excretion rates were not normally distributed, log transformations were performed before statistical analysis of these parameters. The Pearson correlation coefficient was calculated to assess the strength of linear regressions.

RESULTS

Renal hemodynamic studies. Renal and systemic functional and hemodynamic parameters obtained 30 days after renal ablation are given in Table 1. PDTC treatment had no effect on S rats. Food intake was similar among groups, although it tended to be lower in untreated Nx rats (data not shown). In the Nx group, growth was limited, and body weight was reduced by about 12% relative to S. PDTC treatment promoted no further growth limitation. Mean arterial pressure (MAP) was significantly elevated in group Nx (137 ± 6 vs. 106 ± 2 mmHg in S, P < 0.05). PDTC treatment lowered MAP in treated Nx rats, which nevertheless exhibited higher levels than observed in sham-operated rats (116 ± 3 mmHg, P < 0.05 vs. Nx). Kidney weight was expectedly smaller in Nx than in S (1.0 ± 0.04 vs. 1.4 ± 0.06 g in S, P < 0.05) and was unaffected by PDTC treatment. Whole kidney glomerular filtration rate (GFR) was 57% lower in untreated Nx rats compared with S, indicating the expected occurrence of hyperfiltration, since renal mass, hence the nephron number, was reduced by over 80% in these animals. PDTC treatment promoted no additional GFR reduction (0.6 ± 0.1 ml/min in group Nx+PDTC, P 0.05 vs. Nx). Renal plasma flow (RPF) was lowered in similar disproportion to nephron number reduction and was equally unaffected by PDTC treatment. Renal vascular resistance (RVR) was increased in Nx rats (45 ± 7 mmHg·ml -1 ·min, P < 0.05 vs. S). PDTC treatment promoted a numerical decrease in RVR ( P 0.05 vs. Nx). As shown previously, glomerular hydraulic pressure (P GC ) was markedly elevated in group Nx (67 ± 3 vs. 53 ± 1 mmHg in S, P < 0.05). PDTC treatment had no significant effect on P GC (64 ± 2 mmHg in group Nx+PDTC, P < 0.05 vs. S+PDTC and P 0.05 vs. Nx).

Table 1. Renal and systemic functional parameters 30 days after 5/6 nephrectomy

Follow up studies (60 days of ablation). Body weight, TCP, albuminuria, and serum creatinine at 60 days after 5/6 renal ablation are given in Table 2. Again, no effect of PDTC treatment was observed in S rats. Weight gain was significantly lower in both untreated and PDTC-treated Nx rats compared with the respective S. However, growth stunting was less severe in rats treated with PDTC. TCP was markedly elevated in Nx rats (232 ± 6 vs. 135 ± 1 mmHg in S, P < 0.05). Treatment with PDTC lowered TCP, which nevertheless remained elevated in group Nx+PDTC (174 ± 10 mmHg vs. Nx, P < 0.05). Marked albuminuria was observed in group Nx (81 ± 6 vs. 2.1 ± 0.4 mg/24 h in S, P < 0.05). PDTC treatment significantly reduced U alb V compared with Nx (54 ± 6 mg/24 h in group Nx+PDTC, P < 0.05 vs. S+PDTC and vs. Nx). S creat was elevated in group Nx compared with S (1.2 ± 0.1 vs. 0.5 ± 0.1 mg/dl in S, P < 0.05). Treatment with PDTC significantly reduced S creat (0.9 ± 0.1 mg/dl in group Nx+PDTC, P < 0.05 vs. Nx and S+PDTC).

Table 2. Renal and systemic functional parameters 60 days after 5/6 nephrectomy

Renal histomorphometric and immunohistochemical studies (60 days after renal ablation). The GSI was negligible in S and S+PDTC rats ( Fig. 1 A ). Expectedly, GSI was markedly elevated in group Nx compared with S (43 ± 10 vs. 0.9 ± 0.2 in S, P < 0.05). Glomerular injury was clearly attenuated in group Nx+PDTC, although GSI remained significantly higher than in group S+PDTC (12 ± 2 vs. 0.30 ± 0.18 in S+PDTC, P < 0.05 vs. S and Nx). Accordingly, the fraction of the renal cortex occupied by interstitium ( Fig. 1 B ) was sharply increased by renal ablation (4.8 ± 0.6% in group Nx vs. 0.2 ± 0.1 in S, P < 0.05). PDTC treatment limited interstitial expansion compared with Nx (2.1 ± 0.4% in group Nx+PDTC, P < 0.05 vs. both Nx and S+PDTC).

Fig. 1. Glomerulosclerosis index (GSI; top ) and percent interstitial area (%INT; bottom ) 60 days after renal ablation.

Table 3 shows data obtained after immunohistochemical analysis of the renal tissue. As shown previously ( 10, 12 ), renal infiltration by macrophages, evaluated by the density of ED-1-positive cells, was intense in Nx rats (133 ± 16 in group Nx vs. 20 ± 3 cells/mm 2 in S, P < 0.05), concentrating mostly at areas of interstitial inflammation. PDTC treatment limited macrophage infiltration, although the ED-1 density remained significantly above normal (85 ± 8 cells/mm 2 in Nx+PDTC, P < 0.05 vs. Nx and S+PDTC). The density of cells staining positively for ANG II was insignificant in S. The same finding was obtained in group S+PDTC. ANG II-positive cells were much more numerous in group Nx (5.7 ± 0.4 vs. 0.7 ± 0.2 cells/mm 2 in S, P < 0.05) and tended to distribute in a pattern similar to that observed for macrophages, accumulating at inflamed interstitial areas, in consistency with our previous observations ( 12 ). The number of ANG II-positive cells was reduced in Nx rats receiving PDTC treatment, although it remained significantly higher than in S+PDTC (2.0 ± 0.3 cells/mm 2, P < 0.05 vs. Nx and S+PDTC).

Table 3. Immunohistochemical analysis 60 days after 5/6 nephrectomy

The p65 subunit of the NF- B complex was sparsely expressed in the tubules of S and S+PDTC rats, virtually no cells appearing at the glomerular or interstitial areas ( Fig. 2, A - D ). In Nx rats, tubular p65 expression was also scant. Nevertheless, p65-positive cells, many of which exhibiting morphology and location characteristic of podocytes, were observed in glomeruli ( Fig. 2 E ). A more numerous contingent of p65-positive cells distributed at the interstitium, following a pattern that was identical to that observed for ANG II-positive cells and macrophages, assembling mostly at inflamed areas ( Fig. 2 G ). PDTC treatment reduced the number of both glomerular and interstitial p65-positive cells ( Fig. 2, F and H ). Parameters quantifying the immunohistochemical expression of p65 are shown in Table 3.

Fig. 2. Representative microphotographs of the immunohistochemical expression of the p65 subunit 60 days after renal ablation or sham operation. Little glomerular ( A, B ) or interstitial ( C, D ) staining for the p65 subunit was observed in untreated ( A, C ) and PDTC-treated ( B, D ), sham-operated rats. E : glomerular cells staining positively for the p65 subunit (appearing in dark tones) in an Nx rat. F : much less staining for p65 at a glomerular tuft of an Nx rat receiving PDTC. G : cells staining positively for the p65 subunit (in dark tones) at an inflamed interstitial area of an Nx rat. H : little interstitial expression of the p65 subunit in a PDTC-treated Nx rat. All images were captured under x 400 magnification. To improve the visibility of cells staining positively for p65 (originally in red), all images shown in this figure were processed using Adobe PhotoShop, version 5.0, to dim structures staining in blue (mostly nuclei of cells negative for p65) and then converted to grayscale.

A significant positive correlation was observed between GSI and the frequency of glomerular p65-positive cells ( Fig. 3 A ). Likewise, the percent cortical interstitial area correlated strongly with the intensity of renal interstitial infiltration by p65-positive cells ( Fig. 3 B ). A significant positive correlation was also seen between the density of p65-positive and ANG II-positive cells at the renal interstitium ( Fig. 3 C ). Significant correlation was also observed between the frequencies of either p65-positive or ANG II-positive cells and that of macrophages at the renal interstitium (data not shown).

Fig. 3. Positive linear correlation, observed in groups Nx ( ) and Nx+PDTC ( ), between GSI and the frequency of p65+ glomerular cells ( top ), between percent cortical interstitial area and the frequency of p65+ interstitial cells ( middle ), and between the frequency of p65+ interstitial cells and the frequency of ANG II+ interstitial cells ( bottom ). Values for the respective Pearson correlation coefficient, r, and for the statistical significance of the correlation are shown in each panel.

In S and S+PDTC rats, AT 1 R was expressed almost exclusively in vessels and proximal tubules. This pattern was entirely changed in Nx rats, in which most of the AT 1 R expression appeared at the interstitium, chiefly at areas where expansion and inflammation were conspicuous. Accordingly, the ratio between the parameters measuring the interstitial and tubular AT 1 R expressions (AT 1 R int /AT 1 R tub ) was markedly increased (2.3 ± 0.3 vs. S, P < 0.05). PDTC treatment had no effect on the AT 1 R int /AT 1 R tub ratio.

DISCUSSION

As shown previously ( 10 ), systemic and glomerular hypertension were readily observed 30 days after renal ablation in untreated Nx rats, in association with structural and functional renal hypertrophy. Sixty days after renal ablation, GS and interstitial expansion were evident in untreated Nx rats, also reproducing previous findings obtained in this laboratory ( 10 ). These structural abnormalities were associated with intense renal infiltration by macrophages, along with expansion of the extracellular matrix, most notably at the interstitial area, indicating the development of a chronic inflammatory reaction. The participation of locally produced ANG II in the pathogenesis of inflammation and renal damage in Nx rats is suggested by the finding of cells staining positively for this peptide, as well as by anomalous AT 1 R expression, at inflamed interstitial areas. Moreover, the intensity of interstitial macrophage infiltration correlated significantly with that of cells staining positively for ANG II, further suggesting the participation of ANG II in the pathogenesis of interstitial inflammation. Cultured mesangial cells and podocytes generate ANG II, whereas mesangial cells upregulate AT 1 R when subjected to mechanical strain ( 3, 7 ), suggesting that a similar behavior might follow exposure of the glomerular wall to heightened intracapillary pressures such as observed in this study. Abundant evidence now indicates that this anomalous ANG II generation is unrelated to sodium homeostasis and that, instead, it stimulates the cellular immune response and the development of chronic inflammation ( 27, 32 ). These effects can be mediated by several intracellular signaling pathways, among which the NF- B pathway occupies a prominent position. ANG II has been shown to activate the NF- B system in inflammatory as well as in resident renal parenchymal and vascular cells ( 19, 32 ). Of interest, the NF- B system can be directly activated in cultured mesangial cells subjected to mechanical stress ( 14 ). The importance of the NF- B system in the pathogenesis of chronic renal disease is illustrated by the observation that it is activated, by ANG II and/or by other stimuli, in such diverse experimental models as renal ablation ( 6, 13 ), diabetic nephropathy ( 21 ), and protein overload nephropathy ( 11 ), as well as in such clinical conditions as glomerulonephritis ( 24 ), human diabetic nephropathy ( 23 ), and atherosclerosis ( 4 ). Accordingly, inhibition of the NF- B system is associated with amelioration of renal and vascular injury and inflammation ( 17, 37 ).

In the present study, interstitial and glomerular cells staining positively for the p65 subunit were readily detected by immunohistochemistry in the untreated Nx Group, indicating that normally hidden specific epitopes of the p65 subunit were exposed, most likely after degradation of the I- B inhibitory subunit ( 16 ). Although this finding does not prove that nuclear translocation of the p65-p50 dimer with stimulation of proinflammatory genes actually happened, it is consistent with activation of the NF- B system, shown previously to occur in the Nx model ( 6, 13 ). This notion is strengthened by the finding that the frequency of p65-positive glomerular cells correlated significantly with the GS index, even though the frequency of p65-positive cells at the glomerular area was much lower than at the interstitium. The association between p65 expression and renal injury was even more evident at the interstitium, since the density of p65-positive interstitial cells, observed most frequently at areas of inflammation, correlated strongly with the percent cortical interstitial area. Accordingly, the density of p65-positive interstitial cells correlated significantly with the intensity of interstitial infiltration by ANG II-positive cells, in keeping with the concept that abnormally produced ANG II activates the NF- B system. Collectively, these findings suggest that a complex sequence of pathogenic events was set in motion in Nx rats, in which mechanical strain to the glomerular wall was translated into local ANG II generation and stimulation of the NF- B system, with development of intense inflammatory infiltration, inordinate synthesis of extracellular matrix components, and progressive glomerular and interstitial injury.

The identity of the p65-positive glomerular and interstitial cells was not determined in the present study. It is conceivable that at least part of these cells, especially at the interstitium, were activated macrophages, shown to express the p65 subunit under ANG II stimulation ( 19 ). At the glomerular tuft, some morphologic characteristics of the p65-positive cells strongly resembled those of podocytes, which were shown to possess the ability to activate the NF- B system ( 25 ), although the expression of the p65 subunit might also occur in mesangial cells ( 14 ), as well as in macrophages.

PDTC has been shown to prevent or attenuate inflammation and progressive injury of the renal tissue in such diverse experimental models of CKD as the spontaneously hypertensive rat ( 31 ), ureteral obstruction ( 8 ), the renin double-transgenic model ( 26 ), and adriamycin nephropathy ( 29 ). In the present study, PDTC treatment, used for the first time in the renal ablation model, was associated with substantial attenuation of systemic hypertension, albuminuria, renal inflammation, and renal injury. These beneficial effects of PDTC might be ascribed to a hemodynamic effect, since systemic arterial pressure, observed both directly and through measurement of TCP, was substantially lowered, although not normalized, by PDTC treatment. This favorable hemodynamic effect could derive from a direct effect of PDTC on renal and systemic vessels, and/or an effect on renal sodium handling, although these possibilities are essentially speculative at present. Alternatively, blood pressure reduction could reflect the anti-inflammatory properties of PDTC, in consistency with the findings of Rodríguez-Iturbe and co-workers ( 31 ), who showed that hypertension was prevented in the spontaneously hypertensive rat by early treatment with PDTC. Whatever the underlying mechanism, blood pressure reduction was not transmitted to the glomerular microcirculation, since glomerular hypertension persisted in PDTC-treated Nx rats. The reason why PDTC treatment failed to reduce glomerular hypertension in Nx rats is unclear. However, since systemic blood pressure was attenuated, persistence of glomerular hypertension must reflect an adjustment of the glomerular arteriolar resistances, possibly dictated by a complex interaction between humoral factors and reflex myogenic dilatation of the afferent arteriole ( 2 ). As alternative possibilities, PDTC may have increased the pressure in the Bowman's space, thus reducing the transcapillary pressure, or may have altered the ratio of glomerular to systemic pressure under anesthesia, thus falsely elevating P GC in the experimental setting of this study.

Given the persistence of glomerular hypertension in PDTC-treated rats, the observed renoprotective effect must have involved events occurring downstream to glomerular hemodynamic stress. PDTC treatment had no effect on AT 1 R expression but did decrease the density of cells staining positively for ANG II. It is unclear whether this effect was direct or simply reflected mitigation of renal inflammation. An effect on NF- B appears as a highly plausible possibility, given the pivotal role of this system in the propagation and perpetuation of chronic inflammation. PDTC blocks degradation of the I- B inhibitory subunit ( 34 ), hence hindering the activation and nuclear translocation of the p65/p50 dimer. In this manner, even without lowering P GC, PDTC can prevent the translation of glomerular strain into multiple gene activation and enhanced production of inflammatory mediators driven by the NF- B system. In addition to this direct effect, PDTC can inhibit the activation of the NF- B system through its antioxidant properties. Abundant evidence suggests that oxidative stress may exert an important role in the pathogenesis of CKD. Plasma levels and urinary excretion of well-known markers of oxidative stress, such as malondialdehyde, isoprostanes, and lipid peroxides, are increased in CKD patients and in experimental models of CKD, including the renal ablation model ( 1, 35 ). Accordingly, increased plasma levels of free isoprostanes were reported after chronic infusion of ANG II ( 15 ), a maneuver associated with the development of severe renal injury. Conversely, treatment with ACE inhibitors or AT 1 R blockers was associated with a simultaneous decrease in oxidative stress and renal injury in rats with CKD ( 20, 28 ). Consistent with these findings, renal injury was ameliorated in the Nx model by antioxidant therapy ( 35 ). Oxidative stress is believed to promote tissue inflammation through activation of several intracellular transcription pathways, including the NF- B system ( 34 ). Therefore, the renoprotective effect afforded by PDTC in the present study can also be explained by an additional, indirect inhibition of the NF- B system through amelioration of oxidative stress.

Whatever the mechanisms involved, the protective effect of PDTC was only partial, since significant proteinuria, GS, and interstitial inflammation persisted in treated rats. Failure to entirely prevent renal injury may reflect incomplete inhibition of I- B phosphorylation, oxidative stress, or both, although verification of these hypotheses may require assessment of a possible dose-response effect. Alternatively, even complete inhibition of the NF- B system may be insufficient to prevent damage to the renal tissue because other intracellular signaling systems are likely activated in CKD ( 36 ).

Considering the urgent need for new therapeutic strategies in the quest to control the current CKD pandemics, the potential clinical application of the present findings is unquestionable. However, no data on the clinical pharmacology of PDTC are presently available. Although no major collateral effect was observed in treated Nx rats, studies on the clinical toxicity and safety of PDTC and of other inhibitors of the NF- B system are obviously necessary before they can be tested for efficacy and, hopefully, incorporated into the armamentarium employed against the progression of CKD.

In summary, the results of the present study are consistent with the notion that the NF- B system participates in the pathogenesis of renal injury in the 5/6 renal ablation model. Treatment with PDTC, used for the first time in this model, strongly attenuated renal damage and inflammation without lowering glomerular blood pressure, suggesting that inhibition of the NF- B system, directly or through anti-oxidant activity, may have been determinant for the observed renoprotective effect. PDTC and other NF- B inhibitors may constitute a new option for the treatment of CKD, although studies of safety and toxicity are required before such drugs can be considered for clinical use.

GRANTS

During these studies, R. Zatz was the recipient of a Research Award (no. 326.429/81) from the Brazilian Council of Scientific and Technologic Development (CNPq).

Portions of this study were presented at the 36th Congress of the American Society of Nephrology, San Diego, CA, November 12-17, 2003, and published in abstract form ( J Am Soc Nephrol 14: 635A, 2003).

ACKNOWLEDGMENTS

We thank C. R. Sena and L. Faria de Carvalho for expert technical assistance.

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作者单位：Renal Division, Department of Clinical Medicine, Faculty of Medical Sciences, University of São Paulo, São Paulo, Brazil