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B attenuates renal injury in the 5/6 renal ablation model
Renal Division, Department of Clinical Medicine, Faculty of Medical Sciences, University of São Paulo, São Paulo, Brazil
Submitted 25 May 2006 ; accepted in final form 31 July 2006
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ABSTRACT |
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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, AT1. 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. chronic kidney disease; inflammation; glomerulosclerosis; angiotensin II
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 AT1 receptor (AT1R), 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.
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METHODS |
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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 (PGC) 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 (UalbV) 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 (Screat). 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, AT1R, 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, AT1R, 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 AT1R), 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 AT1R), 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 x250 magnification and expressed as cells per millimeter squared. For each section, 25 microscopic fields, each corresponding to an area of 0.06 mm2, were examined. For AT1R, 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.
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RESULTS |
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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. 2E). 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. 2G). 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.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 PGC 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 AT1R 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 PGC, 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 AT1R 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.
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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).
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ACKNOWLEDGMENTS |
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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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B pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol 15: 1514–1529, 2004.B in tubular epithelial cells of rats with intense proteinuria: role of angiotensin II and endothelin-1. Hypertension 37: 1171–1178, 2001.B-dependent monocyte chemoattractant protein-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone. J Am Soc Nephrol 16: 688–696, 2005.B. Nature 365: 182–185, 1993.[CrossRef][Medline]B-dependent pathways in modulating macrophage infiltration in experimental diabetic nephropathy. J Am Soc Nephrol 15: 2139–2151, 2004.B activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol Dial Transplant 19: 2505–2512, 2004.B and AP-1 activation in human proteinuric renal disease. Kidney Int 60: 1366–1377, 2001.[CrossRef][Web of Science][Medline]B inhibition ameliorates angiotensin II-induced inflammatory damage in rats. Hypertension 35: 193–201, 2000.B activation. Ren Fail 23: 773–780, 2001.[CrossRef][Web of Science][Medline]B in experimental glomerulonephritis in rats. Biochim Biophys Acta 1316: 132–138, 1996.[Medline]This article has been cited by other articles:
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) 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.