Change in Renal Heme Oxygenase Expression in Cyclosporine A-induced Injury
Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy (RR,LR,BB,RB); Department of Pharmacology, New York Medical College, Valhalla, New York (AAG,NGA); and Department of Medicine, Robert Wood Johnson Medical School, New Brunswick, New Jersey (EAL)
Correspondence to: Prof. Rita Rezzani, Department of Biomedical Sciences and Biotechnology, Division of Human Anatomy, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. E-mail: rezzani{at}med.unibs.it
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Summary |
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Key Words: cyclosporine A heme oxygenase renal injury
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Introduction |
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The most common feature of CsA use is the development of interstitial fibrosis, which can be patchy or "striped" (Verpooten et al. 1986; Rezzani et al. 2001b
). This may promote development of chronic renal failure in organ transplant recipients, although long-term studies have shown that, with the dose regimens now employed, renal function can remain stable but impaired for many years (Bantle et al. 1990
). The mechanism of CsA-induced interstitial fibrosis has been attributed to an angiotensin-dependent upregulation of molecules that promote scarring, such as TGF-ß1 (Ahmed et al. 2004
) and osteopontin (Lim et al. 2004
). CsA may also impair the regenerative capacity of microvascular endothelial cells and induce apoptosis (Li et al. 2004
). To this end, thrombotic microangiopathy is a distinct form of CsA-induced vascular toxicity that may result from a direct effect on vascular endothelium, possibly interfering with generation of prostacyclin (Rezzani et al. 2001b
).
It is unknown whether renal tissue possesses a defense mechanism against CsA-induced injury. Heme oxygenase (HO), the rate-limiting enzyme in heme catabolism, has been shown to attenuate the extent of various forms of renal injury, most notably injury due to ischemia and to inflammation (Wolf et al. 1994; Datta et al. 1999
). HO is a microsomal enzyme that catalyzes the NADPH-, O2-, and CYP450 reductase-dependent oxidation of heme to carbon monoxide (CO), iron, and biliverdin, which is reduced to bilirubin by biliverdin reductase (Abraham et al. 1996
). Two catalytically active HO isoenzymes have been characterized, HO-1 and HO-2. HO-1 is induced by a host of stimuli that have in common the ability to cause oxidative stress. HO-2 is a constitutive form and, to date, only the adrenal glucocorticoids have been identified as inducers of its gene (Abraham et al. 1996
). In the rat kidney the HO-1 isoform is distributed in proximal convoluted tubules and loops of Henle (Da Silva et al. 2001
). HO controls the initial and rate-limiting step in heme catabolism, thereby regulating cellular heme levels and generation of CO. In this dual capacity, HO controls both the availability of heme for synthesis of heme-containing enzymes and the generation of CO, which binds to these enzymes and thus modulates their activity. Examples of heme-containing enzymes that have been implicated in the pathophysiology of CsA-induced nephrotoxicity are CYP450 (Mayer et al. 1989
), the arachidonate cyclo-oxygenases (COXs) (Jenkins et al. 2001
), TxA2 synthase (Perico et al. 1986
), and NOS (Amore et al. 2000
). It follows that changes in HO expression or enzyme activity in response to CsA would impact on the activity of heme-containing enzymes. The ensuing changes in product synthesis, i.e., prostaglandin, TxA2, and NO, might modulate the extent of CsA-induced renal vasoconstriction and cell injury.
The hypothesis tested is that HO expression is associated with the attenuation of CsA-induced nephrotoxicity. Moreover, we examined by electron microscopic analysis whether the negative CsA effects on intracellular structures might be related to their major susceptibility to drug metabolism and the anatomic structures that were considered to be the intrarenal target for CsA damage.
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Materials and Methods |
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Renal Morphology
For tubulointerstitial fibrosis, the quantitative scorings used were similar to those reported by Li et al. (2004). A minimum of 20 fields per section was assessed and graded using a color image analyzer (Image-Pro Plus, Media Cybernetics Inc.; Silver Spring, MD). The extent of tubulonterstitial fibrosis was estimated by counting the percentage of areas injured per field using a scoring scale of 0 to 3+, where 0 = normal interstitium, 0.5 = <5% injured area, 1 = 515% injured area, 1.5 = 1625% injured area, 2 = 2635% injured area, 2.5 = 3645% injured area, and 3 = >45% injured area.
Western Blot Analyses
All cortical homogenates (protein lysates), obtained by transverse cutting near the renal apex, were processed for Western blot analysis and protein levels were visualized by immunoblotting with rabbit antibodies against human HO-1 or HO-2 (Stressgen Biotechnologies; Victoria, BC, Canada). Briefly, 30 µg of lysate was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Amersham; Piscataway, NJ) using a semidry transfer apparatus (Bio-Rad; Hercules, CA). The membranes were incubated with 5% milk in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween-20 (TBST) buffer at 4C overnight. After washing with TBST, the membranes were incubated with a 1:2000 dilution of anti-HO-1 or anti-HO-2 antibodies for 1 hr at room temperature (RT) under constant shaking. Membranes were then washed and probed with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) at a dilution of 1:2000. Chemiluminescence detection was performed with the Amersham ECL detection kit according to the manufacturer's instructions. The bands were analyzed densitometrically using an image analyzer (Photo-Capt V.99 software).
Statistical Analysis
The results are presented as mean ± SEM of the number of triplicate determinations. Statistical significance of differences between the experimental groups was estimated using the ANOVA and Bonferroni test, with p<0.01 considered as significant.
Measurement of HO Enzyme Activity
Kidneys were perfused with ice-cold HEPE's solution, sliced, and cortical tissue was homogenized in 10 mM Tris buffer, pH 7.5, containing 0.25 M sucrose. Homogenates were centrifuged at 27,000 x g for 20 min at 4C. The supernatant was centrifuged at 105,000 x g for 1 hr at 4C and the resulting microsomal pellet was resuspended in 0.1 M potassium phosphate buffer, pH 7.6. Protein concentration was measured according to the method of Bradford (BioRad). Homogenates were used for measurement of HO activity, which was assayed with an NADPH generating system as previously described (Morimoto et al. 2001). The amount of bilirubin generated was determined by a scanning spectrophotometer (Lambda 17 UV/VIS; Perkin-Elmer Cetus Instruments; Norwalk, CT) and was defined as the difference between 460 and 530 nm (extinction coefficient 40 mM1 for bilirubin). Results were expressed as nanomoles of bilirubin per milligram of protein per 30 min.
Immunohistochemical Analysis for HO-1, HO-2, and ET-1 Proteins
The serial sections were immersed in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. The sections were then incubated with goat serum (Dakopatts, Glostrup, Denmark; diluted 1:5) for 40 min, and successively with rabbit polyclonal anti-HO-1 and HO-2 antibodies (diluted 1:100) and rabbit polyclonal anti-ET-1 antibody (diluted 1:50) for 2 hr. The sections were washed in Tris-buffered saline (0.1 M, pH 7.4) and incubated with biotinylated goat anti-rabbit immunoglobulin and avidinbiotinhorseradish peroxidase complex according to the manufacturer's instructions (ABC kit; Dakopatts).
Specificities of antibody labeling were investigated using appropriate controls, incubating the tissue sections with Tris-buffered saline instead of the primary or secondary antibody.
Semiquantitative Analysis of HO-1, HO-2, and ET-1 Immunostaining
Intensity of the immunostaining within the glomeruli, proximal tubules, and distal tubules was evaluated separately and blindly. Proximal tubules were differentiated from distal tubules as follows: proximal tubules had larger diameters than distal tubules. The nuclei of proximal tubule epithelial cells were usually arrayed at the base of the cells. Most proximal tubule epithelium had obvious brush borders. Based on the intensity and distribution of HO staining, the degree of HO staining was graded as ± when the staining was very weak, + when the staining was weak, ++ when the staining was moderately positive, and +++ when the staining was strong.
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Results |
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All semiquantitative data are reported in Table 3.
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All semiquantitative data are reported in Table 3.
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Discussion |
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The vexing issue of CsA nephrotoxicity has prompted clinical and experimental strategies to attenuate this toxicity, particularly the vasoconstrictor component. Approaches such as low-dose dopamine infusion (Sabbatini et al. 1989), use of calcium channel blockers (Rodicio 2000
), and omega-3 fatty acid (Torras et al. 1994
)-enriched diets to reverse the CsA-induced imbalance between synthesis vasodilator and vasoconstrictor prostaglandins have been employed without apparent benefit in long-term studies. Prostaglandin agonists, such as misoprostol (a PGE analogue; Nast et al. 1995
) and TxA2 synthase inhibitors (Kim et al. 1997
), are also ineffective.
Our studies point to HO both as an underlying mechanism of CsA-induced nephrotoxicity and as a target for novel strategies to ameliorate this toxicity. In our model, 21 days of daily CsA treatment produced histopathological lesions, tubule interstitial fibrosis, and lack or swelling of epithelial cell nuclei resembling those seen in kidneys of organ recipients after long-term CsA treatment. In CsA-treated rats, renal HO-1 protein levels and HO renal enzyme activity were reduced. This observation, coupled with the well-established renoprotective effect of HO in various forms of renal injury, i.e., ischemia and inflammation, indicates that CsA depletes the kidney of an important defense system. The beneficial effects of HO activation in renal injury have been attributed to production of heme degradation products, mainly the vasodilator CO and bilirubinbiliverdin, which scavenge reactive oxygen radicals. With respect to CO, its cellular production is regulated by HO. Therefore, the depletion of HO caused by CsA might reduce CO production, thereby allowing action by unopposed vasoconstrictors, such as TxA2 and ET-1, according to the data reported in this work. On the other hand, a reduced production of biliverdinbilirubin may allow accumulation of reactive oxygen radicals.
Our observations also show that the CsA-induced decrease in HO expression and activity is partial and that it can be restored by established HO inducers such as CoPP. This indicates that attempts to preserve or augment HO activity during administration of CsA treatment is a reasonable strategy to attenuate CsA-induced nephrotoxicity. To this end, augmentation of HO-1 activity would be beneficial by increasing production of CO and biliverdinbilirubin and by decreasing levels of heme. Reduction of cellular heme levels would reduce activity of heme-containing enzymes such as inducible NOS and TxA2 synthase. The byproduct of inducible NOS, high-output NO production, can be toxic, whereas the byproduct of TxA2 was shown to mediate the CsA-induced constriction of the renal microvasculature.
In summary, our observations demonstrate that downregulation of HO is a putative mechanism of CsA-induced nephrotoxicity. This effect is partial and reversible. Strategies to preserve or augment HO activity during CsA treatment may attenuate its nephrotoxicity.
With regard to the structures considered targets for CsA nephrotoxicity, we showed that the proximal tubules were mainly involved in these negative effects. These data were confirmed by our previous works (Rezzani et al. 2001a,b
) and by the results of other authors (Dekant and Vanvakas 1996
) showing that proximal tubules were the first intrarenal targets for most nephrotoxic compounds. Proximal tubules, in fact, were responsible for drug biodegradation and for accumulation of its metabolite (Schaaf et al. 2001
). CsA degradation was due, in part, to its oxidation by CYP 450 mixed-function oxidases, which were strongly present in mitochondria of proximal tubules (Burke and Whiting 1986
; Pahan et al. 1997
; Rezzani et al. 1999
). The metabolites obtained from CsA biodegradation in the mitochondria were altered and toxic (Quesniaux et al., 1987
). Therefore, we suggest that the metabolites produced alterations in the same organelles with lack of normal morphological cytoarchitecture and then fibrosis in the epithelial cells of proximal tubules, leading to enhanced Na+ reabsorption and thus decreasing fractional excretion rates (Mason 1989
).
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Footnotes |
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Literature Cited |
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