GAS-GENERATING SYSTEMS IN ACUTE RENAL ALLOGRAFT REJECTION IN THE RAT: Co-Induction of Heme Oxygenase and Nitric Oxide Synthase: 1: ,: 2 : Transplantation

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Co-Induction of Heme Oxygenase and Nitric Oxide Synthase1,2

Agarwal, Anupam3; Kim, Youngki4; Matas, Arthur J.5; Alam, Jawed6; Nath, Karl A.3,7

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During acute renal transplant rejection, antigen-presenting cells, such as macrophages, dendritic cells, and B cells, interact with alloreactive T lymphocytes to instigate humoral and cellular mechanisms that impair allograft function (1,2). Mononuclear cells prominently infiltrate the interstitium of the acutely rejecting graft(3-7), and such infiltration may incite tissue injury by several mechanisms, including the generation of cytokines, proteolytic enzymes, autacoids, and reactive oxygen species(1-3,6,8,9). While such mediators clearly provoke injury, there is increasing recognition that, in response to potentially injurious insults, cells draw upon and summon protective responses (10, 11). One such response in cells so exposed is the induction of the heme oxygenase(HO*), an enzyme induced by heme, oxidants, and a variety of cytokines(12-18).

HO was initially recognized as the microsomal enzyme involved in the rate-limiting degradation of heme (19). This enzyme converts heme to biliverdin, releases iron, and produces carbon monoxide. Biliverdin is converted to bilirubin by biliverdin reductase.Figure

The induction of HO by a diverse array of stimuli, which all, nonetheless, share the common linkage of exerting oxidative stress, led to the suggestion that the induction of HO is an antioxidant response(13-16). By degrading heme, a prooxidant contained in a multiplicity of intracellular proteins(20), and by generating bilirubin, an antioxidant, induction of HO may provide an antioxidant protection(21, 22). In support of this view are our studies in the glycerol model of heme protein-induced renal injury and the cisplatin model of toxic nephropathy, wherein induction of HO confers a protective response (23-25). We thus undertook the present study to examine whether HO was expressed in acute renal allograft rejection (AR), a disease state that exhibits an abundance of stimuli for HO.

While conducting our studies, we noted a review by Dawson and Synder(26) that called attention to the involvement of nitric oxide and carbon monoxide in signaling processes in the brain. Nitric oxide resembles carbon monoxide in several ways: nitric oxide is a gaseous product, is generated by an enzyme (nitric oxide synthase) that is rapidly induced by cytokines, and, like carbon monoxide, stimulates the production of cGMP(26). Such a precedent, as provided by these observations in neural tissue, led us to extend our studies to determine whether the enzymes, HO and nitric oxide synthase, are co-expressed in the rejecting renal allograft.


Renal transplantation. Studies were performed in male Lewis and Brown Norway rats weighing 275-350 g (Harlan Sprague Dawley, Indianapolis, IN). Renal transplantation was performed as described previously(27). For allografts (n=17), Brown Norway kidneys were transplanted to Lewis rats and for isografts (n=17), Lewis kidneys were transplanted to Lewis rats. The donor renal artery and vein were anastomosed end to side to the recipient aorta and inferior vena cava, respectively. The ureter was anastomosed end to end with the recipient ureter. Recipients underwent bilateral native nephrectomy at the time of renal transplantation. No immunosuppressives were administered. Ticarcillin disodium (100 mg/kg body weight, s.c.) was administered for the first 3 days after transplantation. Rats were maintained on standard Purina rat chow and tap water ad libitum. Renal function was assessed by serum creatinine values on a tail vein sample and measured by the Jaffe reaction (Creatinine Analyzer II, Beckman Instruments, Brea, CA). Kidneys were harvested at different time intervals after transplantation and processed for histology, immunofluorescence studies, Northern and Western analyses, and enzyme activity.

Histology and immunohistochemistry. Antibodies: Polyclonal(rabbit) antibody against rat HO was developed by one of the authors(25). Polyclonal (rabbit) antibody against mouse macrophage inducible nitric oxide synthase (iNOS) was obtained from Affinity Bioreagents (Neshanic Station, NJ) and ED1 anti-rat macrophage monoclonal(mouse) antibody was from Serotec (Indianapolis, IN). FITC-conjugated goat anti-rabbit IgG was obtained from Tago (Burlinghame, CA); FITC-conjugated goat anti-mouse IgG and rhodamine isothiocyanate-conjugated goat anti-mouse IgG was from Caltag Laboratories (South San Francisco, CA). All secondary antibodies were absorbed with normal rat serum.

The transplanted kidneys were harvested and sliced horizontally with a razor blade. Two 1-mm slices were placed in cassettes and fixed in 10% buffered formaldehyde for light microscopic studies. Immunofluorescence studies were performed as described previously (28). Cryostat sections (4 μm) of frozen kidney tissue were sectioned in a Lipshaw cryostat in a constant temperature (25°C) and humidity (30%) room, and fixed for 10 min with acetone. The slides were stained with primary antibodies followed by FITC-conjugated secondary antibodies. Dual fluorochrome labeling was performed using rabbit polyclonal anti-HO or anti-iNOS detected by FITC-conjugated anti-rabbit IgG absorbed with normal rat serum followed by mouse monoclonal ED1 antibody and rhodamine isothiocyanate-conjugated goat anti-mouse IgG absorbed with normal rat and rabbit serum. Reversal of staining order in dual label studies did not cause any appreciable change in the staining pattern. Appropriate controls were used as described previously(28). To retard fluorescent quenching, p-phenylenediamine in PBS glycerol was applied to fluorochrome-stained tissue sections. Sections were examined by indirect immunofluorescence using a Zeiss epifluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany).

The dual-labeled slides were coded and used to quantify the number of interstitial cells positive for HO, iNOS, and ED1. Cells positive for anti-HO or anti-iNOS were counted in 10 random fields using a 10×10-mm grid and results were expressed as a percentage of ED1+ cells in the same fields.

Northern analysis. RNA extraction, Northern analysis, and hybridization against cDNA probes were performed as described previously(29). The cDNA probe for human HO was kindly provided by Dr. Rex Tyrrell and the cDNA probe for iNOS was obtained from Dr. Carl Nathan. Autoradiograms were quantified by video densitometry and expressed as corrected OD units using previously described methods(30).

HO enzyme activity. HO activity was measured by bilirubin generation in kidney microsomes, as described previously(23, 31). Kidney microsomes were incubated with rat liver cytosol, a source of bilirubin reductase (3 mg), hemin (20 μM), glucose 6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 U), and NADPH (0.8 mM) for 1 hr at 37°C in the dark. The formed bilirubin was extracted with chloroform and ΔOD 464-530 nm was measured (extinction coefficient, 40 mM-1 cm-1 for bilirubin). Enzyme activity was expressed as pmol of bilirubin formed/60 min/mg protein.

Western analysis. Kidneys were harvested and microsomal membrane fractions were prepared as described previously (25). Portions equivalent to 15 μg of protein were electrophoresed on a denaturing 15% polyacrylamide gel. The protein samples were electrophoretically transferred to nitrocellulose membranes and Western blot analysis was performed using a rabbit anti-rat HO-1 antibody (Stress Gen, Victoria, Canada) and a chemiluminescent detection system (Tropix, Inc., Bedford, MA) according to the manufacturer's recommendation.

Statistical analysis. Data are expressed as mean ± SEM. For comparisons involving two groups, the unpaired t test was used. For comparisons involving more than two groups, analysis of variance and the Student-Newman-Keuls test were applied. All results were considered significant at P<0.05.


Renal function and histology. Isografts displayed intact renal function as assessed by normal serum creatinine values, whereas rats with allografts showed a 4-fold rise in serum creatinine at day 5 after transplantation (isografts, 0.4±0.1 mg/dl; allografts, 1.6±0.1 mg/dl, P<0.001). In addition to elevations in serum creatinine, the allografts exhibited histologic evidence of acute rejection, the latter reflected by dense mononuclear cellular infiltration in the interstitium, tubulitis, and glomerular hypercellularity at day 5 after transplantation. The isografts showed essentially normal histology with minimal tubular swelling, rare casts, and scant numbers of mononuclear cells in the interstitium.

Induction of HO. A marked induction of HO mRNA occurred in allograft kidneys at day 5 after transplantation (Fig. 1), while no induction was seen in the control isografts. No induction was observed at earlier time points, i.e., at days 1 and 3 after transplantation(data not shown). The mean densitometric value, factored for the amount of RNA transferred onto the nylon membrane, for the isografts was 0.1±0.1 corrected OD units; for allografts, the mean densitometric value was 35±8 corrected OD units. Measurement of HO enzyme activity in kidney microsomes revealed significantly increased activity in the allografts compared with isografts (Fig. 2). In addition to HO mRNA, we also confirmed by Western blot analysis the presence of HO protein in the allograft kidneys. As shown in Figure 3, increased expression of HO protein was detectable in allografts at day 5 after transplantation. Thus, activity, mRNA, and protein were increased for HO in kidneys with allograft rejection.

To localize the site of expression of HO, we performed immunofluorescence studies using a specific antibody. Intense staining for HO was present in interstitial cells encircling tubules in rat renal allografts(Fig. 4A), while no such staining was present in isografts. Staining was absent in the glomeruli. Thus, HO is induced in infiltrating cells in the rejecting kidney. To identify the cell staining positively for HO, we used a rat monocyte-specific antibody, ED1. Using dual labeling, these infiltrating cells were also ED1+, and were thus identified as macrophages (Fig. 4B); these findings led us to conclude that, in acute rejection, HO is induced in infiltrating macrophages. Cells in the control isografts that were positive for ED1 were quite few in number and did not stain for HO. The number of cells staining positively for HO was less than the number of cells identified as macrophages in the allografts and represented 83±1.4% of the total macrophage cell population (Fig. 5).

Induction of nitric oxide synthase. Striking expression of iNOS mRNA occurred in allografts at day 5 after transplantation(Fig. 6), while control isografts showed no such induction. The mean densitometric value, factored for the amount of RNA transferred onto the nylon membrane, for the isografts was 0.5±0.3 corrected OD units; for allografts, the mean densitometric value was 52±19 corrected OD units. Similar to induction of HO, iNOS mRNA was not induced at earlier time points, i.e., at days 1 and 3 after transplantation. To localize the site of induction of nitric oxide synthase in the kidney, we performed immunofluorescence studies using a specific antibody for iNOS. Intense staining for iNOS was present in interstitial cells encircling tubules in rat renal allografts (Fig. 4C), while no such staining was present in isografts. Occasional cells stained positively in the glomeruli. Thus, iNOS is induced in infiltrating cells in the acutely rejecting kidney. These infiltrating cells were again identified as ED1+ macrophages using dual labeling (Fig. 4D), and 65±7.6% of the macrophages were positive for iNOS(Fig. 5).


Our data show that induction of HO, as measured by mRNA, protein, and enzyme activity, occurs in AR in the rat. Accompanying such induction of HO is greatly enhanced expression of iNOS, the latter reflected by marked expression of mRNA and protein. Expression of both HO and iNOS emanates from the cells infiltrating the interstitium during the rejection process, and the specific cellular locus in this infiltrate that is overwhelmingly responsible for expression of these proteins is the macrophage.

Our studies provide the first in vivo demonstration of expression of HO by macrophages in any acute inflammatory state in general, and in acute allograft rejection in particular. We were surprised to find expression of HO in macrophages, since our original hypothesis posited that intrinsic renal cells, exposed to cytokines and other forms of oxidative injury, would induce HO. Yet it was the infiltrating macrophage that exhibited such induction. HO resides mainly in tissues of the reticuloendothelial system, ones charged with dismantling aged red blood cells, degrading hemoglobin, and detoxifying the heme ring (11, 12); for this latter purpose, HO is indispensable because it converts heme to biliverdin(19). It is conceivable that the expression of this enzyme by infiltrating macrophages allows macrophages to degrade heme proteins derived from damaged erythrocytes at sites of inflammation; heme is also contained in multiple intracellular proteins in nucleated cells, and as these cells are destroyed, these proteins present a burden of heme that necessitates degradation. Expression of HO in infiltrating macrophages may also represent an antioxidant response. HO is induced in response to a diverse array of oxidative insults, and such induction may confer antioxidant capability(14, 16, 23, 24). Oxidative stress may destabilize heme proteins, leading to the release of heme(20); by decreasing this burden of heme and by generating bilirubin, an antioxidant metabolite capable of effectively scavenging peroxy radicals and inhibiting lipid peroxidation(21, 22), enhanced HO activity may protect against oxidative insults. It is conceivable that the induction of HO in infiltrating macrophages in the rejecting kidney may provide an antioxidant capability to the macrophage against oxidative injury encountered in this disease state.

The induction of HO in acute rejection would predict that its product, carbon monoxide, would appear in increased amounts. Carbon monoxide is a potent vasodilator, an effect, based to a large part, upon its ability to stimulate the generation of cGMP (32, 33). Such vasodilatory effects may serve to preserve renal blood flow rates in the face of potent vasoconstrictors such as thromboxanes elaborated by the rejecting kidney (34) or endothelin generated in large amounts by cyclosporine (35). cGMP may also exert immune modulatory effects (36). Additionally, carbon monoxide may contribute to tissue toxicity in acute rejection by a number of effects, including its ability to stimulate mitochondrial production of reactive oxygen species (37).

Carbon monoxide, along with nitric oxide, was recently recognized as a signaling molecule in neural tissue (26, 38). This perception led us to examine expression of iNOS, the enzyme responsible for the production of nitric oxide. Remarkably, as demonstrated by our data, these two systems are also generated by infiltrating macrophages in the rejecting kidney (Fig. 7). The macrophage exhibiting co-induction of HO and iNOS, in all likelihood, represents an activated macrophage, since HO and iNOS were expressed at day 5 but not at earlier time points in acute rejection and the majority of ED1+ cells, but not all, expressed these systems. Expression of HO and iNOS in infiltrating macrophages may reflect the relatively high concentrations of cytokines to which the macrophage is exposed, since studies in other tissues provide ample evidence attesting to cytokine-driven induction of HO(17, 18, 39, 40).

Our findings that infiltrating macrophages express nitric oxide synthase are consistent with recent observations in states of transplant rejection. In cardiac allograft rejection in the rat, there is marked induction of the mRNA for iNOS and iNOS protein, which originate overwhelmingly in infiltrating macrophages (41). In another study of this disease model, urinary excretion of nitrate markedly increases in untreated allograft rejection and decreases as rejection is treated with dexamethasone and cyclosporine (42). In patients with liver allograft rejection, plasma concentrations of nitroso-compounds are elevated and correlate with such markers of rejection as tumor necrosis factor-α and interleukin-2 receptor-positive lymphocytes; conversely, these plasma concentrations diminish after administration of high doses of glucocorticoids; treatment with FK506 as compared with cyclosporine led to fewer rejection episodes and reduced generation of nitric oxide (43). Just recently, the induction of iNOS has been reported in macrophages in a rat model of AR (44).

Nitric oxide generated from iNOS may affect the rejection process through multiple mechanisms. Like carbon monoxide, nitric oxide is widely recognized for its vasodilatory effects, the latter elicited by activating soluble guanylate cyclase, and, together with carbon monoxide, it may preserve renal blood flow (26, 45-47). Nitric oxide may inhibit leukocyte adherence to the endothelium and thus its migration into the interstitium (48); the anti-aggregatory effects of nitric oxide on platelets may reduce the involvement of platelet-dependent pathways in renal injury(40). Interestingly, in in vitro studies, nitric oxide inhibits proliferation and cytotoxic T lymphocyte generation(49); in in vivo studies, utilizing the sponge matrix allograft model, allograft-infiltrating cells exhibited nitric oxide-dependent, donor-specific cytolytic activity (50). Subcellular targets vulnerable to inactivation by nitric oxide include enzymes containing iron-sulfur clusters such as aconitase and assorted mitochondrial electron transport enzymes; through such effects of nitric oxide, activated macrophages may inhibit mitochondrial respiration in target cells(40). Nitric oxide in large amounts is also antiproliferative in nature, possibly due to inhibitory effects on ribonucleotide reductase, an enzyme critical to cell proliferation(51). An added effect of nitric oxide is its capacity to nitrosylate free thiols in enzymes with an ensuing decrease in enzyme activity, as occurs in the interaction of nitric oxide with glyceraldehyde-3-phosphate dehydrogenase (51). Nitric oxide may also interact with the superoxide anion, forming the peroxynitrite radical, which can be cytotoxic (52). Thus, through such multiplicity of actions, enhanced generation of nitric oxide could prove damaging to tissues, and indeed has led to the incrimination of this gas in chronic tissue injury (53). While inhibition of the generation of nitric oxide did not appear to influence cardiac allograft rejection (54), the effect of this mediator on renal allograft rejection is unknown and would be of interest.

Our studies provide the first demonstration of the induction of HO in the rejecting renal allograft as well as the first demonstration in vivo for the induction of HO in macrophages at the site of an inflammatory response. Such expression, linked as it is to the expression of iNOS, indicates that the macrophage in the rejecting kidney generates gaseous mediators, and to this extent, mimics the behavior of neural cells (26); neural cells are thus neither alone nor unique in their deployment of these mediators, since, as uncovered by our findings, the macrophage in the rejecting kidney also summons these gaseous signaling molecules. Finally, we also suggest that indices reflecting induction of these systems may provide clinical clues for the presence of acute rejection.

Acknowledgments. The authors acknowledge the technical assistance of Linhong Sun, A.J. Croatt, Kathy Divine, and Kimberly Woller.

Figure 1:
(A) Northern blot shows induction of HO mRNA in acute renal allograft rejection at day 5 after transplantation. Lanes 1 through 4 represent RNA from isografts. Lanes 5 through 8 represent RNA from allografts. Each lane represents RNA (20 μg) extracted from the transplant kidney of a single animal. (B) Photograph of the nylon membrane showing ethidium bromide staining of the 18s and 28s rRNA.
Figure 2:
Graph shows HO enzyme activity in kidney microsomes prepared from allografts (n=8) and isografts (n=5) at day 5 after transplantation. Values are expressed as mean ± SEM. □, isografts; ▪, allografts.* P<0.01, activity in allografts vs. isografts.
Figure 3:
Western analysis demonstrates induction of HO protein in rat renal allografts at day 5 after transplantation. Each lane represents kidney microsomal protein (15 μg) prepared from a single animal. HO migrates as a 31-kDa protein. The migration and size (kDa) of the molecular mass standards are indicated.
Figure 4:
Immunofluorescence with HO, iNOS, and ED1 antibodies demonstrates expression in infiltrating cells in rat renal allografts. (A) Intense staining for HO was present in interstitial cells encircling tubules. (B) Using dual labeling, these infiltrating cells were identified as macrophages(ED1+) (arrows). This is the same field as in (A), observed using a different filter to visualize rhodamine staining. (C) Intense staining for iNOS was present in infiltrating cells in rat renal allografts. (D) These cells were identified as ED1+ macrophages (arrows). Note that not all cells that stain positive with ED1 (arrowheads) stain for HO or iNOS.
Figure 5:
Graph shows the percentage of infiltrating cells staining positively for HO/iNOS and ED1 in rat renal allografts at day 5 after transplantation (n=7). Values are expressed as mean ± SEM.
Figure 6:
(A) Northern blot shows induction of iNOS mRNA in AR at day 5 after transplantation. Lanes 1 through 4 represent RNA from isografts. Lanes 5 through 8 represent RNA from allografts. Each lane represents RNA (20 μg) extracted from the transplanted kidney of a single animal. (B) Photograph of the nylon membrane shows ethidium bromide staining of the 18s and 28s rRNA.
Figure 7:
Schema demonstrates induction of HO and the iNOS in infiltrating macrophages in AR, which increases carbon monoxide (CO) and nitric oxide (NO) production, respectively.


These studies were supported by NIH grants to K.A.N. (RO1-DK 47060), A.J.M. (RO1-DK 13083-27), Y.K. (RO1-AI 010704), and J.A. (RO1-DK 43135).

This work was presented in part at the American Society of Nephrology meeting in Orlando, Florida, October 1994, and was published in abstract form in the Journal of the American Society of Nephrology(1994; 5: 977).

Abbreviations: AR, acute renal allograft rejection; HO, heme oxygenase; iNOS, inducible nitric oxide synthase.


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