EFFECT OF BIOFLAVONOIDS QUERCETIN AND CURCUMIN ON ISCHEMIC... : Transplantation (original) (raw)
*Abbreviations: AIF, allograft inflammatory factor; MCP-1, monocyte chemoattractant protein-1; PCR, polymerase chain reaction; RANTES, regulated upon activation, normal T cell expressed and secreted.
The last two decades in renal transplantation have seen a dramatic increase in short-term allograft survival, but this has not been mirrored by a proportional increase in long-term graft survival. This situation may be at least partially due to therapeutics being focused primarily on immunosuppression at the expense of other forms of renal injury. Indeed, there is an emerging paradigm that long-term allograft survival is determined by both immunological and nonimmunological mechanisms(1), which may act synergistically(2).
Current immunosuppressive protocols utilize drugs and treatments that might worsen nonimmunological mechanisms, such as ischemia, hypertension, hyperlipidemia, and direct drug nephrotoxicity. The response to renal injury, whether ischemic or immunologic, results in a common idiosyncratic molecular response with up-regulation of MHC antigens, inflammatory cytokines, and adhesion molecules (3). Furthermore, some cytokines(e.g., transforming growth factor-β) up-regulated by agents such as cyclosporine (4) and tacrolimus have local immunosuppressive effects short term but also promote glomerulosclerosis(5), fibrosis, and chronic allograft nephropathy(6) long term.
It would be of great potential benefit, therefore, to develop agents that are immunosuppressive but also renoprotective in order to break the cycle of renal injury and injury response that leads to chronic allograft nephropathy. An interesting class of compounds that might fill these criteria are the bioflavonoids, such as quercetin and curcumin. They have a common polyphenolic structure and are broad-acting inhibitors of both cytosolic and membranal tyrosine kinases. Bioflavonoids are found in various food products and plants, including fruits, seeds, vegetables, tea, and wine(7). Although their effects have not been studied in renal or transplant models, data from other experimental systems suggest they may have antioxidant, anti-inflammatory, and tyrosine kinase-inhibiting properties.
We wished, therefore, to study the effects of these agents in a model of renal injury to assess their renoprotective properties and whether they modulate expression of genes important in ischemia-reperfusion injury and rejection.
MATERIALS AND METHODS
Surgery. Unilateral ischemia-reperfusion was produced in the left kidney as reported previously (8). Male F344 rats between 180 and 225 g were used. After anesthetization with ketamine (35 mg/kg i.m.) and xylazine (18 mg/kg i.m.), the animal was placed on a warming pad. The left renal pedicle was identified through a midline incision and occluded with a micro-bulldog clamp for 30 min. Reperfusion was confirmed visually after clamp removal. The right kidney was removed using mass ligature of the pedicle with 4-0 silk. The incision was closed with 3-0 chromic suture, and the animals were allowed to recover from anesthesia. Rats were allowed free access to water and standard chow diet. At various times, blood was collected for serum creatinine (Sigma Chemical Co., St. Louis, MO) determination via the tail vein. At the times specified, animals were killed and their left kidneys were removed and sectioned. One portion was fixed in formalin for hematoxylin and eosin staining while the rest was snap-frozen in liquid nitrogen and stored at -70°C. All groups contained at least five animals.
Bioflavonoid treatment. Quercetin or curcumin (Sigma) dissolved in dimethylsulfoxide (they are not water soluble) was given intraperitoneally 2 hr before surgery. Treatment with up to 100 mg of both agents without surgery did not prove nephrotoxic by histology or serum creatinine at 2 days(data not shown). In pilot studies, dose ranges from 0.5 to 100 mg were tested, and the range of 1-30 mg was found to be most effective in preservation of renal function following ischemia-reperfusion; therefore, this dose range was used in these experiments with at least five animals per group.
Semiquantitative polymerase chain reaction. RNA from kidney homogenates was extracted with Trizol. Total RNA (2-5 µg) samples were added to oligo d(T), and a reverse transcription reaction was carried out with nucleotides dATP, dCTP, dGTP, and dTTP and AMV reverse transcriptase. Primers constructed based on published sequences or on our own primer selections were as follows. Oligonucleotides for rat monocyte chemoattractant protein-1 (MCP-1*), chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) (9), and allograft inflammatory factor (AIF) (10) were made. A polymerase chain reaction (PCR) assay was performed with cDNA, Taq polymerase(Perkin-Elmer Cetus, Norwalk, CT), and the appropriate 5′ and 3′ primers at 94°C, 50°C (reannealing temperatures varied for different primers), and 72°C, for 30 cycles, followed by a final incubation at 72°C for 10 min. An aliquot of the PCR product was run on an agarose gel(2%) with ethidium bromide to identify the specific band, and the gels were scanned to estimate relative band density. Semiquantitative RNA estimation was carried out with rat actin (amplified by rat actin primers from Clontech, Palo Alto, CA) as an internal control. The PCR conditions were standardized for sensitivity, reliability, and specificity with the negative and positive controls.
RESULTS
Effect of bioflavonoids on renal injury following ischemia-reperfusion. The effects of quercetin or curcumin following renal ischemia-reperfusion were assessed by serial serum creatinine measurements and histology. As seen in Figure 1, pretreatment with 1 mg of quercetin lowered the serum creatinine level on day 2 from 6.5±1.4 mg/dl to 3.3±0.5 mg/dl (_P_=0.06). Pretreatment with curcumin or a combination of quercetin and curcumin did not improve the creatinine level on day 2. On day 7, however, there was a statistically significant reduction in the serum creatinine level of the animals pretreated with curcumin, quercetin, or both compared with the untreated control group (7.5±1.5 mg/dl).
Effect of quercetin and curcumin on serum creatinine following ischemia-reperfusion injury. Drugs were given intraperitoneally 2 hr before surgery. *_P_=0.06;**P<0.05
Improvement in the serum creatinine level was mirrored by histological changes in tissue sections. As seen in Figure 2, on day 2, untreated animals exhibited extensive tubular necrosis and obstruction(Fig. 2A), which was much reduced in animals pretreated with 1 mg of quercetin (Fig. 2B). By day 7, the untreated kidneys began to exhibit an interstitial inflammatory infiltrate which became pronounced by day 14 (Fig. 2C). Pretreatment with 1 mg of quercetin prevented the appearance of this infiltrate on day 7 or 14(Fig. 2D).
Renal histology following ischemia-reperfusion injury with or without quercetin pretreatment(hematoxylin and eosin stain). (A) Untreated group, day 2: note tubular necrosis and dilatation; (B) quercetin group, day 2: note relative preservation of tubular architecture; (C) untreated group, day 14: note representative interstitial infiltrate; (D) quercetin group, day 14: note paucity of interstitial inflammatory cells.
Effect of bioflavonoids on gene expression following ischemia-reperfusion. To determine how the bioflavonoids prevented the accumulation of an interstitial infiltrate following ischemia-reperfusion injury, we measured the level of gene expression of the chemokines RANTES and MCP-1 (important in the chemotaxis of T cells and macrophages[11]) by reverse transcription PCR corrected for actin gene expression. No RANTES or MCP-1 gene expression was detected in normal control kidneys, with or without bioflavonoid treatment. As seen inFigure 3, a high level of RANTES and MCP-1 expression was detected 2 and 7 days after ischemia-reperfusion injury. Pretreatment with curcumin and quercetin (30 mg each) reduced the expression of both chemokines to very low levels on both day 2 and day 7. Pretreatment with 1 mg of quercetin slightly reduced the expression of RANTES and MCP-1 on days 2 and 7.
Expression of chemokine genes in ischemia-reperfusion injury. Kidneys were collected on days 2 and 7, and mRNA expression for the stated gene was measured by reverse transcription PCR and corrected by actin expression under identical conditions. Animals were pretreated with curcumin or quercetin (30 mg i.p.) 2 hr before surgery. Representative experiments are shown. (A) RANTES gene expression; (B) MCP-1 gene expression; (C) typical agarose gel from PCR for RANTES. Row A is actin and row B is RANTES under identical PCR conditions. Column 1 is normal kidney, column 2 is ischemia-reperfusion day 2 with curcumin pretreatment, and column 3 is ischemia-reperfusion day 2 with dimethylsulfoxide pretreatment only.
Ischemic renal injury has been identified as a potential nonimmune risk factor for chronic renal allograft rejection. AIF is a gene discovered by differential mRNA display specific to chronic renal allograft rejection in rodents. As seen in Figure 4, AIF expression, which was undetectable in normal control kidneys, increased to high levels 2 and 7 days after ischemia-reperfusion injury alone. This increase was attenuated by pretreatment with 30 mg of quercetin or curcumin.
Expression of AIF gene in ischemia-reperfusion injury. Kidneys were collected on days 2 and 7, and mRNA expression of AIF was measured by reverse transcription PCR and corrected by actin expression under identical conditions. Animals were pretreated with curcumin or quercetin (30 mg i.p.) 2 hr before surgery. Representative experiments are shown.
DISCUSSION
Current immunosuppressive protocols afford excellent short-term allograft survival, but long-term graft loss from chronic allograft nephropathy continues to be a barrier. Risk factors for chronic allograft nephropathy include both immune (12) (e.g., rejection) and nonimmune(13) (e.g., ischemia, infection, hyperlipidemia, hypertension) factors. Unfortunately, immunosuppressive agents, while very effective at preventing acute immune renal injury, can often exacerbate nonimmune injury through their nonimmune side effects (ischemia, direct nephrotoxicity, hyperlipidemia). Furthermore, tissue typing with mandatory national sharing of zero-mismatch kidneys can lead to significantly increased cold ischemia times.
We have proposed that new therapeutic strategies are required in renal transplantation to simultaneously minimize both immune and nonimmune renal injury, as well as the stereotypical response to that injury which leads to a vicious cycle of inflammation, further injury, fibrosis, loss of functional renal mass, hyperfiltration, and progressive chronic allograft nephropathy(1,14). We therefore need therapeutic agents that can prevent all forms of renal injury without provoking an inflammatory or apoptotic injury response and loss of nephron mass. Based on epidemiological and in vitro data, the bioflavonoids, a group of plant-derived compounds with a polyphenolic structure, would appear to have multiple effects that should be beneficial in renal transplantation. As a group, there is in vitro evidence that bioflavonoids are antioxidants, prevent lipid peroxidation(15), inhibit tyrosine kinase pathways(16), suppress NF-κB expression(17), lower serum lipid levels(18), inhibit smooth muscle proliferation(19), and block T-cell proliferation(20). Epidemiological studies provide evidence that they can reduce the incidence of cardiac mortality (21).
For these experiments, we chose to study quercetin and curcumin because of their superior antioxidant effects among the flavonoids (15,22). We found that serum creatinine levels were significantly improved 2 days after ischemia-reperfusion injury following pretreatment with 1 mg of quercetin and at 7 days following treatment with quercetin, curcumin, or both. Reperfusion injury produces irreversible lipid peroxidation by providing oxygen that reacts with xanthene oxidase and hypoxanthine to produce the highly toxic hydroxyl radicals in the presence of iron(23). Quercetin can prevent lipid peroxidation by blocking the action of xanthene oxidase (24), chelating iron (25), and directly scavenging hydroxyl radical(26). Curcumin can prevent lipid peroxidation through iron chelation (15), free radical scavenging(27), and the blockade of tyrosine kinase enzymes responsible for apoptosis in renal epithelial cells triggered by oxidative stress (28). Furthermore, both quercetin and curcumin could reduce ongoing reperfusion injury mediated through infiltrating macrophages by interfering with inducible nitric oxide synthase activity(29). Although the early release of nitric oxide through the activity of constitutive nitric oxide synthase is important in the maintenance of intrarenal dilatation of blood vessels, which reduces ischemic injury (8), the much higher levels of nitric oxide produced by inducible nitric oxide synthase in macrophages can produce oxidative damage in their own right (30). The combination of reactive oxygen and reactive nitrate intermediaries can produce peroxynitrites, which are highly cytotoxic in ischemia-reperfusion(31), have been shown to play a role in human chronic renal allograft rejection (32), and can also be inhibited directly by bioflavonoids (33).
That the lower dose of quercetin was more effective at preserving renal function at 2 days was surprising but could reflect pro-oxidant mechanisms at higher doses. Quercetin has been shown to prevent the induction of heat shock proteins following thermal stress (34), although whether the induction of these genes is sufficient for protection from subsequent ischemic damage is not clear (35). Another explanation is that quercetin is a weak inhibitor of endothelial nitric oxide synthase(36), whose activity we have shown to be important in early recovery from ischemic injury (8). At higher doses, therefore, quercetin may induce further ischemia through local renal vasoconstriction, which would counteract the beneficial antioxidant effects. Finally, quercetin at high doses may reduce the production of other natural renal antioxidants, such as glutathione (37).
Recovery from ischemic renal injury is associated with an interstitial inflammatory infiltrate (38) including lymphocytes and macrophages. Chemokines such as RANTES and MCP-1 are known chemoattractants for these cells (11). Renal RANTES expression is detectable in human allografts undergoing acute cellular rejection, but not cyclosporine toxicity (39), and is localized to infiltrating mononuclear cells and vascular endothelium and renal tubule epithelium. In rodent allograft models, RANTES and MCP-1 expression is induced during acute (40) and chronic(9) rejection. Reduction of ischemic injury with CTLA4-Ig pretreatment prevents the up-regulation of these chemokines(41), which suggests that they are regulated by T cell-derived cytokines. The macrophage-associated cytokine AIF has been detected only in human and rodent heart transplants in the setting of chronic rejection (10,42). Similarly, pretreatment with anti-CD4 and anti-CD8 monoclonal antibodies reduced the expression of AIF(42). That these three cytokines were up-regulated following renal ischemia-reperfusion injury alone provides further molecular evidence for the interplay between ischemic and immune factors in renal transplantation. Inflammatory infiltration with cell activation and cytokine release is a common feature of any form of renal injury and may be one way that ischemic injury can increase the incidence, severity, and consequences of acute rejection (1,2).
Pretreatment with flavonoids prevented or attenuated the induction of the chemokines and reduced the renal inflammatory infiltrate. Although this effect may simply be due to less initial renal injury, reducing the induction of inflammatory cytokines from renal tubule cells (43), there is evidence for direct chemokine inhibition as well(44). Alternatively, the flavonoids can reduce chemokine expression by blocking production of known inflammatory cytokines through blockade of NF-κB (17). While AIF induction is well characterized in allograft rejection of rodent and human hearts, it is not surprising to find it associated with ischemia-reperfusion injury, since macrophages have been found as antigen-presenting cells and effector cells in both processes.
In conclusion, we have shown that administration of the bioflavonoids quercetin and curcumin can reduce renal ischemia-reperfusion injury and reduce the inflammatory chemokine response to that injury. We have preliminary data that these flavonoids can prolong allograft survival in rodent skin and renal transplant models (manuscript in preparation). This, coupled with the ability of bioflavonoids to reduce serum lipid levels and other risk factors for cardiac mortality, suggests that these compounds have the clinical potential to reduce both immune and nonimmune forms of renal injury, thereby minimizing the risk factors for chronic allograft nephropathy. That these compounds are naturally occurring plant products that can be delivered through dietary manipulation could add to their potential utility and cost effectiveness. Whether toxicities or side effects in renal transplant patients at higher doses could limit the therapeutic index requires clinical trials to address.
Acknowledgments. The author thanks N. Aguirre and I. Ryndin for technical assistance. The author also thanks Dr. Satyanarayana for his contributions to this study.
REFERENCES
1. Shoskes DA, Halloran PF. Delayed graft function in renal transplantation: etiology, management and long-term significance. J Urol 1996; 155(6): 1831.
2. Shoskes DA, Churchill BM, McLorie GA, Khoury AE. The impact of ischemic and immunologic factors on early graft function in pediatric renal transplantation. Transplantation 1990; 50(5): 877.
3. Shoskes DA, Parfrey NA, Halloran PF. Increased major histocompatibility complex antigen expression in unilateral ischemic acute tubular necrosis in the mouse. Transplantation 1990; 49(1): 201.
4. Prashar Y, Khanna A, Sehajpal P, Sharma VK, Suthanthiran M. Stimulation of transforming growth factor-beta 1 transcription by cyclosporine. FEBS Lett 1995; 358(2): 109.
5. Border WA, Noble NA, Ketteler M. TGF-β: a cytokine mediator of glomerulosclerosis and a target for therapeutic intervention. Kidney Int 1996; 47 (suppl 49): S59.
6. Sharma VK, Bologa RM, Xu G, et al. Intragraft TGF-β1 mRNA: a correlate of interstitial fibrosis and chronic allograft nephropathy. Kidney Int 1996; 49: 1297.
7. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996; 20(7): 933.
8. Shoskes DA, Xie Y, Cadavid-Gonzales NF. Nitric oxide synthase activity in ischemia-reperfusion injury in the rat: implications for renal transplantation. Transplantation 1997; 63(4): 495.
9. Nadeau KC, Azuma H, Tilney NL. Sequential cytokine dynamics in chronic rejection of rat renal allografts: roles for cytokines RANTES and MCP-1. Proc Natl Acad Sci USA 1995; 92(19): 8729.
10. Utans U, Quist WC, McManus BM, et al. Allograft inflammatory factory-1: a cytokine-responsive macrophage molecule expressed in transplanted human hearts. Transplantation 1996; 61(9): 1387.
11. Luster AD. Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med 1998; 338(7): 436.
12. Almond PS, Matas A, Gillingham K, et al. Risk factors for chronic rejection in renal allograft recipients. Transplantation 1993; 55(4): 752.
13. Yilmaz S, Paavonen T, Hayry P. Chronic rejection of rat renal allografts. II. The impact of prolonged ischemia time on transplant histology. Transplantation 1992; 53(4): 823.
14. Satyanarayana K, Shoskes DA. A molecular injury-response model for the understanding of chronic disease. Mol Med Today 1997; 3(8): 331.
15. Sreejayan, Rao MN. Curcuminoids as potent inhibitors of lipid peroxidation. J Pharm Pharmacol 1994; 46(12): 1013.
16. Singhal RL, Yeh YA, Praja N, Olah E, Sledge GW Jr, Weber G. Quercetin down-regulates signal transduction in human breast carcinoma cells. Biochem Biophys Res Commun 1995; 208(1): 425.
17. Singh S, Aggarwal BB. Activation of transcription factor NF-kappa B is suppressed by curcumin (diferulolylmethane). J Biol Chem 1995; 270(42): 24995.
18. Babu PS, Srinivasan K. Hypolipidemic action of curcumin, the active principle of turmeric (Curcuma longa) in streptozotocin induced diabetic rats. Mol Cell Biochem 1997; 166(1-2): 169.
19. Huang HC, Jan TR, Yeh SF. Inhibitory effect of curcumin, an anti-inflammatory agent, on vascular smooth muscle cell proliferation. Eur J Pharmacol 1992; 221(2-3): 381.
20. Namgoong SY, Son KH, Chang HW, Kang SS, Kim HP. Effects of naturally occurring flavonoids on mitogen-induced lymphocyte proliferation and mixed lymphocyte culture. Life Sci 1994; 54(5): 313.
21. Knekt P, Jarvinen R, Reunanen A, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study. BMJ 1996; 312(7029): 478.
22. Chen ZY, Chan PT, Ho KY, Fung KP, Wang J. Antioxidant activity of natural flavonoids is governed by number and location of their aromatic hydroxyl groups. Chem Phys Lipids 1996; 79(2): 157.
23. Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1988; 255 (6 Pt 2): H1269.
24. Chang WS, Lee YJ, Lu FJ, Chiang HC. Inhibitory effects of flavonoids on xanthine oxidase. Anticancer Res 1993; 13(6A): 2165.
25. Ferrali M, Signorini C, Caciotti B, et al. Protection against oxidative damage of erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating activity. FEBS Lett 1997; 416(2): 123.
26. Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med 1994; 16(6): 845.
27. Soudamini KK, Unnikrishnan MC, Soni KB, Kuttan R. Inhibition of lipid peroxidation and cholesterol levels in mice by curcumin. Indian J Physiol Pharmacol 1992; 36(4): 239.
28. Hagar H, Ueda N, Shah SV. Tyrosine phosphorylation in DNA damage and cell death in hypoxic injury to LLC-PK1 cells. Kidney Int 1997; 51(6): 1747.
29. Brouet I, Ohshima H. Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 1995; 206(2): 533.
30. Langrehr JM, White DA, Hoffman RA, Simmons RL. Macrophages produce nitric oxide at allograft sites. Ann Surg 1993; 218(2): 159.
31. Liu P, Hock CE, Nagele R, Wong PY. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rats. Am J Physiol 1997; 272 (5 Pt 2): H2327.
32. MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS, Thompson JA. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci USA 1996; 93(21): 11853.
33. Haenen GR, Paquay JB, Korthouwer RE, Bast A. Peroxynitrite scavenging by flavonoids. Biochem Biophys Res Commun 1997; 236(3): 591.
34. Nagata K. Regulation of thermotolerance and ischemic tolerance. EXS 1996; 77: 467.
35. Joannidis M, Cantley LG, Spokes K, et al. Induction of heat-shock proteins does not prevent renal tubular injury following ischemia. Kidney Int 1995; 47(6): 1752.
36. Chiesi M, Schwaller R. Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin. Biochem Pharmacol 1995; 49(4): 495.
37. Zhang K, Yang EB, Tang WY, Wong KP, Mack P. Inhibition of glutathione reductase by plant polyphenols. Biochem Pharmacol 1997; 54(9): 1047.
38. Olsen S, Burdick JF, Keown PA, Wallace AC, Racusen LC, Solez K. Primary acute renal failure ("acute tubular necrosis") in the transplanted kidney: morphology and pathogenesis. Medicine 1989; 68(3): 173.
39. Pattison J, Nelson PJ, Huie P, et al. RANTES chemokine expression in cell-mediated transplant rejection of the kidney. Lancet 1994; 343(8891): 209.
40. Kondo T, Novick AC, Toma H, Fairchild RL. Induction of chemokine gene expression during allogeneic skin graft rejection. Transplantation 1996; 61(2): 1750.
41. Chandraker A, Takada M, Nadeau KC, Peach R, Tilney NL, Sayegh MH. CD28-b7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury. Kidney Int 1997; 52(6): 1678.
42. Autieri MV. cDNA cloning of human allograft inflammatory factor-1: tissue distribution, cytokine induction, and mRNA expression in injured rat carotid arteries. Biochem Biophys Res Commun 1996; 228(1): 29.
43. Prodjosudjadi W, Gerritsma JS, Klar-Mohamad N, et al. Production and cytokine-mediated regulation of monocyte chemoattractant protein-1 by human proximal tubular epithelial cells. Kidney Int 1995; 48(5): 1477.
44. Xu YX, Pindolia KR, Janakiraman N, Gautam SC. Curcumin, a compound with anti-inflammatory and anti-oxidant properties, down-regulates chemokine expression in bone marrow stromal cells. Exp Hematol 1997; 25(5): 413.
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