EFFECTS OF INTRAVENOUSLY ADMIN ISTERED BILIRUBIN ON RENAL ISCHEMIA REPERFUSION INJURY By KRISTIN ANN KIRKBY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006
Copyright 2006 by Kristin Ann Kirkby, DVM
iii ACKNOWLEDGMENTS I thank my family for their love, su pport, and encouragement throughout my education. I would also like to thank Drs. Christopher Adin and Chris Baylis for their guidance and mentorship throughou t the pursuit of this degree and Linda Archer for her technical assistance with the project.
iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 THE PRODUCTS OF HEME OXYGENASE: A REVIEW......................................1 Introduction................................................................................................................... 1 Carbon Monoxide.........................................................................................................4 Bilirubin...................................................................................................................... ..9 Biliverdin....................................................................................................................1 5 Iron........................................................................................................................... ...16 Conclusion..................................................................................................................17 2 INTRAVENOUS BILIRUBIN PR OVIDES INCOMPLETE PROTECTION AGAINST ISCHEMIA REPERFUSION INJURY...................................................18 Introduction.................................................................................................................18 Materials and Methods...............................................................................................19 Animals................................................................................................................19 Bilirubin Treatment.............................................................................................19 Reagents..............................................................................................................20 Surgical Procedures.............................................................................................20 Treatment Groups................................................................................................21 Serum and Urine Analysis...................................................................................21 Measurement of Free Radical ProductionÂ—TBAR............................................22 Histologic Grading..............................................................................................23 Statistical Analysis..............................................................................................23 Results........................................................................................................................ .24 Serum Bilirubin Concentration............................................................................24 Renal Hemodynamics..........................................................................................24
v Oxidative Damage...............................................................................................29 Light Microscopy................................................................................................30 Discussion...................................................................................................................33 3 CONCLUSION...........................................................................................................37 LIST OF REFERENCES...................................................................................................40 BIOGRAPHICAL SKETCH.............................................................................................51
vi LIST OF TABLES Table page 1 Histologic scores following treatment with 5 mg/kg BR.........................................33 2 Histologic scores following treatment with 20 mg/kg BR.......................................33
vii LIST OF FIGURES Figure page 1 Heme metabolism by heme oxygenase......................................................................2 2 The physiologic structure of bilirubin IX ................................................................9 3 The three ionic forms of BR.....................................................................................10 4 Serum BR concentrations following IV in fusion (5mg/kg) for one hour prior to and during ischemia and 6 hours follow ing, and IV bolus administration (20 mg/kg) for one hour prior to and during ischemia...................................................24 5 Serum blood urea nitrogen (BUN)...........................................................................25 6 Serum creatinine levels............................................................................................26 7 Fractional excretion of sodium (FENa).....................................................................27 8 Estimated glomerular filtration rate (GFR)..............................................................28 9 Renal vascular resistance (RVR)..............................................................................29 10 Tissue TBAR increased si gnificantly in all treatment groups at 6 hours post ischemia....................................................................................................................30 11 Histologic samples from BR bolus (20 mg/kg BR) and control bolus (0 mg/kg BR) rats....................................................................................................................31
viii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF INTRAVENOUSLY ADMIN ISTERED BILIRUBIN ON RENAL ISCHEMIA REPERFUSION INJURY By Kristin Ann Kirkby May 2006 Chair: Christopher A. Adin Major Department: Veterinary Medicine Heme oxygenase 1 (HO-1) is induced in response to cellular stress, and is responsible for converting the heme molecule into equimolar quantities of biliverdin (BV), carbon monoxide (CO), and iron. Biliverdi n is then converted to bilirubin (BR) by the enzyme biliverdin reductase. Experimental evidence suggests that induction of the HO system is an important endogenous m echanism for cytoprotection and that the downstream products of heme degradation, CO, BR and BV, may mediate these powerful beneficial effects. These molecules, which were once considered to be toxic metabolic waste products, have recently been shown to have dose-dependent vasodilatory, antioxidant, and anti-inflammatory properties th at are particularly desirable for tissue protection during organ transplantation. In fact, recent work has demonstrated that administration of exogenous CO, BR or BV may offer a simple, inexpensive method to substitute for the cytoprotective effects of HO-1 in a variety of clinically applicable models. The objectives of this thesis were to review the li terature regarding HO-1 and, in
ix particular, the potential ther apeutic applications of th e byproducts, and to develop a model for exogenous BR administration and renal ischemia reperfusion injury (IRI) in the rat. Four groups of male Sprague Dawley rats (n = 6 rats/group) were treated: (1) 5 mg/kg IV BR, 1 h prior to ischemia and 6 h re perfusion, (2) Vehicle 1 h prior to ischemia and 6 h reperfusion, (3) 20 mg/kg IV BR, 1 h prior to and during ischemia, (4) Vehicle 1 hr prior to and during ischemia. Bilatera l renal clamping (30 min) was followed by 6 h reperfusion. IV infusion of 5 mg/kg BR achieved target levels in the se rum at 0 (13.5 +/-3.5 uM), 3 (22.4+/-7.7 mol/L), and 6 h (30.75+/-9.1 mol/L) post ischemia; however, glomerular filtration rate (GFR) was not preserved compared to vehicle (0.72+/-.41 mL/min vs 0.84+/-0.36 mL/min at 6 h, p= 0.64). Infusion of 20 mg/kg BR achieved higher serum concentrations, reaching 50 +/-21.7 mol/L at the end of ischemia, and renal function tended to be improved in these rats compared to controls (GFR at 6 hrs; 1.28+/-0.88 ml/min vs.80+/-0.42 ml/min, p= 0.56); however si gnificance was only achieved for serum creatinine at 6 hours (20mg/kg: 1.07+/-0.28 mg/dL; vehicle 1.38+/0.18 mg/dL, p=0.043). No significant improvements were noted fo r fractional excretion of electrolytes or renal vascular resistance. Hist ologic grading demonstrated a trend toward preservation of cortical proximal tubules in rats receiving 20 mg/kg IV BR compared to vehicle control; however, this was not statistically signif icant. Neither BR dose provided protection against IRI to the renal medulla. It is likel y that the combined properties of BR and CO are necessary to protect the kidney from IRI.
1 CHAPTER 1 THE PRODUCTS OF HEME OXYGENASE: A REVIEW Introduction Heme proteins play a critical role in many physiologic processes including oxygen transport, mitochondrial respiration, and signal transduction (90). The majority of heme is present in hemoglobin, while other sour ces of heme proteins include myoglobin, mitochondrial and microsomal cytochromes, and various catalytic enzymes such as nitric oxide synthases, catalase, and respiratory burst oxidase (37). Free heme exerts cytotoxic effects through formation of oxygen free ra dicals and lipid peroxidation (8, 9, 35, 39, 75, 111). The kidney is particularly sensitive to free heme molecules, and heme induced injury appears to be an important compone nt of rhabdomyolyis (74), nephrotoxin(3, 100, 122), and ischemia-reperfusion (58, 99) indu ced acute renal failure (ARF) in animal models. Heme is degraded by the enzyme heme oxygenase (HO) to produce equimolar quantities of carbon monoxide (C O), iron, and biliverdin (BV). The latter is reduced to bilirubin (BR) by biliverdin reductase (106) (F igure 1). Two isoforms of HO have been described: the inducible form HO-1 and the constitutively expressed form HO-2 (57). While HO-2 regulates normal physiologic cell function, HO-1 is induced in response to tissue injury and has been the focus of cons iderable interest in recent years (2-4, 12, 20, 24, 26, 37, 45, 59, 76, 91, 102, 107, 115, 117, 118, 121). Studies have indicated that HO-1 expressi on is increased in response to various forms of injury or insult; and upregulation of the gene is associated with marked
2 cytoprotection (37, 74, 107). Studies using HO de ficient mice (HO-1 -/ -) have confirmed that the heme oxgenase system is indispensa ble to survival and, in particular, to protection from oxidant st ress (3, 43, 91). The HO-1 -/mi ce exhibited a decreased birth rate, growth retardation, microcytic h ypochromic anemia, tissue iron deposition, hepatosplenomegaly, lymphadenopathy, leukoc ytosis, and glomerulonephritis (91). Interestingly, not long after these find ings were reported in mice, Yachie et al. documented the first human case of HO-1 defici ency in a 6-year-old boy. The child suffered from similar symptoms as the (-/-) mice, including stunted growth, anemia, iron deposition, and leukocytosis. Other findings included persistent proteinuria and hematuria, coagulation defects, hyperlip idemia, and hypobilirubinemia. Microscopic examination of renal biopsy samples reveal ed mesangial cell proliferation, lymphocyte infiltration, and detachment of the gl omerular capillary endothelium (118). Fe N N N N HOOC COOH N H N NH HOOC COOH O N H O N H O COOH N H COOH N H N H O Heme IX O2 NADPH Fe CO Heme oxygenase Biliverdin IX a b c d Bilirubin IX Biliverdin Reductase Figure 1. Heme metabolism by heme oxygenase.
3 Further investigations employing HO1 knockout mice have indicated the importance of this gene in mediating ARF. Shiraishi et al. administered cisplatin, a potent nephrotoxin, to HO-1 -/mice and found significantly greater renal injury and more severe renal failure compared to w ild-type mice. Conversely, overexpression of HO-1 caused a substantial reduction of cisplatininduced renal injury ( 100). In a similar experiment, Nath et al. noted severe, irreversible rena l failure and 100% mortality in HO1 -/mice after heme-protein-induced renal in jury, while HO-1 +/+ mice displayed mild, reversible renal injury and 0% mortality (76). Although the cytoprotective e ffects of HO-1 induction ha ve been confirmed in a number of experimental models, the mech anism of action has not been completely elucidated. Degradation of the pro-oxidant he me molecule by HO-1 is likely to aid in tissue protection; however, recen t evidence suggests that it is the downstream products of HO-1 activity (BV, BR and CO) that mediat e many of the anti-inflammatory, antiapoptotic, anti-oxidant, and immune modulator y effects associated with induction of HO1. Iron is also liberated duri ng the breakdown of heme. While it has been shown that the induction of ferritin is enhan ced in conjunction with HO-1 upr egulation (7, 74), iron is an extremely pro-oxidative molecu le. A number of studies have demonstrated prooxidative effects following HO-1 induction, pr imarily through the accumulation of iron and endothelial cell damage (18, 47, 50, 94, 95, 111) . For this reason, the administration of CO and BV/BR, rather than the induction of HO-1, may prove to be a safer and more practical means of conferring cytoprotection in a clinical setting. Administration of micromolar doses of BV, BR and CO have now been shown to substitute for the effects of HO-1, providing dose-dependent cytoprotection (1, 5, 25, 54, 77, 85).
4 This review will focus on the down-str eam products of HO-1, including pertinent aspects of biochemistry, the mechanisms of cytoprotection in the kidney and in other organ systems, and current challenges in the therapeutic application of these molecules. Carbon Monoxide Carbon monoxide was first discovered in the late 18th century and, until recently, was regarded solely as a toxic air pollutant. It is a colorless, odorle ss gas that is liberated by natural sources, or through incomplete combustion of or ganic material including wood, coal, and natural gas. The toxic eff ects of CO lie in its strong affinity to hemoglobin (Hb), which is nearly 245 times that of oxygen (93). CO displaces oxygen from Hb, shifting the oxygen dissociation cu rve to the left, resulting in tissue hypoxia (30, 93). Symptoms of CO t oxicity begin to occur at ca rboxyhemoglobin (CO Hb) levels of 20% and include dizziness, shortness of breath, and headache. CO-Hb levels of 5080% may result in death (93). The dangers of CO exposure have led the Environmental Protection Agency to publish recommendati ons for allowable exposure levels (9 ppm averaged over 8 hours or 35 ppm in one hour) and to recommend the routine installation of CO detectors in homes and businesses. In healthy non-smoking adults, basal leve ls of CO-Hb range between 1-3% (93), and exhalation of CO varies from 0-6 pp m, depending on the amount of environmental exposure and normal endogenous production. (In smokers, CO-Hb can reach 10-15% and exhaled CO 7-70 ppm). HO cleavage of the -meso carbon bridge of b-type heme accounts for greater than 85% of endogenous CO , with the remainder originating from other metabolic pathways (66, 93). Interesti ngly, elevated levels of exhaled CO are associated with chronic disease states such as asthma, bronchitis, and diabetes (88, 119).
5 Intuitively, it seems that this increase in CO would be deleterious, yet experimental evidence now suggests that this Â“toxicÂ” gas ma y exert cytoprotective effects in response to cellular stress. Several inve stigators have supplied exogenous CO to rats at levels far exceeding the EPAÂ’s recommended exposure levels for humans (e.g., 250 ppm for 25 hours) and have noticed signifi cant improvements in transplanted organ survival (77). Thus, despite the irrefutable toxicity that occurs following prolonged exposure to high concentrations of CO, it is apparent that a physiologic dose range exists wherein CO exerts vasodilatory, an ti-apoptotic and anti-inf lammatory effects. Initial investigations into the beneficial physiologic eff ects of CO revealed that this molecule exerts vasodilato ry effects through cyclic guanosine monophosphate (cGMP)dependent smooth muscle relaxation. Similar to the well-established vasodilator nitric oxide (NO), CO binds to the heme moiety of soluble guanylyl cyclase (sGC), causing activation of cGMP and resultant vasc ular relaxation (21, 29, 41, 48, 53, 92, 97, 109). While the affinity of CO for sGS is equiva lent to NO, the potency of NO-stimulated cGMP production is 30-100 times greater than CO (84, 93). Both nitric oxide synthase (NOS) and HO-1 are co-induced in times of stre ss, and the majority of evidence suggests that HO may serve both to regulate and to continue the effects of NOS following the initial stress response. NO has been de monstrated to induce HO-1 and subsequent production of CO (23), while HO-1 and CO app ear to serve as a feedback mechanism, inhibiting NOS activity (62, 116, 127). Since NO is both a potent vasorelaxant and a potential free radical (through the formation of peroxynitrite radicals) these results imply that HO-1 regulation of NOS functions to limit NO free radical production, while maintaining the vasodilatory properties of the molecule by means of CO-stimulated
6 cGMP production. Additional cGMP-mediated effects of CO include neurotransmission (101, 112), inhibition of plat elet aggregation (14) a nd vascular smooth muscle proliferation (64, 65), protection of pancrea tic beta cells from apoptosis (31), and bronchodilation (15, 28). COmediated cytoprotection has also b een described through interaction with mitogen-activated protein kinase (MAPK) signaling pathways. There are three subfamilies of MAPK pathways that modulate gene transcription: extracellular signalregulated kinase (ERK), c-Jun N-terminal ki nase (JNK), and p38. Several aspects of the protective effects of CO a ppear to be mediated thr ough the p38 MAPK pathway, a system that is involved in the physiologic res ponse to stress signals (49). For example, exogenously supplied CO (300 ppm) was shown to decrease portal ve nous resistance via stimulation of the p38 MAPK pathway and pr eserve hepatic function in the isolatedperfused rat liver following 24 hours of cold ischemia (5). The significant antiinflammatory (86) and anti-apoptotic (124) effects of CO following lung injury also appear to be mediated through p38 MAPK dependent mechanisms. CO has been shown to mitigate the infl ammatory response to sepsis and IRI through interaction with the MAPK pathways and displays immunomodulatory effects that may decrease the development of dela yed graft function associated with organ transplantation. Following organ harvest and transplantation, a casca de of inflammatory events occur: leukocyte (WBC) margination and extravasation, increased expression of endothelial cell adhesion protei ns such as ICAM-1, secretio n of inflammatory cytokines (IL-1 , IL-6, TNF ) by local WBC which in turn recruit additional WBC to the area, and smooth muscle proliferation. Th e end result of these processe s is vessel lumen occlusion
7 and graft failure. A number of studies have demonstrated COÂ’s ability to modulate the inflammatory pathway by reducing the produc tion of inflammatory cytokines and preventing smooth muscle proliferation, while increasing the production of antiinflammatory cytokines (IL10) through interaction with the MAPK pathways. For example, CO has been shown to depress T cell proliferation and IL-2 production in vitro via inhibition of the ERK pathway (87) and inhibit TNF , IL-1 , and macrophage inflammatory protein-1 production while increasing IL-10 in vivo and in vitro (83). Additionally, CO has been shown to decrease IL-6 production in vivo through the JNK pathway in response to sepsis (67). In addition to its role in response to ti ssue injury, CO also appears to have a regulatory role in vascular tone. CO is e ndogenously produced by re nal arterioles, where it acts as a depressor of vascular sensitivity to vasoconstrictors (40). Similarly, angiotensin II (Ang II)induced CO expression has been demonstrated in the rat renal vasculature, where it counteracted the pressor e ffects of Ang II (52). The ability of CO to offset such vasoconstrictive substances illustrates that HO and its metabolites exist as an endogenous regulatory system, offering cy toprotection in times of stress. The beneficial vasodilator y, anti-inflammatory, and i mmunomodulatory effects of CO suggest that this molecule may have pot ential therapeutic applications. Recently, several investigators have presented eviden ce of cytoprotection in the lungs, kidneys, small intestines, liver, and pancreatic islet cells of rats following CO inhalation or induction (70-72, 77, 82, 103, 114, 127). Song et al. performed orthotopic lung transplantations and exposed the recipien t animals to 500 ppm CO following surgery, demonstrating marked reduction of apoptosis, suppression of proinflammatory genes, and
8 preservation of tissue architecture in the CO-treated versus co ntrol rats (103). Neto, et al. demonstrated similar protective effects in a syngeneic rat kidney transplant model. Recipient rats were exposed to CO (250 ppm) for 1 hour prior to and 24 hours following orthotopic kidney transplantati on. Results indicated a marked decrease in inflammatory mediators, improved renal cortical blood flow , preservation of glomerular and tubular architecture, and increased survival in CO-t reated rats versus c ontrol (77). Exogenous CO administration has also been shown to produce an analogous reduction of proinflammatory molecules and improved surviv al in rats undergoing orthotopic small intestinal transplantation (70, 71) and protec t against intestinal inflammation in a rat model of necrotizing enterocolitis (127). Most recently, CO induction, following oral administration of methylene ch loride, was shown to protect against chronic rejection of rat renal allografts when administered to th e donor 4 hours prior to organ harvesting (60). It is evident that CO is not merely an injurious byproduct of heme catabolism, but serves a clear physiologic role in cellular de fense. While exogenous supplementation of the molecule effectively ameliorates inflam matory and ischemic injury in rats, the logistics (i.e., dose, timing of delivery, and safe ty) of supplying this potentially toxic gas to human patients have not been determined and could complicate the clinical application of this technique. Recently, novel watersoluble CO-releasing molecules are being investigated which may facil itate therapeutic delivery of CO (27, 68). Unfortunately, CO therapy is most effective when administered prior to the ons et of renal injury in rats (110), and clinical applications may be lim ited to preconditioning of organs prior to elective procedures that i nvolve known tissue injury.
9 Bilirubin In adults, approximately 250-350 mg of BR are produced daily, primarily through the breakdown of hemoglobin. HO opens th e heme (Fe-protoporphyrin IX) molecule ring, liberating iron (Fe) and CO and forming the linear tetrapyrrole molecule BV, which is then reduced to BR. The BR molecule c onsists of two rigid pl anar dipyrrole units joined by a methylene bridge at carbon 10 and can exist as three isomers: III X and XIII , with X being the natural structure formed from heme catabolism (Figure 2) (80). O O H N N N N O C H H O C H O H H O a b c d Bilirubin IX Figure 2. The physiologic structure of bilirubin IX. Bilirubin also exists as three differe nt pH-dependent ionic species, and the proportions of each species are determined by the pKa values of the Â–COOH groups on the carboxymethyl sidechains: 8.1 and 8.4 (33) . At physiologic pH (7.4), approximately 83% of BR is present as the protonated diacid (H2B), while 16% is monoanion (HB-) and <1% dianion (B2-) (Figure 3) (80). The solubility of BR (H2B) at neutral pH is low, approximately 70 nM, due to internal hydrogen bonding of the polar groups. However, if pH rises above the pKa values for the carboxyl groups, ionization of these groups leads to
10 greater proportions of HBand B2-, and the solubility of BR increases dramatically, reaching 1mM at pH 9 and 60 mM above 9.5 (80). O O H N N N N O C H H O C H O H H O O O N N N N O C H H O C H O H H O O O N N N N O C H H O C O H H O a b c d a b c d a b c d BR Diacid BR Monoanion BR Dianion Figure 3. The three ionic forms of BR. The solubility of unconjugated BR at neutra l pH is improved by the high degree of protein binding that occurs between BR (IX ) and albumin in the plasma, while
11 intracellular proteins, such as glutathione-S -transferase (GST), are bound to BR in the cytosol. Albumin has one high-affinity bindi ng site and one or more lower-affinity sites for BR (80). In adults, the normal plasma c oncentration of unconjugated BR is between 5-15 mol/L, and greater than 99% is bound to albumin. The association of BR with albumin serves not only to improve solubility at physiologic pH, but al so to sequester the potentially toxic molecule since unbound, nonionized BR is capabl e of crossing cell membranes and can interfere w ith mitochondrial respirati on when concentrations are greater than 50 M (69). Toxicity can develop when albumin binding becomes saturated and BR concentrations are >200300 M (81). The potential for bi lirubin toxicity is also increased at pH <7.4, as BR dissociates readily from albumin and is able to bind to cell and mitochondrial membranes, leading to cell lysis or disrupt ion of mitochondrial function (13, 69). Unconjugated, albumin-bound BR is transporte d to the liver where it dissociates from albumin and spontaneously diffuses through phospholipids bilayers (126) into hepatocytes. Within the he patocyte, BR is bound to cytosolic proteins and glucuronic acid is attached to one or both of the pr opionic side chains of BR by the microsomal enzyme BR uridine-diphos phate glucuronosyltransferase (UDPGT), forming watersoluble BR monoglucuronide and di glucuronide which are then ex creted into the bile and eliminated from the body. Like CO, unconjugated BR has long been c onsidered solely as a toxic waste product of heme metabolism. Indeed, hyperbilir ubinemia is responsible for diseases such as neonatal jaundice and kernicterus, and BR is capable of contributi ng to other forms of cytotoxicity (42). The question of why BV , a non-toxic and water-soluble compound, is
12 reduced to the potentially toxic and insolubl e BR molecule has long been unanswered. However, in the past three decades, benefici al properties of BR have been identified which begin to elucidate th e physiologic role of BR. The primary mechanism for BR-mediated cy toprotection in various types of stress appears to be due to the powerful antioxida nt activity of this molecule. In 1987, a landmark study by Stocker et al. introduced the idea that bili rubin served as one of the most important endogenous anti-oxidants in the serum (104). In fact, at physiologic oxygen concentration (2%), micr omolar amounts of BR were able to scavenge peroxyl radicals more effectively than -tocopherol, which had previo usly been considered the most powerful serum anti-oxidant. Subseque nt studies have dem onstrated that superinduction of HO leads to BR-mediated reduc tions in oxidative stress following renal ischemia (59) and provides cytoprotection in cardiomyocytes (26) and neurons (22) subjected to oxidative stress. New evidence indicates that BR may also serve as an important mediator of nitrosative injury ( 46) through a similar mechanism. Bilirubin and BV were shown to scavenge pe roxynitrite, an extremely poten t and stable oxidant formed by the interaction of NO and superoxide an ion (46, 63, 108), and NO has been revealed as an inducer of HO-1 expression. These re sults are particularly noteworthy, since NO production is often increased in the kidney dur ing ischemic injury or oxidative stress. While NO is a well-recognized signaling molecu le with numerous beneficial properties (21, 29, 48, 53, 56), excessive production of th e molecule can contribute to oxidative damage through the formation of peroxynitrite (123). Along with potent anti-oxidant propertie s, BR also exerts anti-inflammatory effects. In 1999, a study by Hayashi et al. explored the relationship between HO-1 and
13 endothelial cell-leuko cyte inte ractions in vivo . The investigators induced HO-1 expression in mesenteric tissues by intraper itoneal injection of hemin and subsequent oxidative stress by either hydrogen peroxide infusion or IRI. Leukocyte adhesion and rolling were inhibited in the HO-1 induced rats compared to control rats. Inhibition of HO-1 expression using zinc pr otporphyrin-IX reversed these findings. However, further supplementation of BR or BV, but not CO, ag ain prevented leukocyte adhesion (34). The effects of BR appear to be particular ly valuable in preventing cardiovascular disease. Mildly increased serum BR levels have been shown to decrease risk for the development of coronary artery disease (CAD) and atherosclerosis in humans (61). A report by Schwertner et al. indicated that a 50% decrease in total BR was associated with a 47% increase in the chance of having severe CAD (96). A similar study compared the protective effects of BR to that of HDL choles terol (38). Furthermore, the prevalence of ischemic heart disease in individuals aff ected by Gilbert Syndrome, a deficiency of UDPGT resulting in sustained un conjugated hyperbilir ubinemia, is 2% compared to 12% in the general population (113). A similar UDPGT deficiency in the Gunn rat confers resistance to the pressor effects of a ngiotensin II (Ang II), presumably through scavenging of reactive oxygen species (ROS) by BR (89). High serum BR levels have also been associated with decreased can cer mortality (105), resolution of asthma symptoms (78), and a decreased inciden ce of retinopathy of prematurity (36). The aforementioned properties of BR sugge st that the molecule may be a vital factor in mediating acute renal failure due to toxic or ischemic injury, which are characterized by varying degrees of cell inju ry, leukocyte infiltrati on, and generation of inflammatory mediators and ROS. Base d on documented antioxidant and anti-
14 inflammatory properties of BR, several inve stigators have pursued the direct use of exogenous bilirubin therapy to minimize the effects of IRI associated with organ transplantation. One such study compared the protective effects of heme-induced HO-1 versus administration of micromolar amounts of BR in a rat liver transplantation model (44). Results indicated that flushing the liver with BR wa s equally as effective at defending against oxidative stress as HO-1 induction. These results suggest that supplementation of BR may provide a simple means of organ protection during graft harvest, which is inevitably associated with a period of ischemia and oxidative injury. Recently, work in our laboratory demonstrat ed that micromolar doses of exogenous bilirubin offered similar protective effects in the isolated-perfused rat kidney during ischemia reperfusion injury (1). Rat kidneys flushed with 10 mol/L demonstrated significant improvements in urine output, GFR, tubular function and mitochondrial integrity after 20 minutes of warm ischemia. A study by Leung et al. described the marked reduction of glycerol-induced ARF due to induction of HO-1 in the kidney following ligation of the common bile duct. This group also demonstrated that micromolar concentrations of BR were prot ective against heme-induc ed renal cell injury in vitro (51) . Exogenous BR supplementation has al so been shown to ameliorate oxidative injury in the spinal cord in a rat model of multiple sclerosis (55) and to preserve mucosal integrity in a rat model of intestinal ischemia (16). The most effective dose and timing of admi nistration of BR for pr evention of IRI is unclear and appears to vary widely between different organs and experimental models. One method to establish a proposed dose of exogenous BR is to approximate the physiologic levels of BR produced after i nduction of HO-1. Previous studies have
15 reported that in the glycerol model of ARF, heme content increases in the kidney to approximately 10 mol/L within 3 hours (75), a time point that corresponds to maximal induction of HO-1. Since BV is produced in equimolar ratio to the amount of heme degraded by HO-1, 10 mol/L of BR could potentially be generated in this model. The exogenous BR concentrations used in the pr eviously mentioned studies (1, 19, 44, 51) ranged from 0.05 to 10 mol/Land fell within the reference range for serum BR concentrations in the rat (00.64 mg / dL) (98). In the rat kidney, 10 mol/L BR provided maximal protective effect in vitro (1). Similar to CO, it a ppears that supplying BR prior to an ischemic insult results in superior protection (19). Th e requirement for pretreatment with BR may limit the clinical applicab ility of this protective agent to elective situations in which renal injury is anticipate d. Even with this caveat, use of BR may have wide therapeutic application in organ transplantation or prior to administration of nephrotoxic drugs and radiogr aphic contrast agents. Biliverdin Unlike BR, BV is a soluble and non-t oxic compound; however, it is quickly reduced to BR by the enzyme biliverdin reducta se (BVRA). The conversion of BV to BR is a powerful redox cycle that results in augmentation of the BR molecule. Baranano et al. demonstrated that 10 nM BR was able to protect cells against 10,000-fold higher concentrations of hydrogen peroxide. This was possible since BR is oxidized to BV, which is recycled back to BR by BVRA, resulting in a 10,000-fold amplification of BR available to sequester ROS (10). Recently, several investigators have chosen to supply exogenous BV as a means of harnessing the antioxidative properties of BR. Yamashita et al. found that pre-treatment
16 with BV (50 mol/kg) resulted in an increased surv ival of heart allografts (120). Similarly, Nakao et al. described significantly decreased inflammatory mediators, leukocyte infiltration and organ injury in BV-t reated (50 mg/kg) transplanted small bowel (73). Moreover, Fondevila et al. revealed a dramatic decr ease in apoptosis, iNOS expression, leukocyte infiltration, and pro-in flammatory cytokine expression with a profound increase in hepatic func tion, anti-apoptotic genes and animal survival when BV (10 and 50 mol/L) was added to the perfusate of an ex vivo orthotopic liver transplantation model (25). While administration of BV alone was ineffective in rat syngeneic kidney and heart transplant models , co-administration of BV and CO provided significant protection of orga n function and improved survival (72). Biliverdin may provide a safer and potentially more effectiv e means of BR delivery; further studies are needed to determine the most approp riate dose and timing of BV delivery. Iron Along with the generation of CO and BV , iron is also liberated from the degradation of heme. Free iron is an ex tremely pro-oxidative molecule, primarily through its role in the Fenton reaction (32) . Ferritin is a ubiquitously existing intracellular protein that is able to effectiv ely sequester intracellular iron and, hence, limit its pro-oxidative capacity. While no cytoprot ective properties of free iron have been described, the induction of HO-1 has been linke d to the upregulation of ferritin (7, 74). Some suggest that the induction of ferritin is equally, if not more, advantageous than the induction of HO-1, and that the anti -oxidative property of ferritin is superior to that of BR (7). Recently, Berberat et al. found that over-expression of heavy chain ferritin was associated with the inhibition of endothelia l cell and hepatocyte apoptosis following IRI
17 in vivo and ex vivo (11). This finding may offer anothe r potentially ther apeutic method of diminishing oxidative damage in organ transplantation. Conclusion Induction of HO-1 by cellular stress leads to the production of molecules with antioxidant, anti-apoptotic, and i mmunomodulatory properties. While it is apparent that administration of exogenous CO, BR or BV alone can lead to potent cytoprotective effects, experimental evidence suggests that the coordinated response of all elements may be necessary for maximal cellular defense ( 72). Further research involving HO and its down-stream products will certai nly provide important insights and relevant information. As our understanding of the cellular and mo lecular events surrounding IRI, ARF, and transplant rejection continues to expand, so should our capability of manipulating the involved pathways for the preservation of orga n function and, ultimately, patient survival.
18 CHAPTER 2 INTRAVENOUS BILIRUBIN PROVIDES INCOMPLETE PROTECTION AGAINST ISCHEMIA REPERFUSION INJURY Introduction Along with carbon monoxide (CO), BR is produced through the metabolism of heme by the enzyme heme oxygenase (HO). The inducible form of this enzyme, HO-1, has been associated with ma rked cytoprotection, and the be neficial effects have been attributed to the production of BR and CO. The primary mechanism for BR-mediated cytoprotection in various types of stress appe ars to be due to the powerful antioxidant activity of this molecule (104) . Studies have demonstrated that super-induction of HO-1 leads to BR-mediated reductions in oxidative stress following renal ischemia (59) and provides cytoprotection in cardi omyocytes (19) and neurons (22) subjected to oxidative stress. Along with potent anti-oxidant pr operties, BR also exerts anti-inflammatory effects (34). The cytoprotective properties of BR suggest that the molecule may be a vital factor in mediating acute renal failure due to toxic or ischemic injury, which are characterized by varying degrees of cell injury, leukocyte infiltration, and generation of inflammatory mediators and ROS. Several investigators have pursued the direct use of exogenous bilirubin therapy to minimize the effects of IRI associated with or gan transplantation. One such study compared the protective effects of heme-induced HO-1 versus administration of micromolar amounts of BR in a rat liver transplantation model (44). Results indicated that flushi ng the liver with BR was equally as effective at defending
19 against oxidative stress as HO-1 induction. Thes e results suggest that supplementation of BR may provide a simple means of organ pr otection during graft harvest, which is inevitably associated with a period of ischemia and oxidative injury. Recent work has demonstrated that mi cromolar doses of exogenous bilirubin offered similar protective effects in the is olated-perfused rat ki dney during ischemia reperfusion injury (1). Rat kidneys flushed with 10 mol/L BR demonstrated significant improvements in urine output, GFR, tubular function and mitochondrial integrity after 20 minutes of warm ischemia. The objectives of this study were to develop a protocol for intravenous administration of exogenous BR and to investigate the pr otective effects of intravenous BR on IRI in the rat kidney, in vivo. Materials and Methods Animals This study was approved by the University of Florida Institutiona l Animal Care and Use Committee and was performed in accordance with the Institute for Lab Animal Research Guide for the Care and Use of Labor atory Animals. Male Sprague Dawley rats weighing 250-350 g were purchased from Harl an Sprague Dawley, Inc (Indianapolis, IN) and maintained in a temperature controlled room with alternating 12 hr/12 hr light/dark cycles in an animal facility at the University of Florida. An imals were fed a standard diet and allowed free access to water. Bilirubin Treatment Bilirubin solutions were prepared at con centrations of 0.25 mg/ml and 1 mg/ml, to be given at 6 ml/ hr in order to achie ve target doses of 5 mg/kg and 20 mg/kg, respectively. To make 0.25 mg/ml soluti on, 0.0125 g BR (bilirubin mixed isomers, 94% IX , Frontier Scientific, Logan, UT) was dissolved in 1.25 ml 100 mM NaOH and added
20 to 38.436 ml distilled water and 10.314 ml phosphate buffer (0.2 M); micromolar amounts of HCl were added to reach a final pH of 8 +/1. For the 20 mg/ kg treatment group, 0.033 g BR was dissolved in 833 L 100 mM NaOH and added to 25.622 mL distilled water and 6.875 mL phosphate buffer (0.2 M) to make a 1 mg/ml BR solution of pH 8 +/-1. Reagents p-Aminohippuric acid (PAH) sodium salt (S igma, St. Louis, MO) was dissolved in distilled water to form a 0.22 g/mL solution. 75 mg of fluorescein isothiocyanateinulin (Inulinfitc, Sigma, St. Louis, MO) was disso lved in 29.85 ml sterile saline and added to 150 L of the 0.22 g/mL PAH solution. These solu tions were prepared prior to each experiment and were protected from light at all times by cove ring with aluminum foil. Surgical Procedures Rats were anesthetized using 5% in halant isoflurane in 100% oxygen and maintained with 1.5-2% isoflurane in 100% oxygen through a tracheostomy tube. Animals were placed on a heating pad and body temperature was monitored using a rectal thermometer (Control Company, Friendsw ood, TX) and maintained at 37 +/1 o C. The left femoral vein and artery were cath eterized using polyethylene tubing (PE 50, Intramedic, Clay Adams, Parsippany, NJ ) for treatment administration and blood sampling, respectively. All animals received fluid support including PAH/ inulin (inulin 2.5 mg/ ml; PAH 0.001 g/ml) at a rate of 3 ml/hr intravenously for the duration of surgery. A polyethylene tubing T-port was constructed and attached to the venous catheter in order to administer bilirub in or vehicle and PAH/inulin solution simultaneously. Blood pressure was monito red via the arterial catheter (Transonic
21 Systems Inc, Ithaca, NY)) and recorded (Iox software, Emka Technologies, Falls Church, VA) for the length of the experiment. Follo wing a ventral midline celiotomy, the urinary bladder was catheterized using PE 160 (Intram edic) tubing and the renal pedicles were isolated. Renal ischemia was induced by clamping both renal pedicles for 30 minutes using vascular microclamps (Accurate Surgi cal and Scientific Instruments, Co., Westbury, NY) . After clamp removal, the abdomen was sutured closed using 4-0 Polyglyconate (Maxon, Sherwood, Davis, & Ge ck, St. Louis, MO) for the duration of reperfusion. After 6 hours of reperfusion, the kidneys were harvested and the rats were euthanized by an overdose of sodium pent obarbital (Euthasol, Di amond Animal Health, Inc., Des Moines, IA). A portion of each kidney was frozen to -80oC for subsequent analyses; the remainder of th e kidney was preserved in 10% formalin for histological analysis. Treatment Groups Four groups of male Sprague Dawley rats (n = 6 rats/group) were treated: (1) 5 mg/kg IV BR, 1 h prior to ischemia and 6 h reperfusion (con tinuous infusion), (2) Vehicle 1 h prior to ischemia and 6 h reperf usion, (3) 20 mg/kg IV BR, 1 h prior to and during ischemia (bolus administration), (4) Vehicle 1 hr prior to and during ischemia. Serum and Urine Analysis Blood was collected at the time of catheter placement (baseline, 1 mL), 15 minutes prior to ischemia (200 L), at clamp removal (0 h, 300 L), and 3 (1 mL) and 6 (2 mL) hours post clamp removal. Serum blood ur ea nitrogen (BUN), crea tinine, and total bilirubin were measured at baseline, 0, 3, and 6, and serum sodium and potassium were measured at baseline, 3, and 6 hours. Se rum PAH and inulin concentrations were measured 15 minutes prior to ischemia, a nd 3 and 6 hours post ischemia. Urine was
22 collected for 30 minutes prio r to ischemia, and collectiv ely for 0-3 hours and 3-6 hours post clamp removal. Urine volumes were r ecorded and used for urine flow rate determination. Urine sodium and potassium were measured from the baseline (pre ischemia), 0-3, and 3-6 hour samples. Urine and serum samples were stored at -20oC until the completion of the experiment. Samp les for thiobarbituric acid reaction (TBAR), inulin, and PAH determinati on were transferred to -80oC until analyses were performed. Glomerular filtration rate (GFR), estimate d renal plasma flow (ERPF), and fractional excretion of electrolytes (FENa, FEK) were calculated using sta ndard formulas based on urine and plasma inulin, PAH, and sodium and potassium concentrations, respectively, pre-ischemia and at 3 and 6 hours post clamp removal. Measurement of Free Radical ProductionÂ—TBAR Free radical activity was assessed in plasma samples collected at 0 and 6 hours post ischemia and in kidney tissue samples ac quired at the completion of the 6 hour reperfusion period using the thiobarbituric acid reaction (TBAR), a method used to quantify lipid peroxidation (6). Frozen tissue samples weighing between 0.0125 and 0.0228 grams were added to 100 L of HPLC grade water and homogenized using a variable speed homogenizer (Ti ssue Tearor, Dremel, Inc., Racine, WI) for 60 seconds. 50 L of this homogenate was combined with 50 L of HPLC water to produce the 100 L sample volume required for the assay. Each tissue sample was run twice and measurements were repeated until the co efficient of variance between the two measurements was <10%. Tissue TBAR levels were expressed in nmol/g tissue protein and plasma TBAR levels were expressed in nmol/ml. Tetraethoxypropane was used as the external standard.
23 Histologic Grading Transverse sections of the kidney taken at the level of the hylus were processed using hematoxylin and eosin (HE) staining and periodic-acid-Sc hiff (PAS) staining. Histological examination was performed by a renal pathologist who was blinded to treatment groups. Renal tissue was divided into 4 regions for analysis : cortical proximal convoluted tubules (CPT), outer stripe of outer medullary proximal tubule (OSOMPT), medullary thick ascending limb in inner stripe (ISOM mTAL), and collecting duct (CD). Tubular injury was graded in 5 different categories: normal, cellular swelling/ vacuolization, loss of brush bor der, nuclear condensation, a nd karyolysis, karyorrhexis, cell sloughing. Each category was assigned a nu merical score: 1= 1-24 %, 2= 25-49%, or 3= >50% based on the per centage of cells in each re gion displaying the described injury. Statistical Analysis Statistical calculations were performe d using a computer software program (Statview, SAS Institute, Inc, Cary, NC). Comparisons of serum bilirubin, BUN, creatinine levels, GFR, ERPF, fractional excr etion of sodium and potassium, and renal vascular resistance were made over time a nd between treatment groups using a one-way repeated measures analysis of variance. Individual analyses of variance were then performed to determine relationships between treatment groups at each time point. Tissue and plasma TBAR scores were compared between groups using analyses of variance. Histologic scores were comp ared between groups using a Mann Whitney U test. A p< 0.05 was considered significant.
24 Results Serum Bilirubin Concentration Continuous infusion of BR at 5 mg/kg led to a steady increase in serum BR concentrations over the period of administration, reaching 31 +/9 mol/L at 6 hours post ischemia (Figure 4). Bolus administrati on of 20 mg/kg BR for one hour prior to and during ischemia resulted in higher serum concen trations at the end of ischemia, reaching 50 +/22 mol/L; however, by 6 hours post ischemia serum BR had decreased to within normal range (Figure 4). Figure 4. Serum BR concentrations following IV infusion (5mg/kg) for one hour prior to and during ischemia and 6 hours follow ing, and IV bolus administration (20 mg/kg) for one hour prior to and during ischemia. Renal Hemodynamics Ischemia-reperfusion injury caused signifi cant decreases in GFR and increases in RVR in all treatment groups. Bolus admini stration of BR did not improve GFR (1.28 +/0.88 ml/min vs. 0.80 +/0.42 ml/m in, p= 0.56 at 6 hours), FENa (0.016 +/0.02 vs 0.024 +/0.01 , p=0.68 at 6 hours ) , or RVR (143 +/133 vs. 61 +/14, p= 0.48 at 6 hours), and significance was only achieved for serum crea tinine at 6 hours post ischemia ( 1.07 +/Serum BR 0 10 20 30 40 50 60 70 80 20 5 Pre-Isch Re p erfusion 3 h 6 hSerum Bilirubin ( mol/L)
25 0.28 vs. 1.38 +/0.18 mg/dL, p= 0.043). There wa s no difference in animals treated with continuous infusion of BR vs. untreated rats in BUN (35.3 +/3.5 vs. 33.2 +/3.3 mg/dL, p= 0.37 at 3 hours), creatinine (1.4 +/0.18 vs. 1.13 +/0.29 mg/dL, p= 0.14 at 6 hours), and FENa (0.10 +/0.04 +/0.07 +/0.02, p= 0.07 at 6 hours) (Figures 5-9). Fractional excretion of K decreased in all groups and wa s not statistically different between groups (data not shown). Figure 5. Serum blood urea nitrogen (BUN) A) Continuous infusion (5 mg/kg) BR. B) Bolus administration (20 mg/kg) BR. Pre-Isch Reperfusion 3 h 6 h 0 10 20 30 40 50 60 BUN mg/dL 20 0 Pre-Isch Reperfusion 3 h 6 h 60 0 10 20 30 40 50 BUN mg/dL 5 0 A B
26 Figure 6. Serum creatinine levels. A) Conti nuous (5mg/kg) infusion of BR. B) Bolus (20 mg/kg) administration of BR. * Serum creatinine was significantly decreased in the bolus administration group when compared to control at 6 hours post ischemia. While creatinine tended to be lower in both treatment groups at other time periods, significance was not achieved 20 0 0 .2 .4 .6 .8 1 1.2 1.4 1.6 Serum Creatinine mg/dL Pre-Isch Reperfusion 3 h 6 h * Serum Creatinine mg/dL 0 .2 .4 .6 .8 1 1.2 1.4 1.6 Pre-Isch Reperfusion 3 h 6 h 5 0 A B
27 Figure 7. Fractional excretion of sodium (FENa). A) Continuous infusion (5 mg/kg) of BR. Fractional excretion of Na incr eased in both treatment and control groups, suggesting tubular damage. Wh ile administration of BR tended to improve FENa, no significant difference was f ound between groups. B) Bolus (20 mg/kg) administration of BR. Du e to technical errors, pre-ischemia samples were not available for the bolus treatment group, however based on the similar signalment and experimental conditions, it can be assumed that pre-ischemia values would be similar to those of the continuous infusion groups. Bolus administration resu lted in slightly improved FENa, however this difference was not significant. 0 .03 .05 .08 .1 .13 .15 .17 .2 .23 FE Na Pre-Isch 3 h 6 h 5 0 20 0 0 .005 .01 .015 .02 .025 .03 .035 .04 3 h 6 h FE Na B A
28 Figure 8. Estimated glomerular filtration rate (GFR). A) Continuous infusion (5 mg/kg) of BR. GFR decreased significantly in both groups following ischemia, and continuous infusion of BR did not impr ove GFR when compared to control groups. B) Bolus (20 mg/kg) administra tion of BR. Due to technical errors, pre-ischemia samples were not ava ilable for the bolus treatment group, however based on the similar signalmen t and experimental conditions, it can be assumed that pre-ischemia values would be similar to those of the continuous infusion groups. Administrati on of BR tended to improve GFR, however no significant difference wa s found between BR treatment groups and control groups. GFR mL/min Pre-Isch 3 h 6 h 0 .5 1 1.5 2 2.5 3 3.5 5 0 0 .2 .4 .6 .8 1 1.2 1.4 3 h 6 h GFR mL/min 20 0 B A
29 Figure 9. Renal vascular resistance (RVR). A) Continuous infusion (5 mg/kg) of BR. RVR increased significantly over time in both groups and no significant difference was found between BR treated and control groups. B) Bolus (20 mg/kg) administration of BR. Due to t echnical errors, pre-ischemia samples were not available for the bolus treatment group, however based on the similar signalment and experimental conditions, it can be assumed that pre-ischemia values would be similar to those of the continuous infusion groups. Bolus administration of BR resulted in slightly lower RVR at 6 hours, however this difference was not significant. Oxidative Damage Lipid peroxidation increased af ter renal ischemia in all gr oups. Administration of a continuous infusion or bolus of BR failed to prevent oxidative injury with no significant Pre-Isch 3 h 6 h 0 25 50 75 100 125 150 175 200 225 5 0 3 h 6 h 0 20 40 60 80 100 120 140 20 0 RVR mmHg/ml/min RVR mmHg/ml/min B A
30 difference in tissue or plasma TBAR levels between treatment groups at 3 and 6 hours after reperfusion (Figure 10). Figure 10. Tissue TBAR increased significantly in al l treatment groups at 6 hours post ischemia. No significant differen ce was found between BR and control groups. Light Microscopy Thirty minutes of renal ischemia caused varying degrees of tubular cell swelling, loss of brush border, nuclear condensation, ka ryorrhexis, karyolysis and cell sloughing. No significant differences we re seen in any area of the kidney between treatment groups; however, consistent with prev ious reports, the most severe changes occurred in the S3 segment of outer stripe of the medulla (OSO MPT) (Figure 11). Kidneys treated with a bolus administration of BR prior to IRI s howed a trend towards improved histologic scores in the renal cortical pr oximal tubules compared to cont rol, with a particular effect on preservation of nuclear characteristics in this region (p= 0.06, Tables 1, 2). Interestingly, neither intravenous bolus nor continuous infusion of BR provided significant protection to the renal medulla, s uggesting a differential anatomical effect (Figure 11). 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000Tissue TBARS Control 20mg/kg Control 5mg/kg 5mg/kg 20mg/kg
31 Figure 11. Histologic samples from BR bolus (20 mg/kg BR) and control bolus (0 mg/kg BR) rats. A) Renal cortical proxima l tubules from control rat. Marked nuclear condensation, loss of brush borde r, and tubular cell sloughing is noted. B) Renal cortex from rat treated with bolus BR. A trend towards preservation of nuclear characteristics is seen compar ed to control (p= 0.06). C) Renal medulla from control rat. D) Renal me dulla from rat treated with bolus BR. The protective effect observed in the re nal cortex was not t detected in the medulla, with more severe tubular inju ry occurring in bot h control (C) and BR treated kidneys (D). A B
32 Figure 11. Continued C D
33 Table 1. Histologic scores following treatment with 5 mg/kg BR. Median Â± standard deviation histologic scores following continuous infusion of 5 mg/kg BR Tissue region Treatment Cell swelling/ vacuolization Brush border Nuclear condensation Karyolysis, karyorhexis, cell sloughing Control 1.4 Â± .9 2.6 Â± .9 1.2 Â± 1.1 0.4 Â± .6 CPT 5 mg/kg 2.0 Â± .8 2.7 Â± .8 1.6 Â± 1.1 0.3 Â± .5 Control 2.4 Â± .9 3 Â± 0 3 Â± 0 2.2 Â± .8 OSOMPT 5 mg/kg 2.7 Â± .5 2.5 Â± 1.1 3 Â± 0 2.1 Â± .7 Control 1.0 Â± .7 1.4 Â± .6 1.2 Â± .5 ISOM mTAL 5 mg/kg 1.4 Â± .8 1.6 Â± 1.0 1.1 Â± .4 Control 2.2 Â± .8 2.6 Â± .9 2.6 Â± .9 CD 5 mg/kg 3 Â± 0 2.9 Â± .4 3 Â± 0 Table 2. Histologic scores following treatment with 20 mg/kg BR. Median Â± standard deviation histologic scores following bolus infusion of 20 mg/kg BR. Tissue region Treatment Cell swelling/ vacuolization Brush border Nuclear condensation Karyolysis, karyorhexis, cell sloughing Control 2.5 Â± .8 2 Â± 1.3 2 Â± .9 0.8 Â± .8 CPT 20 mg/kg 1.8 Â± 1.0 1.7 Â± 1.2 1 Â± .6 0.3 Â± .5 Control 2.2 Â± .8 2.8 Â± .4 3 Â± 0 2.7Â± .5 OSOMPT 20 mg/kg 2.3 Â± .8 3 Â± 0 2.5 Â± .8 2 Â± 1.1 Control 1.5 Â± .6 1.3 Â± .5 1 Â± 0 ISOM mTAL 20 mg/kg 1.5 Â± 1.2 1.2 Â± 1.0 1.2 Â± .7 Control 2.5 Â± .8 1.8 Â± .8 1.5 Â± .8 CD 20 mg/kg 2 Â± 1.1 1.8 Â± 1.0 1.5 Â± .8 Discussion The first objective of this experiment was to establish a method of exogenous BR administration and concurrent physiological mon itoring in a rodent model of renal IRI. There is an abundance of information regardi ng the toxic properties of BR, particularly referring to kernicterus a nd neonatal hyperbilirubinemia (13, 33, 69, 81), however there are few references to the use of BR therapeu tically. The solubility of BR is poor at physiologic pH due to the internal hydrogen bonds of the polar groups. In vivo this is overcome by the binding of BR to albumin or in tracellular proteins su ch as glutathione-Stransferase (80). In our model, BR was dissolv ed in a solution of pH 8 and administered
34 intravenously without the addition of albumin. This protocol was successful at reaching target serum levels of 10-80 M, which have previously been reported as optimal concentrations for harnessing the anti oxidative properties of BR (1, 51, 74). Serum BR levels did not appear to plateau with the administration of BR in the continuous infusion treatment group, and reached 30 M by 6 hours. However, the bolus administration group demonstrated that the se rum BR levels returned to normal within 3 hours of discontinuing administration. Because BR concentrations >50M have been shown to impair mitocho ndrial respiration, and toxicity occurs at >200-300 M (69, 81), prolonged administration of BR my not be feasib le without quickly reaching toxic levels. Therefore, therapeutic administration using th ese protocols may be reserved for models of acute injury such as prior to cont rast administration, ch emotherapy, or organ transplantation. The acute model used in this study allo wed for invasive physiologic monitoring and data collection with the placement of intr a-venous, intra-arterial, and urinary bladder catheters. However, the duration of the expe riment and amount of data collected was limited by length of time the animals could rema in at a physiologically stable plane of anesthesia. Previous studies have shown th at following ischemia/reperfusion, significant renal injury is eviden t within 6 hours (17). In our mode l, considerable renal injury was apparent immediately following ischemia and continued to be obser ved at 3 and 6 hours post ischemia. Although this acute model is advantageous for the study of early changes renal hemodynamics during the initial pe riod of reperfusion, it may not predict differences in renal function that would be detected in a recovery model.
35 The second aim of this study was to eval uate the effects of exogenously supplied BR on renal IRI in vivo . The protective effects of HO-1 induction have been well established in multiple organ systems (19, 26, 44, 51, 59, 76) and many of these cytoprotective effects are attributed the anti -oxidative properties of BR. Although subtle protective effects on renal functi on and cortical architecture we re noted at isolated time points, our results suggest that intravenously administered BR is unable to completely substitute for the protective effects of HO-1 in renal IRI, in vivo . In fact, our current study and those of previous investigators sugg est that the protective effects of exogenous bilirubin are variable and may be organ-specific as well as model-specific. For example, BR has been shown to effectively substitute for the protective effects of HO-1 in the liver (44), intestine (25, 73) and ne ural tissue (22); however, our study and that of Nakao, et al. have been unable to reproduce thes e results in the kidney or heart, in vivo (72). Interestingly, our lab has previously show n that BR does have a significant protective effect in the kidney when using an acellular pe rfusate in the isolated perfused rat kidney model (1) and Clark, et al. showed similar protective effects in the isolated perfused heart (19). A possible explanation for the organ-specifi c and model-specific effects of BR may be related to differences in tissue micr ocirculatory hemodynamics and regional oxygen tension that become crucial in the reperf usion phase and Â“no-reflowÂ” phenomenon that occurs immediately after ischemic injury. Fo r example, outer medullary blood flow in the kidney is particularly sensitive to ischemic injury, decreasing to 16% of baseline flow rate in comparison to 60% in the renal cortex (79). This specific hemodynamic effect in the kidney is obviated by hemodilution, which completely restored outer medullary blood
36 flow and prevented erythroc yte trapping (79). It is po ssible that the use of nonsanguineous perfusate in the is olated perfused organ model produced a similar result, preventing the no-reflow phenomenon by elimina ting the possibility of erythrocyte and leukocyte trapping in this model. Histologic findings in our study were similar to those in previous rodent models of IRI, with severe injury occurring in the mTAL and S3 se gment of the proximal tubules in the outer stripe of the outer medulla. Zou a nd others have documented the importance of the cGMP-mediated effects of CO in maintaining blood flow to this region of the medulla (125). The anatomic location of the histol ogic injury noted in our model and the concurrent increase in RVR would support that the vasodilatory properties of CO may be essential to maintain renal hemodynam ics in the kidney and that combined supplementation of both BR and CO may be required to mimic the anti-oxidant and vasodilatory properties conf erred by upregulation of HO-1, in vivo . In conclusion, intravenous infusion of BR was successful in producing short-term elevations in serum bilirubin concentrations ranging from 10-80 ÂµM. In this acute model of IRI, BR treatment had only mild protectiv e effects on renal cortical architecture and was not successful in restoring renal hem odynamics immediately following reperfusion. Our findings are supportive of previous studies, which suggest that CO-mediated vasodilation may be a particul arly important component of the reno-protective effects of HO-1 in the kidney.
37 CHAPTER 3 CONCLUSION Kidney transplants are routinely performed in humans for the treatment of end stage renal failure; however, the number of patients needing kidney transplants is far greater than available donors. An effort has been made to in crease the number of available kidneys by using less than ideal dono rs, or non-heart-beati ng donors, in which the organs have undergone a period of warm is chemia. Unfortunately, IRI is recognized as a significant cause of dela yed graft function, and has rece ntly been associated with chronic allograft rejection. Renal injury following IRI is due to seve ral factors, includi ng the production of reactive oxygen species (ROS), inflammati on, vasoconstriction, and apoptosis. Heme oxygenase-1 appears to be an important e ndogenous protective mechanism against renal IRI, and manipulation of this system provi des an exciting opport unity to decrease morbidity and mortality in renal allograft recipients. Chapter 1 described, in detail, the concept of supplying exogenous dos es of the by-products HO-1 (CO, BV, BR) in order to provide a clinically applicable means of cytoprotection. Numerous studies have demonstrated protection of cells and organs following upregulation of the HO-1 enzyme (and conversely, the adverse effects of inhibi ting HO-1), and mounting evidence suggests that this cytoprotection is due , in part, to the individual and combined effects of the products of heme degradation by HO-1 (CO and BR). While both CO and BR are potentially toxic molecules, and have traditionally been regarded solely for those properties, it is clea r that both also serve clear physiologic roles.
38 Carbon monoxide possesses potent anti-apoptot ic, anti-inflammatory, and vasodilatory properties, and has demonstrat ed beneficial affects both in-vitro and in-vivo. Bilirubin is known to be a powerful anti-oxidant, and wh ile research has shown bilirubin to be protective in the heart, brain, intestines, a nd kidney, no studies have definitively shown exogenous bilirubin to protect against IRI in a renal transplant model. Chapter 2 of this thesis describes an expe riment designed to in vestigate the effects of intravenously administered BR on renal IRI in the rat. Either a bolus or continuous infusion of BR was administer ed, and both kidneys were s ubjected to 30 minutes of warm ischemia followed by 6 hours of reperfusion. Markers of renal function and hemodynamics were examined and compared between BR treated and vehicle control groups. In addition, renal tissue TBAR (an indi cator of free radicals) were measured and histologic samples of the kidneys were obt ained at the completion of reperfusion. Results of this study demonstrated an inco mplete protective effect of BR on renal function and tissue architecture. At 6 hours post ischemia, serum creatinine levels were significantly lower in the bolus administra tion group compared to the control bolus group. Furthermore, it appeared that bolus administration of BR protected renal proximal tubular cells from significant damage compared to the control group. Yet, neither bolus administration nor continuous infusion of BR was able to protect cells of the renal medulla or preserve renal hemodynamic function. It was concluded that the anti-oxidant effects of BR alone are insufficient to prot ect the kidney from IRI. The vasodilatory properties of CO may be necessary, in additi on to BR, in order maintain renal medullary blood flow and preserve renal function.
39 Ischemia reperfusion injury and the pr oducts of HO are an exciting and evolving area of research with many clinical implicati ons, and the results obtai ned in this thesis have provided some insights into future appl ications of the products of HO-1 in renal injury. In addition, we describe a murine model for the study of renal hemodynamics and architecture in the acute phase of reperfusion; a model that may prove helpful in analysis of other protective techniques for renal IRI. Future studies admini stering CO alone, CO and BR, and inducing HO-1 in this model are warranted. Additionally, supplementation of BR (+/CO) in a cold ischemia model may also provide valuable information.
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51 BIOGRAPHICAL SKETCH Kristin Kirkby began her coll egiate education in 1996 at the University of Florida and received her Bachelor of Science in animal science in May 2000. She was accepted to the University of Florida College of Ve terinary Medicine, and in May, 2003 Kristin graduated magna cum laude with her Doctor of Veterinary Medicine . Kristin completed a one year internship at a small animal referral hospital in Loveland, Colorado, and returned to the University of Florida to pur sue a combined Master of Science and small animal surgical residency at the College of Veterinary Medicine. Kristin will complete her surgical residency in July, 2008.