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【摘要】 Exogenous bilirubin (BR) substitutes for the protective effects of heme oxygenase (HO) in several organ systems. Our objective was to investigate the effects of exogenous BR in an in vivo model of ischemia-reperfusion injury (IRI) in the rat kidney. Four groups of male Sprague-Dawley rats were anesthetized using isoflurane in oxygen and treated with 1 ) 5 mg/kg intravenous (iv) BR, 1 h before ischemia and 6-h reperfusion; 2 ) vehicle 1 h before ischemia and 6-h reperfusion; 3 ) 20 mg/kg iv BR, 1 h before and during ischemia; and 4 ) vehicle 1 h before and during ischemia. Bilateral renal clamping (30 min) was followed by 6-h reperfusion. Infusion of 5 mg/kg iv BR achieved target levels in the serum at 6 h postischemia (31 ± 9 µmol/l). Infusion of 20 mg/kg BR reached 50 ± 22 µmol/l at the end of ischemia, and a significant improvement was seen in serum creatinine at 6 h (1.07 ± 28 vs. 1.38 ± 0.18 mg/dl, P = 0.043). Glomerular filtration rate, estimated renal plasma flow, fractional excretion of electrolytes, and renal vascular resistance were not significantly improved in BR-treated groups. Histological grading demonstrated a trend toward preservation of cortical proximal tubules in rats receiving 20 mg/kg iv BR compared with control; however, neither BR dose provided protection against injury to the renal medulla. At the doses administered, iv BR did not provide complete protection against IRI in vivo. Combined supplementation of both BR and carbon monoxide may be required to preserve renal blood flow and adequately substitute for the protective effects of HO in vivo.
【关键词】 heme oxygenase carbon monoxide biliverdin
HEME OXYGENASE 1 (HO-1)is induced in response to cellular stress and is responsible for converting the prooxidant heme molecule into equimolar quantities of biliverdin (BV), carbon monoxide (CO), and iron. BV is then converted to bilirubin (BR) by the enzyme biliverdin reductase ( 34 ). Experimental evidence suggests that induction of the HO system is an important endogenous mechanism for cytoprotection and that these beneficial effects may be mediated by the downstream products of heme degradation: CO, BR, and BV ( 1, 3, 4, 7, 11, 19, 25, 31, 32, 35, 36 ). 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 that 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 ( 1a, 5, 13, 21, 22, 23, 26, 30, 31a ).
The primary mechanism for BR-mediated cytoprotection in various types of stress appears to be due to the powerful antioxidant activity of this molecule ( 33 ). Studies have demonstrated that superinduction of HO leads to BR-mediated reductions in oxidative stress following renal ischemia ( 19 ) and provides cytoprotection in cardiomyocytes ( 9 ) and neurons ( 12 ) subjected to oxidative stress. Along with potent antioxidant properties, BR also exerts anti-inflammatory effects ( 16 ).
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 is characterized by varying degrees of cell injury, leukocyte infiltration, and generation of inflammatory mediators and reactive oxygen species. Several investigators have pursued the direct use of exogenous BR therapy to minimize the effects of IRI associated with organ transplantation. One such study compared the protective effects of heme-induced HO-1 vs. administration of micromolar amounts of BR in a rat liver transplantation model ( 17 ). Results indicated that flushing the liver with BR was 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 demonstrated that micromolar doses of exogenous BR offered similar protective effects in the isolated, perfused rat kidney during IRI ( 1a ). Rat kidneys flushed with 10 µmol/l BR demonstrated significant improvements in urine output, glomerular filtration rate (GFR), tubular function, and mitochondrial integrity after 20 min of warm ischemia. The objectives of this study were to develop a protocol for intravenous administration of exogenous BR and to investigate the protective 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 Institutional Animal Care and Use Committee and was performed in accordance with the Institute for Lab Animal Research Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats, weighing 250-350 g, were purchased from Harlan Sprague Dawley (Indianapolis, IN) and maintained in a temperature-controlled room with alternating 12:12-h light-dark cycles in an animal facility at the University of Florida. Animals were fed a standard diet and allowed free access to water.
BR treatment. BR solutions were prepared at concentrations of 0.25 and 1 mg/ml, to be given at 6 ml/h to achieve target doses of 5 and 20 mg/kg, respectively. To make a 0.25 mg/ml solution, 0.0125 g BR (BR mixed isomers, 94% bilirubin IX, 3% bilirubin III, and 3% bilirubin XIII, Frontier Scientific, Logan, UT) was dissolved in 1.25 ml 100 mM NaOH and added 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 µl 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. PAH sodium salt (Sigma, St. Louis, MO) was dissolved in distilled water to form a 0.22 g/ml solution. Then, 75 mg of fluorescein isothiocyanate-inulin (Inulin-FITC, Sigma) was dissolved in 29.85 ml sterile saline and added to 150 µl of the 0.22 g/ml PAH solution. These solutions were prepared before each experiment and were protected from light at all times by a covering of aluminum foil.
Surgical procedures. Rats were anesthetized using 5% inhalant 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, Friendswood, TX) and maintained at 37 ± 1°C. The left femoral vein and artery were catheterized 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/h intravenously for the duration of surgery. A polyethylene tubing T port was constructed and attached to the venous catheter to administer BR or vehicle and PAH/inulin solution simultaneously. Blood pressure was monitored via the arterial catheter (Transonic Systems, Ithaca, NY) and recorded (Iox software, Emka Technologies, Falls Church, VA) for the length of the experiment. Following a ventral midline celiotomy, the urinary bladder was catheterized using PE-160 (Intramedic) tubing and the renal pedicles were isolated. Renal ischemia was induced by clamping both renal pedicles for 30 min using vascular microclamps (Accurate Surgical and Scientific Instruments, Westbury, NY). After clamp removal, the abdomen was sutured closed using 4-0 Polyglyconate (Maxon, Sherwood, Davis, & Geck, St. Louis, MO) for the duration of reperfusion. After 6 h of reperfusion, the kidneys were harvested and the rats were euthanized by an overdose of pentobarbital sodium (Euthasol, Diamond Animal Health, Des Moines, IA). A portion of each kidney was frozen to -80°C for subsequent analyses; the remainder of the kidney was preserved in 10% formalin for histological analysis.
Treatment groups. Four groups of male Sprague-Dawley rats ( n = 6 rats/group) were treated: group 1, 5 mg/kg iv BR, 1 h before ischemia and 6-h reperfusion (continuous infusion); group 2, vehicle 1 h before ischemia and 6-h reperfusion; group 3, 20 mg/kg iv BR, 1 h before and during ischemia (bolus administration); and group 4, vehicle 1 h before and during ischemia.
Serum and urine analysis. Blood was collected at the time of catheter placement (baseline, 1 ml), 15 min before ischemia (200 µl), at clamp removal (0 h, 300 µl), and 3 (1 ml) and 6 (2 ml) h post-clamp removal. Blood urea nitrogen (BUN), creatinine, and total BR were measured at baseline, 0, 3, and 6 h, and serum sodium and potassium were measured at baseline, 3, and 6 h. Serum PAH and inulin concentrations were measured 15 min before ischemia and 3 and 6 h postischemia. Urine was collected for 30 min before ischemia and collectively for 0-3 h and 3-6 h post-clamp removal. Urine volumes were recorded and used to determine urine flow rate. Urine sodium and potassium were measured from the baseline (preischemia), 0- to 3-, and 3- to 6-h samples. Urine and serum samples were stored at -20°C until the completion of the experiment. Samples for thiobarbituric acid reaction (TBAR), inulin, and PAH determination were transferred to -80°C until analyses were performed.
GFR, estimated renal plasma flow (ERPF), and fractional excretion of electrolytes (FE Na, FE K ) were calculated using standard formulas based on urine and plasma inulin, PAH, and sodium and potassium concentrations, respectively, preischemia and at 3 and 6 h after clamp removal.
Measurement of free radical production TBAR. Free radical activity was assessed in plasma samples collected at 0 and 6 h postischemia and in kidney tissue samples acquired at the completion of the 6-h reperfusion period using TBAR, a method used to quantify lipid peroxidation ( 6 ). Frozen tissue samples weighing between 0.0125 and 0.0228 g were added to 100 µl of HPLC-grade water and homogenized using a variable speed homogenizer (Tissue Tearor, Dremel, Racine, WI) for 60 s. Fifty microliters of this homogenate were combined with 50 µl of HPLC water to produce the 100-µl sample volume required for the assay. Each tissue sample was run twice, and measurements were repeated until the coefficient of variance between the two measurements was <10%. Tissue TBAR levels were expressed in nanomoles per gram tissue protein, and plasma TBAR levels were expressed in nanomoles per milliliter. Tetraethoxypropane was used as the external standard.
Histological grading. Transverse sections of the kidney taken at the level of the hylus were processed using hematoxylin and eosin staining and periodic-acid-Schiff staining. Histological examination was performed by a renal pathologist who was blinded to treatment groups. Renal tissue was divided into four regions for analysis: cortical proximal convoluted tubules, S3 segment of outer stripe of outer medullary proximal tubule, medullary thick limb in inner stripe, and collecting duct. Tubular injury was graded in five different categories: normal; cellular swelling/vacuolization; loss of brush border; nuclear condensation; and karyolysis, karyorrhexis, and cell sloughing. Each category was assigned 50%) based on the percentage of cells in each region displaying the described injury.
Statistical analysis. Statistical calculations were performed using a computer software program (Statview, SAS Institute, Cary, NC). Comparisons of serum BR, BUN, creatinine levels, GFR, ERPF, fractional excretion of sodium and potassium, and renal vascular resistance (RVR) were made over time and between treatment groups using a two-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. Histological scores were compared using a Mann-Whitney U -test. P < 0.05 was considered significant.
RESULTS
Serum BR 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 µmol/l at 6 h postischemia ( Fig. 1 ). Bolus administration of 20 mg/kg BR for 1 h before and during ischemia resulted in higher serum concentrations at the end of ischemia, reaching 50 ± 22 µmol/l; however, by 6 h postischemia serum BR had decreased to within the normal range ( Fig. 1 ).
Fig. 1. Serum bilirubin (BR) concentrations following continuous intravenous (iv) infusion (5 mg/kg) for 1 h before and during ischemia and 6 h following, and iv bolus administration (20 mg/kg) for 1 h before and during ischemia. Both doses achieved target serum BR concentrations of 10-80 µmol/l, which have previously been shown to provide antioxidant protection.
Renal hemodynamics. IRI caused significant decreases in GFR and increases in RVR in all treatment groups. Bolus administration of BR did not improve GFR (1.28 ± 0.88 vs. 0.80 ± 0.42 ml/min, P = 0.56 at 6 h), FE Na (0.016 ± 0.02 vs. 0.024 ± 0.01, P = 0.68 at 6 h), or RVR (143 ± 133 vs. 61 ± 14 mmHg·ml·min -1, P = 0.48 at 6 h), and significance was only achieved for serum creatinine at 6 h postischemia (1.07 ± 0.28 vs. 1.38 ± 0.18 mg/dl, P = 0.043). There was no difference in animals treated with continuous infusion of BR vs. untreated rats for BUN (35.3 ± 3.5 vs. 33.2 ± 3.3 mg/dl, P = 0.37 at 3 h), creatinine (1.4 ± 0.18 vs. 1.13 ± 0.29 mg/dl, P = 0.14 at 6 h), and FE Na (0.10 ± 0.04 vs. 0.07 ± 0.02, P = 0.07 at 6 h) ( Figs. 2 - 6 ). FE K decreased in all groups and was not statistically different among groups (data not shown).
Fig. 2. Estimated glomerular filtration rate (GFR) following continuous infusion ( A ) or bolus administration ( B ) of BR. A : GFR decreased significantly in both groups following ischemia, and continuous infusion of BR did not improve GFR compared with control groups. B : due to technical errors, preischemia samples were not available for the bolus treatment group; however, based on the similar signalment and experimental conditions, it can be assumed that preischemia values would be similar to those of the continuous infusion groups. Administration of BR tended to improve GFR; however, no significant difference was found between BR treatment groups and control groups.
Fig. 6. Blood urea nitrogen (BUN) following continuous infusion ( A ) or bolus administration ( B ) of BR. Serum BUN was increased at all time points following ischemia, and no significant difference was found between BR administration and control groups.
Oxidative damage. Lipid peroxidation increased after renal ischemia in all groups. Administration of a continuous infusion or bolus of BR failed to prevent oxidative injury with no significant difference in tissue or plasma TBAR levels between treatment groups at 3 and 6 h after reperfusion (plasma TBAR data not shown) ( Fig. 7 ).
Fig. 3. Fractional excretion of sodium (FE Na ) following continuous infusion ( A ) or bolus administration ( B ) of BR. A : FE Na increased in both treatment and control groups, suggesting tubular damage. While administration of BR tended to improve FE Na, no significant difference was found between groups. B : due to technical errors, preischemia samples were not available for the bolus treatment group; however, based on the similar signalment and experimental conditions, it can be assumed that preischemia values would be similar to those of the continuous infusion groups. Bolus administration resulted in slightly improved F Na; however, this difference was not significant.
Fig. 4. Renal vascular resistance (RVR) following continuous infusion ( A ) or bolus administration ( B ) of BR. A : RVR increased significantly over time in both groups, and no significant difference was found between BR-treated and control groups. B : due to technical errors, preischemia samples were not available for the bolus treatment group; however, based on the similar signalment and experimental conditions, it can be assumed that preischemia values would be similar to those of the continuous infusion groups. Bolus administration of BR resulted in slightly lower RVR at 6 h; however, this difference was not significant.
Fig. 5. Serum creatinine levels following continuous infusion ( A ) or bolus administration ( B ) of BR. While creatinine tended to be lower in both treatment groups at other time periods, significance was not achieved. *Serum creatinine was significantly decreased in the bolus administration group compared with control at 6 h postischemia.
Fig. 7. Tissue thiobarbituric acid reactions (TBARs) increased significantly in all treatment groups at 6 h postischemia. No significant difference 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, karyorrhexis, karyolysis, and cell sloughing. No significant differences were seen in any area of the kidney between treatment groups; however, consistent with previous reports, the most severe changes occurred in the S3 segment of outer stripe of the medulla ( Fig. 8 ). Kidneys treated with a bolus administration of BR before IRI showed a trend toward improved histological scores in the renal cortical proximal tubules compared with control, with a particular effect on preservation of nuclear characteristics in this region ( P = 0.06, Tables 1 and 2 ). Interestingly, neither an intravenous bolus nor continuous infusion of BR provided significant protection to the renal medulla, suggesting a differential anatomic effect ( Fig. 8 ).
Fig. 8. Histological grading did not show significant preservation of renal architecture, although a trend toward preservation of nuclear characteristics in the cortical proximal tubules was observed in rats treated with bolus administration of BR compared with vehicle control ( P = 0.06). Note the marked nuclear condensation, loss of brush border, and tubular cell sloughing in the cortical kidney of a rat treated with vehicle control [periodic acid-Schiff (PAS) stain, x 400; A ] compared with a rat treated with 20 mg/kg BR (PAS stain, x 400; B ). No evidence of a differential effect was noted in the medulla, with more severe tubular injury occurring in both control (PAS stain, x 400; C ) and BR-treated kidneys (PAS stain, x 400; D ).
Table 1. Histological scores following continuous infusion of 5 mg/kg BR
Table 2. Histological scores following bolus infusion of 20 mg/kg BR
DISCUSSION
The first objective of this experiment was to establish a method of exogenous BR administration and concurrent physiological monitoring in a rodent model of renal IRI. There is an abundance of information regarding the toxic properties of BR, particularly referring to kernicterus and neonatal hyperbilirubinemia ( 8, 15, 20, 29 ); however, there are few references to the use of BR therapeutically. The solubility of BR is poor at physiological pH due to the internal hydrogen bonds of the polar groups. In vivo this is overcome by the binding of BR to albumin or intracellular proteins such as glutathione- S -transferase ( 28 ). In our model, BR was dissolved in a solution of pH 8 and administered intravenously without the addition of albumin. This protocol was successful at reaching target serum levels of 10-80 µmol/l, which have previously been reported as optimal concentrations for harnessing the antioxidative properties of BR ( 1a, 18, 25 ).
Serum BR levels did not plateau with the administration of BR in the continuous infusion treatment group, and reached 30 µmol/l by 6 h. However, the bolus administration group demonstrated that the serum BR levels returned to normal within 3 h of discontinuing 50 µmol/l have been shown to impair mitochondrial respiration, and toxicity 200-300 µmol/l ( 20, 29 ), prolonged administration of BR may not be feasible without quickly reaching toxic levels. Therefore, therapeutic administration using these protocols may be reserved for models of acute injury such as before contrast administration, chemotherapy, or organ transplantation.
The acute model used in this study allowed for invasive physiological monitoring and data collection with the placement of intravenous, intra-arterial, and urinary bladder catheters. However, the duration of the experiment and amount of data collected were limited by the length of time the animals could remain at a physiologically stable plane of anesthesia. Previous studies have shown that following ischemia-reperfusion, significant renal injury is evident within 6 h ( 10 ). In our model, considerable renal injury was apparent immediately following ischemia and continued to be observed at 3 and 6 h postischemia. Although this acute model is advantageous for the study of early changes in renal hemodynamics during the initial period of reperfusion, it may not predict differences in renal function that would be detected in a recovery model.
The second aim of this study was to evaluate 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 ( 9, 14, 17, 18, 19, 25 ), and many of these cytoprotective effects are attributed the antioxidative properties of BR. Previous investigators have induced HO-1 and measured serum BR and BR production in target organs at various time points. A study by Hayashi et al. ( 16 ) induced HO-1 following 40 µmol/kg intraperitoneal hemin administration and found plasma BR levels to be 2.8 ± 0.6 µmol/l at 6 h postinduction. BR levels increased to 3.7 ± 1.1 µmol/l 12 h post-HO-1 induction and returned to normal (0.9 µmol/l) by 18 h. This study showed that leukocyte adhesion following oxidative stress decreased following HO-1 induction and was lowest at 12 h, corresponding to the time point at which BR levels were highest ( 16 ). Another study using the glycerol model combined with ligation of the common bile duct showed that serum BR levels reached 148 µM 7 days after bile duct ligation (1 day after HO-1 induction via glycerol injury) and that this was associated with markedly reduced acute renal injury ( 18 ). Results of theses studies suggest that elevated serum and tissue BR levels are associated with a protective effect against oxidative or toxic injury. It is evident that a wide range of BR levels can be associated with cytoprotection and that the most effective level of endogenously induced or exogenously administered BR has yet to be determined.
Although subtle protective effects on renal function and cortical architecture were 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 suggest that the protective effects of exogenous BR 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 ( 17 ), intestine ( 13, 24 ), and neural tissue ( 12 ); however, our study and that of Nakao et al. ( 23 ) have been unable to reproduce these results in the kidney or heart in vivo. Interestingly, our laboratory has previously shown that BR does have a significant protective effect in the kidney when an acellular perfusate in the isolated, perfused rat kidney model ( 1a ) is used, and Clark et al. ( 9 ) showed similar protective effects in the isolated, perfused heart.
A possible explanation for the organ-specific and model-specific effects of BR may be related to differences in tissue microcirculatory hemodynamics and regional oxygen tension that become crucial in the reperfusion phase and "no-reflow" phenomenon that occurs immediately after ischemic injury. For example, outer medullary blood flow in the kidney is particularly sensitive to ischemic injury, decreasing to 16% of baseline flow rate compared with 60% in the renal cortex ( 27 ). This specific hemodynamic effect in the kidney is obviated by hemodilution, which completely restored outer medullary blood flow and prevented erythrocyte trapping ( 27 ). It is possible that the use of nonsanguineous perfusate in the isolated, perfused organ model produced a similar result, preventing the no-reflow phenomenon by eliminating the possibility of erythrocyte and leukocyte trapping in this model.
Histological findings in our study were similar to those in previous rodent models of IRI, with severe injury occurring in the medullary thick ascending limb and S3 segment of the proximal tubules in the outer stripe of the outer medulla. Zou and others ( 37 ) have documented the importance of the cGMP-mediated effects of CO in maintaining blood flow to this region of the medulla. The anatomic location of the histological 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 hemodynamics in the kidney and that combined supplementation of both BR and CO may be required to mimic the antioxidant and vasodilatory properties conferred by upregulation of HO-1 in vivo.
In conclusion, intravenous infusion of BR was successful in producing short-term elevations in serum BR concentrations ranging from 10-80 µmol/l. In this acute model of IRI, BR treatment had minimal protective effects on renal cortical architecture and was not successful in restoring renal hemodynamics immediately following reperfusion. Our findings are supportive of previous studies, which suggest that CO-mediated vasodilation may be a particularly important component of the renoprotective effects of HO-1 in the kidney.
GRANTS
This work was supported by a University of Florida Consolidated Faculty Research Development Award and by the International Renal Interest Society Award, funded by Novartis Animal Health (grants awarded to C. Adin).
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作者单位：1 Comparative Nephrology and Transplantation Laboratory, Department of Small Animal Clinical Sciences, and 2 Department of Physiology and Functional Genomics, University of Florida, Gainesville; 3 Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Alabama; and 4 Pat