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Insulin-like growth factor-I attenuates delayed graft function in a canine renal autotransplantation model
Insulin-like growth factor-I attenuates delayed graft function in a canine renal autotransplantation model Drazen Petrinec, MD, Jeffrey M. Reilly, MD, Gregorio A. Sicard, MD, Jeffrey A. Lowell, MD, T o d d IL Howard, MD, Daniel R. Martin, MS, Daniel C. Brennan, MD, and Steven B. Miller, MD, St. Louis, Mo. Background. Insulin-like growth factor-I (IGF-I) has been shown to accelerate recovery in animal models of ischemic or toxic acute renal injury. Ischemic renal injury is frequently encountered after cadaveric transplantation manifested as delayed graft function. This study was performed to determine whether perfusion of kidneys with preservation solution supplemented with IGF-I would improve the course of renal injury in a canine autotransplantation model of delayed graft function. Methods. Dogs underwent unilateral nephrectomy with kidneys perfused and stored in Euro-Collins solution supplemented with vehicle (n = 11) or IGF-I (n = 8). After 24 hours of kidney preservation, a contralateral nephrectomy was performed and the stored kidney was autotransplanted. Renal function was examined for 5 days after the transplantation, and an inulin clearance was obtained at the time of death. Results. Compared with dogs that received kidneys preserved in the vehicle, dogs receiving the IGF-I preserved kidneys had significantly lower daily serum creatinine and blood urea nitrogen levels during the course of 5 days after transplantation. Inulin clearance at death was nearly double in the IGF-I treated animals compared with the vehicle-treated controls (1.37 +-- O.16 ml/min/kg versus O. 77 +- 0.13 ml/min/kg; p < 0.05). Conclusions. Perfusion and storage of kidneys with preservation solution supplemented with IGF-I can attenuate the course of delayed graft function in a canine renal autotransplantation model. IGF-I may have potential for use in cadaveric human renal transplantation. (Surgery 1996;120:221-6.) Prom the Washington University School of Medicine, St. Louis, Mo.
POLYPEPTIDE GROWTH FA(~ORS regulate kidney development, growth, and function and participate in processes o f repair after renal injury, l' 2 Recently the use o f o n e o f these factors, insulin-like growth factor-I (IGF-I), has b e e n shown to accelerate recovery from acute ischemic kidney failure when administered at the time o f injury, 24 hours after the injury, when kidney failure is "established," or before injury in a prophylactic m a n n e r . 3' 4 T h e mechanisms by which IGF-I acts in acute kidney failure include stimulation o f anabolism, the mainten a n c e o f g i o m e r u l a r filtration, and the e n h a n c e m e n t o f tubular regeneration. 5 An acute renal injury is frequently e n c o u n t e r e d in the setting o f kidney procureSupported in part by a grant from the MissouriKidneyProgram. S.B.M. is supported by a Clinician-ScientistAward from the American Heart Association. Presented at the Fifty-seventhAnnual Meeting of the Societyof University Surgeons, Washington, D.C., Feb. 8-10, 1996. Reprint requests: Steven B. Miller, MD, Washington UniversitySchool of Medicine, 660 S. Euclid Avenue, Box 8126, St. Louis, MO 63110. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/6/73849
ment, storage, and revascularization for cadaveric transplantation. In fact, approximately 30% of all recipients o f cadaveric renal allografts suffer from acute injury severe e n o u g h to require dialysis after transplantation. 6 This condition is referred to as delayed graft function a n d is a major complication o f cadaveric transplantation. Patients suffering delayed graft function have longer a n d m o r e expensive hospitalization, an increased incidence of rejection, a n d a significantly lower 1-year graft survival rate. 7 Because o f the potential use o f IGF-I as a p h a r m a c o logic agent to treat acute renal injury, we carried o u t studies to d e t e r m i n e w h e t h e r IGF-I might ameliorate the course o f delayed graft function in a canine renal autotransplantation model. T h e data show that perfusion and cold storage o f kidneys in preservation solution s u p p l e m e n t e d with IGF-I hasten the recovery o f renal function a n d ameliorate the course of delayed graft function. O u r findings establish the potential for the use of IGF-I as a therapeutic agent for the prevention o f delayed graft function in h u m a n cadaveric renal transplantation. SURGERY
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MATERIAL AND METHODS Operative procedure. The experimental protocol was approved by the Animal Studies Committee of Washington University School of Medicine and was performed in accordance with their guidelines. Female mongrel dogs weighing 25 to 35 kg were obtained 72 hours before initiation into the study and allowed to acclimate to the animal facility. On day 0 the dogs received premedication with atropine 0.04 m g / k g and acepromazine 0.05 m g / k g , and anesthesia was induced with pentothal 18 m g / k g and continued with a combination of isoflurane (0.4% to 1.5%) and oxygen. Respiration was maintained through an endotracheal tube with mechanical ventilation. Fluid balance was maintained with lactated Ringer's solution at 10 m l / k g / m i n during the first hour and 5 m l / k g / m i n thereafter. A broad-spectrum antibiotic (cefazolin 1 gm) was administered for prophylaxis before laparotomy incision. The dogs did not receive any anticoagulation. Under sterile conditions a left nephroureterectomy was performed by using a median laparotomy. The renal artery was cannulated within 2 minutes of excision, and the organ was flush perfused (gravity perfusion from a height of 100 cm) with 350 ml perfusate at 4 ~ C. The perfusate was EuroCollins solution supplemented with either an acetic acid vehicle (n--11) or IGF-I ( n = 8 ) at a concentration of 10 -7 m o l / L (generously provided by Genentech, Inc., South San Francisco, Calif.). After flush perfusion the organs were stored in the same supplemented EuroCollins solution for 24 hours at 4 ~ C. Assignment of the preservation solution was random, and the team involved in the pertbrmance of the surgery and the care of the animal were blinded. On day 1, at 24 hours "alter harvest a second operation was performed. Under similar conditions the dog underwent a contralateral nephrectomy with autotransplantation of the preserved kidney. The kidney was placed in the pelvis with end-toside anastomoses of the renal vessels to the c o m m o n iiiac vessels. The ureter was anastomosed to the bladder in standard fashion. Before reperfusion of the transplanted kidney, furosemide 2 m g / k g and mannitol 0.5 g m / k g were administered. All subjects received the same amount of intravenous hydration per kilogram of body weight during the operative procedure. Before the operation animals were allowed free access to water and standard laboratory food. Measurement of renal function. Daily serum creatinine and blood urea nitrogen (BUN) levels were obtained from the day of the initial operation (day 0) until death on day 6. Creatinine and BUN levels were measured in plasma by a Cobas Mira Autoanalyser (Roche Diagnostic Systems, Inc., Brancbburg, N.J.). Glomerular filtration rate was determined on the day of death by the clearance of exogenous inulin. The dog was placed in a sling and venous, arterial, and bladder
Surgery August 1996 catheters were placed. A loading dose of inulin 30 m g / k g (Cypros Pharmaceuticals, Carlsbad, Calif.) was administered during a period of 5 minutes, followed by a constant infusion at 400 m g / h r . After a 60 minute equilibration period three consecutive 60-minute collections of urine and blood were obtained for estimation of inulin clearance. Inulin concentration in blood and urine was measured by means of standard techniques as before. 3, 4 Clearance rates were calculated and expressed per kilogram o f body weight. Mean body weight was similar between the two groups and did not change substantially during the course of the experiment. Data reflect the mean of three calculated clearances per dog. Histopathologic assessment. All transplanted kidneys were removed at the time of death. The kidneys were placed in phosphate-buffered formalin for fixation. They were then paraffin embedded and sliced at 5 pm. Slides were stained with hematoxylin-eosin (H&E) and periodic acid/Schiff (PAS). A pathologist blinded to the treatments examined and graded all the specimens by using a scoring system that considered the pathologic changes consistent with ischemic injury and recovery as described previously. 3's The pathologic scoring system grades four features consistent with acute tubular necrosis and recovery: epithelial calcification, tubular dilatation, proximal tubular papillary proliferation, and interstitial infiltration. Twelve microscopic fields per section were chosen randomly to be scored for each parameter. The details of the pathologic scoring system are as tbllows: (1) epithelial calcification; (0) no epithelial calcification, (i) 1 to 4 fields with epithelial calcification, (ii) 5 to 8 fields with epithelial calcifications, (iii) 9 to 12 fields with epithelial calcifications; (2) tubular dilatation: (0) normal by H&E and PAS, (i) suggestive tubular dilatation by H&E with partial loss of PAS staining along the brush border; (ii) unequivocal tubular dilatation by H&E with widespread loss of PAS staining along the brush border, (iii) unequivocal tubular dilatation by H&E with loss of tubular specificity and total absence of PAS staining along the brush border; (3) proximal tubular papillary proliferation: (0) no proliferation; (i) 1 to 4 fields with proliferative changes, (ii) 5 to 8 fields with proliferative changes, (iii) 9 to 12 fields with proliferative changes; (4) interstitial infiltration: (0) no infiltration, (i) 1 to 4 fields with an infiltrate, (ii) 5 to 8 fields with an infiltrate, (iii) 9 to 12 fields with an infiltrate. The total score for each kidney examined was divided by 4, to give a final score between 0 and 3. Statistics. Levels of BUN and creatinine were compared by Dunnett's multiple comparisons test. 9 Inulin clearance rates and pathologic scores in the two groups were compared by two-tailed Student's t test. Differences were considered significant if p < 0.05.
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RESULTS
Unilateral nephrectomy resulted in a small increase in the serum creatinine level between days 0 and 1 in both the vehicle and IGF-I treated animals, as shown in Fig. 1. In contrast, the contralateral nephrectomy and transplantation of the stored kidney on day 1 of the protocol resulted in a significant injury. In animals that received kidneys stored in preservation solution supplemented with the vehicle, the mean serum creatinine level increased to a peak of 7.23 - 1.24 m g / d l on day 5 of the protocol and only came down to 6.90 -+ 1.58 m g / d l at the time of death on day 6. Animals that received a kidney perfused and stored with preservation solution supplemented with IGF-I exhibited less functional renal injury, with the mean serum creatinine level peaking on day 4 of the protocol at 5.56 +- 0.64 mg/dl. At the time of death on day 6 of the protocol the mean serum creatinine level had decreased to 3.53-+ 0.34
mg/dl. The day 6 creatinine levels for the IGF-I treated animals were statistically lower when c o m p a r e d with the levels obtained from the vehicle-treated animals (p < 0.05). A similar finding was observed when examining the daily BUN levels from animals receiving kidneys stored in preservation solution supplemented with the vehicle or IGF-I, as shown in Fig. 2. The levels of BUN did not increase after unilateral nephrectomy between days 0 and 1 of the protocol. However, after contralateral nephrectomy and transplantation of the stored kidney, there was a dramatic rise in levels of BUN, especially in the animals receiving kidneys stored with the vehicle. In the vehicle group the BUN level was still rising on the final day of the protocol and had reached a level of 100.9-+ 14.9 mg/dl. Animals receiving kidneys preserved with supplemental IGF-I became less uremic, and the BUN levels peaked on day 4 of the protocol at
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Table. Renal function at day 6 as determined by levels of creatinine
Creatinine (mg/dl)
BUN (mg/dl)
E-C + veh 6.9_+1.9 101_+15 E-C + IGF-1 3.5 -4-0.3 68 -+ 4 p Value <0.05 <0.05
Inulin clearance (ml/min/kg body weight) 0.77-+0.13 1.37 -+ 0.16 <0.05
Values are mean -+ SEM. E-C + veh, Euro-Collins supplemented with vehicle; E-C + IGF-1, Euro-Collins supplemented with IGF-I.
79.8 _+ 7.0 mg/dl. The BUN levels were statistically lower in the IGF-I group when compared with the vehicle group on days 5 and 6 of the protocol (p < 0.05). To ascertain whether the changes in levels of creatinine and BUN were reflective of changes in glomerular filtration, we measured inulin clearance on day 6 before death. The inulin clearance rate was nearly twice as high in the IGF-I treated dogs compared with the animals that received the vehicle, 1.37 -+ 0.16 versus 0.77 + 0.13 m l / m i n / k g , respectively (p < 0.05) (Table). Morphologic analysis was performed on all transplanted kidneys harvested at the time o f death. Kidneys stored in solution supplemented with IGF-I had a score of 1.4 -+ 0.4. The pathologic score obtained from grading the kidneys stored in solution supplemented with the vehicle was 1.9 -+ 0.5. These scores were not significantly different.
DISCUSSION This study shows in a canine model of renal autotransplantation that the addition of IGF-I to Euro-Collins solution attenuates renal autograft dysfunction. This finding is of potential clinical significance because delayed graft function in h u m a n cadaveric renal transplantation remains a significant problem despite improvements in organ preservation solutions, l~ The frequency of delayed graft function varies from center to center; however, the United Network for Organ Sharing (UNOS) reports an incidence of 30% nationwide, l! Not only does delayed graft function result in an early increase in the direct cost of transplantation because of prolonged hospitalization and the need for dialysis, but it is also associated with a higher incidence of acute rejection, poorer long-term graft survival, and higher long-term patient mortality, a2 Therefore any strategy that decreases the incidence of this complication should decrease the cost of transplantation and improve graft and patient survival rates. The injury induced by renal procurement and preservation is similar to the injury seen with acute ischemia.13 Ischemic renal injury is characterized by damage to the most distal ($3) segment of the proximal tubule and the medullary thick ascending limb of the loop of
Henle. 14 The ability of tubular cells to regenerate and reline the damaged n e p h r o n segments determines the degree of recovery after injury. 1'~ Because of their potential to act as growth-promoting agents for renal tissue, pharmacologic roles for polypeptide growth factors have been proposed in the setting of acute kidney failure. 15 IGF-I is a proinsulin-like peptide and is the major growth h o r m o n e dependent growth factor, l~ The kidney both produces and responds to IGF-I. 16 In the kidney, receptors for IGF-I are located in glomeruli and on the basolateral membranes of the proximal tubular cells. Although IGF-I regulation is incompletely understood, IGF-I production is increased in certain pathologic conditions including compensatory renal hypertrophy, experimental diabetes mellitus, and renal infarction. 16 Most important, however, is that renal IGF-I production increases during the regenerative process that follows ischemic renal injury. 17 The ability of IGF-I to attenuate renal ischemic injury has been previously d o c u m e n t e d in our laboratory. '~ When IGF-I is administered to rats immediately after renal ischemia and reperfusion, there is a marked attenuation of the injury. Renal function as assessed by inulin clearance was 100% greater in treated rats at 48 hours after injury, and serum creatinine level was statistically significantly lower each day for 1 week after injury in treated animals. 3 Furthermore, there was a marked decrease in mortality (7% versus 37%) in the treated animals. The renal histologic characteristics of the treated animals 1 week 'after injury were nearly normal, whereas those of controls showed marked injury. :~These findings have subsequently been confirmed by others.5. 18 Further studies from our laboratory have used IGF-I prophylactically by administering it 24 hours before ischemic injury. Once again, there was a marked attenuation in the severity of injury as determined by renal function and histologic characteristics. The observation that IGF-I is efficacious when it is administered either before or after injury may be particularly important in the setting of h u m a n cadaveric renal transplantation. In this setting the organ can be injured before procurement or during cold storage or reperfusion. Renal autotransplantation in dogs is a model commonly used to study the influence of therapeutic interventions on the severity of delayed graft function.19 Cold storage of kidneys in this model has been associated with significant renal injury with a subsequent sixfold to eightfold increase in serum creatinine level. 19-21 Because IGF-I had proved so effective in the rat renal ischemia model, it seemed reasonable that it would be efficacious in attenuating delayed graft function in this model. Fortunately for our purposes, canine kidneys are responsive to h u m a n recombinant IGF-I (the peptide
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c u r r e n t l y available).17 T h e p u r p o s e o f this s t u d y was to d e t e r m i n e w h e t h e r IGF-I w o u l d a t t e n u a t e t h e a c u t e renal injury associated with cold storage and autotransp l a n t a t i o n in t h e c a n i n e m o d e l . I n this s t u d y we d o c u m e n t e d i n a p r o s p e c t i v e b l i n d e d f a s h i o n t h a t IGF-I a t t e n u a t e d t h e r e n a l i n j u r y a s s o c i a t e d with 24 h o u r s o f c o l d s t o r a g e in a c a n i n e m o d e l o f r e n a l a u t o t r a n s p l a n t a t i o n . T h i s was e v i d e n c e d by t h e statistically significantly l o w e r s e r u m c r e a t i n i n e levels (3.5 + 0.3 versus 6.9 -+ 1.9 m g / d l ) (p < 0.05) a n d B U N levels (68 -+ 4 v e r s u s 101 -+ 15 m g / d l ) i n t h e t r e a t m e n t g r o u p ( T a b l e ) . Also, a t 6 days a f t e r t r a n s p l a n t a t i o n r e n a l f u n c t i o n as d e t e r m i n e d by i n u l i n c l e a r a n c e was statistically g r e a t e r (1.37 -+ 0.16 versus 0.77 -+ 0.13 m l / m i n / k g ) i n t h e t r e a t m e n t g r o u p (p < 0.05) ( T a b l e ) . A l t h o u g h r e n a l f u n c t i o n was c o n v i n c i n g l y i m p r o v e d in t h e t r e a t m e n t group, the histologic studies showed only a trend toward i m p r o v e d h i s t o l o g i c characteristics. I n o u r p r e v i o u s r a t studies t h e h i s t o l o g i c d a t a were m o r e c l e a r cut. However, t h e fact r e m a i n s t h a t r e n a l f u n c t i o n was b e t t e r in t h e t r e a t m e n t g r o u p u p to 6 days a f t e r a u t o t r a n s p l a n t a tion. T h i s r e p o r t is t h e first to d o c u m e n t t h a t t h e a d d i t i o n o f IGF-I to o r g a n p r e s e r v a t i o n fluid c a n a t t e n u a t e delayed graft function. T h e m e c h a n i s m by w h i c h IGF-I a t t e n u a t e d t h e c o u r s e o f d e l a y e d g r a f t f u n c t i o n was n o t d e l i n e a t e d in this study. B e c a u s e k i d n e y s w e r e n o t p e r f u s e d b e f o r e transp l a n t a t i o n , r e c i p i e n t s m a y h a v e r e c e i v e d a small b o l u s o f IGF-I. A systemic effect o f IGF-I is u n l i k e l y b u t c a n n o t b e excluded. T h e s e d a t a p r o v i d e p r e l i m i n a r y e v i d e n c e t h a t IGF-I m a y b e a p p l i c a b l e to a c u t e r e n a l i n j u r y in c a d a v e r i c ren a l t r a n s p l a n t a t i o n . IGF-I h a s b e e n safely a d m i n i s t e r e d to h u m a n b e i n g s with n o r m a l a n d r e d u c e d r e n a l f u n c t i o n w i t h o u t a n y s i g n i f i c a n t a d v e r s e s e q u e l a e . ~2 A h u m a n trial o f IGF-I in c a d a v e r i c r e n a l t r a n s p l a n t a t i o n o r in a n a c u t e r e n a l i n j u r y s e t t i n g ( s u p r a r e n a l c r o s s - c l a m p ) is t h e n e x t step in d e t e r m i n i n g its efficacy.
REFERENCES 1. Hammerman MR, Miller SB. Therapeutic use of growth thctors in renal failure. J Am Soc Nephrol 1994;5:1-11. 2. Humes HD, Liu S. Celhdar and molecular basis of renal repair in acute renal failure. J Lab Clin Med 1994;124:749-54. 3. Miller SB, Martin DR, Kissane J, Hammerman MR. Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. Proc Natl Acad Sci USA 1992;89:11876-80. 4. Miller SB, Martin Dr, KissaneJ, Hammerman MR. Rat models for the clinical use of insulin-like growth factor I in acute renal failure. Am J Physiol 1994;266:F949-56. 5. Ding H, Kopple JD, Cohen A, Hirschberg R. Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure. J Clin Invest 1993;91:2281-7. 6. Lim EC, Terasaki PI. Early graft function. In: Terasaki PI, ed. Clinical transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1991:401-7. 7. Rosenthal JT, Danovitch GM, Wilkinson A, Ettenger RB. The
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high cost of delayed graft function in cadaveric renal transplantations. Transplantation 1991;51:1115-39. 8. Miller SB, Martin DR, Kissane J, Hammerman MR. Hepatocyte growth factor accelerates recovery from acute renal injury in rats. Am J Physiol 1994;266:F129-34. 9. Dunnett CW. A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 1955;50:1096121. 10. Ploeg RJ, van Bockel JH, Langendijk PTH, et al. Effect of preservation solution on results of cadaveric kidney transplantation. Lancet 1992;340:129-37. 11. Pierce GA. Facts about transplantation in the United States. UNOS Update 1993;9:35-8. 12. Koyama H, CeckaJM. Race effects. In: Terasaki PI, ed: Clinical transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1991; 269-80. 13. Olsen S, BurdickJF, Keown PA, et al. Primary acute renal failure ("acute tubular necrosis") in the transplanted kidney: morphology and pathogenesis. Medicine 1989;68:173-87. 14. Brezis M, Rosen S, Silva P, Epstein FH. Renal ischemia: a new perspective. Kidney Int 1984;26:375-83. 15. Toback GF. Regeneration after acute tubular necrosis. Kidney Int 1992;41:226-46. 16. Hammerman MR, Miller SB. The growth hormone insulin-like growth factor axis in kidney revisited. AmJ Physiol 1993;265:F114. 17. Matejka GL, Jennische E. IGF-1 mRNA expression in the post-ischemic regenerating kidney. Kidney Int 1992;42:111~23. 18. Noguchi S, Kachihara Y, Ikegami Y, et al. Insulin-like growth factor I ameliorates transient ischemia-induced acute renal failure in rats. J Pharmacol Exp Ther 1993;267:919-26. 19. Ploeg RJ, Gossens D, McAnultyJF, et al. Successful 72-hour cold storage of dog kidneys with UW solution. Transplantation 1988;46:191-6. 20. Belzer FO, Ashby BS, DunphyJE. 24-hour and 72-hour preservation of canine kidneys. Lancet 1967;2:536-9. 21. Collins GM, Brovo-Shugarman M, Terasaki, PI. Kidney preservation for transportation. Lancet 1969;1:1219-23. 22. O'Shea MH, Miller SB, Hammerman MR. Effects of IGF-I on renal function in patients with chronic renal failure. Am.l Physiol 1993;264:F917-22.
DISCUSSION Dr. Mitchell H. G o l d m a n (Knoxville, Tenn.). The BUN and creatinine levels do not seem to fall until day 4 or 5. What is the IGF doing up to that time? Do you have data on how long the IGF remains in the kidney? Does it lock o n to receptors during the reperfusion phase? I was a little curious as to why you stopped at 6 days. Does the effect last longer? We know that you can preserve kidneys for 48 to 72 hours, and ultimately they will regain almost normal function. Would both curves have joined together at 10 days or 12 days further down the road? Along the lines of the length of preservation time, what would be the effect at a 48-hour or 72-hour preserved kidney? You did answer a little bit a b o u t the mechanism of action in terms of the histologic characteristics and the proximal tubule. Do you have any functional information as to how those particular sections of the kidney were working, by using some uptake experiments, for instance? Dr. Petrinec. In terms of the mechanism of action, we believe that the effect of IGF-1 occurs with reperfusion. There are clearly defined receptors in the basolateral m e m b r a n e s of the
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proximal tubules that take up the IGF-1. We suspect that they are not very active at 4 ~ C and that with reperfusion the IGF stimulates the receptor activity. We have not really examined the exact mechanisms involved; we suspect it is a combination of all the ones that we mentioned, occurring on reperfusion of the kidney and activation of the IGF-1 receptor. In terms of the length of storage of the kidneys, our whole goal was to actually induce an ischemic injury; that is why we used the Euro-Collins solution and did not use any systemic heparin. We found that when we originally let these dogs go out to 48 hours, many of the vehicle-treated dogs were lost to acute kidney failure before the end of the study. We shortened storage time to 24 hours to be able to have histologic studies and inulin clearances on all the animals. Finally, in regard to how long the IGF-1 lasts, I suspect that after a single dose circulating levels are only around for about 4 hours, which I believe is the half-life in human beings. After the initial saturation of the IGF-1 receptors, ongoing levels of renal IGF-1 are unknown. Dr. Mark L. Jordan (Pittsburgh, Pa.). University of Wisconsin solution obviously may have certain advantages over EuroCollins; as you know, that has been shown in the canine model as well. Have you looked at whether IGF might add to Univer-
Surgery August 1996 sity of Wisconsin solution as far as its preservation effects in your model? Dr. Petrinec. We have not looked at that specifically. That is something we are actually looking at now to do, and I think that would be a good experiment to do. But originally we were designing it to actually induce an ischemic injury rather than to see how much we can prolong the preservation time. Dr. Mitchell L. Henry (Columbus, Ohio). You noted that IGF was hemodynamically active, and I wonder whether the effects that you are seeing are simply as a vasodilator. Did you measure renal resistance at the time of unclamping of these kidneys? One might be able to reproduce the exact same thing with a simple vasodilator, resulting in better reperfusion and less damage at the time of the operation. Dr. Petrinec. That is a very good point. No, we did not measure the actual vascular resistance, although as an observation we did notice that after reperfusion the kidneys that were treated with IGF-1 did not show a vasoconstrictive effect that we often saw with reperfusion of the vehicle-treated controls. In addition, IGF-1 does have some effects on the heart as well, although we suspect not at the doses that we use, but we did not really control for systemic hemodynamic effects or did not specifically measure the resistances in the kidney.
POLYPEPTIDE GROWTH FA(~ORS regulate kidney development, growth, and function and participate in processes o f repair after renal injury, l' 2 Recently the use o f o n e o f these factors, insulin-like growth factor-I (IGF-I), has b e e n shown to accelerate recovery from acute ischemic kidney failure when administered at the time o f injury, 24 hours after the injury, when kidney failure is "established," or before injury in a prophylactic m a n n e r . 3' 4 T h e mechanisms by which IGF-I acts in acute kidney failure include stimulation o f anabolism, the mainten a n c e o f g i o m e r u l a r filtration, and the e n h a n c e m e n t o f tubular regeneration. 5 An acute renal injury is frequently e n c o u n t e r e d in the setting o f kidney procureSupported in part by a grant from the MissouriKidneyProgram. S.B.M. is supported by a Clinician-ScientistAward from the American Heart Association. Presented at the Fifty-seventhAnnual Meeting of the Societyof University Surgeons, Washington, D.C., Feb. 8-10, 1996. Reprint requests: Steven B. Miller, MD, Washington UniversitySchool of Medicine, 660 S. Euclid Avenue, Box 8126, St. Louis, MO 63110. Copyright 9 1996 by Mosby-YearBook, Inc. 0039-6060/96/$5.00 + 0 11/6/73849
ment, storage, and revascularization for cadaveric transplantation. In fact, approximately 30% of all recipients o f cadaveric renal allografts suffer from acute injury severe e n o u g h to require dialysis after transplantation. 6 This condition is referred to as delayed graft function a n d is a major complication o f cadaveric transplantation. Patients suffering delayed graft function have longer a n d m o r e expensive hospitalization, an increased incidence of rejection, a n d a significantly lower 1-year graft survival rate. 7 Because o f the potential use o f IGF-I as a p h a r m a c o logic agent to treat acute renal injury, we carried o u t studies to d e t e r m i n e w h e t h e r IGF-I might ameliorate the course o f delayed graft function in a canine renal autotransplantation model. T h e data show that perfusion and cold storage o f kidneys in preservation solution s u p p l e m e n t e d with IGF-I hasten the recovery o f renal function a n d ameliorate the course of delayed graft function. O u r findings establish the potential for the use of IGF-I as a therapeutic agent for the prevention o f delayed graft function in h u m a n cadaveric renal transplantation. SURGERY
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MATERIAL AND METHODS Operative procedure. The experimental protocol was approved by the Animal Studies Committee of Washington University School of Medicine and was performed in accordance with their guidelines. Female mongrel dogs weighing 25 to 35 kg were obtained 72 hours before initiation into the study and allowed to acclimate to the animal facility. On day 0 the dogs received premedication with atropine 0.04 m g / k g and acepromazine 0.05 m g / k g , and anesthesia was induced with pentothal 18 m g / k g and continued with a combination of isoflurane (0.4% to 1.5%) and oxygen. Respiration was maintained through an endotracheal tube with mechanical ventilation. Fluid balance was maintained with lactated Ringer's solution at 10 m l / k g / m i n during the first hour and 5 m l / k g / m i n thereafter. A broad-spectrum antibiotic (cefazolin 1 gm) was administered for prophylaxis before laparotomy incision. The dogs did not receive any anticoagulation. Under sterile conditions a left nephroureterectomy was performed by using a median laparotomy. The renal artery was cannulated within 2 minutes of excision, and the organ was flush perfused (gravity perfusion from a height of 100 cm) with 350 ml perfusate at 4 ~ C. The perfusate was EuroCollins solution supplemented with either an acetic acid vehicle (n--11) or IGF-I ( n = 8 ) at a concentration of 10 -7 m o l / L (generously provided by Genentech, Inc., South San Francisco, Calif.). After flush perfusion the organs were stored in the same supplemented EuroCollins solution for 24 hours at 4 ~ C. Assignment of the preservation solution was random, and the team involved in the pertbrmance of the surgery and the care of the animal were blinded. On day 1, at 24 hours "alter harvest a second operation was performed. Under similar conditions the dog underwent a contralateral nephrectomy with autotransplantation of the preserved kidney. The kidney was placed in the pelvis with end-toside anastomoses of the renal vessels to the c o m m o n iiiac vessels. The ureter was anastomosed to the bladder in standard fashion. Before reperfusion of the transplanted kidney, furosemide 2 m g / k g and mannitol 0.5 g m / k g were administered. All subjects received the same amount of intravenous hydration per kilogram of body weight during the operative procedure. Before the operation animals were allowed free access to water and standard laboratory food. Measurement of renal function. Daily serum creatinine and blood urea nitrogen (BUN) levels were obtained from the day of the initial operation (day 0) until death on day 6. Creatinine and BUN levels were measured in plasma by a Cobas Mira Autoanalyser (Roche Diagnostic Systems, Inc., Brancbburg, N.J.). Glomerular filtration rate was determined on the day of death by the clearance of exogenous inulin. The dog was placed in a sling and venous, arterial, and bladder
Surgery August 1996 catheters were placed. A loading dose of inulin 30 m g / k g (Cypros Pharmaceuticals, Carlsbad, Calif.) was administered during a period of 5 minutes, followed by a constant infusion at 400 m g / h r . After a 60 minute equilibration period three consecutive 60-minute collections of urine and blood were obtained for estimation of inulin clearance. Inulin concentration in blood and urine was measured by means of standard techniques as before. 3, 4 Clearance rates were calculated and expressed per kilogram o f body weight. Mean body weight was similar between the two groups and did not change substantially during the course of the experiment. Data reflect the mean of three calculated clearances per dog. Histopathologic assessment. All transplanted kidneys were removed at the time of death. The kidneys were placed in phosphate-buffered formalin for fixation. They were then paraffin embedded and sliced at 5 pm. Slides were stained with hematoxylin-eosin (H&E) and periodic acid/Schiff (PAS). A pathologist blinded to the treatments examined and graded all the specimens by using a scoring system that considered the pathologic changes consistent with ischemic injury and recovery as described previously. 3's The pathologic scoring system grades four features consistent with acute tubular necrosis and recovery: epithelial calcification, tubular dilatation, proximal tubular papillary proliferation, and interstitial infiltration. Twelve microscopic fields per section were chosen randomly to be scored for each parameter. The details of the pathologic scoring system are as tbllows: (1) epithelial calcification; (0) no epithelial calcification, (i) 1 to 4 fields with epithelial calcification, (ii) 5 to 8 fields with epithelial calcifications, (iii) 9 to 12 fields with epithelial calcifications; (2) tubular dilatation: (0) normal by H&E and PAS, (i) suggestive tubular dilatation by H&E with partial loss of PAS staining along the brush border; (ii) unequivocal tubular dilatation by H&E with widespread loss of PAS staining along the brush border, (iii) unequivocal tubular dilatation by H&E with loss of tubular specificity and total absence of PAS staining along the brush border; (3) proximal tubular papillary proliferation: (0) no proliferation; (i) 1 to 4 fields with proliferative changes, (ii) 5 to 8 fields with proliferative changes, (iii) 9 to 12 fields with proliferative changes; (4) interstitial infiltration: (0) no infiltration, (i) 1 to 4 fields with an infiltrate, (ii) 5 to 8 fields with an infiltrate, (iii) 9 to 12 fields with an infiltrate. The total score for each kidney examined was divided by 4, to give a final score between 0 and 3. Statistics. Levels of BUN and creatinine were compared by Dunnett's multiple comparisons test. 9 Inulin clearance rates and pathologic scores in the two groups were compared by two-tailed Student's t test. Differences were considered significant if p < 0.05.
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12 ,,, 10 z...
O
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Fig. 1. Mean levels of creatinine in animals that received kidney autografts preserved in Euro-Collins supplemented with vehicle (Q)) (n = 11) or Euro-Collins supplemented with IGF-1 ([---]) (n = 8). *=p < 0.05.
120
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Fig. 2. Mean levels of BUN in animals that received kidney autografts preserved in Euro-Collins supplemented with vehicle (Q) or Euro-Collins supplemented with IGF-1 (['-']). *p = <0.05.
RESULTS
Unilateral nephrectomy resulted in a small increase in the serum creatinine level between days 0 and 1 in both the vehicle and IGF-I treated animals, as shown in Fig. 1. In contrast, the contralateral nephrectomy and transplantation of the stored kidney on day 1 of the protocol resulted in a significant injury. In animals that received kidneys stored in preservation solution supplemented with the vehicle, the mean serum creatinine level increased to a peak of 7.23 - 1.24 m g / d l on day 5 of the protocol and only came down to 6.90 -+ 1.58 m g / d l at the time of death on day 6. Animals that received a kidney perfused and stored with preservation solution supplemented with IGF-I exhibited less functional renal injury, with the mean serum creatinine level peaking on day 4 of the protocol at 5.56 +- 0.64 mg/dl. At the time of death on day 6 of the protocol the mean serum creatinine level had decreased to 3.53-+ 0.34
mg/dl. The day 6 creatinine levels for the IGF-I treated animals were statistically lower when c o m p a r e d with the levels obtained from the vehicle-treated animals (p < 0.05). A similar finding was observed when examining the daily BUN levels from animals receiving kidneys stored in preservation solution supplemented with the vehicle or IGF-I, as shown in Fig. 2. The levels of BUN did not increase after unilateral nephrectomy between days 0 and 1 of the protocol. However, after contralateral nephrectomy and transplantation of the stored kidney, there was a dramatic rise in levels of BUN, especially in the animals receiving kidneys stored with the vehicle. In the vehicle group the BUN level was still rising on the final day of the protocol and had reached a level of 100.9-+ 14.9 mg/dl. Animals receiving kidneys preserved with supplemental IGF-I became less uremic, and the BUN levels peaked on day 4 of the protocol at
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Table. Renal function at day 6 as determined by levels of creatinine
Creatinine (mg/dl)
BUN (mg/dl)
E-C + veh 6.9_+1.9 101_+15 E-C + IGF-1 3.5 -4-0.3 68 -+ 4 p Value <0.05 <0.05
Inulin clearance (ml/min/kg body weight) 0.77-+0.13 1.37 -+ 0.16 <0.05
Values are mean -+ SEM. E-C + veh, Euro-Collins supplemented with vehicle; E-C + IGF-1, Euro-Collins supplemented with IGF-I.
79.8 _+ 7.0 mg/dl. The BUN levels were statistically lower in the IGF-I group when compared with the vehicle group on days 5 and 6 of the protocol (p < 0.05). To ascertain whether the changes in levels of creatinine and BUN were reflective of changes in glomerular filtration, we measured inulin clearance on day 6 before death. The inulin clearance rate was nearly twice as high in the IGF-I treated dogs compared with the animals that received the vehicle, 1.37 -+ 0.16 versus 0.77 + 0.13 m l / m i n / k g , respectively (p < 0.05) (Table). Morphologic analysis was performed on all transplanted kidneys harvested at the time o f death. Kidneys stored in solution supplemented with IGF-I had a score of 1.4 -+ 0.4. The pathologic score obtained from grading the kidneys stored in solution supplemented with the vehicle was 1.9 -+ 0.5. These scores were not significantly different.
DISCUSSION This study shows in a canine model of renal autotransplantation that the addition of IGF-I to Euro-Collins solution attenuates renal autograft dysfunction. This finding is of potential clinical significance because delayed graft function in h u m a n cadaveric renal transplantation remains a significant problem despite improvements in organ preservation solutions, l~ The frequency of delayed graft function varies from center to center; however, the United Network for Organ Sharing (UNOS) reports an incidence of 30% nationwide, l! Not only does delayed graft function result in an early increase in the direct cost of transplantation because of prolonged hospitalization and the need for dialysis, but it is also associated with a higher incidence of acute rejection, poorer long-term graft survival, and higher long-term patient mortality, a2 Therefore any strategy that decreases the incidence of this complication should decrease the cost of transplantation and improve graft and patient survival rates. The injury induced by renal procurement and preservation is similar to the injury seen with acute ischemia.13 Ischemic renal injury is characterized by damage to the most distal ($3) segment of the proximal tubule and the medullary thick ascending limb of the loop of
Henle. 14 The ability of tubular cells to regenerate and reline the damaged n e p h r o n segments determines the degree of recovery after injury. 1'~ Because of their potential to act as growth-promoting agents for renal tissue, pharmacologic roles for polypeptide growth factors have been proposed in the setting of acute kidney failure. 15 IGF-I is a proinsulin-like peptide and is the major growth h o r m o n e dependent growth factor, l~ The kidney both produces and responds to IGF-I. 16 In the kidney, receptors for IGF-I are located in glomeruli and on the basolateral membranes of the proximal tubular cells. Although IGF-I regulation is incompletely understood, IGF-I production is increased in certain pathologic conditions including compensatory renal hypertrophy, experimental diabetes mellitus, and renal infarction. 16 Most important, however, is that renal IGF-I production increases during the regenerative process that follows ischemic renal injury. 17 The ability of IGF-I to attenuate renal ischemic injury has been previously d o c u m e n t e d in our laboratory. '~ When IGF-I is administered to rats immediately after renal ischemia and reperfusion, there is a marked attenuation of the injury. Renal function as assessed by inulin clearance was 100% greater in treated rats at 48 hours after injury, and serum creatinine level was statistically significantly lower each day for 1 week after injury in treated animals. 3 Furthermore, there was a marked decrease in mortality (7% versus 37%) in the treated animals. The renal histologic characteristics of the treated animals 1 week 'after injury were nearly normal, whereas those of controls showed marked injury. :~These findings have subsequently been confirmed by others.5. 18 Further studies from our laboratory have used IGF-I prophylactically by administering it 24 hours before ischemic injury. Once again, there was a marked attenuation in the severity of injury as determined by renal function and histologic characteristics. The observation that IGF-I is efficacious when it is administered either before or after injury may be particularly important in the setting of h u m a n cadaveric renal transplantation. In this setting the organ can be injured before procurement or during cold storage or reperfusion. Renal autotransplantation in dogs is a model commonly used to study the influence of therapeutic interventions on the severity of delayed graft function.19 Cold storage of kidneys in this model has been associated with significant renal injury with a subsequent sixfold to eightfold increase in serum creatinine level. 19-21 Because IGF-I had proved so effective in the rat renal ischemia model, it seemed reasonable that it would be efficacious in attenuating delayed graft function in this model. Fortunately for our purposes, canine kidneys are responsive to h u m a n recombinant IGF-I (the peptide
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c u r r e n t l y available).17 T h e p u r p o s e o f this s t u d y was to d e t e r m i n e w h e t h e r IGF-I w o u l d a t t e n u a t e t h e a c u t e renal injury associated with cold storage and autotransp l a n t a t i o n in t h e c a n i n e m o d e l . I n this s t u d y we d o c u m e n t e d i n a p r o s p e c t i v e b l i n d e d f a s h i o n t h a t IGF-I a t t e n u a t e d t h e r e n a l i n j u r y a s s o c i a t e d with 24 h o u r s o f c o l d s t o r a g e in a c a n i n e m o d e l o f r e n a l a u t o t r a n s p l a n t a t i o n . T h i s was e v i d e n c e d by t h e statistically significantly l o w e r s e r u m c r e a t i n i n e levels (3.5 + 0.3 versus 6.9 -+ 1.9 m g / d l ) (p < 0.05) a n d B U N levels (68 -+ 4 v e r s u s 101 -+ 15 m g / d l ) i n t h e t r e a t m e n t g r o u p ( T a b l e ) . Also, a t 6 days a f t e r t r a n s p l a n t a t i o n r e n a l f u n c t i o n as d e t e r m i n e d by i n u l i n c l e a r a n c e was statistically g r e a t e r (1.37 -+ 0.16 versus 0.77 -+ 0.13 m l / m i n / k g ) i n t h e t r e a t m e n t g r o u p (p < 0.05) ( T a b l e ) . A l t h o u g h r e n a l f u n c t i o n was c o n v i n c i n g l y i m p r o v e d in t h e t r e a t m e n t group, the histologic studies showed only a trend toward i m p r o v e d h i s t o l o g i c characteristics. I n o u r p r e v i o u s r a t studies t h e h i s t o l o g i c d a t a were m o r e c l e a r cut. However, t h e fact r e m a i n s t h a t r e n a l f u n c t i o n was b e t t e r in t h e t r e a t m e n t g r o u p u p to 6 days a f t e r a u t o t r a n s p l a n t a tion. T h i s r e p o r t is t h e first to d o c u m e n t t h a t t h e a d d i t i o n o f IGF-I to o r g a n p r e s e r v a t i o n fluid c a n a t t e n u a t e delayed graft function. T h e m e c h a n i s m by w h i c h IGF-I a t t e n u a t e d t h e c o u r s e o f d e l a y e d g r a f t f u n c t i o n was n o t d e l i n e a t e d in this study. B e c a u s e k i d n e y s w e r e n o t p e r f u s e d b e f o r e transp l a n t a t i o n , r e c i p i e n t s m a y h a v e r e c e i v e d a small b o l u s o f IGF-I. A systemic effect o f IGF-I is u n l i k e l y b u t c a n n o t b e excluded. T h e s e d a t a p r o v i d e p r e l i m i n a r y e v i d e n c e t h a t IGF-I m a y b e a p p l i c a b l e to a c u t e r e n a l i n j u r y in c a d a v e r i c ren a l t r a n s p l a n t a t i o n . IGF-I h a s b e e n safely a d m i n i s t e r e d to h u m a n b e i n g s with n o r m a l a n d r e d u c e d r e n a l f u n c t i o n w i t h o u t a n y s i g n i f i c a n t a d v e r s e s e q u e l a e . ~2 A h u m a n trial o f IGF-I in c a d a v e r i c r e n a l t r a n s p l a n t a t i o n o r in a n a c u t e r e n a l i n j u r y s e t t i n g ( s u p r a r e n a l c r o s s - c l a m p ) is t h e n e x t step in d e t e r m i n i n g its efficacy.
REFERENCES 1. Hammerman MR, Miller SB. Therapeutic use of growth thctors in renal failure. J Am Soc Nephrol 1994;5:1-11. 2. Humes HD, Liu S. Celhdar and molecular basis of renal repair in acute renal failure. J Lab Clin Med 1994;124:749-54. 3. Miller SB, Martin DR, Kissane J, Hammerman MR. Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. Proc Natl Acad Sci USA 1992;89:11876-80. 4. Miller SB, Martin Dr, KissaneJ, Hammerman MR. Rat models for the clinical use of insulin-like growth factor I in acute renal failure. Am J Physiol 1994;266:F949-56. 5. Ding H, Kopple JD, Cohen A, Hirschberg R. Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure. J Clin Invest 1993;91:2281-7. 6. Lim EC, Terasaki PI. Early graft function. In: Terasaki PI, ed. Clinical transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1991:401-7. 7. Rosenthal JT, Danovitch GM, Wilkinson A, Ettenger RB. The
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high cost of delayed graft function in cadaveric renal transplantations. Transplantation 1991;51:1115-39. 8. Miller SB, Martin DR, Kissane J, Hammerman MR. Hepatocyte growth factor accelerates recovery from acute renal injury in rats. Am J Physiol 1994;266:F129-34. 9. Dunnett CW. A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 1955;50:1096121. 10. Ploeg RJ, van Bockel JH, Langendijk PTH, et al. Effect of preservation solution on results of cadaveric kidney transplantation. Lancet 1992;340:129-37. 11. Pierce GA. Facts about transplantation in the United States. UNOS Update 1993;9:35-8. 12. Koyama H, CeckaJM. Race effects. In: Terasaki PI, ed: Clinical transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1991; 269-80. 13. Olsen S, BurdickJF, Keown PA, et al. Primary acute renal failure ("acute tubular necrosis") in the transplanted kidney: morphology and pathogenesis. Medicine 1989;68:173-87. 14. Brezis M, Rosen S, Silva P, Epstein FH. Renal ischemia: a new perspective. Kidney Int 1984;26:375-83. 15. Toback GF. Regeneration after acute tubular necrosis. Kidney Int 1992;41:226-46. 16. Hammerman MR, Miller SB. The growth hormone insulin-like growth factor axis in kidney revisited. AmJ Physiol 1993;265:F114. 17. Matejka GL, Jennische E. IGF-1 mRNA expression in the post-ischemic regenerating kidney. Kidney Int 1992;42:111~23. 18. Noguchi S, Kachihara Y, Ikegami Y, et al. Insulin-like growth factor I ameliorates transient ischemia-induced acute renal failure in rats. J Pharmacol Exp Ther 1993;267:919-26. 19. Ploeg RJ, Gossens D, McAnultyJF, et al. Successful 72-hour cold storage of dog kidneys with UW solution. Transplantation 1988;46:191-6. 20. Belzer FO, Ashby BS, DunphyJE. 24-hour and 72-hour preservation of canine kidneys. Lancet 1967;2:536-9. 21. Collins GM, Brovo-Shugarman M, Terasaki, PI. Kidney preservation for transportation. Lancet 1969;1:1219-23. 22. O'Shea MH, Miller SB, Hammerman MR. Effects of IGF-I on renal function in patients with chronic renal failure. Am.l Physiol 1993;264:F917-22.
DISCUSSION Dr. Mitchell H. G o l d m a n (Knoxville, Tenn.). The BUN and creatinine levels do not seem to fall until day 4 or 5. What is the IGF doing up to that time? Do you have data on how long the IGF remains in the kidney? Does it lock o n to receptors during the reperfusion phase? I was a little curious as to why you stopped at 6 days. Does the effect last longer? We know that you can preserve kidneys for 48 to 72 hours, and ultimately they will regain almost normal function. Would both curves have joined together at 10 days or 12 days further down the road? Along the lines of the length of preservation time, what would be the effect at a 48-hour or 72-hour preserved kidney? You did answer a little bit a b o u t the mechanism of action in terms of the histologic characteristics and the proximal tubule. Do you have any functional information as to how those particular sections of the kidney were working, by using some uptake experiments, for instance? Dr. Petrinec. In terms of the mechanism of action, we believe that the effect of IGF-1 occurs with reperfusion. There are clearly defined receptors in the basolateral m e m b r a n e s of the
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proximal tubules that take up the IGF-1. We suspect that they are not very active at 4 ~ C and that with reperfusion the IGF stimulates the receptor activity. We have not really examined the exact mechanisms involved; we suspect it is a combination of all the ones that we mentioned, occurring on reperfusion of the kidney and activation of the IGF-1 receptor. In terms of the length of storage of the kidneys, our whole goal was to actually induce an ischemic injury; that is why we used the Euro-Collins solution and did not use any systemic heparin. We found that when we originally let these dogs go out to 48 hours, many of the vehicle-treated dogs were lost to acute kidney failure before the end of the study. We shortened storage time to 24 hours to be able to have histologic studies and inulin clearances on all the animals. Finally, in regard to how long the IGF-1 lasts, I suspect that after a single dose circulating levels are only around for about 4 hours, which I believe is the half-life in human beings. After the initial saturation of the IGF-1 receptors, ongoing levels of renal IGF-1 are unknown. Dr. Mark L. Jordan (Pittsburgh, Pa.). University of Wisconsin solution obviously may have certain advantages over EuroCollins; as you know, that has been shown in the canine model as well. Have you looked at whether IGF might add to Univer-
Surgery August 1996 sity of Wisconsin solution as far as its preservation effects in your model? Dr. Petrinec. We have not looked at that specifically. That is something we are actually looking at now to do, and I think that would be a good experiment to do. But originally we were designing it to actually induce an ischemic injury rather than to see how much we can prolong the preservation time. Dr. Mitchell L. Henry (Columbus, Ohio). You noted that IGF was hemodynamically active, and I wonder whether the effects that you are seeing are simply as a vasodilator. Did you measure renal resistance at the time of unclamping of these kidneys? One might be able to reproduce the exact same thing with a simple vasodilator, resulting in better reperfusion and less damage at the time of the operation. Dr. Petrinec. That is a very good point. No, we did not measure the actual vascular resistance, although as an observation we did notice that after reperfusion the kidneys that were treated with IGF-1 did not show a vasoconstrictive effect that we often saw with reperfusion of the vehicle-treated controls. In addition, IGF-1 does have some effects on the heart as well, although we suspect not at the doses that we use, but we did not really control for systemic hemodynamic effects or did not specifically measure the resistances in the kidney.