Experimental and clinical assessment of preservation-induced reperfusion injury comparing renal transplant blood flow and renal endothelin concentrations

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Experimental and Clinical Assessment of Preservation-Induced Reperfusion Injury Comparing Renal Transplant Blood Flow and Renal Endothelin Concentrations P.N. Bretan, Jr., J. Chang, E. Lobo, 0. Dumitrescu, B. Miller, and T.S.-Benedict Yen

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T HAS BEEN well established that a graded increase in preservation time adversely affects long-term renal allograft survival, as illustrated from cumulative data of 23,000 patients from the UCLA Transplant Registry.’ Of the total preservation and ischemic injuries suffered by the transplant kidney around the time of transplantation, it is hypothesized that the proportion of this injury, described as reperfusion injury, may be reversible, thus great interest has been focused on the mechanisms and pathology of this type of injury. The current working hypothesis of reperfusion injury is that ischemia is sustained from donor conditions, organ removal, or organ preservation. This elicits an acute endothelial injury via the upregulation of DR antigens, and adhesion antigen expression, leading to a susceptibility to subsequent acute rejection. In addition, this “endothelitis” sets up a chronic endothelial injury leading to a secondary response often described as “vessel wall remodeling,” which can be seen histologically with smooth muscle cell proliferation. It is believed that this leads to chronic rejection, loss of function, and hyperfiltration.’ Previously, we studied reperfusion during the first hour after revascularization of a transplanted kidney. Freeze clamping a renal tissue specimen showed equivalent and viable tissue of kidneys after 50 hours of cold storage and at 5 minutes postreperfusion; however, the injury seemed to establish itself in terms of decreased ATP and ADP regeneration after 45 minutes of reperfusion. A mannitol-based preservation solution was shown to prevent such injury compared with a Collins -2 solution.3 Based on simultaneous electron-microscopy studies, it was seen that increased cell adhesiveness seemed to be associated with reperfusion injury leading to thrombosis within the microvasculature of the transplanted kidney and to secondary parenchymal destruction. Our previous post-renal-transplant reperfusion injury studies concentrated on the microvasculature aspects of this process.4 The causes of renal transplant reperfusion injury are numerous and multifactorial. Biochemical and vasoactive substances and their effect on this process have recently become the subject of great debate. One of the most potent vasoconstrictive substances known is endothelin-1, which has 10 times the effect of angiotensin-2. Endothelin-1 is a

21-amino-acid peptide that may be implicated in acute renal failure because of its depressive effects on renal blood flow secondary to increased arterial resistance and mesangial cell contraction.s,6 It remains unclear whether this molecule is a cause or effect of posttransplant reperfusion injury. To further define post-renal-transplant reperfusion injury the following combination of microvascular, biochemical, and macrovascular studies was performed. The purpose of these studies was to develop a unique model for studying renal blood flow, enabling simultaneous collection of renal vein blood specimens from the same transplanted kidney, and also to analyze the significance of endothelin-1 in the determination of reperfusion injury in both animals and humans. METHODS-STUDY

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In this animal surgical model, porcine kidneys were removed and flushed with either UW-1 (N = 11) or Collins-2 (N = 11) solution and cold stored for 72 hours. An in situ native kidney was used as a control. Perfusion cannulas were placed in the aorta and vena cava in the recipient animals, which enabled immediate reperfusion with no anastomotic warm ischemia time. Direct blood specimens were obtained in the corresponding renal vein enabling no systemic dilution or contamination. Renal blood flow measurements and specimens were taken serially up to 90 minutes postreperfusion for endothelin-1 assays7 Histologic injury scores were assessed for all kidneys after 90 minutes. Blood flow was analyzed with a perivascular flow probe that surrounded the aorta proximal to the renal arteries. A Transonic transit time flow probe and animal flowmeter were used. The probe emits a bidirectional ultrasonic wave at angles of 60” to the axis of blood flow in which a tight fit of the artery is not required. The requirement for tight fit was a factor of inaccuracy in the older electromagnetic flow probe devices. These waves are reflected by a bracket on the opposite side of the vessel and transducers of the probe receive and relay the signals to the flowmeter.

From the University of California at San Francisco Renal Transplant Service, San Francisco, California, USA. Address reprint requests to Dr Peter N. Bretan, Jr, University of California, San Francisco, Renal Transplant Service, M884, San Francisco, CA 94143-0116.

0041-1345/97/$17.00 PII so041 -1345(97)01004-x

0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

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Transplantation

Proceedings,

29, 3520-3521

(1997)

PRESERVATION-INDUCEDREPERFUSION INJURY RESULTS

The flow probe was calibrated with absolute blood flow measurements that were taken simultaneously from the renal vein yielding a correlation coefficient of 0.962. The decrease in renal blood flow in the transplanted allograft kidneys compared with control autograft kidneys was significantly greater with kidneys flushed and stored with Collins-2 compared with those using UW-1 (41.75 2 5.69 vs 11.18 ‘I 13.99, P < .OOS). Endothelin concentrations of blood samples taken during the same blood flow measurement showed no significant differences in increases from baseline (19.0 2 3 pg/mL) for UW versus the Collins -2 Histologic group (8.35 2 5.7 vs 3.39 2 1.6 pg/mL, P = .lO). examinations taken from the kidneys after reperfusion showed no significant differences in ischemic injury between the groups. METHODS-STUDY

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Based on the results of the animal studies, clinical human studies with a similar design were performed. IRB approval and informed consent were obtained. Renal vein samples were taken at time intervals similar to those of the animal studies. Core renal transplant biopsies using a Tru-Cut needle were performed at 40 minutes postreperfusion and injury was assessed histologically. Endothelin-1 assays were likewise taken using similar techniques in animal studies. An S-series renal blood flow probe system (Transonic) was used. Again, this was similar to the animal probe. RESULTS

The 5-minute postreperfusion renal blood flow was used as the baseline flow. Reperfusion injury (RPI) was defined as flow not recovered to the baseline value in less than 1 hour after revascularization. A total of nine patients were entered into this study. Biopsies showed no evidence of ischemic injury, indicating that this technique is not sensitive enough to detect reperfusion injury changes clinically. Postreperfusion endothelin levels did not correlate with the postoperative course of patients. However, renal blood flow did. Four of nine patients had defined reperfusion injury by persistent decreases in renal blood flow 1 hour after reperfusion. In these patients, serum creatinine was significantly higher after 1 week posttransplant compared with the other five patients (6.75 + 3.03 vs 2.08 + 1.28 mg/dL, P < .015). RPI patients had more frequent rejections (SO%, N = 2 vs 20%, N = 1) and lower graft survival (75% vs 100%) at 1 year follow-up compared with the non-RPI patients. After observing delayed graft function in the first two patients with reperfusion injury criteria, the subsequent two

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patients were started on sequential therapy based on similar intraoperative blood flow patterns. All four patients who had reperfusion injury were not suspected by any demographic donor data such as cold storage preservation time, terminal serum creatinine, cause of death, or revascularization time. The latter two patients treated with sequential therapy had appreciably more urine output and faster recovery of renal function at 1 week postoperatively. Histologic parameters did not differ in any of these patients. DISCUSSION

Unfortunately, mild-to-moderate injuries could not be determined clinically using biopsies as seen in our study. In addition, there are probably other vasoactive factors more important than endothelin that may have a role in the depression of renal blood flow. Alternatively, reperfusion mechanisms may be completely unrelated to these types of vasoactive factors. In conclusion, Collins-Zpreserved kidneys have significantly more depressed renal blood flow postreperfusion compared with UW-l-preserved kidneys consistent with previous microvascular studies, in the absence of elevated endothelin levels; therefore, vasoactive factors other than endothelin are likely to contribute more appreciably to reperfusion injury. The ultrasonic transit time flow probe accurately measures postreperfusion renal blood flow and offers a practical and noninvasive method for assessing renal reperfusion injury after transplantation. This can help optimize immunosuppressive strategies to maximize renal recovery. REFERENCES 1. Zhou YC, Cecka JM: In Cecka JM, Terasaki PI (eds): Clinical Transplants 1992. Los Angeles, Calif: UCLA Tissue Typing Laboratory; 1992, p 386 2. Alejandro VS, Nelson WJ, Huie P, et al: Kidney Int 48:1308, 1995 3. Bretan PN, Paul G, Sharma J: Transplant Int 7 (suppl 1): S469, 1994 4. Bretan PN, Baldwin N, Martinez A, et al: Urol Res 19:73, 1991 5. Gianello P, Fishbein J, Besse T, et al: Transplant Int 7:11, 1994 6. Yamada K, Gunji Y, Hishikawa E. et al: Transplantation 57:1137, 1994 7. Yanagisawa M, Masaki T: Biochem Pharmacol 38:1877, 1989 8. Transonic Systems, Inc, Product Catalog, Ithaca, NY, 1993, P 77