Organ preserving fluid for protecting vascular endothelium and application thereof

Abstract

The invention provides organ preserving fluid for protecting vascular endothelium and application of the organ preserving fluid, and belongs to the technical field of organ transplantation. The organ preserving fluid for protecting the vascular endothelium comprises sulodexide. A rat heart death donor kidney in-vitro low-temperature oxygen-carrying mechanical perfusion combined kidney transplantation model is adopted, and experiments show that when the organ preserving fluid containing sulodexide is used for conducting low-temperature oxygen-carrying mechanical perfusion on the rat heart death donor kidney, vascular endothelial glycocalyx disengagement and vascular endothelial injury can be remarkably relieved, perfusion parameters are improved, and the survival rate of the rat heart death donor kidney is increased. Vascular endothelial cells and kidney functions after kidney transplantation are remarkably protected, and inflammatory factor levels (TNF-alpha and IL-6) and kidney tissue structure damage are relieved. The organ preserving fluid is used for protecting heart death donor organ vascular endothelium, blood vessel and tissue damage caused by ischemia is effectively relieved, renal fibrosis induced by ischemia reperfusion injury after transplantation is prevented, and the organ preserving fluid has a great clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ transplantation, and particularly relates to an organ preservation solution for protecting vascular endothelium and its application. Background Art

[0002] Renal transplantation remains the best option for patients with end-stage renal disease. However, the transplanted kidney inevitably undergoes ischemia-reperfusion injury, which is an important cause of acute kidney injury after renal transplantation. Acute kidney injury often leads to rapid loss of renal function and is the main harmful factor limiting the success of transplantation.

[0003] Today, with the increasing shortage of organs globally, organs from donors after cardiac death are considered the main method to expand the source of donor organs. However, kidneys from donors after cardiac death experience a long period of warm ischemia injury, resulting in poor organ quality. Severe ischemia-reperfusion injury after transplantation can lead to primary renal non-function and delayed recovery of renal function.

[0004] The main pathophysiological changes after renal ischemia-reperfusion injury include direct cell damage caused by ischemia and delayed damage caused by inflammation after reperfusion, mainly involving vascular endothelial damage and tubular injury. Acute tubular injury will develop into tubular atrophy and renal interstitial fibrosis due to vascular endothelial changes, leading to graft loss.

[0005] Hypothermic oxygenated machine perfusion, as a new organ preservation method, can also be used as a means of in vitro repair and treatment. A large number of clinical studies have proven that hypothermic oxygenated machine perfusion can effectively reduce renal ischemia-reperfusion injury, significantly extend the preservation time of donor kidneys, improve the success rate of transplantation, and reduce the occurrence of serious postoperative complications. In addition, by adding therapeutic drugs to the organ perfusion preservation solution to intervene in specific pathophysiological processes, the quality of donor kidneys can be further repaired and improved. Currently, the commonly used organ perfusion preservation solutions for kidneys in clinical practice mainly include KPS-1 solution, UW solution or HTK solution, which have played a certain role in extending the preservation of donor kidneys, but have limited effects on kidneys from donors after cardiac death with a longer ischemic time.

[0006] The inventors' recent research published in Kidney International, a renowned journal in the field of nephrology (Salvaging donated kidneys from prolonged warm ischemia during ex vivo hypothermic oxygenated perfusion. 2024. 106(2). 273 - 290), found that for kidneys donated after cardiac death, warm ischemia injury leads to severe vascular endothelial injury in the donor kidney, massive thrombosis and vascular occlusion within the blood vessels, exacerbating post - transplantation ischemia - reperfusion injury and resulting in the loss of function and death of the transplanted kidney. Although hypothermic oxygenated mechanical perfusion has been used to repair kidneys with severe vascular endothelial injury in vitro, the post - transplantation effect is still very poor. Apparently, the protection of vascular endothelium and the repair of tubular injury are key steps in the recovery of renal function after transplantation. Unfortunately, there is no organ preservation perfusion fluid targeting the protection of vascular endothelium in clinical practice.

[0007] Therefore, the development of an organ preservation solution that can effectively protect and repair the vascular endothelium of organs is crucial for reducing ischemia - reperfusion injury after kidney transplantation and improving the quality of donor kidneys after cardiac death. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide an organ preservation solution for protecting vascular endothelium and its application. This organ preservation solution can effectively improve the vascular endothelial injury of donor kidneys after cardiac death and ischemia - reperfusion injury after transplantation. When used as an in vitro hypothermic oxygenated mechanical perfusion fluid for donor kidneys after cardiac death, it greatly improves the quality of the donor kidney and the success rate of transplantation.

[0009] To achieve the above - mentioned invention purpose, the present invention provides the following technical solutions:

[0010] The present invention provides an organ preservation solution for protecting vascular endothelium, which includes sulodexide.

[0011] Preferably, the concentration of sulodexide is 10 - 500 LSU / mL.

[0012] Preferably, it further includes KPS - 1 solution, UW solution or HTK solution.

[0013] Preferably, the organ is a donor kidney after cardiac death.

[0014] The present invention also provides the application of the above - mentioned organ preservation solution in repairing kidney injury.

[0015] Preferably, the kidney injury is kidney warm ischemia - reperfusion injury.

[0016] Preferably, the method for the organ preservation solution to repair kidney warm ischemia-reperfusion injury comprises the following steps: placing the organ preservation solution in a low-temperature oxygen-carrying mechanical system to perfuse the kidneys of cardiac death donors.

[0017] Preferably, the temperature of the perfusion is 4-10 °C, and the time of the perfusion is 2-6 h.

[0018] The present invention also provides the application of the organ preservation solution in organ preservation or organ transplantation.

[0019] Preferably, the organ preservation solution reduces the inflammatory level and tissue damage after organ transplantation.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] In the present invention, sulodexide is evenly distributed in the organ preservation solution. By virtue of the characteristics of reconstructing and maintaining the structure and function of the glycocalyx, sulodexide reduces the interaction between leukocytes and endothelial cells after transplantation, reduces the release of pro-inflammatory factors and chemokines, inhibits C-reactive protein, and further inhibits the activation of the complement system, alleviates the inflammatory process, and reduces the damage to the endothelial glycocalyx. It provides a possibility for clinical application, reduces kidney injury, improves the quality of grafts, and alleviates the contradiction between organ supply and demand.

[0022] In the present invention, sulodexide is added to the organ preservation solution to improve the low-temperature mechanical perfusion solution. Among them, sulodexide can promote the production of endothelium-dependent nitric oxide to dilate blood vessels. Nitric oxide protects vascular endothelial cells by exerting anti-inflammatory, vasodilating, inhibiting platelet adhesion and aggregation, preventing leukocytes from adhering to the blood vessel wall, and inhibiting endothelial cell apoptosis, so that the kidneys maintain good and stable perfusion parameters and functional states during ex vivo perfusion.

[0023] The present invention can effectively increase the perfusion flow of the kidneys and reduce the renal resistance, not solely relying on the effect of low-temperature oxygen-carrying mechanical perfusion or the vasodilating effect of sulodexide, but the result of the organic combination of the two to play complementary advantages. Diffuse thrombosis in blood vessels and contraction of vascular endothelial cells after cardiac death are the reasons for hindering the effect of mechanical perfusion. The activation of the production of vascular endothelial nitric oxide by sulodexide can effectively promote the dilation of microcirculation blood vessels, while low-temperature oxygen-carrying mechanical perfusion effectively flushes out the thrombus in the microcirculation blood vessels by simulating the normal fluid flow in blood vessels, enabling the whole organ to obtain effective perfusion and remove metabolic wastes. At the same time, oxygen and sulodexide can be smoothly delivered to the renal tissue, evenly covering the surface of renal vascular endothelium, more effectively restoring the function and vitality of the kidneys from cardiac death donors, and preventing kidney fibrosis induced by ischemia-reperfusion injury after transplantation.

[0024] In summary, adding sulodexide to the organ preservation solution can regulate the vasodilation function of the kidney blood vessels and improve the effective perfusion of various regions of the kidney. Uniformly covering the vascular endothelial surface with sulodexide before transplantation has achieved breakthrough technical effects compared with the current general organ preservation solution, making a substantial contribution to the existing organ preservation and repair technologies for donors after cardiac death. Description of the Drawings

[0025] Figure 1 Shows the effects of sulodexide on the parameters of hypothermic oxygenated mechanical perfusion of kidneys with 30 minutes of warm ischemia (where A is the perfusion flow rate and B is the renal resistance during perfusion);

[0026] Figure 2 Shows that sulodexide reduces vascular injury in kidneys from donors with 30 minutes of warm ischemia (where A is the immunohistochemical staining result of von Willebrand factor (vWF), B is the expression level of the vascular injury marker CD31, C is the immunohistochemical staining result of the vascular injury marker CD31, and D is the expression level of von Willebrand factor (vWF));

[0027] Figure 3 Shows that sulodexide reduces the inflammatory level and tissue damage after transplantation of kidneys from donors with 30 minutes of warm ischemia (where A is the concentration of the inflammatory factor TNF-α, B is the concentration of the inflammatory factor IL-6, C is the HE staining diagram, and D is the tubular injury score);

[0028] Figure 4 Shows the effects of sulodexide on the parameters of hypothermic oxygenated mechanical perfusion of kidneys with 60 minutes of long-term warm ischemia (where A is the perfusion flow rate and B is the renal resistance during perfusion);

[0029] Figure 5 Shows that sulodexide reduces vascular injury in kidneys from donors with 60 minutes of long-term warm ischemia (where A is the immunohistochemical staining result of von Willebrand factor (vWF), B is the expression level of the vascular injury marker CD31, C is the immunohistochemical staining result of the vascular injury marker CD31, and D is the expression level of von Willebrand factor (vWF));

[0030] Figure 6 Shows that sulodexide reduces the inflammatory level and tissue damage after transplantation of kidneys from donors with 60 minutes of long-term warm ischemia (where A is the concentration of the inflammatory factor TNF-α, B is the concentration of the inflammatory factor IL-6, C is the HE staining diagram, and D is the tubular injury score). Detailed Embodiments

[0031] The present invention provides an organ preservation solution for protecting vascular endothelium, which includes sulodexide.

[0032] In the present invention, sulodexide is a highly purified natural glycosaminoglycan extracted from porcine intestinal mucosa, composed of 80% heparin sulfate and 20% dermatan sulfate. Sulodexide has multiple biological effects on the vascular system, including anti-inflammatory, antithrombotic, antifibrotic, and vasoregulatory effects, and can reduce ischemia-reperfusion injury through these effects. Most importantly, the vascular protection function of sulodexide plays a key role in restoring the physiological activity and regulating the function of kidneys from donors after cardiac death. Sulodexide is the only drug currently approved for clinical use to inhibit the activity of heparanase, and its safety and effective concentration have been verified and fully studied, which can also accelerate the clinical translational application of sulodexide. Its composition is similar to that of the glycocalyx on the surface of vascular endothelium, providing a material source for the repair of the glycocalyx. The concentration of the sulodexide is preferably 10 - 500 LSU / mL, more preferably 15 - 400 LSU / mL; the organ preservation solution further includes KPS-1 solution, UW solution, or HTK solution; the organ is preferably a kidney from a cardiac death donor, and the kidney from a cardiac death donor is a kidney with vascular endothelial injury caused by warm ischemia and hypoxia after cardiac arrest.

[0033] The present invention also provides the application of the organ preservation solution in repairing kidney injury.

[0034] In the present invention, the kidney injury is kidney warm ischemia-reperfusion injury; the method for the organ preservation solution to repair kidney warm ischemia-reperfusion injury includes the following steps: placing the organ preservation solution in a low-temperature oxygen-carrying mechanical system to perfuse the kidney from a cardiac death donor; the temperature of the perfusion is preferably 4 - 10 °C, more preferably 4 - 6 °C; the time of the perfusion is preferably 2 - 6 h, more preferably 2 - 3 h.

[0035] The present invention also provides the application of the organ preservation solution in organ preservation or organ transplantation.

[0036] In the present invention, the organ preservation solution reduces the inflammatory level and tissue injury after organ transplantation.

[0037] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Sulodexide was purchased from Alfresa Pharma Italia S.p.A.

[0039] Example 1

[0040] Verification of the effect of the organ preservation solution containing sulodexide in reducing the reperfusion injury of the transplanted kidney with 30 min of warm ischemia

[0041] 1. Experimental animals: Healthy male Sprague-Dawley rats (aged 10 - 11 weeks, weighing 350 - 380 g), purchased from Hubei Provincial Center for Disease Control and Prevention. All animals were housed in a pathogen-free environment at a temperature of 22 - 24 °C and a humidity of 60% - 70%, and followed a 12-hour light / 12-hour dark cycle. They were placed in ventilated cages and provided with a standard diet as rat feed. Water was available ad libitum during the experiment, and the cage bedding was changed regularly to maintain cleanliness and dryness. Food was withheld for 12 hours before and after surgery.

[0042] 2. Experimental grouping and model

[0043] 2.1 Experimental grouping: Twenty Sprague-Dawley rats were randomly divided into the following 2 groups using a random number table method:

[0044] Control group: The donor kidney after 30 minutes of warm ischemia was perfused in KPS-1 solution for 2 hours and then orthotopic kidney transplantation was performed (WIT30min group);

[0045] Experimental group: The donor kidney after 30 minutes of warm ischemia was perfused in KPS-1 solution containing sulodexide for 2 hours and then orthotopic kidney transplantation was performed (WIT 30min + SDX group);

[0046] KPS-1 solution containing sulodexide: 20,000 LSU of sulodexide was contained in 1000 mL of conventional organ preservation solution KPS-1.

[0047] 2.2 Establishment of the donor kidney model after cardiac death in rats and kidney acquisition: The donor kidney model after cardiac death was established by inducing cardiac arrest by thoracotomy under heparin-free pretreatment. After anesthesia, the chest cavity and bilateral diaphragms were incised to induce cardiac arrest. The cardiac arrest time was observed within 6 - 7 minutes. The donor rats were placed on a 37 °C heating pad to control the warm ischemia time at 30 minutes. The left kidney was pre-rinsed in situ via the abdominal aorta with 4 °C sterile heparinized saline. After the renal venous effluent became clear, the kidney was removed, a renal artery stent (24G) was placed, and it was stored in 4 °C KPS-1 solution.

[0048] 2.3 Hypothermic oxygenated mechanical perfusion model: The rat kidney normothermic mechanical preservation and reperfusion system constructed according to the previous patent (CN213099890U rat kidney normothermic mechanical preservation and reperfusion system) was modified so that the organ perfusion pool and perfusion pipeline were placed in an ice-water bath for hypothermic oxygenated mechanical perfusion. The arterial end of the donor kidney was connected to the kidney mechanical perfusion system for perfusion, and oxygen was passed through the oxygenator for oxygenation. When starting hypothermic oxygenated mechanical perfusion, the temperature was controlled at 4 °C, and the mean arterial pressure was monitored and controlled at 30 mmHg through a bioinformation acquisition system. Perfusion was carried out for 2 hours, and perfusion parameters such as pressure, temperature, and flow rate were continuously monitored and recorded.

[0049] Experimental results: As Figure 1As shown in the figure. During the 2-hour low-temperature oxygenated machine perfusion, the perfusion flow and the renal resistance during perfusion were continuously monitored. Compared with the WIT 30-minute group, in the WIT 30-minute + SDX group, low-temperature oxygenated machine perfusion was performed using an organ perfusion solution containing sulodexide, and the renal perfusion flow increased significantly and the renal resistance during perfusion decreased significantly.

[0050] 2.4 Renal transplantation model: The recipient rats were anesthetized with sodium pentobarbital (30 mg / kg). After the left kidney of the recipient rats was removed, orthotopic renal transplantation was performed using end-to-end anastomosis of the renal artery, cannulation of the renal vein, and ureterovesical anastomosis, and at the same time, the right kidney of the recipient was removed. After the operation, the recipient rats were subcutaneously injected with meloxicam (1 mg / kg) for postoperative analgesia. All transplanted recipients were intraperitoneally injected with cyclosporine A (5 mg / kg) daily after the operation.

[0051] 3. Sample detection

[0052] 3.1 Immunohistochemical staining examination of the kidney after perfusion: The donor kidney tissue samples after perfusion were collected for pathological analysis. They were fixed with 10% paraformaldehyde for 12 hours, embedded in paraffin and sectioned. The expression levels of the vascular injury markers CD 31 and von Willebrand factor (vWF) were examined by immunohistochemical staining to evaluate the renal vascular injury after low-temperature oxygenated machine perfusion.

[0053] Experimental results: As Figure 2 shown in the figure. After 2 hours of low-temperature oxygenated machine perfusion, the expression levels of the vascular injury markers vWF and CD 31 were detected. Compared with the WIT 30-minute group, the vWF expression level in the WIT 30-minute + SDX group was significantly lower, indicating a lower degree of renal vascular endothelial injury; on the contrary, the CD 31 expression level was significantly increased, indicating better vascular integrity and patency. The results of the vWF and CD 31 expression levels both suggest that sulodexide significantly reduces the vascular endothelial injury of the donor kidney with 30 minutes of warm ischemia.

[0054] 3.2 Serum inflammatory factors after transplantation: Blood was collected from the tail vein, and the concentrations of inflammatory factors (TNF-α and IL-6) in the serum 1 day after transplantation were detected by enzyme-linked immunosorbent assay (ELISA) to evaluate the inflammatory response.

[0055] 3.3 Histopathological examination of the transplanted kidney: One day after kidney transplantation, a tissue sample of the transplanted kidney was collected for pathological analysis. The sample was fixed with 10% paraformaldehyde for 12 hours, embedded in paraffin, sectioned, stained with HE, and observed under a light microscope (×400) for pathological changes, and ATI scoring was performed. Scoring criteria: 0 points, normal histological structure; 1 point, swelling of renal tubular epithelial cells, loss of brush border, pyknosis or loss of nuclei; 2 points, swelling of renal tubular epithelial cells, loss of brush border, pyknosis or loss of nuclei, but the damaged area accounts for 1 / 3 - 2 / 3; 3 points, swelling of renal tubular epithelial cells, loss of brush border, pyknosis or loss of nuclei, and the damaged area accounts for the entire section.

[0056] Experimental results: As Figure 3 shown. Orthotopic kidney transplantation was performed after 2 hours of hypothermic oxygenated machine perfusion. The levels of inflammatory factors TNF-α and IL-6 in serum were detected 1 day after transplantation in both groups, and the kidneys were subjected to pathological histological examination 1 day after transplantation to evaluate the ischemic reperfusion injury of the transplanted kidney. Compared with the WIT 30min group, the levels of TNF-α and IL-6 in serum were significantly lower 1 day after transplantation in the WIT 30min + SDX group, and the kidney injury score was also significantly lower.

[0057] Example 2

[0058] Verification of the effect of sulodexide-containing organ preservation solution on reducing reperfusion injury of transplanted kidneys with 60 min of long-term warm ischemia

[0059] As the warm ischemia time prolongs, the kidney injury and vascular endothelial injury caused by warm ischemia become more severe, which will lead to severe ischemic reperfusion injury after transplantation.

[0060] 1. Experimental animals: The same as in Example 1.

[0061] 2. Experimental grouping and model

[0062] 2.1 Experimental grouping: Twenty SD rats were randomly divided into the following 2 groups according to the random number table method:

[0063] Control group: The donor kidney was perfused in KPS-1 solution for 2 hours after 60 min of warm ischemia and then orthotopic kidney transplantation was performed (WIT60min group);

[0064] Experimental group: The donor kidney was perfused in KPS-1 solution containing sulodexide for 2 hours after 60 min of warm ischemia and then orthotopic kidney transplantation was performed (WIT 60min + SDX group);

[0065] KPS-1 solution containing sulodexide: It contains 20,000 LSU of sulodexide in 1000 mL of conventional organ preservation solution KPS-1.

[0066] 2.2 Establishment of the kidney donor model after the death of rats' hearts and kidney acquisition: After anesthesia, the chest cavity and bilateral diaphragms were incised to induce cardiac arrest. The cardiac arrest time was observed within 6 - 7 minutes. The donor rats were placed on a heating pad at 37°C, and the warm ischemia time was controlled at 60 minutes. The remaining methods were the same as those in Example 1.

[0067] Experimental results: As Figure 4 shown. During the 2-hour hypothermic oxygenated mechanical perfusion, the perfusion flow rate and the renal resistance during perfusion were continuously monitored. Compared with the WIT 60min group, in the WIT 60min + SDX group, an organ perfusion fluid containing sulodexide was used for hypothermic oxygenated mechanical perfusion, and the renal perfusion flow rate increased significantly and the renal resistance during perfusion decreased significantly.

[0068] 2.3 Hypothermic oxygenated mechanical perfusion model: The same as that in Example 1.

[0069] 2.4 Kidney transplantation model: The same as that in Example 1.

[0070] 3. Sample detection

[0071] 3.1 Immunohistochemical staining examination of the kidneys after perfusion: The same as that in Example 1.

[0072] Experimental results: As Figure 5 shown. After 2-hour hypothermic oxygenated mechanical perfusion, the expression levels of the vascular injury markers vWF and CD 31 were detected. Compared with the WIT 60min group, the vWF expression level in the WIT 60min + SDX group was significantly lower, indicating a lower degree of renal vascular endothelial injury; on the contrary, the CD 31 expression level was significantly increased, indicating better vascular integrity and patency. The results of the vWF and CD 31 expression levels both suggest that sulodexide significantly reduces the vascular endothelial injury of the kidneys with 60-minute warm ischemia.

[0073] 3.2 Serum inflammatory factors after transplantation: The same as that in Example 1.

[0074] 3.3 Histopathological examination of the kidneys after transplantation: The same as that in Example 1.

[0075] Experimental results: As Figure 6 shown. Orthotopic kidney transplantation was performed after 2-hour hypothermic oxygenated mechanical perfusion. The levels of the inflammatory factors TNF-α and IL-6 in the serum 1 day after transplantation were detected in both groups, and the kidneys 1 day after transplantation were subjected to pathological histological examination to evaluate the ischemic reperfusion injury of the transplanted kidneys. Compared with the WIT 60min group, the levels of TNF-α and IL-6 in the serum 1 day after transplantation in the WIT 60min + SDX group were significantly lower, and the kidney injury score results were also significantly lower.

[0076] As can be seen from the above embodiments, the present invention provides an organ preservation solution for protecting vascular endothelium and its application. The organ preservation solution of the present invention can be used for in vitro hypothermic oxygen-carrying mechanical perfusion repair of kidneys from donors after cardiac death. In particular, the addition of the active ingredient sulodexide has a significant protective effect on vascular endothelium, can effectively reduce vascular endothelial damage after warm ischemia injury, increase perfusion flow, reduce perfusion resistance, and show significant improvement effects in reducing inflammatory reactions and tissue pathological structure damage caused by ischemia-reperfusion injury after transplantation, thereby improving the success rate of organ preservation and transplantation. The present invention solves the problem that there is currently no organ preservation solution specifically for protecting the blood vessels of kidneys from donors after cardiac death, and has important clinical application value especially in the preservation, repair and transplantation of donor organs after cardiac death.

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An organ preservation solution for protecting vascular endothelium, characterized in that: Including sulodexed.

2. The organ preservation solution according to claim 1, characterized in that: The concentration of sulodexide is 10 to 500 LSU / mL.

3. The organ preservation solution according to claim 1, characterized in that: Also included are KPS-1 solution, UW solution or HTK solution.

4. The organ preservation solution according to any one of claims 1 to 3, characterized in that: The organ is a kidney from a donor following cardiac death.

5. Use of the organ preservation solution according to any one of claims 1 to 4 in repairing kidney damage.

6. The use according to claim 5, characterized in that: The kidney injury is kidney warm ischemia-reperfusion injury.

7. The use according to claim 6, characterized in that: The method for repairing kidney warm ischemia-reperfusion injury with the organ preservation solution comprises the following steps: placing the organ preservation solution in a low-temperature oxygen-carrying mechanical system to perfuse the kidney of a donor with cardiac death.

8. The use according to claim 7, characterized in that: The perfusion temperature is 4-10° C., and the perfusion time is 2-6 hours.

9. Use of the organ preservation solution according to any one of claims 1 to 4 in organ preservation or organ transplantation.

10. The use according to claim 9, characterized in that: The organ preservation solution reduces inflammation levels and tissue damage after organ transplantation.

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