Rev Nefrol Dial Traspl. ene.-mar. 2020; 40(1):14-24.

Amniotic fluid in cold renal storage

ARTÍCULO ORIGINAL

PDF

Can amniotic fluid be an alternative organ preservation solution for cold renal storage?

¿Puede el líquido amniótico ser una solución alternativa de conservación de órganos para el almacenamiento renal en frío?

Başak Büyük¹ ORCID , Tuba Demirci² ORCID , Yasemen Adalı³ ORCID , Hüseyin Avni Eroğlu⁴ ORCID

1) Department of Histology and Embryology, Faculty of Medicine, İzmir Democracy University, İzmir, Turkey drbasakbuyuk@hotmail.com
2) Department of Histology and Embryology, Faculty of Medicine, Atatürk University, Erzurum, Turkey
3) Department of Pathology, Faculty of Medicine, Çanakkale Onsekiz Mart University, Çanakkale, Turkey
4) Department of Physiology, Faculty of Medicine, Çanakkale Onsekiz Mart University, Çanakkale, Turkey

Recibido en su forma original: 14 de octubre de 2019
En su forma corregida: 27 de octubre de 2019
Aceptación final: 4 de noviembre de 2019

Cómo citar este artículo (How to cite this article): Büyük B, Demirci T, Adalı Y, Eroğlu HA. Can amniotic fluid be an alternative organ preservation solution for cold renal storage? Rev Nefrol Dial Traspl. 2020; 40(1):14-24.

Materials and experimental design

Apoptosis assessment with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method

Apoptosis causes fractures in DNA. In the TUNEL method, terminal deoxynucleotidyl transferase (TdT) adds numerous dUTP-labeled deoxynucleotides to the 3’ end of a DNA strand (at 37 °C for 1 h). To detect apoptosis, in situ DNA fragmentation analysis was performed with the TUNEL (TdT-mediated dUTP nick end labeling) method. Sections with 4 µm thickness were cut from each paraffin block. After dewaxing, hydration, and serum blocking, the ApopTag Peroxidase in situ Apoptosis Detection Kit (S7100, Merck Millipore, Darmstadt, Germany) was used according to the manufacturer's protocol. After several washes, anti-digoxigenin-peroxidase was applied to the sections for 30 min at room temperature. The reaction was revealed using DAB for 6 min. After the washes, the sections were counterstained with hematoxylin for core staining. Then, the sections were examined and photographed under the light microscope, and the brown-black stained nuclei of apoptotic cells were observed. Apoptotic index (AI) was calculated in percent, using the following formula:

Statistical analysis

The Statistical Package for the Social Sciences (SPSS, version 22, IBM, Armonk, New York 10504, NY, USA) was used for the statistical analysis. Bivariate comparisons were done with the paired samples t-test and one-way ANOVA. The statistical significance level (p) was set to <0.05.

RESULTS

Histopathological evaluation

When tubular necrosis data belonging to the 6th hour is evaluated, there was significant statistical increase in Control RL) group compared to UW and HTK groups (p values are 0.010 and 0.038, respectively). When tubular necrosis data from the 12^th hour is evaluated, there was a statistically significant decrease in the RL group compared to UW and AF groups (p values are 0.035 and 0.044, respectively). There was no significant difference between the other groups.

When vacuolization data belonging to the 6^th hour is evaluated, there was significant statistical increase in the RL group compared to HTK and AF groups (p values are 0.021 and 0.021, respectively). When vacuolization data from the 12th hour is evaluated, there was statistically significant increase in the RL group compared to HTK and AF groups (p values are 0.013, and 0.005, respectively). There was no significant difference between the other groups.

When dilatation in Bowman’s space data from the 6^th hour is evaluated, there was statistically significant increase in the RL group compared to HTK, UW, and AF groups (p values are 0.009, 0.009 and 0.000, respectively). When dilatation in Bowman’s space data belonging to the 12^th hour is evaluated, there was statistically significant increase in the RL group compared to UW and AF groups (p values are 0.008 and 0.000, respectively). There was a statistically significant increase in the HTK group compared to the AF group (p=0.012). There was no significant difference between the other groups.

Histopathology changes and micrographs from the experimental groups can be seen in Table 1 and Figure 1.

Table 1. Semi-quantitative assessment of histopathological findings in rat kidney tissue

none: 0; mild: 1; moderate: 2; severe: 3; more severe: 4

Figure 1. Histological sections of the kidney tissues, which were taken in the 12^th hour of the experiment, showing RL, HTK, UW, and AF groups. Blue arrows indicate vacuolization and the black arrows point to tubular necrosis (Magnification x200)

Immunohistochemical evaluation

When the immunohistochemical results for the 6^th hour for IL-18 are evaluated, there was no significant difference between the experimental groups. When the immunohistochemical results from the 12^th hour for IL-18 are evaluated, there was a significant increase in the Control group (RL) compared to UW and AF groups (p values are 0.007 and 0.031, respectively). There was no significant difference between the other groups.

When the immunohistochemical results for the 6^th hour for TNF-α are evaluated, there was a significant increase between Control Group (RL) compared to UW and HTK groups (p values are 0.024, and 0.021, respectively). When the immunohistochemical results from the 12^th hour for TNF-α are evaluated, there was a significant increase in the Control group (RL) compared to HTK, UW, and AF groups (p values are 0.001, 0.044, and 0.003, respectively). There was no significant difference between the other groups.

When the immunohistochemical results from the 6^th hour for iNOS are evaluated, there was a significant increase in the RL group compared to the UW group (p = 0.008). When the immunohistochemical results for the 12^th hour for iNOS are evaluated, there was a significant increase in the RL group compared to UW and AF groups (p values are 0.014 and 0.003, respectively). There was no significant difference between the other groups.

Immunohistochemical results and micrographs for the experimental groups can be seen in Figure 2 and Graphic 1.

Figure 2. Immunohistochemical staining of the kidney tissues for TNF-alpha, IL-18, and iNOS showing RL, HTK, UW, and AF groups in the 12^th hour of the experiment (Magnification x200)

Graphic 1. Immunohistochemical results of all experimental groups. Letters show statistically significant differences between groups

Apoptotic cell detection via TUNEL method

A significant increase in terms of AI results was demonstrated in the RL group compared to UW and AF groups in the 6^th hour (p values are 0.002, and 0.001, respectively). There was no significant difference between the other groups.

A significant increase in terms of AI results was demonstrated in the RL group compared to HTK, UW, and AF groups in the 12^th hour (p values are 0.004, 0.004, and 0.001, respectively). There was no significant difference between the other groups.

There was no statistically significant difference between the AI values of HTK, UW, and AF groups in the 6^th and 12^th hours. TUNEL staining micrographs of experimental groups can be seen in Figure 3.

Figure 3. Immunohistochemical staining of kidney tissues by TUNEL showing RL, HTK, UW, and AF groups. Black arrows point to apoptotic cells (Magnification x400)

DISCUSSION

This study demonstrated that AF provides kidney protection similar to UW and HTK, which are standard organ preservation solutions used in kidney transplantation, at the immunohistochemical and microscopic level. Histopathologically, parameters evaluated in the AF group presented decreased injury compared to the control group, and similar results compared to UW and HTK groups, showing that AF can be an alternative preservation solution for kidneys. In addition, a significant decrease in the number of apoptotic cells in the AF group showed the positive effect of AF on organ viability. Immunohistochemical data also supported these results.

Cold storage of kidney grafts for transplantation includes four main components. In the order of occurrence, these are hypothermia, ischemia, hypoxia, and reperfusion. Kidneys harvested from the donor are subject to prolonged ischemia. To prevent possible injury, various organ preservation solutions were developed and improved to maintain the viability of kidneys.⁽¹¹⁾ Simple cold storage is the most prevalent and effective technique for kidney preservation. Protective effects of different organ preservation solutions have been widely studied.^{(4, 12)} The UW solution has high-viscosity and osmotically active substances. Additionally, it contains high-potassium and low-sodium, and is the gold standard solution for the preservation of liver, kidney, and pancreas.^(13-14) An alternative organ preservation solution to UW is the Histidine-Tryptophan-Ketoglutarate (HTK) solution, which is cheaper and has low-viscosity compared to UW. As with UW, HTK is also being used increasingly for intra-abdominal organ preservation including kidney, pancreas, and liver. It provides rapid cooling and has fewer complications than the UW solution.^{(13, 15)}

Ringer’s Lactate was one of the most preferred organ preservation solutions in early transplantation practices, but it was abandoned afterwards due to several disadvantages. It also has a capacity for rapid cooling of organs and thereby leads to a decrease in metabolism. However, after using RL solution, cellular metabolism and oxygen demand still remains ‎around 50%.^(16-17) In our study, RL was used for kidney perfusion of the control group.

Studies have shown that UW is the gold standard organ preservation solution, with its alternative being the HTK solution. Most of the literature has focused on the preserving effect of these solutions for abdominal organs and pointed out that the UW solution provides better preservation for renal grafts. It was mentioned that UW and HTK are equivalent for renal preservation.⁽¹⁴⁾ Other studies suggested that no significant differences exist between HTK and UW concerning the preservation of kidneys.^{(13, 15, 18)} However, HTK is preferred more because of its low-viscosity and lower costs.⁽¹⁰⁾ In our study, these two solutions proved to be similar; there was no statistical difference between UW and HTK solutions. In addition, AF was found to be as effective as UW and HTK for preservation of kidneys during the cold ischemic storage period. AI level is the parameter considered for organ viability, and it was found to be lower than the RL group. Comparing the AI values of the groups, the lowest apoptosis values in the AF group indicate that AF increases organ viability and provides effective protection. Decreased apoptotic cell counts which were obtained in the AF group can be evidence for valuable protection properties of AF, and it is predicted that postransplant success rates for kidney transplantation with AF perfusion may be high.

Recently, various potential chemical solutions have been studied for organ preservation all over the world to get better results. Schreinemachers et al.⁽¹⁹⁾ used Polysol as an alternative organ preservation solution in their study. Also, N-acetylcysteine, L-arginine, alpha-ketoglutarate, nitroglycerin, vasosol, hyperbranched polyglycerol and prostaglandin E-1 were used for organ preservation and were shown to be promising compared to the known solutions such as UW, HTK, Celsior and Collins.^(20-21)

High viscosity and high potassium concentrations may be possible reasons for abnormal delivery of organ preservation solutions into the renal segments. It is known that HTK has a lower viscosity than UW and the vasoconstriction process may be induced by high potassium concentrations. Studies reported that the preservation solutions, including high-sodium/low-potassium, were considered to be effective in stopping vasoconstriction and were better for renal preservation than low sodium-high potassium ones.^(22-23)

AF has normal sodium and normal-to-low potassium concentrations similar to serum, which is promising as an alternative preserving solution for renal grafts. However, the protective effects of AF on the renal cold ischemic injury were not studied before. It was reported that AF can be used for the cryopreservation of cell culture media.⁽²⁴⁾ Also, AF was compared with the Roswell Park Memorial Institute media and saline for skin graft preservation.^{(8, 29)} AF was used as preservation solution for the liver previously, and it was demonstrated that AF decreased apoptotic cell counts after 12 hours cold storage period.⁽⁸⁾ Histopathological, injury grades of AF group, such as hydropic degeneration and sinusoidal dilatation, were lower than in the RL group.⁽⁸⁾ In this study, we demonstrated the protective effect of AF on cold storage of kidneys, and the results showed that using AF as preservation solution for kidney transplantation can increase the organ viability. These results are compatible with the literature.

TNF-α is a proinflammatory cytokine secreted by various cells in the inflammation process.⁽²⁶⁾ IL-18 is another proinflammatory cytokine, and it was shown to be involved in renal inflammation. IL-18 is known to increase tubular necrosis and other ischemic damage parameters, especially in renal ischemia-reperfusion injury.⁽²⁷⁾ Increased IL-18 is an important parameter for evaluating renal viability.^(27-29) In our study, TNF-α and IL-18 were evaluated immunohistochemically and there was a decrease in UW, HTK and AF groups compared to the RL group. This decrease was also statistically significant. Results for the AF group were similar to the UW and HTK groups, and there was no statistically significant difference between groups. This suggests that AF reduces proinflammatory cytokine secretion as effectively as UW and HTK for renal preservation.

iNOS is expressed in many cells, such as Kupffer cells, hepatocytes and vascular smooth muscle cells.⁽³⁰⁾ It is a marker that is frequently used in the determination of tissue injury levels, since its release increases especially during the SCS process.⁽¹⁰⁾ In our study, iNOS values were significantly lower in UW, HTK and AF groups compared to the RL group, suggesting that AF provides kidney protection similar to standard preservation solutions.

As a limitation of this study, biochemical, molecular and immunological parameters such as catalase, lipid peroxidase, glutathione peroxidase, and endothelial nitric oxide synthase could be studied. The ultimate benefit of AF as an organ-preservation solution could be shown after transplantation to a receiver and by demonstrating its clinical performance.

In conclusion; our results show that AF has similar effects compared to the UW and HTK solutions and is superior to RL solution concerning the histopathological renal injury parameters, TNF-alpha, iNOS, IL-18, and TUNEL markers. Thus, as a natural tissue preservation solution, AF may be an inexpensive, promising, and readily available alternative for organ preservation.

Acknowledgements: The authors thank to Dr. Fatma Beyazit for collection amniotic fluid.
Conflict of interest: Authors declare no conflict of interest.

BIBLIOGRAPHY

1) McAnulty JF, Reid TW, Waller KR, Murphy CJ. Successful six-day kidney preservation using trophic factor supplemented media and simple cold storage. Am J Transplant. 2002;2(8):712-8.

2) Mahendran AO, Barlow AD. Kidney transplantation. Surgery (Oxford). 2014;32(7):364-70.

3) Wang S, Gumpper K, Tan T, Luo X, Guo H, Ming C, et al. A novel organ preservation solution with efficient clearance of red blood cells improves kidney transplantation in a canine model. Cell Biosci. 2018;8(1):28.

4) Marcén R, Burgos FJ, Ocaña J, Pascual J, Perez-Sanz P, Galeano C, et al. Wisconsin and Celsior solutions in renal preservation: a comparative preliminary study. Transplant Proc. 2005;37(3):1419-20.

5) Büyük B, Karakoç E. Effects of thiopental in cold ischemia in liver transplantation: An experimental study. J Surg Med. 2019;3(1):143-8.

6) Turhan-Haktanır N, Dilek FH, Köken G, Demir Y, Yılmaz G. Evaluation of amniotic fluid as a skin graft storage media compared with RPMI and saline. Burns. 2011;37(4):652-5.

7) Sterne GD, Titley OG, Christie JL. A qualitative histological assessment of various storage conditions on short term preservation of human split skin grafts. Br J Plast Surg. 2000;53(4):331-6.

8) Büyük B, Demirci T, Adalı Y, Eroğlu HA. A new organ preservation solution for static cold storage of the liver. Amniotic fluid. Acta Cir Bras. 2019;34(4):e201900402.

9) Estados Unidos. National Research Council. Guide for the Care and Use of Laboratory Animals [Internet]. 8th ed. Washington, DC: The National Academies Press, 2011. Disponible en: https://www.nap.edu/catalog/12910/guide-for-the-care-and-use-of-laboratory-animals-eighth (Consulta: 10/08/2019).

10) Akbulut S, Sevmis S, Karakayali H, Bayraktar N, Unlukaplan M, Oksuz E, et al. Amifostine enhances the antioxidant and hepatoprotective effects of UW and HTK preservation solutions. World J Gastroenterol. 2014;20(34):12292-300.

11) Janssen H, Janssen PH, Broelsch CE. UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury. Liver Transpl. 2004;10(12):1514-23.

12) Wang L, Wei J, Jiang S, Li HH, Fu L, Zhang J, et al. Effects of different storage solutions on renal ischemia tolerance after kidney transplantation in mice. Am J Physiol Renal Physiol. 2018;314(3):F381-7.

13) Parsons RF, Guarrera J V. Preservation solutions for static cold storage of abdominal allografts: Which is best? Curr Opin Organ Transplant. 2014;19(2):100-7.

14) Nunes P, Mota A, Figueiredo A, Macário F, Rolo F, Dias V, et al. Efficacy of renal preservation: comparative study of Celsior and University of Wisconsin solutions. Transplant Proc. 2007;39(8):2478-9.

15) Agarwal A, Goggins WC, Pescovitz MD, Milgrom ML, Murdock P, Fridell JA. Comparison of histidine-tryptophan ketoglutarate and University of Wisconsin solutions as primary preservation in renal allografts undergoing pulsatile perfusion. Transplant Proc. 2005;37(5):2016-9.

16) Caban A, Dolińska B, Budziński G, Oczkowicz G, Ostróżka-Cieślik A, Cierpka L, et al. The effect of HTK solution modification by addition of thyrotropin and corticotropin on biochemical indices reflecting ischemic damage to porcine kidney. Transplant Proc. 2013;45(5):1720-2.

17) Bessems M, Doorschodt BM, Albers PS, van Vliet AK, van Gulik TM. Wash-out of the non-heart-beating donor liver: a comparison between ringer lactate, HTK, and polysol. Transplant Proc. 2005;37(1):395-8.

18) Mangus RS, Fridell JA, Vianna RM, Milgrom MA, Chestovich P, Chihara RK, et al. Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution in extended criteria liver donors. Liver Transpl. 2008;14(3):365-73.

19) Schreinemachers MC, Doorschodt BM, Florquin S, Tolba RH. Comparison of preservation solutions for washout of kidney grafts: an experimental study. Transplant Proc. 2009;41(10):4072-9.

20) Li S, Constantinescu I, Guan Q, Kalathottukaren MT, Brooks DE, Nguan CY, et al. Advantages of replacing hydroxyethyl starch in University of Wisconsin solution with hyperbranched polyglycerol for cold kidney perfusion. J Surg Res. 2016;205(1):59-69.

21) Singh M, Odeniyi DT, Apostolov EO, Savenka A, Fite T, Wangila GW, et al. Protective effect of zinc-N-acetylcysteine on the rat kidney during cold storage. Am J Physiol Physiol Renal Physiol. 2013;305(7):1022-30.

22) Moen J, Claesson K, Pienaar H, Lindell S, Ploeg RJ, McAnulty JF, et al. Preservation of dog liver, kidney, and pancreas using the Belzer-UW solution with a high-sodium and low-potassium content. Transplantation. 1989;47(6):940-5.

23) Ramella SG, Hadj-Aïssa A, Barbieux A, Steghens JP, Colpart JJ, Zech P, et al. Evaluation of a high sodium-low potassium cold-storage solution by the isolated perfused rat kidney technique. Nephrol Dial Transplant. 1995;10(6):842-6.

24) Thomasen H, Pauklin M, Noelle B, Geerling G, Vetter J, Steven P, et al. The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane. Curr Eye Res. 2011;36(3):247-55.

25) Turhan-Haktanir N, Sahin Ö, Yagmurca M, Köken G, Demir Y, Cosar E. Histological assessment of skin grafts stored in amniotic fluid and saline. J Plast Surg Hand Surg. 2010;44(4):226-30.

26) Qi QA, Yang ZY, Ma KS, Lu Q, Wang SG, Li XW, et al. Impact of cold ischemia on cytokines after partial liver transplantation in rats. Gen Molec Res. 2013;12(3):4003-8.

27) Zhang H, Hile KL, Asanuma H, Vanderbrink V, Franke EI, Campbell MT, et al. IL-18 mediates proapoptotic signaling in renal tubuler cells through a Fas ligand-dependent mechanism. Am J Physiol Renal Physiol. 2011;301(1):171-8.

28) Matsui F, Rhee A,  Hile KL, Zhang H, Meldrum KK. IL-18 induces profibrotic renal tubular cell injury via STAT3 activation. Am J Physiol Renal Physiol. 2013;305(7):F1014-F1021.

29) Parikh CR, Jani A, Mishra J, Ma Q, Kelly C, Barasch J, et al. Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation. Am J Transplant. 2006 Jul;6(7):1639-45.

30) Meguro M, Katsuramaki T, Nagayama M, Kimura H, Isobe M, Kimura Y, et al. A novel inhibitor of inducible nitric oxide synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig liver. Transplantation. 2002;73(9):1439-46.

Esta obra se encuentra bajo la Licencia de Creative Commons Reconocimiento-No-Comercial 4.0 Internacional. Reconocimiento - Permite copiar, distribuir y comunicar públicamente la obra. A cambio se debe reconocer y citar al autor original. No comercial - Esta obra no puede ser utilizada con finalidades comerciales, a menos que se obtenga el permiso.

Revista de Nefrología, Diálisis y Trasplante
ISSN 2346-8548 (electrónico) - ISSN 0326-3428 (impreso)