041 Consequences and control of ice formation in the renal inner medulla

Abstract

The mammalian kidney provides a valuable model of the requirements for and the biological benefits of suppressing ice formation in complex living systems during attempted cryopreservation by vitrification. The structure of the renal vasculature limits the flow of blood or of perfusate through the renal medulla, which limits delivery of cryoprotective agents to the outer and especially to the inner medulla by vascular perfusion. Despite this problem, it is relatively easy to deliver sufficient cryoprotectant to prevent ice formation on cooling, but it is difficult to deliver enough to prevent ice formation during warming (devitrification), and the latter phenomenon is currently perhaps the central challenge to successful cryopreservation by vitrification. Transplantation of previously vitrified rabbit kidneys has indicated that if >2% of the inner medullary mass is transformed into ice during devitrification, the vascular system of the medulla is selectively destroyed, and upon transplantation, blood entering the medulla becomes trapped there. If the quantity of inner medullary ice (IMI) is limited to 1–2% w/w, the kidney as a whole can survive after vitrification and transplantation and support the life of the recipient indefinitely, although with significant functional consequences. Therefore, the goal must be to reduce IMI formation during warming to less than 1% w/w. Fortunately, it is possible to approach this goal by taking advantage of the fact that the renal medulla also receives cryoprotectants by means of glomerular filtration, and the delivery of cryoprotectants by filtration can be influenced by adjusting, for example, the perfusion pressure profile and the perfusion time at peak cryoprotectant concentration. Previous studies indicated that IMI can be avoided during warming at 40 °C/min when glomerular perfusate ultrafiltrate (“urine”) concentrations approach 95% of the full molarity of the M22 vitrification solution (95% M22) when kidneys are perfused at −3 °C. Current results indicate that, when kidneys are perfused at −22 °C, a urinary concentration of at least 96% M22, or about 9M, is needed to reduce IMI to ∼0.05% w/w at the same warming rate, presumably due to lower permeability at the lower temperature. This concentration requirement, however, may be relaxed somewhat by utilizing still higher warming rates. Our laboratory has recently been able to achieve uniform electromagnetic warming of rabbit kidney sized M22 samples at over 100 °C/min over the temperature range from −75 to −35 °C, which extends from well below typical inner medullary devitrification temperatures to well above typical inner medullary melting points. The rabbit kidney can presently tolerate urinary concentrations equaling at least 95% M22 with complete long-term functional recovery following transplantation. This suggests that it should be possible to completely or almost completely prevent ice formation in rabbit kidneys during both cooling and warming after using a cryoprotection method that is compatible with complete functional recovery of the kidney after transplantation. Therefore, careful analysis and control of the behavior of water in the renal inner medulla at low temperatures appears to be pointing the way to a method of renal cryopreservation by vitrification that can preserve renal viability by avoiding ice crystal damage during rewarming. Source of funding: This research was supported by 21st Century Medicine, Inc. Conflict of interest: None declared. gfahy@21cm.com