Research Article: Characterization of injury in isolated rat proximal tubules during cold incubation and rewarming

Date Published: July 3, 2017

Publisher: Public Library of Science

Author(s): Anja Bienholz, Björn Walter, Gesine Pless-Petig, Hana Guberina, Andreas Kribben, Oliver Witzke, Ursula Rauen, Edward J. Lesnefsky.


Organ shortage leads to an increased utilization of marginal organs which are particularly sensitive to storage-associated damage. Cold incubation and rewarming-induced injury is iron-dependent in many cell types. In addition, a chloride-dependent component of injury has been described. This work examines the injury induced by cold incubation and rewarming in isolated rat renal proximal tubules. The tissue storage solution TiProtec® and a chloride-poor modification, each with and without iron chelators, were used for cold incubation. Incubation was performed 4°C for up to 168 h, followed by rewarming in an extracellular buffer (3 h at 37°C). After 48, 120 and 168 h of cold incubation LDH release was lower in solutions containing iron chelators. After rewarming, injury increased especially after cold incubation in chelator-free solutions. Without addition of iron chelators LDH release showed a tendency to be higher in chloride-poor solutions. Following rewarming after 48 h of cold incubation lipid peroxidation was significantly decreased and metabolic activity was tendentially better in tubules incubated with iron chelators. Morphological alterations included mitochondrial swelling and fragmentation being partially reversible during rewarming. ATP content was better preserved in chloride-rich solutions. During rewarming, there was a further decline of ATP content in the so far best conditions and minor alterations under the other conditions, while oxygen consumption was not significantly different compared to non-stored control tubules. Results show an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and reveal a benefit of chloride for the maintenance of tubular energy state during cold incubation.

Partial Text

Solid organ transplantation in general, and kidney transplantation in particular are torn between the conflicting areas of organ supply and demand. The currently existing shortage of kidney donations and the steadily rising number of potential recipients dying while being on the waiting list encourages transplantation of marginal organs by accepting older and expanded criteria donors [1, 2]. Marginal organs are particularly sensitive to storage-associated damage with an increasing risk for delayed or poor graft function affecting long-term organ survival with effects on both patients`morbidity and mortality [3, 4]. Therefore, improvements of currently used storage and transport conditions are urgently needed to maintain and enhance organ quality and thereby patient outcome.

Chemicals and Materials. Cold incubation solutions (composition see Table 1) are based on the tissue storage solution TiProtec® [23], a derivative of the new preservation solution Custodiol-N. This tissue preservation solution was used here, as it can easily be modified with regard to the chloride content, yielding a high chloride and a low chloride cold storage solution [16, 23]. The term “iron chelators” refers to 0.5 mM deferoxamine plus 20 μM LK 614. This combination of the strong, but large and hydrophilic and thus relatively poorly membrane-permeable chelator deferoxamine (that, however, does enter cells [25]) and the small lipophilic hydroxamic acid derivative LK 614 [23, 26] has been shown previously to provide optimal protection and to affect intracellular iron-dependent processes [16, 27]. The extra-cellular buffer was used in previous experiments with isolated renal proximal tubules [28, 29].

Hypothermic injury. Hypothermic injury to isolated proximal renal tubules, as assessed by LDH release, a quantitative marker of cell death, was followed during cold incubation (4°C) for 168 h. In solution 1 LDH release of isolated proximal renal tubules during cold incubation at 4°C continuously increased during the course of the experiment reaching a maximum of 77 ± 7% after 168 h (Fig 1). No significant differences in LDH release could be found between solution 1 and the chloride-poor variant solution 2. Addition of iron chelators resulted in a significantly lower LDH release in both solutions after 48 h of cold incubation (p <0.01; n = 5) with further increase of differences over time (after 120 h and 168 h: p <0.001; n = 5). No significant differences in LDH release between iron chelator-containing solutions with high and low chloride concentrations could be found. Results of the present study confirmed an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and revealed a benefit of chloride for the maintenance of tubular energy state during cold incubation. The present study suggests that cellular integrity (membrane integrity) is only one point that has to be addressed in the attempt to decrease storage-associated damage. Once early cell death is prevented, cellular and especially mitochondrial functionality gain importance. While LDH release was only 32% after rewarming in tubules incubated for 48 h in the chloride-rich solution containing iron chelators, reflecting about 68% of intact cells, ATP content was decreased to as little as 40% of baseline values. Iron-independent components of damage appear to impact especially mitochondria and thereby energy state. This should be considered with regard to reconditioning of organs after cold storage in which the re-establishment of normal energy metabolism is a major focus. The contrary behavior of tubules cold-incubated in chloride-poor solutions to show a slight tendency to increase energy content during rewarming, while energy loss proceeded to decline in tubules incubated in chloride-rich solutions, in this context requires further studies.   Source:


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