Mitochondrial alterations after cold incuabtion and rewarming.

<p>Freshly isolated rat proximal renal tubules (A, F: control) were incubated at 4°C in chloride-rich (B, D, G, I) or chloride-poor (C, E, H, J) cold storage solution with addition of iron chelators for 48 h. Mitochondria were stained with 300 nM MitoTracker Red at the beginning of cold storage and confocal laser scanning images were taken after fixation at the end of cold storage (B, C; detail: G, H) or after 3 h of rewarming (D, E; detail: I, J). Arrows: filamentous mitochondria; arrow heads: mitochondrial fragmentation; stars: mitochondrial swelling.</p

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

<div><p>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.</p></div

Lipid peroxidation in isolated proximal renal tubules after cold incubation and rewarming.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 (intermediate grey bars) or its chloride-poor counterpart solution 2 (dark grey bars) each in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM; striped bars) for 48 h under gentle agitation. Thereafter, tubules were spun down, resuspended in pre-warmed extracellular buffer and incubated under gentle motion at 37°C for two hours. Thiobarbituric acid-reactive substances (TBARS) were determined in the supernatants at the end of cold incubation (darker part of the bars) and at the end of rewarming (light grey part of the bars). *p<0.05 and ***p<0.001 vs. respective solution without iron chelators at the end of cold incubation; ##p<0.01 and ###p<0.001 vs. respective solution without iron chelators at the end of rewarming (n = 7).</p

Composition of cold incubation solutions and of extra-cellular buffer used during rewarming.

<p>Composition of cold incubation solutions and of extra-cellular buffer used during rewarming.</p

Oxygen consumption of renal proximal tubules.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 in the presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM) for 48 h. Tubules were rewarmed in extra-cellular buffer at 37°C for up to three hours. Oxygen consumption was measured at baseline (control) and after 48 h of cold incubation followed by 2 h or 3 h of rewarming in extra-cellular buffer. Respiration was measured in extra-cellular buffer at 37°C under continuous stirring using an O2K-oxygraph. Results summarize data from 20 measurements. Two measurements each were performed with utilization of tubules from 10 individual preparations. *p <0.05.</p

LDH release of isolated proximal renal tubules during cold incubation at 4°C for 168 h.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 or its chloride-poor counterpart solution 2 in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM). Results display released LDH activity as a percentage of total LDH activity. Values are means ± standard deviation of tubules of five preparations. **p <0.01 and ***p <0.001 vs. respective solution without iron chelators.</p

ATP content of isolated renal proximal tubules after cold incubation and subsequent rewarming.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 or its chloride-poor counterpart solution 2 in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM) for 48 h (A) and 120 h (B). Tubules were rewarmed in extra-cellular buffer at 37°C for three hours. ATP content was normalized for protein content and is expressed as percentage of the respective non-stored control tubules. (A) *p <0.05 vs. solution 1; ###p <0.001 vs. respective chloride-rich solution. (B) ##p <0.01 vs. solution 1+chelators. Values are means ± standard deviation of eight (A) and four (B) preparations.</p

Metabolic activity of isolated renal proximal tubules following rewarming after 48 h of cold incubation.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 or its chloride-poor counterpart solution 2 in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM) for 48 h. Tubule suspensions were rewarmed in extra-cellular buffer at 37°C for three hours; thereafter, metabolic activity was assessed by the resazurin reduction assay. Reduction of resazurin to fluorescent resorufin was followed at λ_exc = 560 nm, λ_em = 590 nm. Reduction rates are given as percentage of non-stored control tubules. Values are means ± standard deviation of tubules of four preparations.</p

LDH release of isolated proximal renal tubules during rewarming after 24 h, 48 h and 120 h of cold incubation.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 or its chloride-poor counterpart solution 2 in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM) for 24 h (A), 48 h (B) or 120 h (C). For gentle rewarming cell suspensions in the respective cold solutions were kept at room temperature for 10 min followed by 10 min at 37°C (transition period). Afterwards, tubules were spun down, resuspended in pre-warmed extra-cellular buffer and incubated under gentle motion at 37°C for three hours. Arrows mark time of buffer exchange. Results display released LDH activity as a percentage of total LDH activity. Values are means ± standard deviation of tubules of three (A), eight (B) and eight (C) preparations. (A) *p <0.05, **p <0.01 and ***p <0.001 vs. respective solution without iron chelators. (B) ###p <0.001 vs. solution 1. **p <0.01 and ***p <0.001 vs. respective solution without iron chelators.</p

Managing the risk of low falling numbers in wheat

Grain is purchased at a discount when falling numbers are below 300 seconds (sec). This can result in serious financial losses for farmers. This article addresses many commonly asked questions about the Hagberg-Perten Falling Number test, and provides some suggestions for reducing losses due to low falling numbers