Effects of glutamate and rotenone on succinate-supported respiration of normoxic and H/R rabbit tubules.

<p>A. Measurements of energization using safranin O uptake done in parallel with the respiration studies on the same preparations. Abbreviations for the experimental groups testing different conditions during safranin O uptake are as for Fig. 8. Tubules were subjected to 67.5±SEM for N = 4. Figure symbols indicate #P<0.01 or +P<0.001 vs. corresponding normoxic values. Values for glutamate and rotenone-treated H/R groups without dBSA were significantly different from the corresponding group with succinate alone, P<0.05. Delipidated albumin (dBSA) significantly improved energization under all the H/R conditions (P<0.001). B. Measurements of respiration. Oxygen consumption was assessed sequentially under basal conditions, then after addition of ADP to stimulate oxidative phosphorylation, then during suppression of the ADP-induced oxidative phosphorylation by oligomycin (OLIGO), then during maximally-stimulated uncoupled respiration produced by carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Studies were done with and without addition of dBSA on tubules previously subjected to either normoxic incubation or H/R. Values are means±SEM for N = 4. There were no significant effects of glutamate and/or rotenone under any condition except for a small decrease of the ADP rate in the normoxic glutamate+rotenone+dBSA group (P<0.05). Basal and oligomycin rates did not significantly differ between normoxic and H/R tubules. dBSA significantly lowered the basal and oligomycin rates in all groups (P<0.001 normoxic, P<0.01 hypoxic). ADP and CCCP rates of H/R tubules were significantly lower than the corresponding normoxic at *P<0.05 or #P<0.01 under all conditions. dBSA did not significantly affect the ADP rates, but it significantly increased the CCCP rates in the normoxic S and SR groups and the H/R S, SG, and SR groups, Ps<0.05 or better.</p

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

Effects of oleate on energization and respiration of permeabilized tubules: comparison of complex I and complex II-dependent substrates.

<p>Direct effects of oleate on energization of permeabilized rabbit tubules measured with safranin O uptake (panels A–C) and respiration (D) supported by either succinate or the combination of complex I dependent substrates, α-ketoglutarate, malate, and glutamate (AMG). Sets of typical safranin O uptake tracings (inverted fluorescence) are shown in panels A and B and group averages for those studies are in panel C. Numbers adjacent to each tracing in panels A are the concentrations of oleate added in µM. In panel C, “Peak” indicates the maximal uptake compared to the uptake seen without added oleate using succinate as substrate measured in the presence of delipidated albumin to eliminate the effect of endogenous fatty acids. “End” indicates the final level reached at the end of the 600 second measurement period, which can be less than the peak if there has been decay of ΔΨm. The panel D respiratory rates (RR) are given as percentages relative to “Control” rates without added oleate using succinate as substrate. Shown are both the initial rate produced by oleate and then the rate at the end of 600 seconds of measurement. Values in panels C and D are means±SEM for N = 3–5 except 4 µM oleate with AMG where the N was 2. *P<0.05, #P<0.01, +P<0.001 vs. corresponding succinate group.</p

Energization and respiration after H/R of unprotected and substrate-protected rabbit tubules supported by succinate or complex I-dependent substrates.

<p>These experiments were done similarly to those in Fig. 9, except they compared the behavior of complex II-dependent respiration with complex I and also tested tubules that were protected by dBSA+αKG/MAL in the incubation flasks during the 60 min. reoxygenation period. A. Measurements of energization using safranin O uptake. Abbreviations for the experimental groups testing different conditions during safranin O uptake are as for Fig. 8. Values are means±SEM for N = 4. All H/R groups except protected tubules with succinate+BSA were significantly different from the corresponding normoxic groups at P<0.001 for unprotected flasks without dBSA and P<0.05 for all other groups. Statistical symbols shown in the figure indicate: *P<0.05, #P<0.01, +P<0.001 vs. corresponding protected flask. dBSA significantly increased energization in all AMG groups and in succinate groups from unprotected tubules, P<0.01. SG, SR, and SGR without dBSA had significantly better uptake than S alone in the unprotected tubules (P<0.05), and AMG without dBSA supported energization after H/R less strongly than S alone in both protected and unprotected tubules, P<0.01. B. Measurements of respiration. Oxygen consumption was measured with either succinate+glutamate+rotenone (SGR) or α-ketoglutarate+malate+glutamate (AMG) following the same experimental sequence as described for Fig. 9. Values are means±SEM for N = 4. SGR rates were significantly greater than the corresponding AMG rates under all conditions, P<0.01. Statistical symbols shown in the figure indicate: *significantly different from normoxic at P<0.01 (SGR studies) or P<0.05 (AMG), #significantly different from corresponding unprotected group at P<0.01 (SGR studies) or P<0.05 (AMG studies). All basal and oligomycin rates with dBSA were significantly lower than the corresponding rates without dBSA (P<0.02) except for the oligomycin rate of the AMG H/R group. dBSA significantly increased the succinate-supported CCCP rates for all groups and the AMG-supported CCCP rate of the normoxic tubules.</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

Effect of glutamate and rotenone on energization and respiration supported by succinate in rabbit and mouse tubules.

<p>Safranin O uptakes and respiratory rates (RR) of permeabilized tubules were measured either under control conditions or in the presence of the indicated concentrations of oleate with either no further additions (NFA) or glutamate (G), rotenone (R) or glutamate+rotenone (G+R). “Peak” indicates the maximal uptake or RR during the measurement period. “End” indicates the uptake level or RR reached at the end of the second measurement period, which is less than the peak for conditions where there has been decay of ΔΨm or RR. Concentrations of oleate were chosen to give moderate deenergization (3 µM for rabbit, 4 µM for mouse) or severe deenergization (8 µM for rabbit, 10 µM for mouse). The high oleate concentration used for the rabbit RR studies was increased to 10 µM to provide more consistent decreases of the end RR to allow assessment of agents that ameliorate it. Values are means±SEM for N = 3 for both types of rabbit studies, 5–7 for the mouse safranin O uptakes and 4–5 for the mouse respiratory rates. *P<0.05, #P<0.01, +P<0.001 vs. corresponding NFA group.</p

Effects of oleate and malate on energization of mouse tubules.

<p>A. Concentration dependence of effects of oleate on energization of permeabilized mouse tubules measured with safranin O uptake supported by either succinate or the combination of complex I dependent substrates, α-ketoglutarate, malate, and glutamate. “Peak” indicates the maximal uptake compared to the uptake seen without added oleate using succinate as substrate in the presence of delipidated albumin to eliminate the effect of endogenous fatty acids. “End” indicates the final level reached at the end of the 600 second measurement period, which can be less than the peak if there has been decay of ΔΨm. Values are means±SEM for N = 3. *P<0.05, #P<0.01, +P<0.001 vs. corresponding succinate group. B and C. Effects of malate on succinate-supported energization measured using safranin O uptake (Panel B) and respiration (Panel C). Permeabilized tubules were incubated with succinate and the indicated test agents for 350 seconds (Pre-Malate period) followed by addition of either sham malate or malate for 200 seconds with measurement of safranin uptake and respiratory rate (RR) at the end of that period. Values are compared to those measured for the no further addition (NFA) group at the end of the ‘Pre-Malate’ period. Other abbreviations are: G – glutamate, R - rotenone, G+R =  glutamate+rotenone, dBSA – delipidated bovine serum albumin. For the safranin O uptakes, values are means±SEM for N = 2–3 for sham malate and 3–5 for malate. +P<0.001 for conditions with N≥3 vs. the corresponding NFA malate condition. For the respiratory rates, values are means±SEM for N = 4, *P<0.05, #P<0.01, +P<0.001 vs. corresponding NFA group.</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

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