Renal cold storage followed by transplantation impairs expression of key mitochondrial fission and fusion proteins

<div><p>Background</p><p>The majority of transplanted kidneys are procured from deceased donors which all require exposure to cold storage (CS) for successful transplantation. Unfortunately, this CS leads to renal and mitochondrial damage but, specific mitochondrial targets affected by CS remain largely unknown. The goal of this study is to determine whether pathways involved with mitochondrial fusion or fission, are disrupted during renal CS.</p><p>Methods</p><p>Male Lewis rat kidneys were exposed to cold storage (CS) alone or cold storage combined with transplantation (CS/Tx). To compare effects induced by CS, kidney transplantation without CS exposure (autotransplantation; ATx) was also used. Mitochondrial function was assessed using high resolution respirometry. Expression of mitochondrial fusion and fission proteins were monitored using Western blot analysis.</p><p>Results</p><p>CS alone (no Tx) reduced respiratory complex I and II activities along with reduced expression of the primary mitochondrial fission protein, dynamin related protein (DRP1), induced loss of the long form of <u>O</u>ptic <u>A</u>trophy <u>P</u>rotein (OPA1), and altered the mitochondrial protease, OMA1, which regulates OPA1 processing. CS followed by Tx (CS/Tx) reduced complex I, II, and III activities, and induced a profound loss of the long and short forms of OPA1, mitofusin 1 (MFN1), and mitofusin 2 (MFN2) which all control mitochondrial fusion. In addition, expression of DRP1, along with its primary receptor protein, mitochondrial fission factor (MFF), were also reduced after CS/Tx. Interestingly, CS/Tx lead to aberrant higher molecular weight OMA1 aggregate expression.</p><p>Conclusions</p><p>Our results suggest that CS appears to involve activation of the OMA1, which could be a key player in proteolysis of the fusion and fission protein machinery following transplantation. These findings raise the possibility that impaired mitochondrial fission and fusion may be unrecognized contributors to CS induced mitochondrial injury and compromised renal graft function after transplantation.</p></div

Alteration of OPA1 regulator proteases (YME1L and OMA1) after cold storage plus transplantation.

<p>Renal extracts (30 ug) were resolved on SDS-PAGE gels and immunoblotted. Representative YME1L western blot showing distinct protein bands of YME1L (~60 kDa) in Sham, ATx, and CS/Tx kidneys <b>(A)</b> and in control and CS alone kidneys <b>(B).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Representative OMA1 western blot showing distinct protein bands of OMA1 (~40 kDa) after CS/Tx <b>(C)</b> and CS alone <b>(D).</b> Multiple higher and lower molecular weights of OMA1 reactivity (?) were also visible after CS/Tx. Actin was used as a loading control. Densitometry evaluation of 40 kDa OMA1 (normalized to actin) is shown on the right panel. Values were expressed as Mean ± S.E.M. (n = 4). Unpaired Student’s <i>t</i> test was used to compare the means between control and CS kidneys. One-way ANOVA followed by Tukey’s post-hoc test for multiple group comparisons was used to compare the means between sham, ATx, and CS/Tx kidneys; * indicates means are significantly different (P < 0.05) when compared to control or sham and # indicates means are significantly different (P < 0.05) when compared to ATx.</p

Cold storage alone and combined with transplantation reduces mitochondrial function.

<p>Graph showing respiratory complex I, II, and III activity of the electron transport chain using HRR in kidney biopsies from control, sham, CS alone, and CS combined with transplantation (CS/Tx). Values were expressed as Mean ± S.E.M. (n = 5). Difference between the mean of the groups were compared using a one-way factorial analysis of multivariance (ANOVA) followed by Tukey’s post-hoc test for multiple group comparisons; *, indicates means are significantly different (P < 0.05), when compared between control and CS alone or between Sham and CS/Tx. Similarly, #, indicates means are significantly different (P < 0.05), when compared between CS alone and CS/Tx.</p

Cold storage alone and cold storage plus transplantation alters mitochondrial fission proteins, but autotransplantation has no effect.

<p>Renal extracts (30 ug) were resolved on SDS-PAGE gels and immunoblotted. Representative DRP1 western blot showing distinct protein bands of DRP1 (~75 kDa) in Sham, ATx, and CS/Tx kidneys <b>(A)</b> as well as control and CS alone kidneys <b>(B).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Representative MFF western blot showing distinct protein bands of MFF (~38 kDa) in Sham, ATx, and CS/Tx kidneys CS/Tx <b>(C)</b> as well as control and CS alone kidneys <b>(D).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Values were expressed as Mean ± S.E.M. (n = 4). Unpaired Student’s t test was used to compare the means between control and CS kidneys. One-way ANOVA followed by Tukey’s post-hoc test for multiple group comparisons was used to compare the means between sham, ATx, and CS/Tx kidneys; * indicates means are significantly different (P < 0.05) when compared to control or sham and # indicates means are significantly different (P < 0.05) when compared to ATx.</p

Impact of cold storage alone, combined cold storage plus transplantation, and autotransplantation on mitochondrial fusion protein expression.

<p>Renal extracts (30 ug) were resolved on SDS-PAGE gels and immunoblotted. Representative MFN 1 and MFN2 western blots showing distinct protein bands of both MFN1/2 (~75 kDa) in Sham, ATx, and CS/Tx kidneys <b>(A)</b> as well as control and CS alone kidneys <b>(B).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Representative OPA1 western blot showing long form of OPA1 (L, ~95 kDa) and short form of OPA1 (S, ~75 kDa) in Sham, ATx, and CS/Tx kidneys CS/Tx <b>(C)</b> as well as control and CS alone kidneys <b>(D).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Values were expressed as Mean ± S.E.M. (n = 4). Unpaired Student’s t test was used to compare the means between control and CS kidneys. One-way ANOVA followed by Tukey’s post-hoc test for multiple group comparisons was used to compare the means between sham, ATx, and CS/Tx kidneys; * indicates means are significantly different (P < 0.05) when compared to control or sham and # indicates means are significantly different (P < 0.05) when compared to ATx.</p

Cold storage plus transplantation impairs renal function and mitochondrial morphology.

<p><b>(A)</b> Serum creatinine and blood urea nitrogen (BUN) from the sham and 18 hr CS/Tx rats were analyzed using hand-held clinical chemistry analyzer (iSTAT^TM) and Chem8⁺ cassettes as described in materials and methods. Values were expressed as Mean ± S.E.M. (n = 4). The unpaired Student’s t test was used to compare the means between sham and CS/Tx; * indicates means are significantly different from sham (P < 0.05). <b>(B)</b> Electron micrographs revealed normal mitochondria in sham kidneys (cortical region), but 18 hr CS/Tx kidneys show rounded, fragmented mitochondria with dense aggregates. Images are representative of n = 3 in each group; bar, 500 nm. TEM was performed at the Georgia Reagents TEM Core Facility.</p

Schematic of five rat surgical groups used in this study.

<p>The left panel depicts the sham surgery, in which the right kidney was removed (<b>control kidney</b>) and the rat survives with only the left native kidney (<b>sham kidney</b>). The middle panel shows transplant surgery using donor kidneys (left and right), which were harvested and exposed to cold storage solution for 18 hrs. The right kidney was saved as CS control (<b>CS kidney</b>) and the left kidney was transplanted in a new recipient rat, in which both native kidneys were removed so that the kidney function depends on the transplanted donor kidney (<b>CS/Tx kidney</b>). The right panel shows autotransplant surgery, in which both native kidneys were removed in a rat, but the left native kidney was transplanted immediately back to the same rat. This kidney was saved as autotransplanted kidney (<b>ATx kidney</b>) and served as a control transplant kidney without CS for the CS/Tx kidney.</p

Renal cold storage alone or cold storage plus transplantation does not decrease all mitochondrial protein expression.

<p>Renal extracts (30 ug) were resolved on SDS-PAGE gels and immunoblotted. Representative Core2 and MnSOD western blots showing distinct protein bands of Core2 (~49 kDa) and MnSOD (~ 24 kDa) in Sham, ATx, and CS/Tx <b>(A)</b> and comparing control to CS alone <b>(B).</b> Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Values were expressed as Mean ± S.E.M. (n = 4). Unpaired Student’s <i>t</i> test and one-way ANOVA were used to compare the means.</p