Hong Huang

Involvement of the Mitochondrial Pathway in Cold Storage and Rewarming‐Associated Apoptosis of Human Renal Proximal Tubular Cells

The cellular and molecular mechanisms of cold storage‐ATN are not well characterized. In our earlier studies, cold storage caused necrosis of human proximal tubular epithelial (RPTE) cells, whereas apoptosis was prominent during rewarming. An intriguing finding was the pronounced swelling of the mitochondria in the cold, which promoted us to further characterize its role in rewarming‐associated apoptosis. Human proximal tubular epithelial cells were cold stored in University of Wisconsin (UW) solution for 48u2003h followed by 24u2003h of rewarming in regular cell culture medium. During the cold storage, there was no significant change in the Bcl‐2 to Bax protein ratio, mitochondrial location of cytochrome C or caspse‐3 activity. However, during rewarming, the Bcl‐2 to Bax ratio increased, cytochrome C was translocated to cytosol, and caspase‐3 was activated: events and timing were consistent with the occurrence of apoptosis during rewarming. In a time‐course experiment, mitochondrial swelling was discernable by electron microscopy as early as at 2u2003h. Cold storage of isolated‐mitochondria for 2u2003h was attended by an increase in the opening of the permeability transition pores (PTP), suggesting PTP opening as an early mechanism for mitochondrial swelling. Addition of antioxidants (deferoxamine or 2‐methyaminochroman) to the storage solution suppressed mitochondrial pore opening and swelling, Bcl‐2 to Bax ratio increase, cytochrome C translocation, caspase‐3 activation as well as rewarming‐induced apoptosis. Our data demonstrate for the first time that apoptosis following cold storage and rewarming of human renal tubular cells is accompanied by specific mitochondrial events, and that these events and apoptosis can be suppressed by adding antioxidants to the cold storage solution.

Mechanism and prevention of cold storage-induced human renal tubular cell injury.

BACKGROUNDnThe recent observation that cold storage of kidneys and tubular cells causes marked increase in free radical-catalyzed F2-isoprostanes suggests that radicals might be formed during cold storage. As cold temperature is associated with reduced metabolic and enzymic activity, the notion that cold temperature causes free radical production appeared less tenable. The objective was, therefore, to seek direct evidence for the free radical production during the cold storage of human renal tubular cells, and to define the roles of extrinsic and intrinsic antioxidants in cold-induced cell injury.nnnMETHODSnHuman renal tubular cells were cold-stored at 4 degrees C for varying duration in University of Wisconsin solution and subjected to mRNA analysis, biochemical measurements, and cytoprotective studies.nnnRESULTSnCold storage caused a time-dependent reduction in glutathione levels, and an increase in the formation superoxide, hydrogen peroxide, and hydroxyl radicals. Cold-induced lactate dehydrogenase (LDH) release, ATP depletion, DNA damage, and membrane degradation were suppressed with the inclusion of antioxidant 2-methyl aminochroman or deferroxamine. The cells that were structurally protected with antioxidants were also intact functionally, as they had significantly improved cell proliferation. To examine the effect of cold on intrinsic antioxidant gene expression, antioxidant mRNA levels were analyzed using reverse transcription-polymerase chain reaction. The gene expression of mitochondrial Mn-superoxide dismutase (SOD), but not of cytosolic Cu,Zn-SOD or of glutathione peroxidase expression increased with cold exposure. The oxidant-sensitive gene heme oxygenase I increased slightly with 48-hr cold storage.nnnCONCLUSIONSnCold storage of human tubular cells causes marked increase in free radicals. These are likely of mitochondrial origin as there is a differential inducement of Mn-SOD gene, and are causal to cold-induced cell injury as extrinsic antioxidants abrogated the injury. Our findings support the strategy of adding antioxidants to preservation solutions or the strategy of pre-conditioning the organs to oxidative stress to minimize cold storage-induced organ damage.

Deferoxamine Reduces Cold-Ischemic Renal Injury in a Syngeneic Kidney Transplant Model

In cell‐culture models, addition of deferoxamine (DFO) to University of Wisconsin Solution (UW solution) reduces cold‐storage injury. The efficacy of DFO was therefore tested in a kidney transplantation model employing inbred Wistar Furth rats. Donor left kidneys, cold stored for 18 h in UW solution with or without 0.125 mM or 0.625 mM DFO were transplanted to the recipients left renal fosse. Deferoxamine dose‐dependently and significantly increased glomerular filtration rate (GFR) and renal blood flow (RBF), and suppressed renal F2‐isoprostanes (vasoactive lipid peroxidation products) and apoptotic and necrotic injury 3 days post‐transplantation. In a second set of similar experiments, the remaining native kidneys of the recipient rats were removed on day 7 of transplantation. Transplanted kidneys function assessed by serum creatinine was 75% higher in the cold‐stored transplanted kidneys treated with DFO compared with untreated kidneys. Moreover, the DFO treatment was attended by a significant reduction in apoptotic and necrotic tubular injury. Thus, our consistent findings from two sets of studies in a transplant model suggest that a simple strategy of including DFO in the cold‐storage solution reduces cold ischemia‐associated renal transplant damage and improves renal function. Our findings have potentially important ramifications for cold preservation of kidneys, and possibly other organs, in clinical transplantation.

Vitamin E inhibits renal mRNA expression of COX II, HO I, TGFβ, and osteopontin in the rat model of cyclosporine nephrotoxicity

Background. Ina rat model of cyclosporine (CsA) nephrotoxicity, vitamin E preserves renal function and reduces free radicals, vasoconstrictive thromboxanes, and tubulointerstitial fibrosis. We examined the effect of vitamin E on tubule gene expression in this model. Methods. In two of three groups, rats were treated with either CsA, or CsA plus vitamin E, whereas the control group received vehicles. We pooled purified tubules or whole kidney tissue in a novel manner to represent each treatment group, harvested RNA, and performed rigorously controlled qualitative reverse transcription-polymerase chain reaction. Results. Cyclooxygenase (COX) I mRNA was detectable in control animals, was increased by CsA, but was unchanged by vitamin E. COX II mRNA was detected in controls, was inhibited in the CsA group, and was further inhibited with vitamin E. Hemeoxygenase I and TGF-&bgr; and osteopontin mRNA were increased in the CsA-treated group and were inhibited by vitamin E. Conclusions. Our data support the involvement of free radicals, COX pathways, and pro-fibrotic genes in cyclosporine nephrotoxicity and suggest that the salutary effect of vitamin E involves the suppression of some of these genes.

Overexpression of heme oxygenase protects renal tubular cells against cold storage injury: studies using hemin induction and HO-1 gene transfer.

Heme oxygenase-1 (HO-1), a 32-kd microsomal enzyme, is induced as an adaptive response to a wide variety of injurious stimuli. We examined the possible role of HO-1 in cold storage of renal proximal tubular epithelial (RPTE) cells. Hemin, a potent HO-1-inducer, caused a time-dependent increase in HO-1 mRNA and protein expression. Hemin pretreatment of human RPTE cells before cold storage conferred cytoprotection. Increased HO-1 protein was associated with a brisk and early increase in catalytically active iron and a robust increase in cellular ferritin. Deferoxamine, an iron sequestrating antioxidant, prevented hemin-induced iron release and the increase in ferritin, suggesting iron release as an antecedent mechanism for ferritin induction. To verify that the proximate cause of hemin cytoprotection was due to HO-1 induction, we transiently transfected LL-CPK1 porcine kidney cells with a HO-1 expression vector before cold storage. HO-1 transfection resulted in increased expression of HO-1 protein and reduced cell injury during cold storage. The novel observation that prior induction of HO-1 prevents cold storage–induced cell injury suggests that a similar strategy may prove efficacious in preventing cold storage–induced organ damage during transplantation.

Antiapoptotic properties of erythropoiesis-stimulating proteins in models of cisplatin-induced acute kidney injury

Erythropoietin (Epo) induces erythrocytosis by suppressing erythroid progenitor cell apoptosis through the Janus-activated kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Since apoptosis contributes to cisplatin (CP)-induced nephrotoxicity and Epo receptors (EpoR) are expressed in the kidney, we examined the role of antiapoptosis in recombinant human erythropoietin (rHuEpo)-mediated renal protection. In human renal proximal tubular epithelial (RPTE) cells in culture, rHuEpo, but not inactive rHuEpo (I-rHuEpo), the receptor-binding sites of which are mutated, caused a significant reduction in CP-induced apoptosis at > or = 100 U/ml. rHuEpo, but not I-rHuEpo, increased STAT5 and Akt/PKB phosphorylation, demonstrating functional EpoR expression on RPTE cells. Furthermore, the JAK2 inhibitor tyrphostin AG-490 attenuated rHuEpo protection, suggesting a role of the JAK-STAT pathway in rHuEpo-mediated antiapoptosis. In rats, intravenous administration of 5,000 U/kg rHuEpo, but not an equivalent peptide mass of I-rHuEpo, before a single 5.5 mg/kg iv injection of CP, significantly increased hematocrit (Hct) and reduced the CP-induced increase in serum creatinine. Serum creatinine on day 4 was 3.4 +/- 0.3, 1.9 +/- 0.3, and 3.5 +/- 0.4 mg/dl in the CP, CP + rHuEpo, and CP + I-rHuEpo groups, respectively. Similarly, darbepoietin-alpha (DA), a hyperglycosylated analog of rHuEpo with prolonged in vivo activity when injected at 25 microg/kg iv before CP, significantly increased Hct and reduced serum creatinine. Renal clearance studies based on glomerular filtration rate and renal blood flow confirmed the significant renal protection by DA against CP. Tubular apoptosis and necrosis were significantly reduced in the kidneys of the CP + DA vs. the CP + saline group. Moreover, the equalization of Hct by venesection did not abrogate the DA-mediated renal protection. Administration of DA 48 h after CP injection also conferred significant renal protection. Thus our experiments confirm a role for erythropoiesis-stimulating proteins, including the new analog DA, in limiting CP-induced nephrotoxicity and suggest that antiapoptosis via the Epo-EpoR interaction is an important mechanism for renal protection.

Cold Induces Catalytic Iron Release of Cytochrome P‐450 Origin: A Critical Step in Cold Storage‐Induced Renal Injury

Earlier experimental studies have suggested a role for iron in cold‐storage‐induced organ injury. Whether the cytochrome P‐450 enzymes, shown to be a source for iron in several injury models, contribute to cold‐induced iron release is not known. Storage of human proximal tubular epithelial (RPTE) cells at 4u2003°C in the University of Wisconsin (UW) solution caused a significant and time‐dependent increase in bleomycin‐detectable iron (BDI). To identify the cellular source of BDI, RPTE cells were subfractionated and stored at 4u2003°C for 4u2003h. Bleomycin‐detectable iron release was highest in the microsomes, next in the cytosol and none in the mitochondria. As microsomes are rich in iron‐containing cytochrome P‐450 enzymes, microsomes were cold stored with P‐450 inhibitors, cimetidine and piperonyl butoxide. P‐450 inhibitors significantly reduced cold‐induced BDI release. Furthermore, cimetidine and iron chelator deferoxamine (DFO) significantly reduced cold‐induced cell injury, suggesting a role for P‐450‐derived iron in cold‐induced cell injury. In rat kidney experiments, BDI and LDH release were significantly higher in cold‐stored kidneys than in control kidneys. Inclusion of cimetidine and DFO in the cold‐storage solution significantly suppressed the BDI and LDH release, and reduced the ultrastructural changes. Our data demonstrate for the first time that cold‐induced catalytic iron release may be at least in part of microsomal cytochrome P‐450 origin, and that it participates in cold‐storage‐induced renal injury. In the clinical setting, sequestering free iron released during cold storage is possible and may prove to be useful in limiting organ injury.

Fenoldopam preconditioning: Role of heme oxygenase-1 in protecting human tubular cells and rodent kidneys against cold-hypoxic injury

Background. Kidneys from brain-dead donors are cold preserved until transplanted. However, prolonged cold storage can contribute to allograft failure. Studies suggest that donor preconditioning with dopaminergics may reduce cold-ischemic transplant injury, but whether heme oxygenase (HO)-1 induction is an underlying mechanism is not known. Objective. To test whether preconditioning with fenoldopam (FD) induce HO-1 and protect kidneys against cold storage injury and whether HO-1 plays a role in protection. Method. We used human renal proximal tubular epithelial cells, rat kidney transplants, and HO-1(−/−) null mice kidneys. Results. FD preconditioning of cells for 4 hr significantly protected against cell death from 24-hr cold hypoxia and was associated with a dose-dependent increase in HO-1 expression. In a syngeneic rat kidney transplant model, FD preconditioning for 18 hr markedly increased kidney HO-1 expression and protected kidneys against 24-hr cold-ischemic transplant injury. To test the role of HO-1, renal proximal tubular epithelial cells were treated with HO-1 small interfering RNA, followed by FD-preconditioning. Small interfering RNA inhibited the HO-1 messenger RNA expression and reversed the FD protection. Suspension of kidneys of HO-1(−/−) null and wild-type mice preconditioned with FD or saline were subjected to 24- and 48-hr cold storage. N-acetyl glucosaminidase, a specific tubular injury marker, was significantly lower in FD-preconditioned wild-type kidneys, but not in HO-1 null kidneys, suggesting a role for HO-1 in FDs preconditioning. Conclusion. Our data suggest HO-1 induction as an underlying mechanism for FD preconditioning and support the idea of testing FD preconditioning in the clinical setting. Studies are required to determine the optimum FD-preconditioning protocol.