Renal Preservation Injury Research Articles

Mouse IPK: A Powerful Tool to Partially Characterize Renal Reperfusion and Preservation Injury.

Main ProblemThe molecular basis of renal preservation injury is not well understood. Since mouse kidney transplantation models are not useful in this setting, a mouse Isolated Perfused Kidney (IPK) model was developed to take advantage of mouse genetic design capabilities for testing complex biological hypothesis regarding mechanisms of preservation injury in transplanted kidneys.MethodsMouse kidneys were recovered, preserved, and reperfused in-vitro with an acellular physiological crystalloid buffer containing hypo-physiological oncotic pressure. Outcome variables were measured to predict preservation injury. These included perfusate flow, vascular resistance, VO2, urine output, GFR, proteinuria, LDH release, and edema. The model was tested by subjecting mouse kidneys to cold storage in University of Wisconsin (UW) solution for 24, 48, or 72 hours (time-dependent preservation injury), cold storage in Euro-Collins Solution (solution dependent preservation injury), and exposure to prior warm ischemia (DCD dependent preservation injury).ResultsThe model accurately predicted the qualitative and quantitative changes in the readouts based on known responses to preservation injury in kidney transplants in large animals and humans.ConclusionThe mouse IPK accurately predicts many of the variables associated with renal organ preservation injury in the very early phases of reperfusion and may provide an attractive model for studying the molecular basis of renal preservation injury.

Involvement of ezrin in cold storage preservation injury in renal proximal tubule epithelial cells: Possible structural and signaling roles

Renal transplantation is the treatment of choice for patients with end‐stage renal disease. Preservation injury is caused by ischemia/reperfusion and hypothermia during cold storage, which leads to early graft loss and delayed graft function. Advancements in organ preservation are limited by our understanding of basic root mechanisms that cause this injury. We hypothesize that sub‐lamellar cytoskeletal protein failure is involved and studied this in isolated rabbit renal proximal tubules and epithelial cells. Tubules cold stored in UW solution released ezrin from cytoskeletal actin attachments. Ezrin phosphorylation at T567 and Y353 decreased with progressive cold storage ischemia, which controls both ezrin binding to the cytoskeleton and survival signaling through Akt, respectively. Tubule epithelial cell death at reperfusion after cold storage ischemia was almost exclusively by apoptosis (annexin‐V FACS analysis). Transfection of primary renal epithelial cells with wild‐type mammalian ezrin cDNA increased survival and viability after cold storage ischemia and reperfusion and increased Akt phosphorylation (IHC). Conversely, inhibition of ezrin expression with specific siRNA duplexes decreased cell survival and viability. In conclusion, inactivation of ezrin during preservation may cause renal preservation injury by altering membrane structure and by reducing survival signaling.

Cytoskeletal Involvement in Hypothermic Renal Preservation Injury

Cytoskeletal degradation occurs in warm renal ischemia and reperfusion and during hypothermia. The purpose of this study was to determine cytoskeletal changes during cold storage preservation injury in renal tubules and to determine whether these changes contribute to the injury. Isolated canine renal proximal tubules or their primary epithelial cultures were cold-stored in University of Wisconsin solution and reperfused in vitro to simulate renal cold storage preservation injury. Assays for cytoskeletal protein degradation and viability together with biologically active cytoskeletal agents and molecular interventions were used to test this hypothesis. Progressively increasing the cold storage time of isolated renal tubules in University of Wisconsin solution caused proportional disassembly of both ezrin and Na/K adenosine triphosphatase proteins from their cytoskeletal attachments. The sublamellar structural protein, fodrin, was metabolized to products consistent with calpain hydrolysis during preservation. Time-dependent deterioration in tubule membrane function (organic cation transporter-1 transport activity) was observed during cold preservation, and this was mimicked in fresh tubules by chemically induced cytoskeletal disruption with cytochalasin-D treatment. Cold preservation decreased total tubulin content. Taxol slowed this loss by preventing tubulin depolymerization, which improved membrane function and tubule viability. The viability of primary renal epithelial cells was enhanced by overexpression of ezrin (transfection) and diminished with specific ezrin small interfering RNA knockdown during cold preservation injury. Hypothermic storage preservation causes disruption of key cytoskeletal elements in kidney tubules, which contributes causally to the injury at rewarming.

Role of Peroxynitrite Anion in Renal Hypothermic Preservation Injury

Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0-120 h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.

The effects of donor brain death on renal function and arachidonic acid metabolism in a large animal model of hypothermic preservation injury.

Donor brain death (BD) has been implicated as a risk factor for the poor performance of kidneys after transplantation in small but not large animal models. This study determined the effects of donor BD on renal function and lipid mediator metabolism in a large animal model of renal hypothermic preservation injury. Adult beagle donors were subjected to explosive BD for 16 hr. After BD, the kidneys were removed, cold stored for 24 hr in cold University of Wisconsin solution, and allotransplanted into recipient dogs for either 4 hr (group 1) or 7 days (group 2). Controls for both groups consisted of kidneys obtained from living donors. Renal allograft function and tissue arachidonic acid (AA) metabolism were determined after reperfusion. Short-term renal function after transplantation was generally unaffected by BD. Renal blood flow decreased after reperfusion but was not altered during the 16-hr BD period. Neutrophil infiltration significantly increased in kidneys from brain-dead donors before storage and after 4 hr of reperfusion. Renal cortex and medulla AA metabolism were not significantly affected by BD after short-term reperfusion except when thiol-ether leukotrienes (LTC(4)/D(4)/E(4)) were increased with BD. Serum creatinine was elevated during 7 days, but, surprisingly, BD significantly attenuated this injury. BD in large mammals does not significantly affect renal allograft function or AA metabolism after transplantation. The role of BD in human renal preservation injury and inflammation should be reevaluated.

Protective effects of glycine during hypothermic renal ischemia-reperfusion injury

The objective of this investigation was to test the effects of glycine, a cytoprotectant in normothermic in vitro models of renal ischemia, in a model of hypothermic renal preservation injury. This study also probes possible physiological mechanisms of glycine protection during renal hypothermic ischemia-reperfusion injury. Canine kidneys were subjected to 48 h of hypothermic ischemia (4 degrees C) after intravascular flush with cold conventional Collins solution (G. H. Collins, M. B. Bravo-Shugarman, and P. I. Terasaki, Lancet 2: 1219-1223, 1969) and were subsequently revascularized for 1 h. After 1 h of reperfusion, glomerular filtration rate, urine production, and electrolyte excretion were dramatically higher when the Collins flush contained 5 mM glycine, compared with the 0 mM glycine controls. Renal tissue adenine nucleotides and glutathione levels progressively declined with graded cold ischemia times, and glycine had no effect on these levels. However, renal tissue ATP levels (but not glutathione) were significantly higher when kidneys were flushed with glycine, stored for 48 h, and reoxygenated in vitro for 1 h at 37 degrees C, compared with kidneys flushed without glycine. Analysis of CoA esters from ischemic renal tissue indicated altered production of only butyryl CoA after 48 and 72 h of cold ischemia, but no differences were detected in glycine or control kidneys. In conclusion, this study reports dramatic functional preservation with glycine in kidneys subjected to hypothermic ischemia and in vivo reperfusion. The mechanisms of these effects appear not to be attributable to the maintenance of cellular adenine nucleotide or glutathione levels nor to the scavenging of accumulated amphipathic acyl CoA esters.

Prostanoids and hypothermic renal preservation injury

The effect of 48 hours of hypothermic renal ischemia utilizing Euro-Collins flush and short term reperfusion on renal prostaglandin synthesis was studied in dogs. Hypothermic ischemia followed by 60 minutes of reperfusion in-vivo resulted in significant elevations in renal Thromboxane B 2 (TXB 2) production in the outer cortex, inner cortex, and medulla, relative to non-ischemic kidneys. Prostaglandin E 2 (PGE 2) and 6-keto Prostaglandin F 1α (6-K PGF 1α) production were not significantly affected by ischemia and reperfusion. Enhanced TXB 2 saturated buffer, indicating a blood born source or stimuli. Early postreperfusion renal blood flow after hypothermic ischemia followed a biphasic pattern; blood flow increased for the first 10 minutes of reperfusion to achieve normal values, and then steadily declined over the next 20 minutes. This pattern was not altered by the cyclooxygenase inhibitors Indomethacin (5 mg/kg, P.O.) or Mefenamic acid (10 mg/kg, I.V.). Administration of the TXA 2 synthesis inhibitor CGS-12970 (3 mg/kg, I.V.) or the TXA 2/endoperoxide receptor antagonist SQ-29548 (80 μg/min, I.A.) significantly increased renal blood flow during reperfusion but neither agent altered the basic time dependent pattern observed in the control group. These data indicate that 48 hours of hypothermic renal ischemia results in dramatic changes in intrarenal TXA 2 synthesis at the time of reperfusion. Enhanced TXA 2 production is not dependent on reoxygenation per se, but rather requires reperfusion with blood suggesting a circulatory source. Altered renal prostanoid production does not appear to play a role in postreperfusion vasoconstriction but inhibition of TXA 2 synthesis or receptor activation unmasks a hyperdynamic renal blood flow state during reperfusion after 48 hours of hypothermic ischemia.

The physiology of renal preservation injury: I. Freezing injury and its modification

A method of analysis is described which evaluates the biochemical and physiological status of kidneys after preservation experiments and identifies the injury of preservation in a numerical way. These methods have been applied to the study of freezing injury in kidneys through experimental efforts to freeze slices of intact renal cortex in a viable state. Successful freezing as measured in this assay system has not yet been accomplished, but differing patterns of improvement have been seen after cryoprotection with 15% dimethyl sulfoxide and after eryoprotection plus control of freezing rate at −1°C per min. In addition, kidney cortex tolerates 15% DMSO without measurable injury. The techniques of in vitro analysis have been shown to be applicable to the further study of freezing of intact tissue and organs and will be helpful in the solution of the problems in at least two of three major areas involved in the successful freeze preservation of whole organs.