Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model.

BACKGROUND: Prolonged cold ischemia is a risk factor for delayed graft function of kidney transplants, and is associated with caspase-3-mediated apoptotic tubular cell death. We hypothesized that treatment of tubular cells and donor kidneys during cold storage with a caspase inhibitor before transplant would reduce tubular cell apoptosis and improve kidney function after transplant. METHODS: Mouse tubular cells were incubated with either dimethyl sulfoxide (DMSO) or Q-VD-OPh during cold storage in saline followed by rewarming in normal media. For in vivo studies, donor kidneys from C57BL/6 mice were perfused with cold saline, DMSO (vehicle), or QVD-OPh. Donor kidneys were then recovered, stored at 4°C for 60 minutes, and transplanted into syngeneic C57BL/6 recipients. RESULTS: Tubular cells treated with a caspase inhibitor had significantly reduced capsase-3 protein expression, caspase-3 activity, and apoptotic cell death compared with saline or DMSO (vehicle) in a dose-dependent manner. Treatment of donor kidneys with a caspase inhibitor significantly reduced serum creatinine and resulted in significantly less tubular cell apoptosis, BBI, tubular injury, cast formation, and tubule lumen dilation compared with DMSO and saline-treated kidneys. CONCLUSIONS: Caspase inhibition resulted in decreased tubular cell apoptosis and improved renal function after transplantation. Caspase inhibition may be a useful strategy to prevent cold ischemic injury of donor renal grafts.

Role of Donors and Recipients' Glutathione S-Transferase Gene Polymorphisms in Association of Oxidative Stress With Delayed Graft Function in Kidney Allograft Recipients.

INTRODUCTION: Oxidative stress contributes to delayed graft function (DGF). Glutathione S-transferases (GSTs) are polymorphic genes which produce enzymes with protective effect against oxidative stress. This study aimed to investigate the association between donors' and recipients' GSTM1 and GSTT1 polymorphisms and DGF, creatinine clearance, and oxidative stress parameters in kidney allograft recipients. MATERIALS AND METHODS: One hundred and eighty-two donor-recipient pairs were studied. Lipid peroxidation and total antioxidant capacity were measured in the recipients' plasma as the parameters of oxidative stress. Delayed graft function was determined based on at least 10% increase, no change, or less than 10% decrease in the serum creatinine level in 3 consecutive days during the 1st week after transplantation. RESULTS: Lipid peroxidation was significantly greater in the recipients with DGF (P < .001). The frequency of GSTM1 null was significantly higher in the patients with DGF (odds ratio [OR], 0.38; 95% confidence interval [CI], 0.17 to 0.86; P = .02). There was also a significant association between the donors' GSTM1 polymorphism and DGF (OR, 0.31; 95% CI, 0.14 to 0.68; P = .003). A significant association was detected between combination of recipients and donors' GSTM1 polymorphism and DGF (OR, 0.20; 95% CI, 0.07 to 0.64, P = .006). The recipients' GSTM1 polymorphism, alone and in combination with donors' GSTM1 and GSTT1, significantly affected the creatinine clearance on discharge day. CONCLUSIONS: These results suggest that the donors and recipients' GSTM1 polymorphism may be a major risk factor for oxidative stress and poor kidney allograft transplantation outcomes.

The hypoxanthine-xanthine oxidase axis is not involved in the initial phase of clinical transplantation-related ischemia-reperfusion injury.

The hypoxanthine-xanthine oxidase (XO) axis is considered to be a key driver of transplantation-related ischemia-reperfusion (I/R) injury. Whereas interference with this axis effectively quenches I/R injury in preclinical models, there is limited efficacy of XO inhibitors in clinical trials. In this context, we considered clinical evaluation of a role for the hypoxanthine-XO axis in human I/R to be relevant. Patients undergoing renal allograft transplantation were included (n = 40) and classified based on duration of ischemia (short, intermediate, and prolonged). Purine metabolites excreted by the reperfused kidney (arteriovenous differences) were analyzed by the ultra performance liquid chromatography-tandem mass spectrometer (UPLCMS/MS) method and tissue XO activity was assessed by in situ enzymography. We confirmed progressive hypoxanthine accumulation (P < 0.006) during ischemia, using kidney transplantation as a clinical model of I/R. Yet, arteriovenous concentration differences of uric acid and in situ enzymography of XO did not indicate significant XO activity in ischemic and reperfused kidney grafts. Furthermore, we tested a putative association between hypoxanthine accumulation and renal oxidative stress by assessing renal malondialdehyde and isoprostane levels and allantoin formation during the reperfusion period. Absent release of these markers is not consistent with an association between ischemic hypoxanthine accumulation and postreperfusion oxidative stress. On basis of these data for the human kidney we hypothesize that the role for the hypoxanthine-XO axis in clinical I/R injury is less than commonly thought, and as such the data provide an explanation for the apparent limited clinical efficacy of XO inhibitors.

Protection of the Transplant Kidney from Preservation Injury by Inhibition of Matrix Metalloproteinases.

BACKGROUND: Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, play an important role in ischemic injury to the heart, yet it is not known if these MMPs are involved in the injury that occurs to the transplant kidney. We therefore studied the pharmacologic protection of transplant kidneys during machine cold perfusion. METHODS: Human kidney perfusates were analyzed for the presence of injury markers such as cytochrome c oxidase, lactate dehydrogenase, and neutrophil-gelatinase associated lipocalin (NGAL), and MMP-2 and MMP-9 were measured. The effects of MMP inhibitors MMP-2 siRNA and doxycycline were studied in an animal model of donation after circulatory determination of death (DCDD). RESULTS: Markers of injury were present in all analyzed perfusates, with higher levels seen in perfusates from human kidneys donated after controlled DCDD compared to brain death and in perfusate from kidneys with delayed graft function. When rat kidneys were perfused at 4°C for 22 hours with the addition of MMP inhibitors, this resulted in markedly reduced levels of MMP-2, MMP-9 and analyzed injury markers. CONCLUSIONS: Based on our study, MMPs are involved in preservation injury and the supplementation of preservation solution with MMP inhibitors is a potential novel strategy in protecting the transplant kidney from preservation injury.

Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

Delayed graft function (DGF) is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2), as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2) that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC), in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are associated with tubular injury in DGF and offer novel therapeutic targets for the prevention of this disorder.

Glutathione S-transferase iso-enzymes in perfusate from pumped kidneys are associated with delayed graft function.

Accurate and reliable assessment tools are needed in transplantation. The objective of this prospective, multi-center study was to determine the associations of the alpha and pi iso-enzymes of glutathione S-transferase (GST), measured from perfusate solution at the start and end (base and post) of kidney allograft machine perfusion, with subsequent delayed graft function (DGF). We also compared GST iso-enzyme perfusate levels from discarded versus transplanted kidneys. A total of 428 kidneys were linked to outcomes as recorded by the United Network of Organ Sharing. DGF, defined as any dialysis in the first week of transplant, occurred in 141 recipients (32%). Alpha- and pi-GST levels significantly increased during machine perfusion. The adjusted relative risks (95% confidence interval) of DGF with each log-unit increase in base and post pi-GST were 1.14 (1.0-1.3) and 1.36 (1.1-1.8), respectively. Alpha-GST was not independently associated with DGF. There were no significant differences in GST values between discarded and transplanted kidneys, though renal resistance was significantly higher in discarded kidneys. We found pi-GST at the end of machine perfusion to be independently associated with DGF. Further studies should elucidate the utility of GST for identifying injured kidneys with regard to organ allocation, discard and recipient management decisions.

Urinary exosomes as a source of kidney dysfunction biomarker in renal transplantation.

End-stage renal disease (ESRD) requires for its treatment permanent dialysis or kidney transplantation (KT). KT is the best clinical treatment, however, the early function of the allograft varies depending on multiple factors associated with cold ischemia time (CIT) and the allograft rejection process. It is known that serum creatinine is an insensitive and late marker for predicting graft recovery after KT, mainly in patients with delayed graft function (DGF). Neutrophil gelatinase-associated lipocalin (NGAL) is produced in the distal nephron and it is one of the most promising novel biomarkers for acute kidney injury (AKI) and chronic kidney disease (CKD). NGAL has been proposed to be a predictor of organ recovery from DGF after KT from donors after cardiac death. Because nonrenal diseases can also induce NGAL, more information is necessary to validate the sensitivity and specificity of urine and plasma NGAL in clinical samples. The exosomes are vesicles released into the urine from the kidney epithelium and they have been proposed as better source to explore as biomarker of renal dysfunction. The molecular composition of the urinary exosomes could be representative of the physiological or physiopathologic condition of the urinary system. We propose that determination of NGAL in urinary exosomes is a better predictor of kidney dysfunction after KT than other urinary fractions. We analyzed 15 kidney allograft recipients, with a mean age of 36 years (range, 16-60 years) and 75% were male: 11 living donors (LD) and 4 deceased donors (DD). The average length of CIT was 14 hours in DD and less than 1 hour in LD. Three patient developed DGF. Using Western blot analysis, NGAL was detectable in the cellular and exosomal fraction of the urine. The exosomes expressed higher levels of NGAL than the cellular fraction. The expression of NGAL was observed from the first day after transplantation. In the cellular fraction of the urine, no significant differences of NGAL were observed between the patients. However, the median of NGAL expression in the exosomes fraction was significantly higher in DD patient, from the first day after KT (P < .05). Moreover, we noticed that NGAL expression in exosomes remained elevated in the patients with DGF compared with non-DGF patients (P < .05). Considering the highest abundance of NGAL in the urinary exosomes and its correlation with DGF patients, we suggest the exosomal fraction as a more sensitive substrate to evaluate early biomarkers of DGF after KT.

Serum aminoacylase-1 is a novel biomarker with potential prognostic utility for long-term outcome in patients with delayed graft function following renal transplantation.

Early identification and prognostic stratification of delayed graft function following renal transplantation has significant potential to improve outcome. Mass spectrometry analysis of serum samples, before and on day 2 post transplant from five patients with delayed graft function and five with an uncomplicated transplant, identified aminoacylase-1 (ACY-1) as a potential outcome biomarker. Following assay development, analysis of longitudinal samples from an initial validation cohort of 55 patients confirmed that the ACY-1 level on day 1 or 2 was a moderate predictor of delayed graft function, similar to serum creatinine, complementing the strongest predictor cystatin C. A further validation cohort of 194 patients confirmed this association with area under ROC curves (95% CI) for day 1 serum (138 patients) of 0.74 (0.67-0.85) for ACY-1, 0.9 (0.84-0.95) for cystatin C, and 0.93 (0.88-0.97) for both combined. Significant differences in serum ACY-1 levels were apparent between delayed, slow, and immediate graft function. Analysis of long-term follow-up for 54 patients with delayed graft function showed a highly significant association between day 1 or 3 serum ACY-1 and dialysis-free survival, mainly associated with the donor-brain-dead transplant type. Thus, proteomic analysis provides novel insights into the potential clinical utility of serum ACY-1 levels immediately post transplantation, enabling subdivision of patients with delayed graft function in terms of long-term outcome. Our study requires independent confirmation.

Protein kinase C inhibition ameliorates posttransplantation preservation injury in rat renal transplants.

BACKGROUND: Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca²âº-dependent and Ca²âº-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation. METHODS: Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry. RESULTS: Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal-regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action. CONCLUSIONS: The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Lack of association of the rs2476601 PTPN22 gene polymorphism with transplanted kidney function.

BACKGROUND: PTPN22 gene, located on the long arm of chromosome 1, encodes PTPN22 protein, known as lymphoid tyrosine phosphatase (Lyp). The function of Lyp is suggested to be negative regulation of T-cell reaction through dephosphorylation of Src family kinases--Lck and Fyn, negative regulatory kinase Csk and other signaling molecules. Several studies suggested that individuals lacking the C allele of the C1858T PTPN22 gene polymorphism may have reduced capacity to downregulate T-cell response. Therefore, they may present changes in immunological reaction and be more susceptible to autoimmunity. The aim of this study was to examine the association of the rs2476601 (C1858T) PTPN22 gene polymorphism with transplanted kidney function. MATERIAL/METHODS: The study enrolled 269 Caucasian renal transplant recipients (166 males, 103 females, mean age 47.63±12.96 years). Genotyping of the rs2476601 (C1858T) PTPN22 gene polymorphism was performed using the PCR-RFLP method. RESULTS: Comparison of the distribution of genotypes and alleles of the rs2476601 PTPN22 gene polymorphism among patients with delayed graft function (DGF) and without DGF revealed no statistically significant differences (OR=0.69, 95%CI=0.37-1.28, p=0.29) for TT+CT vs. CC genotypes. Similarly, there were no statistically significant differences in regard to the studied polymorphism and acute rejection (OR=0.79, 95%CI=0.41-1.52, p=0.52) or chronic allograft nephropathy (CAN) occurrence (OR=1.22, 95%CI=0.64-2.32, p=0.61). CONCLUSIONS: We found no association between rs2476601 (C1858T) PTPN22 gene polymorphism and transplanted kidney function.

Clinical evidence of the association between serum perioperative changes in xanthine metabolizing enzymes activity and early post-transplant kidney allograft function.

BACKGROUND: Xanthine oxidoreductase (XOR) and its active forms, dehydrogenase (XD) and oxidase (XO), act as double-edged swords during ischemia-reperfusion injury. On the one hand, their action generates antioxidants, such as uric acid (UA); however, they may strongly enhance production of free radicals. In this study, we examined the association between post-transplant graft function and perioperative xanthine metabolizing enzymes (XME) activity in kidney transplant recipients divided into early (EGF), slow (SGF), and delayed graft function (DGF) groups. STUDY DESIGN: XME activity and UA levels were measured in blood samples collected directly before and during the first and fifth minutes of reperfusion. RESULTS: Results demonstrated an increase in XO and XOR activity in all groups; however, these parameters were lower in the EGF than in the DGF group (p < 0.005; p < 0.05). XD activity increased in SGF and DGF patients (p = 0.01); nevertheless, the XD/total XOR coefficient decreased only in DGF individuals (p = 0.0007). XME sensitivity, specificity, and positive and negative predictive values in discriminating SGF/DGF from EGF were 73.3% to 78%, 54% to 62.5%, 76% to 78.6%, and 56.5%, respectively. Moreover, mixed model analysis revealed that recipients classified according to results of XOR(5) and XO(5) significantly differ in 1-year post-transplant allograft function (p = 0.04 and p = 0.02, respectively), but not in the frequency of acute rejection episodes (p = 0.66 and p = 0.90, respectively). CONCLUSIONS: During renal transplantation, significant changes in XME occur that are associated with early post-transplant graft function and have potential value to discern between EGF and SGF/DGF.

The association of -262C/T polymorphism in the catalase gene and delayed graft function of kidney allografts.

BACKGROUND: Catalase is an intracellular antioxidant enzyme that is mainly located in cellular peroxisomes and in the cytosol. This enzyme plays a significant role in the development of tolerance to oxidative stress in the adaptive response of cells and tissues. The aim of the present study was to examine the association between the -262C/T polymorphism in the catalase gene and delayed graft function (DGF), acute rejection and chronic allograft nephropathy of kidney allografts. METHODS: One hundred eighty-seven recipients of first renal transplants were included in the study. The histories of the patients were analysed regarding DGF, acute rejection and chronic allograft nephropathy. The polymorphism -262C/T in the catalase gene was analysed using the polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The risk of DGF was significantly lower in T allele carriers compared with CC homozygotes: odds ratio = 0.34, 95% confidence interval = 0.17-0.67, P = 0.001. There were no statistically significant associations between the studied polymorphism and acute rejection or chronic allograft nephropathy. CONCLUSION: The results of this study suggest that -262C/T polymorphism in the catalase gene is associated with DGF in kidney allograft recipients.

Serum tissue inhibitor of metalloproteinases 1 (TIMP-1) predicts organ recovery from delayed graft function after kidney transplantation from donors after cardiac death.

Donors after cardiac death (DCD) have recently become an important source of renal transplants to alleviate the shortage of renal grafts in kidney transplantation (KTx), although DCD kidneys often have complications associated with a delayed graft function (DGF). A microarray-based approach using renal biopsy samples obtained at 1 h after KTx from DCD identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) gene as a potential predictive marker for DGF. The current study measured serum TIMP-1 in patients undergoing KTx and analyzed the time course after KTx. The average serum TIMP-1 level before KTx was 240 ± 10 ng/ml (n = 34). In patients undergoing KTx from a living donor (n = 23), the serum TIMP-1 levels showed no increase after KTx (POD1: 226 ± 12, POD2: 211 ± 12, and POD3: 195 ± 10 ng/ml), but in one case, the only patient who required post-KTx HD due to DGF, the level on POD1 was the highest among subjects (361 ng/ml). In contrast, patients undergoing KTx from DCDs (n = 11), the serum TIMP-1 levels increased rapidly after a KTx (POD1: 418 ± 32, POD2: 385 ± 42, and POD3: 278 ± 25 ng/ml). However, two patients who avoided post-KTx HD due to the immediate function of the graft did not show increased levels (<370 ng/ml) on either POD1 or POD2. The peak serum TIMP-1 values appeared to correlate to the post-KTx dialysis period. Furthermore, the increment of serum TIMP-1 on the early POD was found to be predictive of immediate or delayed function of the grafts. These data suggest that monitoring of serum TIMP-1 levels allow the prediction of graft recovery and the need for HD after a KTx from a DCD.

[The state of lipid peroxidation and antioxidant defense system in recipients of renal allotransplant].

The peroxidal oxidation of lipids (POL) and antioxidant defense (AOD) system state in the patients blood before renal transplantation performing and on the fourth day after it were studied, depending on kind of familial or potential agonal with beating (DBH) or nonbeating (DNBH) heart donor applied and the presence of complications--delayed renal autotransplant (RAT) function and acute reaction of rejection (ARR). In chronic renal insufficiency, RAT transplantation from cadaver, DNBH and ARR the POL processes intensification and AOD system activity lowering were noted.

Protein levels of heme oxygenase-1 during reperfusion in human kidney transplants with delayed graft function.

INTRODUCTION: Delayed graft function (DGF) as a consequence of ischemia reperfusion injury (IRI) is associated with a decrease in long-term allograft survival. Heme oxygenase-1 (HO-1) is a stress responsive gene that is highly expressed in multiple pathological processes. The aim of our study was to analyze whether HO-1 protein levels in human kidney transplants during IRI correlate with the incidence of DGF. METHODS: Kidney biopsies were obtained from 27 kidney allografts at two time points: at the end of cold storage and shortly after reperfusion. Samples were analyzed for HO-1 protein levels by Western blot. RESULTS: Heme oxygenase-1 protein levels were significantly higher in post-reperfusion biopsies (39.4 vs. 13.7 arbitrary units, p = 0.001). In pre-reperfusion biopsies no association was observed between HO-1 protein levels and DGF. In post-reperfusion biopsies, higher levels of HO-1 protein were measured in kidneys with DGF (53.7 vs. 36.2 arbitrary units, p = 0.064). DGF kidneys showed a significantly higher increase from pre- to post-reperfusion in HO-1 protein (42.0 vs. 18.7 arbitrary units, p = 0.025). CONCLUSION: Heme oxygenase-1 protein levels shortly after allograft reperfusion are closely related with initial graft function. Assessment thereof may be considered a valuable tool to predict DGF.

Activity of CuZn-superoxide dismutase, catalase and glutathione peroxidase in erythrocytes in kidney allografts during reperfusion in patients with and without delayed graft function.

BACKGROUND: Generation of reactive oxygen species (ROS) is the main mechanism involved in the ischemic/reperfusion damage of the transplanted organ. Oxygen burst is a trigger for complex biochemical events leading to generation of oxygenated lipids and changes in microcirculation. Many markers have been researched to prove the presence of ROS in the transplanted tissue. Some of them, like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are considered to play a major role in graft protection against oxygen stress during reperfusion. METHODS: The aim of this study was to examine the changes of SOD1, CAT and GPx activity in erythrocytes during the first minutes after total graft reperfusion. Forty patients undergoing kidney transplantation at our center were assigned to two groups: with or without delayed graft function (DGF). Before anastomosing kidney vessels with recipient's iliac vessels, the '0' blood sample was taken from the iliac vein. Next blood samples I, II and III were taken from the graft's renal vein. The reperfusion of the transplanted kidney was evaluated precisely with the thermovision camera. Erythrocyte SOD1, CAT and GPx activity was measured with a spectrophotometric method. RESULTS: We did not observe statistically significant changes in SOD1, CAT and GPx activity in erythrocytes during the early phase of reperfusion in patients with and without DGF. CONCLUSIONS: Erythrocyte-antioxidative system in graft's vein remain stable during the early phase of reperfusion. The results of the study suggest that further studies on extracellular enzymes are required for the assessment of antioxidant system in the conditions of ischemia/reperfusion.