Kidney transplantation is the most likely therapy for end-stage renal diseases. Despite improvements immunosuppressive protocols, the rate of chronic graft loss is not altered (1). During transplantation, cold preservation and warm reperfusion induce ischemia reperfusion injury (IRI), a complex process involving oxidative stress, mitochondrial uncoupling (2), coagulation cascade (3), and innate immune response, independently of allogenicity (4), characterized by intense immune cells invasion in the kidney (5). IRI correlates with delayed graft function (6) and chronic graft failure (7). Better management of organ preservation could improve IRI, optimize outcome and mitigate early and long-term complications.
During preservation, ATP-depletion compromises the ionic balance between the intracellular (high-K+, low-Na+) and the extracellular (low-K+, high-Na+) compartments. Some preservation solutions such as University of Wisconsin (UW), gold standard in organ transplant, adopt high-K+ and low-Na+ to maintain ionic balance. However, high potassium alters cellular polarization, ATP content and calcium balance (8), participating in the “no reflow” phenomenon (9). Moreover, recent work suggests equal or improved results with low-K+/high-Na+ solutions (10).
Edema, characterized by mitochondrial and cell swelling, is mitigated with colloids. UW contains hydroxyethyl starch (HES), however, it increases red blood cell aggregation (11) and tubular damage (12). New solution are emerging, using polyethylene glycol (PEG), which provides interesting benefits (13): PEG interacts with cell membranes and water molecules, creating layers of “structured water” providing “immunocamouflage” (14).
The first report of PEG benefits in preservation dates back to 1991 in human heart transplantation (15) where PEG 20 kDa (50 g/L) decreased the rate of acute rejection. Several other studies confirmed the benefits of PEG for preservation of liver (16, 17), pancreas (18), pancreatic islets (19–21), small bowel (22), lung (23), and RBCs (24–26). In the kidney, previous studies showed that PEG 20 kDa (30 g/L) (23, 27–32) and 35 kDa (33–35) in low-Na+/high-K+ solution protects kidneys from IRI (for a review, see Ref. 13).
The aim of the present study is to determine which ionic composition and colloid are optimal for organ preservation. We compared three solutions: UW, the gold standard; IGL-1 (Institute George Lopez-1), low potassium solution in which HES is replace by PEG 35 kDa at low dose (1 g/L); and SCOT (Solution de Conservation des Organes et Tissus), of ionic composition similar to plasma, containing a higher dose of PEG 20 kDa (30 g/L). Compositions are exposed in the Supplemental Table (see Supplemental Digital Content 1,http://links.lww.com/TP/A368).
SCOT Improved Cell Resistance to IRI
Cell survival assays MTT and lactate dehydrogenase (LDH) showed a significant superiority of SCOT over other preservation solutions (P<0.05). SCOT-preserved cells had significantly more ATP than cells preserved with UW and IGL-1 (P<0.05). Caspase 3 activity suggested high apoptosis in UW group, while IGL-1 group showed significantly lower caspase 3 activity (P<0.05), SCOT group displayed the lowest activity (P<0.05; Fig. 1A).
Grafts Preserved With SCOT Have Better Function Recovery
In vivo, urine production resumed at day 1 for SCOT grafts, versus day 3 for IGL and UW animals (Fig. 1B). Serum creatinine showed better function recovery for SCOT (P<0.05), followed by IGL grafts. UW animals presented the latest peak and slowest recovery (Fig. 1C). Histology showed the least amount of lesions in SCOT grafts (P<0.05). IGL group displayed better histology on some parameters compared with UW (P<0.05, Fig. 1D and E). Measure of tubular enzymes activity in the urine confirmed the superiority of SCOT in preserving graft integrity, with significant benefits of IGL observed at day 3 (P<0.05, Fig. 1E).
SCOT Mitigated Innate Immune Response
UW grafts displayed a growing invasion of ED1+ cells (Fig. 2A), while IGL1 kidneys showed less invading cells (P<0.05 to UW). SCOT grafts retained the lowest amount of invasion (P<0.05 to UW and IGL-1). Western blot analysis of MCP-1 expression (Fig. 2B) and real time-quantitative polymerase chain reaction (RT-qPCR) assay of β-2 microglobulin, a major histocompatibility (MHC) class I subunit in charge of antigen presentation (Fig. 2C), at 3 months confirmed this trend.
SCOT Reduced Adaptative Immune Response Development
We found intense immunostainings for CD4+ (Fig. 2D) and CD8+ cells (Fig. 2E) in UW grafts, while IGL-1 grafts showed less invasion (P<0.05). SCOT kidneys retained a low number of adaptative immune cells throughout the duration of the experiment. Similar findings were found with evaluation of MHC class II protein expression: swine lymphocytes alloantigen (SLA)-DR (Fig. 2F) and SLA-DQ (Fig. 2G), equivalents to HLA-DR and DQ, respectively.
SCOT Improved Graft Outcome
In our model, animal survival reflects graft survival as dialysis was not used. Euthanasia was performed due to poor-overall conditions. Morphological analysis (Fig. 3A) revealed extensive necrosis and tubule loss at week 1 for cases of primary nonfunction, graft loss at weeks 2 and 4 was due to high rate of inflammation and tubulitis, whereas organs lost at week 6 displayed immune invasion and fibrosis. Biochemical analysis showed high creatinemia (1500–2100 μmol/L) at sacrifice. There was no difference in histology and creatinemia between groups for animals lost at similar time points. Animals lost from surgical complications were excluded, only loss of animal to graft failure was counted.
Measurement of fibrosis (Fig. 3B) showed high level in UW and IGL-1 grafts and low lesions in SCOT grafts (P<0.05). Kaplan-Meier analysis (Fig. 4A) showed low UW group survival (20%), while IGL-1 preservation allowed 40% survival. SCOT allowed 80% survival (P<0.01 to UW). Benefits of SCOT were further evidenced by kidney function: serum creatinine (Fig. 4B) and proteinuria (Fig. 4C) at 3 months were lower in SCOT grafts than in UW and IGL-1 groups (P<0.05). Western blot analysis of transforming growth factor (TGF)-β at 3 months (Fig. 4D) revealed increased expression in both UW and IGL-1 grafts (P<0.05), whereas expression in SCOT graft remained low. Analysis of Smad 4 expression, one of the intracellular effector of TGF-β, showed the same pattern, confirmed by RT-qPCR analysis of TGF-β expression (Fig. 4E).
SCOT Preservation Reduced Long-Term Hypoxic Injury
Chronic fibrosis reduces the blood supply to the tubules, inducing the activation of oxygen-sensitive pathways. To evaluate the hypoxic situation in kidney grafts at 3 months, we performed immunofluorescent staining for HIF1-α, an oxygen-dependent marker, and vascular endothelial growth factor (VEGF), one of HIF1-α effectors (Fig. 5A). Evaluation of the staining in the tubules showed intense HIF1-α expression in UW and IGL-1 grafts. In the interstitium, UW grafts showed important HIF1-α staining with lesser VEGF and costained cells. IGL-1 grafts revealed an opposed staining, with high VEGF staining and lower HIF1-α (P<0.05 to UW). SCOT showed limited staining for both markers. Immunofluorescent staining for VEGF receptors FLT1 (VEGFR1) and FLK1 (VEGFR2) showed loss of tubular staining in UW and IGL grafts, whereas SCOT-preserved kidneys presented a percentage of stained tubules similar to control organs (P<0.05, Fig. 5B). Western Blot and ELISA on whole tissue confirmed these findings (see Figure 1, Supplemental Digital Content 2, http://links.lww.com/TP/A369).
Apoptosis Was Intense in UW and IGL-1 Grafts, Whereas Limited in SCOT Organs
Quantification of TUNEL positive cells (Fig. 5C) showed an important number of apoptotic cells in UW and IGL-1 kidneys, whereas SCOT grafts had limited apoptosis, similar to control levels (P<0.05 to UW and IGL-1).
Herein, we determined that compared with the gold standard in allograft preservation, use of SCOT in organ preservation allows for a higher resistance to acute tissue damage and dramatically improves long-term outcome.
In vitro evaluation revealed little difference between UW and IGL-1 in maintaining cellular integrity, ATP levels and lowering cell death. Cells preserved with SCOT were better able to resume ATP production and showed less death, signs of less IRI. Considering no immune cells was present in this in vitro model, the protection offered by SCOT appears due to a ionic composition closer to plasma and a higher PEG content better able to regulate osmotic stress. Although further studies are necessary to assess the impact of PEG chain length and concentration on cell and graft viability.
The transition to in vivo model confirmed these results, as SCOT optimized graft function recovery and lowered tissue damage at day 7. In this low mismatch allograft model, acute protection is likely due to similar factors than in vitro. IGL-1 was able to provide some level of protection, confirming the superiority of extracellular composition. However, use of PEG in this solution may need to be optimized.
Acute damage often favors the development of immune response. Following immune response for 3 months, we showed an intense invasion of innate cells in UW grafts early on, and a high MHC class II antigen presentation, with growing adaptative immune cell invasion, supported by measurements of MCP1 and β2-microglobulin expression levels. IGL-1 showed some level of protection against immune invasion and the development of a full scale immune response. As there was no differences in vitro between these solutions, this protection may be due to immune camouflage from PEG chains (14, 36). Higher PEG content in SCOT could explain the better protection against immune response in these grafts.
At 3 months, survival and function were greatly improved in SCOT grafts compared with the other groups. Loss of graft function, and ultimately loss of the grafts itself, is often due to the development of tubular atrophy and interstitial fibrosis (IFTA) (37). Evaluation of IFTA development confirmed the superiority of SCOT. TGF-β signaling is involved in cell proliferation, tissue growth, and extracellular matrix remodeling by enhanced collagen synthesis (38). The regulation of the TGF-β pathway during chronic kidney injury is complex and still unclear; however, the correlation between intensity of IRI and chronic injury has been demonstrated (39). Moreover, TGF-β is the most widely involved and likely the most potent inducer of epithelial to mesenchymal transition, because it can initiate and complete the entire course of these processes (40), a mechanism strongly linked to IFTA development (41–44). The essential role of TGF-β is also consistent with the observation that its expression is universally up-regulated in every kind of chronic kidney disease in experimental models and in clinical settings (45). In our in vivo model, free of any effects due to immunosuppression (46), we observe the summated effects of damage sustained by organ preservation and reperfusion (47) and our data outline the central role of preservation condition and its importance for IFTA development
Further evaluations lead us to show differences in tissue oxygenation between the groups. Recently, it was suggested that stabilization of HIF-1α in the cell is a major factor in the tissue adaptation to hypoxia during ischemia and reperfusion (For a review, see Refs. 48–50). However, recent studies showed that hypoxia response through HIF-1α enhanced fibrogenesis (48–54) and epithelial to mesenchymal transition (55, 56). SCOT-preserved kidneys displayed little staining; however, there were differences in the staining pattern between IGL-1 and UW: while both had similar staining in the tubules, UW grafts showed more HIF1-α than VEGF in the interstitium while IGL-1 staining was the opposite. Thus, IGL-1 grafts seemed to have angiogenesis in response to the stress in the tubules, while UW kidney, more advanced in the lesion process as suggested by other markers, show hypoxia in the tissue, unable to coordinate an appropriate response. This is in accordance with recent findings showing VEGF expression attenuated despite an activation of intracellular hypoxia response in progressive renal disease (57). These data suggest that HIF1-α is indeed an important marker of chronic kidney lesions, and furthermore that HIF1-α and VEGF can be valuable markers to further characterize the intensity of graft lesions and discriminate between organs showing similar phenotypes measured through classical methods. Staining of VEGF receptors 1 (FLT1) and 2 (FLK1) was localized in the tubules, and quantification revealed that IGL and UW group displayed less positive tubules compared with SCOT and Control organs. There thus appear to be little relationship between VEGF expression and that of its receptors; however, the tubular localization of this staining is difficult to appreciate as there is extensive tubular atrophy in these groups due to fibrosis development.
We finally evaluated apoptosis level in each groups, and again confirmed the highest graft quality offered by SCOT preservation. Here also, there was no significant difference between UW and IGL-1 grafts although there was a trend towards less apoptosis in IGL-1 grafts, possibly related to the lower level of immune reaction found in these organs. Moreover, high amount of apoptosis in the UW group is in accordance with the high level of hypoxia found in these grafts.
The dichotomy in protection performance between IGL and SCOT suggests that PEG polymers are not all born equal in terms of the benefits they can bring to organ preservation (14) and thorough evaluation of which polymer chain length, and which dosage, to add in organ preservation solution is necessary to obtain optimal outcome.
In conclusion, we show that superior graft preservation with SCOT, through the use of an ionic composition close to plasma in conjunction with PEG, month before the development of fibrosis, has a critical impact on the long-term graft outcome (summarized in Figure 2, Supplemental Digital Content 3, http://links.lww.com/TP/A370). The use of such solutions could be invaluable for the proper optimization of newer donor pools with fragile organs, such as extended criteria donors.
MATERIALS AND METHODS
In Vitro Experiments
Primary kidney Endothelial cells were used as previously described (58), ischemia was achieved by incubation in hypoxic atmosphere (Bactal 2 gaz, Air Liquide, France) 24 hr at 4°C in UW, IGL-1, or SCOT. Reperfusion was performed by culture with DMEM+10%FBS for 24 hr at 37°C in normoxic conditions.
Assays were as follows: Necrosis–ratio supernatant LDH/intracellular LDH (tested on automated analyzer, Modular analytics P, Roche); Mitochondrial Activity XTT kit (Roche, France); Caspase 3 activity–Caspase-3 Fluorometric Assay kit (R&D System, Minneapolis, MN); Intracellular ATP–adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (Sigma, France); following the manufacturer's guidelines. Reactions were quantified by spectrophotometer (Victor3, Perkin-Elmer, France).
In Vivo Surgical Procedures and Experimental Groups
Large white male pigs (INRA/GEPA, Surgères, France) were prepared as previously described (59) in accordance with French guidelines of the Ethical Committee for Human and Animal Studies. The kidney was harvested, cold flushed, and preserved for 24 hr before transplantation, when the left kidney was nephrectomized to mimic nephron mass in transplanted situation. Surgical teams were blinded to protocols. Time for anastomosis was 30±5min, blood loss was minimal, and no postoperative complication was observed. For this allograft, preoperative blood samples analysis ensured compatibility for SLA class I by microlymphotoxicity, the microsatellite technique was used to ensure less that 10% recombination for SLA class II, permitting graft survival with low grade acute rejection without immunosuppression (60). Three groups were studied: 1-UW–UW solution (low-Na+/high-K+, Bristol-Myers Squibb, USA) with 50 g/L HES; 2-IGL–IGL-1 (high-Na+/low-K+, IGL Group, France) with 1 g/L PEG 35 kDa; 3-SCOT–SCOT (high-Na+/low-K+, 30 g/L PEG20 kDa, MacoPharma, France). Controls were sham-operated animals (n=6).
Function and Histopathology
Endogenous creatinine clearance and urinary proteins were measured as previously described (7). Activities of brush border enzyme alanine aminopeptidase and lysosomal enzyme N-acetyl-β-d-glucosaminidase were determined in urine at days 1, 3, and 7 postreperfusion, as previously described (61).
Samples were collected, frozen, or fixed in 10% formalin and embedded in paraffin. All sections were examined under blind conditions by a pathologist. The magnitude of tubular atrophy was scored as previously described (59): 0, no abnormality; 1, less than 10%; 2, 10% to 25%; 3, 26% to 50%; 4, 51% to 75%; 5, more than 75%. A standard procedure was used to estimate the level of tubulointerstitial fibrosis using Picro-Sirius staining (62). Immunohistochemistry was used for ED1+, CD4+, and CD8+ cell invasion measurement (SouthernBiotech, Birmingham, AL).
Apoptosis was assayed using DeadEnd fluorometric TUNEL system (Promega, Madison, WI). The number of positive cells was determined on 10 random high powered fields (200×). For immunofluorescence, antibodies against HIF1-α, Flt1, Flk1 (Santa Cruz Biotech, Santa Cruz, CA) and VEGF (Invitrogen) were used, with secondary antibodies coupled with Alexa488 (green, for HIF1-α) or Cy3 (red, for VEGF). Quantitative evaluation was performed on 5 to 10 high-powered fields (200×).
SLA-DR and SLA-DQ protein expression were evaluated (AbDSerotec, Germany). Five high-powered fields were graded in each compartment of the parenchyma (tubule, inflammatory cells, endothelium) as follows: no staining (−), faint (+), moderate (++), intense (+++) staining.
We used antibodies against MCP-1, TGF-β, Smad4 (Santa Cruz, USA) and loading control β Actin (Sigma). Protein bands intensities were quantified using ImageJ (National Institute of Health, Bethesda, MD).
Mean±standard error of the means are shown. A biostatistician was consulted for proper data processing. Paired Student's t test was used for comparisons within groups. Survivals were compared using the Kaplan Meier. Semiquantitative data were compared by the nonparametric Kruskal-Wallis followed by the Connover test. Significance was accepted for P less than 0.050.
The authors thank Prof. Gerard Mauco for his guidance and support in the conduction of this study.
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