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THE ULTRASTRUCTURE OF ARTERIOSCLEROSIS IN PATIENTS WITH ENDSTAGE RENAL DISEASE
753
754
ROLE OF ENDOTHELIAL CELLS IN SOLID ORGAN TRANSPLANTATION: TECHNIQUE AND FIRST RESULTS FROM A TRANSGENIC MOUSE MODEL
MICROCHIMERISM AND RENAL TRANSPLANTATION: DOUBT STILL PERSISTS
Engeler D.1, Krebs P.2, Bolinger B.2, Schmid H.P.2, Ludewig B.2
Pourmand G.1, Nikbin B.2, Saraji A.3, Mehrsai A.3, Moosavi S.3, Abedi A.R.3 Tehran University, Urology, Tehran, Iran, 2Tehran University of Medical Sciences, Immunology, Tehran, Iran, 3Tehran University of Medical Sciences, Urology Research Centre, Tehran, Iran
1
1
Kantonsspital St. Gallen, Department of Urology, St. Gallen, Switzerland, 2 Kantonsspital St. Gallen, Research Department, St. Gallen, Switzerland INTRODUCTION & OBJECTIVES: Immune recognition of vascular endothelial cells (EC) has been implicated in chronic allograft rejection. In order to assess the role of EC in chronic transplant rejection in vivo, we are using a transgenic mouse model in which the E. coli beta-galactosidase (beta-gal) is selectively expressed in vascular EC (Tie2-LacZ mice). In a first set of experiments, heterotopic heart transplantation of Tie2-LacZ heart onto wild type C57/B6 mice was performed. MATERIAL & METHODS: Microsurgical procedures are done under the microscope. 10-0 running sutures were used for vascular anastomosis. Ischemia time was usually between 45 to 50 min. Transplant function was controlled by palpation of the abdomen. Animals were sacrificed between 14 to 100 days after transplantation. In challenge experiments, mice were immunized with antigenloaded dendritic cells. Immune reactivity was assessed using beta-gal specific MHC class I tretramers and cytotoxic T lymphocytes (CTL) assays. RESULTS: Successfully transplanted hearts (n=40) from transgenic and syngenic donors are continuously beating up to day 100 post transplantation. Inflammatory reactions both in Tie2-LacZ and control C57BL/6 transplants was minimal as determined by immunohistochemical analysis. Priming of recipients with transgenic hearts with beta-gal antigen loaded dendritic cells showed unimpaired CTL expansion and functionality suggesting that the antigen presented by EC in transgenic Tie2-LacZ had remained immunologically ignored. CONCLUSIONS: Our results indicate that, at least in our heart transplantation model, nominal antigens presented by EC can remain “invisible” for the immune system unless appropriate immune activation is provided. We will determine in future experiments how antigen presentation by EC can be induced and enhanced. Furthermore, we will assess whether EC from other organs such as the kidney fail to induce spontaneous immune reactivity, even in the inflammatory context of transplantation procedures.
INTRODUCTION & OBJECTIVES: The presence of donor leukocytes in recipient of organ allografts has been shown even several years after transplantation. However, it remains unclear whether this donor cell microchimerism plays an effective role in allograft acceptance or is simply a consequence of immunosuppression condition in recipients. MATERIAL & METHODS: In this study we retrospectively evaluated the Peripheral Blood Microchimerism (PBM) after renal transplantation in 32 male-to-female recipients of living (unrelated) and cadaveric donor renal transplants. Using a nested Polymerase Chain Reaction (nested–PCR) amplification specific for SRY region of the Y chromosome microchimerism was detected with sensitivity up to 1:1000000. According to the presence of PBM recipients were classified into microchimeric and nonmicrochimeric groups, and then acute and chronic rejection episodes, type of allotransplant (living or cadaveric donor), recipient and donor age at transplantation, previous male labour or blood transfusion, allograft function (serum creatinine level), post-transplant period duration, and body mass index were compared between two groups. RESULTS: Among 32 recipients 7 were positive for PBM in multiple testing at different post-transplantation times. All microchimeric recipients had been received kidney from livingunrelated donors. The mean age of microchimeric group was 36.3±10 year vs. 36.3±11.9 year in non-microchimeric group, the mean transplantation duration was 73±29 month vs. 48±23 month (P-value<0.02), the mean serum creatinine level was similar in both groups 1.07±0.4 mg/dl vs. 1.07±0.38 mg/dl, BMI was 24±5 kg/m2 vs. 23±3.8 kg/m2, 5 (71%) of microchimeric patients had history of blood transfusion vs. 19(76%) patients among nonmicrochimeric group, the history of male labour was 6 (85%) vs. 12 (48%) P-value>0.05 respectively. Regarding to all parameters mentioned above significant difference was not observed. In addition, acute rejection rate in microchimeric group was 3 (42%) versus 4 (16%) in nonmicrochimeric recipients (not significant). CONCLUSIONS: Our results demonstrate better establishment of microchimerism after living donor kidney transplantation. But, concerning true effect of microchimerism after renal transplantation doubt still persists; and it seems that microchimerism alone has no major protective role in renal allograft survival.
755
756
PRECONDITIONING IN RENAL TRANSPLANTATION DEPENDS ON THE MANIPULATION OF ANTI-APOPTOTIC PROTEINS
THE ULTRASTRUCTURE OF ARTERIOSCLEROSIS IN PATIENTS WITH ENDSTAGE RENAL DISEASE
Daly P.1, Healy D.1, O’Connor K.1, Docherty N.1, Taylor C.1, Fitzpatrick J.2, Watson W.1
Rohrmann D.1, Schlieper G.2, Mahnken A.3, Mayer J.4, Ketteler M.2, Jakse G.1, Floege J.2
1
Universitätsklinik Rwth, Urology, Aachen, Germany, 2Universitätsklinik RWTH, Nephrology, Aachen, Germany, 3Universitätsklinik RWTH, Radiology, Aachen, Germany, 4Universitätsklinik RWTH, Electronmicroscopy, Aachen, Germany
Conway Institute, Biomolecular and Biomedicine, Dublin, Ireland, 2Mater Misercordiae, Department of Surgery, Dublin, Ireland INTRODUCTION & OBJECTIVES: Reperfusion and immunosuppressant drugs induce damage to kidneys during transplantation. This contributes to delayed graft function (DGF) and eventual graft failure. Protecting against this damage has clinical implications to the survival of transplanted organs. Our objective was to determine that non-lethal periods of hypoxia confer protection against these injuries and establish the cellular mechanisms by which this protection is induced. MATERIAL & METHODS: Human proximal tubular cells (HK-2) were incubated under normoxic or hypoxic conditions for 24 hours and then placed at 4°C for 6 hours before been returned to 37°C to mimic reperfusion injury and cultured with varying concentrations of Cyclosporine A (CSA). Cell viability and apoptosis were measured by PI staining and flow cytometry. Genechip and western blotting analysis of HK-2 cells cultured in hypoxia was carried out to identify the expression of potential protective proteins. RESULTS: Hypoxia alone for the time periods produced no change in cell viability and apoptosis compared to cells in normoxia. Pre-exposure of cells to hypoxia significantly (p<0.05) protected against CSA across an in vitro model of transplantation. Gene chip analysis identified a number of potential genes but protein validation studies demonstrated the up-regulation of the anti-apoptotic proteins Bcl-2 and HSP 70. Over-expression of HSP-70 mimicked the protective effects of hypoxia to cold storage and reperfusion damage. In addition the protective effect had to be induced prior to cold storage as HIF-1alpha did not mediate a functional response at 4°C. CONCLUSIONS: Exposure of HK-2 cells to reperfusion and immunosuppressant drugs predispose them to injury and death. Preconditioning kidneys, to cause up regulation of hypoxic induced protective proteins, at the time of retrieval from the donor, could reduce injury to the transplanted kidney and decrease the occurrence of DGF resulting in the prolonged life-expectancy of the transplanted organ.
1
INTRODUCTION & OBJECTIVES: Patients with endstage renal insufficiency develop accelerated arteriosclerosis which is characterized by severe calcification of the intima and media. Risk factors such as inflammation, hyperphosphatemia and increased calcium-phospate product are well-known. In order to understand the pathophysiologic mechanism that initiates the rapid progression of calcification at the level of microcalcifications, we applied highly advanced electronmicroscopic techniques that have been used in material sciences. MATERIAL & METHODS: Arterial biopsies of 16 patients (age : 51 +/- 15 yrs) taken at the time of transplantation were examined. Calcification of the coronary arteries was measured by applying ECG-triggered multi-sliceCTs (AgatstonScore). Calcifications of pelvic arteries were determined semiquantitatively by X-ray of the pelvis. Light microscopy (von Kossa stain) was performed to detect calcifications. In addition we used transmission electron microscopy (TEM) like Focussed Ion Beam (FIB) to obtain ultrathin lamellae which were then analysed by Electron-Energy-Loss-Spectroscopy (EELS) and Energy-Dispersive-Spectroscopy (EDS). RESULTS: 65% of patients were found to have media calcifications showing a granular pattern. A high degree of calcification in the iliac compartment was associated with severe coronary calcification. Electronmicroscopy detected multiple microcalcifications of 50 to 200 nm diameter, but no confluent macrocalcifications. The morphology of calcifications could be well defined as a core surrounded by centrifugal crystallisations. EELS and EDS identified calcium and phosphate (hydroxyapatite) as major components of the microcalcifications. CONCLUSIONS: Our study shows that calcifications of the media in arteries of dialysis patients consist of multiple homogeneous microcalcifications and not of irregular macrocalcifications. Moreover, the extent of coronary calcification appears to correlate well with the degree of calcification of peripheral major arteries. Eur Urol Suppl 2006;5(2):211
754
ROLE OF ENDOTHELIAL CELLS IN SOLID ORGAN TRANSPLANTATION: TECHNIQUE AND FIRST RESULTS FROM A TRANSGENIC MOUSE MODEL
MICROCHIMERISM AND RENAL TRANSPLANTATION: DOUBT STILL PERSISTS
Engeler D.1, Krebs P.2, Bolinger B.2, Schmid H.P.2, Ludewig B.2
Pourmand G.1, Nikbin B.2, Saraji A.3, Mehrsai A.3, Moosavi S.3, Abedi A.R.3 Tehran University, Urology, Tehran, Iran, 2Tehran University of Medical Sciences, Immunology, Tehran, Iran, 3Tehran University of Medical Sciences, Urology Research Centre, Tehran, Iran
1
1
Kantonsspital St. Gallen, Department of Urology, St. Gallen, Switzerland, 2 Kantonsspital St. Gallen, Research Department, St. Gallen, Switzerland INTRODUCTION & OBJECTIVES: Immune recognition of vascular endothelial cells (EC) has been implicated in chronic allograft rejection. In order to assess the role of EC in chronic transplant rejection in vivo, we are using a transgenic mouse model in which the E. coli beta-galactosidase (beta-gal) is selectively expressed in vascular EC (Tie2-LacZ mice). In a first set of experiments, heterotopic heart transplantation of Tie2-LacZ heart onto wild type C57/B6 mice was performed. MATERIAL & METHODS: Microsurgical procedures are done under the microscope. 10-0 running sutures were used for vascular anastomosis. Ischemia time was usually between 45 to 50 min. Transplant function was controlled by palpation of the abdomen. Animals were sacrificed between 14 to 100 days after transplantation. In challenge experiments, mice were immunized with antigenloaded dendritic cells. Immune reactivity was assessed using beta-gal specific MHC class I tretramers and cytotoxic T lymphocytes (CTL) assays. RESULTS: Successfully transplanted hearts (n=40) from transgenic and syngenic donors are continuously beating up to day 100 post transplantation. Inflammatory reactions both in Tie2-LacZ and control C57BL/6 transplants was minimal as determined by immunohistochemical analysis. Priming of recipients with transgenic hearts with beta-gal antigen loaded dendritic cells showed unimpaired CTL expansion and functionality suggesting that the antigen presented by EC in transgenic Tie2-LacZ had remained immunologically ignored. CONCLUSIONS: Our results indicate that, at least in our heart transplantation model, nominal antigens presented by EC can remain “invisible” for the immune system unless appropriate immune activation is provided. We will determine in future experiments how antigen presentation by EC can be induced and enhanced. Furthermore, we will assess whether EC from other organs such as the kidney fail to induce spontaneous immune reactivity, even in the inflammatory context of transplantation procedures.
INTRODUCTION & OBJECTIVES: The presence of donor leukocytes in recipient of organ allografts has been shown even several years after transplantation. However, it remains unclear whether this donor cell microchimerism plays an effective role in allograft acceptance or is simply a consequence of immunosuppression condition in recipients. MATERIAL & METHODS: In this study we retrospectively evaluated the Peripheral Blood Microchimerism (PBM) after renal transplantation in 32 male-to-female recipients of living (unrelated) and cadaveric donor renal transplants. Using a nested Polymerase Chain Reaction (nested–PCR) amplification specific for SRY region of the Y chromosome microchimerism was detected with sensitivity up to 1:1000000. According to the presence of PBM recipients were classified into microchimeric and nonmicrochimeric groups, and then acute and chronic rejection episodes, type of allotransplant (living or cadaveric donor), recipient and donor age at transplantation, previous male labour or blood transfusion, allograft function (serum creatinine level), post-transplant period duration, and body mass index were compared between two groups. RESULTS: Among 32 recipients 7 were positive for PBM in multiple testing at different post-transplantation times. All microchimeric recipients had been received kidney from livingunrelated donors. The mean age of microchimeric group was 36.3±10 year vs. 36.3±11.9 year in non-microchimeric group, the mean transplantation duration was 73±29 month vs. 48±23 month (P-value<0.02), the mean serum creatinine level was similar in both groups 1.07±0.4 mg/dl vs. 1.07±0.38 mg/dl, BMI was 24±5 kg/m2 vs. 23±3.8 kg/m2, 5 (71%) of microchimeric patients had history of blood transfusion vs. 19(76%) patients among nonmicrochimeric group, the history of male labour was 6 (85%) vs. 12 (48%) P-value>0.05 respectively. Regarding to all parameters mentioned above significant difference was not observed. In addition, acute rejection rate in microchimeric group was 3 (42%) versus 4 (16%) in nonmicrochimeric recipients (not significant). CONCLUSIONS: Our results demonstrate better establishment of microchimerism after living donor kidney transplantation. But, concerning true effect of microchimerism after renal transplantation doubt still persists; and it seems that microchimerism alone has no major protective role in renal allograft survival.
755
756
PRECONDITIONING IN RENAL TRANSPLANTATION DEPENDS ON THE MANIPULATION OF ANTI-APOPTOTIC PROTEINS
THE ULTRASTRUCTURE OF ARTERIOSCLEROSIS IN PATIENTS WITH ENDSTAGE RENAL DISEASE
Daly P.1, Healy D.1, O’Connor K.1, Docherty N.1, Taylor C.1, Fitzpatrick J.2, Watson W.1
Rohrmann D.1, Schlieper G.2, Mahnken A.3, Mayer J.4, Ketteler M.2, Jakse G.1, Floege J.2
1
Universitätsklinik Rwth, Urology, Aachen, Germany, 2Universitätsklinik RWTH, Nephrology, Aachen, Germany, 3Universitätsklinik RWTH, Radiology, Aachen, Germany, 4Universitätsklinik RWTH, Electronmicroscopy, Aachen, Germany
Conway Institute, Biomolecular and Biomedicine, Dublin, Ireland, 2Mater Misercordiae, Department of Surgery, Dublin, Ireland INTRODUCTION & OBJECTIVES: Reperfusion and immunosuppressant drugs induce damage to kidneys during transplantation. This contributes to delayed graft function (DGF) and eventual graft failure. Protecting against this damage has clinical implications to the survival of transplanted organs. Our objective was to determine that non-lethal periods of hypoxia confer protection against these injuries and establish the cellular mechanisms by which this protection is induced. MATERIAL & METHODS: Human proximal tubular cells (HK-2) were incubated under normoxic or hypoxic conditions for 24 hours and then placed at 4°C for 6 hours before been returned to 37°C to mimic reperfusion injury and cultured with varying concentrations of Cyclosporine A (CSA). Cell viability and apoptosis were measured by PI staining and flow cytometry. Genechip and western blotting analysis of HK-2 cells cultured in hypoxia was carried out to identify the expression of potential protective proteins. RESULTS: Hypoxia alone for the time periods produced no change in cell viability and apoptosis compared to cells in normoxia. Pre-exposure of cells to hypoxia significantly (p<0.05) protected against CSA across an in vitro model of transplantation. Gene chip analysis identified a number of potential genes but protein validation studies demonstrated the up-regulation of the anti-apoptotic proteins Bcl-2 and HSP 70. Over-expression of HSP-70 mimicked the protective effects of hypoxia to cold storage and reperfusion damage. In addition the protective effect had to be induced prior to cold storage as HIF-1alpha did not mediate a functional response at 4°C. CONCLUSIONS: Exposure of HK-2 cells to reperfusion and immunosuppressant drugs predispose them to injury and death. Preconditioning kidneys, to cause up regulation of hypoxic induced protective proteins, at the time of retrieval from the donor, could reduce injury to the transplanted kidney and decrease the occurrence of DGF resulting in the prolonged life-expectancy of the transplanted organ.
1
INTRODUCTION & OBJECTIVES: Patients with endstage renal insufficiency develop accelerated arteriosclerosis which is characterized by severe calcification of the intima and media. Risk factors such as inflammation, hyperphosphatemia and increased calcium-phospate product are well-known. In order to understand the pathophysiologic mechanism that initiates the rapid progression of calcification at the level of microcalcifications, we applied highly advanced electronmicroscopic techniques that have been used in material sciences. MATERIAL & METHODS: Arterial biopsies of 16 patients (age : 51 +/- 15 yrs) taken at the time of transplantation were examined. Calcification of the coronary arteries was measured by applying ECG-triggered multi-sliceCTs (AgatstonScore). Calcifications of pelvic arteries were determined semiquantitatively by X-ray of the pelvis. Light microscopy (von Kossa stain) was performed to detect calcifications. In addition we used transmission electron microscopy (TEM) like Focussed Ion Beam (FIB) to obtain ultrathin lamellae which were then analysed by Electron-Energy-Loss-Spectroscopy (EELS) and Energy-Dispersive-Spectroscopy (EDS). RESULTS: 65% of patients were found to have media calcifications showing a granular pattern. A high degree of calcification in the iliac compartment was associated with severe coronary calcification. Electronmicroscopy detected multiple microcalcifications of 50 to 200 nm diameter, but no confluent macrocalcifications. The morphology of calcifications could be well defined as a core surrounded by centrifugal crystallisations. EELS and EDS identified calcium and phosphate (hydroxyapatite) as major components of the microcalcifications. CONCLUSIONS: Our study shows that calcifications of the media in arteries of dialysis patients consist of multiple homogeneous microcalcifications and not of irregular macrocalcifications. Moreover, the extent of coronary calcification appears to correlate well with the degree of calcification of peripheral major arteries. Eur Urol Suppl 2006;5(2):211