- Email: [email protected]
31 Histological alterations found in the ureter during organ preservation and early phases of renal transplantation
29 MYCOPHENOLATE OF TUMOUR CELLS Beecken
W.D.,
MOFETIL IN VITRO
UP-REGULATES
Engl T., Jonas D., Blaheta
J.W.Goethe-Universit&klinik, am Main, Germany
Klinik
ADHESION
CAPACITY
30 FUNCTIONAL AND HISTOLOGICAL TATION IN THE RAT
RESULTS
OF BLADDER
TRANSPLAN-
Fiaueir-edo A.‘, Cunha F.2, Furtado A.’
R.
fur Urologie
und Kinderurologie,
Frankfurt
‘University Hospital Portugal, ‘University
of Coimbra, Department of Urology and Transplantation, Hospital of Coimbra, Department of Pathology, Coimbra,
INTRODUCTION & OBJECTIVES: This study aimed at evaluating bladder transplantation in the animal, anatomically and functionally.
INTRODUCTION & OBJECTIVES: The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements ICAMand VCAM-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. We speculated that MMF might diminish further receptors of the immunoglobulin super family, which however act as homophilic binding elements. Since down-regulation of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumours. MATERIAL & METHODS: We analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumour cell attachment to an endothelial cell monolayer. Neuroblastoma cells (NB), which self-aggregate via the homophilic binding element NCAM, were used. Effects of MMF on the 140 and 180 kd NCAM isoforms were investigated quantitatively by flow cytometry, Western blot and RT-PCR. The relevance of NCAM for tumour cell binding was proven by treating NB with NCAM antisense oligonucleotides. RESULTS: MMF profoundly increased the number of adherent NB with a maximum effect at 0.1 FM, compared to controls. Down-regulation of NCAM on the cell surface was detected by flow cytometry Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140 and 180 kd isoform. Treatment of NB with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumour cell adhesion. CONCLUSIONS: MMF down-regulates NCAM receptors which is associated with enhanced tumour cell invasiveness. We conclude that MMF based immunosuppressive regimen might increase the risk of tumour metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.
Coimbra, Portugal
the results of
MATERIAL & METHODS: Four experimental models of bladder transplantation were developed using inbred female Wistar rats: A - Orthotopic allotransplantation over sub-total cystectomy with vascular anastomosis (24 animals). B Heterotopic allotransplantation with vascular anastomosis (16 animals). C - Orthotopic segmental allotransplantation without vascular anastomosis over sub-total cystectomy (10 animals). D - Orthotopic segmental auto transplantation
Animals
without
vascular
anastomosis
over sub-total
were kept alive up to 12 months post transplantation.
cystectomy
(I 0 animals).
Blood and urine analysis,
video-urodynamic and bladder permeability studies (using 99”TcDTPA) and, at death or sacrifice, thoroughly histological study was done.
were performed
RESULTS: Mortality was 37.5%, 25%, 70% and 60% in groups A, B, C and D, respectively. Death in groups A and B was related to vascular anastomosis thrombosis in most cases and occurred on the first 10 days in 11 out of 13 rats. In groups C and D, death was dependent of graft necrosis and rupture. Surviving animals grew normally. Blood test did not reveal major alterations. Bladder capacity on urodynamics was 3.40 i 3.96 ml in group A, 0.75 i 0.14 ml in group C, 0.70 * 0.26 ml in group D and 1.50 f 0.41 ml in native bladders of group B animals. Bladder residue at the beginning of cystometry was 1.79 i 3.38 ml in group 4, 0.09 + 0.11 ml in group C, 0.07 i 0.09 ml in group D and 0.10 f 0.08 ml in the native bladders of group B. Bladder compliance was normal in 77%, X7%, 66% and 50% of the group A, B, C and D bladders, respectively, and reduced in the remnant cases. Permeability tests were negative in all group A bladders studied. At sacrifice, grafts from group A and B animals were irrigated by their own vascular pedicle. In group A, the graft constituted most of the bladder in 80% of the cases and the histology revealed urothelium ulceration and oedema in the cases sacrificed at one and two weeks, with progressive recuperation towards normal patterns afterwards. In group B, five grafts were severely retracted and hyalinised, whether the histology of the remaining eight was nearly normal. The most notorious alteration in grafts of groups A and B was at the muscular layer level, often thinner and disorganised. The study of the bladders from groups C and D revealed extensive “quasi-total” retraction of the grafts, the bladder being constituted by the expanded trigonal area. CONCLUSIONS: Bladder transplantation is feasible in the experimental setting and, despite the innervation problems, grafts proved to comply with most requisites of a bladder substitute. They performed as large low pressure reservoirs, impermeable to low weight molecules and maintained almost normal histology over time.
31 HISTOLOGICAL ALTERATIONS ORGAN PRESERVATION TRANSPLANTATION Figueiredo
FOUND IN AND EARLY
A.‘, Cunha F.l, Mota A.‘, Furtado
THE URETER PHASES OF
DURING RENAL
32 RENAL TISSUE TION IN RATS
REGENERATION
USING
CELLULAR
TRANSPLANTA-
ChoY.S.‘,Moo~~H.C.‘,ChoiC.Y.Z,KimS.S.L,KimB.S.3,HanJ.H.4,JooK.J.‘,KwonC.H.‘, Park H-l.’
A.’
Department of Urology and Transplantation, ‘University Hospital of Coimbra, Coimbra, Portugal. ZUniversity Hospital of Coimbra, Department of Pathology, Coimbra, Portugal
‘Sungkyunkwan University School of Medicine, Department of Urology, Seoul, South Korea, ‘Seoul National University, School of Chemical Engineering, Seoul, South Korea, 3Hanyang University, Department of Chemical Engineering, Seoul, South Korea, “Sungkyunkwan University School of Medicine, Department of Pathology, Seoul, South Korea
INTRODUCTION & OBJECTIVES: Despite the extensive investigation on kidney preservation and transplantation, there are no studies on the phenomena that occur on the ureter during organ preservation and immediately after transplantation.
INTRODUCTION & OBJECTIVES: Chronic renal failure (CRF) is a major public health problem and the incidence of end stage of renal disease (ESRD) has been increased continuously over the past years, but there is no other treatment modality except haemodialysis, peritoneal dialysis and kidney transplantation. This study was undertaken to evaluate renal tissue regeneration using cells from neonatal kidneys of rats and fibrin gel and its clinical usefulness.
MATERIAL & METHODS: After the development of criteria for classification of the ureteric lesions, we studied ureteral fragments obtained during organ harvesting in the cadaver (nine cases), immediately after the cold preservation period (18 cases) and after the kidney graft reperfusion (126 cases). Further to the histological analysis, including the detection of signs of rejection, we evaluated the risk factors for the development of lesions (from the donor, the recipient and the transplant) and their relation to the evolution of the transplant, including the occurrence of renal rejection episodes. RESULTS: One hundred and twenty out of the 126 fragments studied after graft reperfusion presented some kind of alterations, namely epithelial exfoliation in 42%, cell vacuolisation in 52.4% and cellular infiltration of the lamina ptopria and urothehum in 74.6% and 77X%, respectively. Global cellular infiltration was considered to be normal, mild and moderate to severe in 34.9%, 41.3% and 23.8%, respectively, consisting mainly in lymphocytes T CDS+. There was an inverse relationship between donor ventilation time and the intensity of the cellular infiltration and between donor age and vacuolisation. Seven and three out of the nine fragments obtained during organ harvesting showed mild cellular infiltration of the lamina propria and urothelium, respectively. Cold storage promoted minor histological changes, apart from lymphocitic migration to the epithelium. This pattern was more pronounced after re-perfusion, when it was detected an increase in urothelium infiltration in 11 out of the 18 cases. There were no detectable relation between the lesions encountered and HLA compatibilities between donor and recipient, the development of rejection lesions in future ureteral biopsies or to the evolution of the graft itself. CONCLUSIONS: The consequences of brain death and/or mechanical ventilation were detected at the ureter level, with abnormal lymphocitic infiltration in most cases. On the other hand, cold storage did not produce any major histological changes. The lesions detected after graft re-perfusion do not seem to involve any immunological phenomena. European
Urology
Supplements
3 (2004)
No. 2, pp.
10
MATERIAL & METHODS: In 16 male Spraque-Dawley rats with a body weight 200. 250gm, two-stage 516 nephrectomy was performed and confirmed CRF state of rats by increased BUN. creatinine 4 weeks after neohrectomv. We resected kidnevsi in neonate rats under sterile condition and chopped renai tissues were incubated in collgenaseidispase (Roche) solution for 40 minutes at 37°C. The incubated renal cells were labelled with fluorescent dye. The labelled renal cells were injected to remnant kidney of nephrectomized rats (n=8) witb fibrin gel matrix. Rats were sacriticed four weeks after injection and kidneys were harvested. Each injection site was analyzed histologically, and BUN, creatinine were measured before and after injection respectively. The nephrectomized rats (n=X) which were not injected were used as control group. RESULTS: All rats were healthy and there were no complications until being sacrificed. The initial BUN and creatinine were 41.78+9.42mg/dL (mean ISD) and 0.910.12mg/dL, and increased to 174+12.6mg/dL, 2.6+1.3mgidL in control group. However, BUN and creatinine were 50.6+8.48mg/dL and l&O.l4mg/dL in cell-injected group, respectively (p
CONCLUSIONS: Improvement of renal function in CRF rats and regeneration of renal tissues after injection of renal cells mixed with fibrin gel were observed. Further investigation will show the possibility of application of renal cell injection for treatment of CRF patients.
W.D.,
MOFETIL IN VITRO
UP-REGULATES
Engl T., Jonas D., Blaheta
J.W.Goethe-Universit&klinik, am Main, Germany
Klinik
ADHESION
CAPACITY
30 FUNCTIONAL AND HISTOLOGICAL TATION IN THE RAT
RESULTS
OF BLADDER
TRANSPLAN-
Fiaueir-edo A.‘, Cunha F.2, Furtado A.’
R.
fur Urologie
und Kinderurologie,
Frankfurt
‘University Hospital Portugal, ‘University
of Coimbra, Department of Urology and Transplantation, Hospital of Coimbra, Department of Pathology, Coimbra,
INTRODUCTION & OBJECTIVES: This study aimed at evaluating bladder transplantation in the animal, anatomically and functionally.
INTRODUCTION & OBJECTIVES: The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements ICAMand VCAM-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. We speculated that MMF might diminish further receptors of the immunoglobulin super family, which however act as homophilic binding elements. Since down-regulation of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumours. MATERIAL & METHODS: We analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumour cell attachment to an endothelial cell monolayer. Neuroblastoma cells (NB), which self-aggregate via the homophilic binding element NCAM, were used. Effects of MMF on the 140 and 180 kd NCAM isoforms were investigated quantitatively by flow cytometry, Western blot and RT-PCR. The relevance of NCAM for tumour cell binding was proven by treating NB with NCAM antisense oligonucleotides. RESULTS: MMF profoundly increased the number of adherent NB with a maximum effect at 0.1 FM, compared to controls. Down-regulation of NCAM on the cell surface was detected by flow cytometry Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140 and 180 kd isoform. Treatment of NB with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumour cell adhesion. CONCLUSIONS: MMF down-regulates NCAM receptors which is associated with enhanced tumour cell invasiveness. We conclude that MMF based immunosuppressive regimen might increase the risk of tumour metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.
Coimbra, Portugal
the results of
MATERIAL & METHODS: Four experimental models of bladder transplantation were developed using inbred female Wistar rats: A - Orthotopic allotransplantation over sub-total cystectomy with vascular anastomosis (24 animals). B Heterotopic allotransplantation with vascular anastomosis (16 animals). C - Orthotopic segmental allotransplantation without vascular anastomosis over sub-total cystectomy (10 animals). D - Orthotopic segmental auto transplantation
Animals
without
vascular
anastomosis
over sub-total
were kept alive up to 12 months post transplantation.
cystectomy
(I 0 animals).
Blood and urine analysis,
video-urodynamic and bladder permeability studies (using 99”TcDTPA) and, at death or sacrifice, thoroughly histological study was done.
were performed
RESULTS: Mortality was 37.5%, 25%, 70% and 60% in groups A, B, C and D, respectively. Death in groups A and B was related to vascular anastomosis thrombosis in most cases and occurred on the first 10 days in 11 out of 13 rats. In groups C and D, death was dependent of graft necrosis and rupture. Surviving animals grew normally. Blood test did not reveal major alterations. Bladder capacity on urodynamics was 3.40 i 3.96 ml in group A, 0.75 i 0.14 ml in group C, 0.70 * 0.26 ml in group D and 1.50 f 0.41 ml in native bladders of group B animals. Bladder residue at the beginning of cystometry was 1.79 i 3.38 ml in group 4, 0.09 + 0.11 ml in group C, 0.07 i 0.09 ml in group D and 0.10 f 0.08 ml in the native bladders of group B. Bladder compliance was normal in 77%, X7%, 66% and 50% of the group A, B, C and D bladders, respectively, and reduced in the remnant cases. Permeability tests were negative in all group A bladders studied. At sacrifice, grafts from group A and B animals were irrigated by their own vascular pedicle. In group A, the graft constituted most of the bladder in 80% of the cases and the histology revealed urothelium ulceration and oedema in the cases sacrificed at one and two weeks, with progressive recuperation towards normal patterns afterwards. In group B, five grafts were severely retracted and hyalinised, whether the histology of the remaining eight was nearly normal. The most notorious alteration in grafts of groups A and B was at the muscular layer level, often thinner and disorganised. The study of the bladders from groups C and D revealed extensive “quasi-total” retraction of the grafts, the bladder being constituted by the expanded trigonal area. CONCLUSIONS: Bladder transplantation is feasible in the experimental setting and, despite the innervation problems, grafts proved to comply with most requisites of a bladder substitute. They performed as large low pressure reservoirs, impermeable to low weight molecules and maintained almost normal histology over time.
31 HISTOLOGICAL ALTERATIONS ORGAN PRESERVATION TRANSPLANTATION Figueiredo
FOUND IN AND EARLY
A.‘, Cunha F.l, Mota A.‘, Furtado
THE URETER PHASES OF
DURING RENAL
32 RENAL TISSUE TION IN RATS
REGENERATION
USING
CELLULAR
TRANSPLANTA-
ChoY.S.‘,Moo~~H.C.‘,ChoiC.Y.Z,KimS.S.L,KimB.S.3,HanJ.H.4,JooK.J.‘,KwonC.H.‘, Park H-l.’
A.’
Department of Urology and Transplantation, ‘University Hospital of Coimbra, Coimbra, Portugal. ZUniversity Hospital of Coimbra, Department of Pathology, Coimbra, Portugal
‘Sungkyunkwan University School of Medicine, Department of Urology, Seoul, South Korea, ‘Seoul National University, School of Chemical Engineering, Seoul, South Korea, 3Hanyang University, Department of Chemical Engineering, Seoul, South Korea, “Sungkyunkwan University School of Medicine, Department of Pathology, Seoul, South Korea
INTRODUCTION & OBJECTIVES: Despite the extensive investigation on kidney preservation and transplantation, there are no studies on the phenomena that occur on the ureter during organ preservation and immediately after transplantation.
INTRODUCTION & OBJECTIVES: Chronic renal failure (CRF) is a major public health problem and the incidence of end stage of renal disease (ESRD) has been increased continuously over the past years, but there is no other treatment modality except haemodialysis, peritoneal dialysis and kidney transplantation. This study was undertaken to evaluate renal tissue regeneration using cells from neonatal kidneys of rats and fibrin gel and its clinical usefulness.
MATERIAL & METHODS: After the development of criteria for classification of the ureteric lesions, we studied ureteral fragments obtained during organ harvesting in the cadaver (nine cases), immediately after the cold preservation period (18 cases) and after the kidney graft reperfusion (126 cases). Further to the histological analysis, including the detection of signs of rejection, we evaluated the risk factors for the development of lesions (from the donor, the recipient and the transplant) and their relation to the evolution of the transplant, including the occurrence of renal rejection episodes. RESULTS: One hundred and twenty out of the 126 fragments studied after graft reperfusion presented some kind of alterations, namely epithelial exfoliation in 42%, cell vacuolisation in 52.4% and cellular infiltration of the lamina ptopria and urothehum in 74.6% and 77X%, respectively. Global cellular infiltration was considered to be normal, mild and moderate to severe in 34.9%, 41.3% and 23.8%, respectively, consisting mainly in lymphocytes T CDS+. There was an inverse relationship between donor ventilation time and the intensity of the cellular infiltration and between donor age and vacuolisation. Seven and three out of the nine fragments obtained during organ harvesting showed mild cellular infiltration of the lamina propria and urothelium, respectively. Cold storage promoted minor histological changes, apart from lymphocitic migration to the epithelium. This pattern was more pronounced after re-perfusion, when it was detected an increase in urothelium infiltration in 11 out of the 18 cases. There were no detectable relation between the lesions encountered and HLA compatibilities between donor and recipient, the development of rejection lesions in future ureteral biopsies or to the evolution of the graft itself. CONCLUSIONS: The consequences of brain death and/or mechanical ventilation were detected at the ureter level, with abnormal lymphocitic infiltration in most cases. On the other hand, cold storage did not produce any major histological changes. The lesions detected after graft re-perfusion do not seem to involve any immunological phenomena. European
Urology
Supplements
3 (2004)
No. 2, pp.
10
MATERIAL & METHODS: In 16 male Spraque-Dawley rats with a body weight 200. 250gm, two-stage 516 nephrectomy was performed and confirmed CRF state of rats by increased BUN. creatinine 4 weeks after neohrectomv. We resected kidnevsi in neonate rats under sterile condition and chopped renai tissues were incubated in collgenaseidispase (Roche) solution for 40 minutes at 37°C. The incubated renal cells were labelled with fluorescent dye. The labelled renal cells were injected to remnant kidney of nephrectomized rats (n=8) witb fibrin gel matrix. Rats were sacriticed four weeks after injection and kidneys were harvested. Each injection site was analyzed histologically, and BUN, creatinine were measured before and after injection respectively. The nephrectomized rats (n=X) which were not injected were used as control group. RESULTS: All rats were healthy and there were no complications until being sacrificed. The initial BUN and creatinine were 41.78+9.42mg/dL (mean ISD) and 0.910.12mg/dL, and increased to 174+12.6mg/dL, 2.6+1.3mgidL in control group. However, BUN and creatinine were 50.6+8.48mg/dL and l&O.l4mg/dL in cell-injected group, respectively (p
CONCLUSIONS: Improvement of renal function in CRF rats and regeneration of renal tissues after injection of renal cells mixed with fibrin gel were observed. Further investigation will show the possibility of application of renal cell injection for treatment of CRF patients.