Ejvind Kemp

Nephrotoxicity of cyclosporin A. A lithium clearance and micropuncture study in rats

Abstract. Renal function was studied in rats treated with cyclosporin A (CyA). Peroral CyA 25 mg kg‐1 day‐1 depressed glomerular filtration rate (GFR) from 1284 ± 429 to 500 ± 228 μl min‐1 g‐1 kidney weight (KW) (P < 0·01). Absolute rate of proximal tubular reabsorption (APR) decreased from 1075 ± 437 to 468 ± 203 μl min gKW‐1 (P < 0·01). Proximal tubular fractional reabsorption (PFR) was 67·7 and 68·5% measured with the TT/OT and fractional lithium‐clearance methods, respectively. Amiloride had no effect on lithium‐clearance in CyA treated rats. Acute isotonic volume expansion increased GFR and APR towards normal, while PFR remained increased. Increased sodium clearance did not normalize renal function. CyA intravenously (12·5 mg kg‐1) depressed GFR and APR acutely, while PFR increased. Proximal intratubular pressures were low normal (mean 11·6 mmHg). Proximal transit times were prolonged (mean 25·2 s, P < 0·01). Renal morphology was normal. The data are evidence against a primary tubular damage of CyA, and makes it less likely that the major lesion is located to the glomerular membrane. The results suggest that CyA nephrotoxicity mainly is due to a haemodynamic effect.

Treatment with total lymphoid irradiation, cyclosporin A and a monoclonal anti-T-cell antibody in a hamster-to-rat heart transplantation model: Graft survival and morphological analysis

Abstract. Treatment with preoperative total lymphoid irradiation and post‐transplant cyclosporin A has been shown to have a synergistic effect on graft survival in alio‐and xenotransplantation. Specific monoclonal antibodies against T cells and T cell subpopulations could offer new ways of preventing graft rejection in xenotransplantation. Graft survival and histology were examined after total lymphoid irradiation plus cyclosporin A treatment versus cyclosporin A plus a monoclonal antibody in a concordant, heterotopic, hamster‐to‐rat heart transplantation model. Preoperative total lymphoid irradiation was given at a dose of 1. 25 Gy, 12 times over a period of 3 weeks. Cyclosporin A at a dose of 12. 5 mg/kg per day was administered perorally and OX‐19, a pan T cell monoclonal antibody, was given as intraperitoneal injections at doses of 100 μg or 500 μg/kg per day from day 0 until graft rejection. While total lymphoid irradiation alone prolonged graft survival to 9. 4 days, total lymphoid irradiation plus cyclosporin A extended graft survival to a mean of 22 days. Cyclosporin alone or combined with the monoclonal antibody could not increase graft survival significantly when compared to untreated animals, which rejected their grafts within 3. 7 days. Vascular rejection was the characteristic morphological finding, even after some weeks of excellent graft function. In conclusion, total lymphoid irradiation and cyclosporin A had a synergistic effect on graft survival in this concordant xenotransplantation model, although recent impressive results from other groups could not be reproduced. Total lymphoid irradiation combined with cyclosporin A appears to delay a primary humoral graft rejection, while the mechanism of rejection, judged by histology, stays the same.

Hope for successful xenografting by immunosuppression with monoclonal antibody against CD4, total lymphoid irradiation and cyclosporine: six months' survival of hamster heart transplanted into rat

Hamster hearts were transplanted to rats, and the effects of combinations of total lymphoid irradiation (TLI), cyclophosphamide, cyclosporine A (CyA) and monoclonal antibodies (MAB) were investigated. Controls not immunosuppressed rejected their xenograft in 3 to 5 days, while combination immunosuppression including MABs against CD4 or IL-2-receptors extended graft survival significantly. In one case, the graft was still functioning 180 days after transplantation, which is the longest survival seen in this model. The use of specific MABs may open a new era for both xeno- and allo-transplantation.

Survival, function, morphology and serological aspects of rat renal allografts. Effect of short‐term treatment with cyclosporine A, anti‐CD4 and anti‐interleukin‐2 receptor monoclonal antibodies

The aim of this study was to investigate the effect of short‐term treatment with cyclosporine A (CyA) combined with anti‐CD4 (OX‐38) and anti‐interleukin‐2 receptor (OX‐39) monoclonal antibodies (MAbs) on graft survival, graft function, morphology, and anti‐donor antibody levels in a BN‐to‐LEW rat kidney transplantation model. Spontaneous rejection occurred at 9.3 days (range 9–10 d). Administration of CyA (12.5 mg/kg/d) for 14 days prolonged graft survival to 33 days (range 23–40 d, P<0.02). Supplementing with OX‐38 and OX‐39 100 μg/kg/d, given i.p. from days 0 to 7, further prolonged graft survival to 70 days (range 38‐> 100 d, P<0.02 vs controls and CyA group). One of seven recipients had good graft function for more than 100 days. A three‐fold increase of the MAb dosage did not improve mean graft survival (53.5 d), but three of eight recipients had well functioning grafts for > 100 days. Kidney function was characterized by reduced creatinine clearance, also in the recipients with long‐term graft survival, and a defect in concentrating urine creatinine with subsequent pronounced increase in urinary output. Graft histology showed a complex pattern of interstitial alterations including mononuclear cell infiltration, fibrosis, tubular atrophy and vascular damage with intimal/endothelial cell hyperplasia and perivascular inflammation. In nine of 10 MAb‐treated recipients with graft survival > 60 days, granular deposits of immunoglobulins and C3 were found by immunofluorescence microscopy (IFM). The deposits were localized in the glomerular capillaries and mesangium. IFM in MAb‐treated control animals could not demonstrate any deposits. Flow cytometric evaluation of posttransplant serum samples against donor target cells showed increasing amounts of anti‐donor antibodies until the time of rejection, while recipients with long‐term graft function had moderately positive cross‐matches up to two months after transplantation. Hereafter antibody titres decreased and cross‐matches at the time of sacrifice were again negative. The morphological findings and the flow cytometric cross‐match results seem to indicate a postponed antibody‐mediated type of rejection. The reason why some kidney recipients showed decreasing antibody titres and stable long‐term graft function is unclear.

Adenosine stimulated postimplantational regeneration of 5-adenine nucleotides in rabbit kidney grafts

Abstract The ability to regenerate the adenylate content in renal tissue appears to fail in rabbit kidney grafts, cold stored for 24 hours. The post implantational adenylate regeneration which did not occur, took place within the first lo minutes of re-established circulation. Thirty and sixty minutes of re-vascularization did not give any additional adenylate synthesis. The presence of adenosine (2.5 mM) in the preservation fluid, and that taken up by the kidney tissue during in vitro storage, brought about a significant increase in adenylate regeneration during the postimplantational period, independennt of a parallel increase in renal blood flow, and irrespective of the fact that adenosine did not show any adenylate preserving effect in vitro. The present results suggest that in long-term stored rabbit kidney grafts there occurs a relative postischaemic lack of intracellular available purine precursors. Owing to the filtration inhibiting effect of adenosine a study of the correlation between kidney function and adenylate regeneration was not possible.

The resistance of delayed xenograft rejection to α(1,3)‐galactosyltransferase gene inactivation and CD4 depletion in a mouse‐to‐rat model

Critical to the prevention of xenograft loss is the prevention of delayed xenograft rejection (DXR), due to its resistance to conventional immunosuppression. The role of the carbohydrate galactose‐α1,3‐galactose (α1,3Gal) has been a matter of great debate and it has been proposed that the reaction between α1,3Gal epitopes on donor endothelial cells and recipient anti‐α1,3Gal antibodies (Abs) may damage the graft during DXR. Recipient anti‐α1,3Gal Abs are produced by CD4‐dependent B cells. To test the above‐mentioned hypothesis, hearts from α1,3Gal‐free mice (GT‐Ko mice), generated by α1,3‐galacto‐syltransferase gene disruption, were transplanted to anti‐α1,3Gal antibody‐free Lew/Mol rats. This model consists of an α1,3Gal/α1,3Gal‐antibody‐free environment, eliminating a possible influence of this specific system on DXR. A subgroup of recipients were furthermore CD4 depleted in order to inhibit CD4‐dependent B‐cell antibody production. Rejected hearts were evaluated by light‐ and immunofluorescence microscopy. Treatment effects on recipient T‐cell subsets and cytokine expression were analyzed by flow cytometry, while antibody production was measured by ELISA. All recipients developed DXR with no differences among the groups. DXR was related to thrombosis with IgG and IgM desposition in vessel walls, as well as macrophage and granulocyte accumulation in the myocardium. No complement C3, CD4 cells or NK cells were found. Flow cytometric analysis confirmed peripheral blood CD4 depletion and IFN‐γ suppression in CD4 Ab‐treated recipients. Finally, ELISA showed that specific anti‐α1,3Gal Ab production was absent. However, Ab(s) against an unidentified Galα 1 were found among recipients. In our model, DXR is resistant to α1,3‐galactosyltransferase gene inactivation and CD4 depletion. However, other Galα 1 epitopes and antibodies may play a role during DXR. Further studies are needed to elucidate the precise pathways leading to DXR.

Pinacidil - Effects on Function and Perfusion of Normal Kidneys and Renal Xe nog rafts

Pinacidil is a new vasodilating compound claimed to increase renal perfusion and function. A preliminary study showed that pinacidil did not sustain renal perfusion through a period of increasing renal vascular resistance in pig-to-rabbit renal xenotransplantation. Accordingly, the effect of pinacidil on renal function in conscious catheterized rats was studied. Pinacidil 0.12-0.18 mg/kg i.v. caused a moderate reduction in mean arterial blood pressure (14-17 mmHg), a decrease in inulin and PAH clearances (50-65%), and sodium and water retention. The sodium clearance/lithium clearance ratio decreased, suggesting increased sodium reabsorption in the distal nephron during pinacidil administration. Thus the results are in accord with those obtained with several other vasodilators, but not with those obtained in mongrel dogs during pinacidil administration.

Xenotransplantation – State of the Art

Rejections caused by xenotransplantation Transplantation between two different species may give rise to two types of graft rejection. Between so-called discordant species a hyperacute rejection (HAR) occurs within minutes or hours. In so-called concordant species this type of rejection does not occur, but over days a delayed xenograft rejection (DXG) (6) will occur. In all transplanted organs, xenoas well as allotransplanted, chronic graft rejection (CRG) will occur after years. Man is concordant with old world primates, but due to several reasons, such as breeding difficulties, risk of retroviral epidemics, concerns for the use of endangered species, etc., the discordant pig is considered the donor of choice. HAR is primarily caused by initiation of the complement cascade. The antigen known to be activating the classical pathway is the alpha-gal epitope (10) (Figure 1); the epitope alternative to the primate AB0 blood group system in most non-primate mammals. In species, in which the antigen is absent, circulating antibodies are present. In humans IgG against the alpha-gal epitope counts for up to a total of 1% of circulating IgG (11). These antibodies probably derive from a reaction to members of the intestinal flora, especially Enterobacteriaceae spp., but also other types of infectious agents possess the alpha-gal epitope as a structural element in their cell walls (10).