Using vital microscopy (tscm) to evaluate living kidneys.

Tandem Scanning Confocal Microscopy (TSCM) is a non-invasive form of vital microscopy which can be used to evaluate superficial uriniferous tubules in living kidneys. Because TSCM has a number of advantages over conventional microscopic examination of renal biopsies, the present study was undertaken to determine whether the histopathological images obtained by TSCM can be correlated with post-transplant renal function. The kidneys of New Zealand male rabbits were harvested, flushed in Euro-Collins solution, and stored at 0-2 degrees C for periods of 24, 48, 67 and 72 hours prior to transplantation. As expected, there was a significant deterioration in post-transplant renal function as the kidneys were stored for longer periods of time. TSCM observations of the kidneys prior to their transplantation revealed characteristic histopathological changes of the proximal convoluted tubules which correlated closely with post-transplant renal function. These observations support the proposed use of TSCM in evaluating human donor kidneys prior to their transplantation.

Normothermic blood perfusion of isolated rabbit kidneys. II. In vitro evaluation of renal function followed by orthotopic transplantation.

This study describes the use of a blood perfusion apparatus to assess the renal function of isolated kidneys. Eight fresh kidneys were obtained from healthy rabbits and perfused with blood at 36 degrees C for 2 hours. Rabbit blood was drawn and diluted to a hematocrit of 25%. The kidneys were evaluated for their capacity to support life in an autograft model. Blood and urine samples were taken at regular time intervals during kidney perfusion. Serum creatinine was measured in surviving rabbits after transplantation. Over the course of the perfusion, arterial pressure was maintained at 87.2 +/- 5.5 mm Hg. The renal blood flow (3.7 +/- 1.0 ml/min per g) and urine output (0.11 +/- 0.04 ml/min per g) were continuously monitored. Glomerular filtration rate (0.29 +/- 0.02 ml/min per g) and fractional reabsorption (FR) of sodium and glucose indicated appreciable tubular function (FR(Na) = 67.9 +/- 8.5%, FR(Glu) = 91.2 +/- 5.8%). Protein was excluded from urine at 99.8% +/- 0.1%. After transplantation, the peak creatinine was 6.8 +/- 3.2 mg/dl at 1.90 +/- 0.92 days for the seven surviving rabbits and was above 16 mg/dl for the only rabbit that died 4 days after operation. The level of free hemoglobin generated at the end of the perfusion (2.6% +/- 2.8%) was correlated with the postoperative peak creatinine (r2 = 0.84). Perfusion of seven additional kidneys by using the roller pump lead to a final hemolysis of only 0.34 +/- 0.14%. Kidneys transplanted after 2 hours of blood perfusion were able to resume normal function and support life. Hemolysis was a measurable stress factor causing delayed function of the kidney after transplantation. Introduction of a roller pump significantly reduced the hemolysis.

Vitreous cryopreservation maintains the function of vascular grafts.

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.

Cryopreservation of the mammalian kidney. I. Transplantation of rabbit kidneys perfused with EC and RPS-2 at 2-4 degrees C.

The requirements of organ cryopreservation differ from those of conventional organ preservation. The encouraging results of Karow's group with dog kidneys transplanted after perfusion with more than 4 M dimethyl sulfoxide were based on an RPS-2 (renal preservation solution 2) vehicle solution, but transplantation of rabbit kidneys after perfusion with RPS-2 has not been reported. We evaluated RPS-2 in comparison to Euro-Collins solution (EC) using a modified technique for rabbit kidney autotransplantation and a computer-based organ perfusion machine designed for the introduction and removal of cryoprotective agents. Consistent success in rabbit kidney transplantation was found to depend on the anesthetic used, the hydration volumes administered, and direct ureter-to-ureter anastomosis. RPS-2 was found to be equivalent to EC for short-term (about 5 h) preservation by either perfusion or simple cold storage. However, good results with EC were associated with perfusion at 4 degrees C, recovery being significantly worse at 2 degrees C. In addition, we found that the solitary rabbit kidney is not able to fully compensate for the loss of the contralateral kidney, the result being persistent (to 3 weeks) mild elevation of serum creatinine, potassium, and calcium and persistent moderate reduction of serum phosphate. These results establish perfusates, perfusion conditions, transplantation techniques, computer-based perfusion control techniques, and a general clinical baseline that are permissive of further direct experiments on cryoprotectant introduction and removal.

Inhibition of coronary artery transplant atherosclerosis in rabbits with angiopeptin, an octapeptide.

Accelerated coronary atherosclerosis of cardiac allograft occurs in 30-40% of cardiac transplant patients and remains an unsolved clinical problem. The etiology is unknown and anti-platelet drugs are used without conspicuous success. The inhibitory effect of the octapeptide, angiopeptin on coronary atherosclerosis was studied in a previously described rabbit heterotopic cardiac transplant model where allograft rejection is prevented by daily administration of cyclosporin A (CsA, 10 mg/kg per day s.c.). Twenty male New Zealand white rabbits (2.6-2.8 kg) received a heterotopic cardiac transplant from rabbits of the same strain. Donors and recipients were fed a 0.5% cholesterol diet 1 week prior to transplantation which was continued for the recipient until death 6 weeks later. The control group (n = 16) received CsA and saline injections twice daily and the treatment group (n = 4) received CsA and angiopeptin (60 micrograms/rabbit daily s.c.) in 2 divided doses. The treatment began after completion of the transplantation. Coronary artery transplant atherosclerosis was uniformly distributed (tubular) in the entire length of the coronary arteries. Angiopeptin inhibited the intimal hyperplasia in the transplanted heart from 47.5 +/- 2.4% (mean +/- SE) to 25.0 +/- 6.9% and in the native heart from 24.2 +/- 1.4% to 15.7 +/- 1.5%. The intimal hyperplasia is expressed as area of intimal hyperplasia/total vessel area x 100%. A similar inhibition by angiopeptin was seen in lipid deposition in the donor ascending aorta which is transplanted with the heart. Angiopeptin attenuated significantly the hyperplasia and the lipid deposition of the native coronary arteries and aorta but to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of prednisolone, thromboxane, and platelet-activating factor receptor antagonists on lymphocyte and platelet migration in experimental cardiac transplantation.

Three agents that significantly prolong cardiac allograft survival were tested in Lewis rats that were recipients of hearts from Lewis X Brown-Norway F1 hybrid donors. In the presence of azathioprine, the effects of daily administration of either the thromboxane antagonist (L 640,035), the platelet-activating factor (PAF) antagonist (BN 52021) or prednisolone were evaluated on the infiltration of cardiac allografts by syngeneic lymphocytes and platelets labeled with 111indium. As anticipated, platelet deposition was reduced by the thromboxane antagonist and unaffected by the PAF antagonist; the latter is likely due to the known absence of PAF receptors in rat platelets. In addition prednisolone had no effect. The increased accumulation of lymphocytes on days 4-5 was also unaffected by all three drugs. These experiments indicate that, in this model, graft survival is not necessarily related to lymphocyte and platelet infiltration of the graft. The data also provide evidence for the efficacy of the thromboxane receptor antagonist L 640,035 in preventing platelet deposition in vivo.

Leukotrienes, thromboxane, and platelet activating factor in organ transplantation.

The antirejection eicosanoids--PGE2, (PGD2), and PGI2--have an attenuating effect on T-cell proliferation by inhibition of IL-1, IL-2, and class II antigen expression on macrophages, and the prorejection eicosanoids--TXA2, LTB4, LTC4, and LTD4--enhance T-cell proliferation. LTB4 stimulates IL-1 and IL-2 formation and expression of IL-2 receptor. The mechanism of enhancement of T-cell proliferation by TXA2 has not been demonstrated. LTC4 and LTD4 promote gamma-interferon release and can replace IL-2 as a stimulator of gamma-interferon. PAF at high concentrations inhibits lymphocyte proliferation. The eicosanoids interfere with the same mechanisms as CsA and corticosteroids on T-cell clonal expansion. In experimental organ transplantation, corticosteroids can be replaced by compounds preventing the formation or expression of the prorejection eicosanoids or analogs of antirejection eicosanoids as well as by PAF antagonists. In addition, these drugs exert synergistic effect with CsA and azathioprine.

Platelet deposition in rat heart allografts and the effect of a thromboxane receptor antagonist.

The effect of a thromboxane antagonist, L640,035 on platelet deposition in heart allografts was studied. Twenty Lewis rats received heterotopic allografts from Lewis x Brown-Norway F1 hybrid. All recipients received azathioprine (5 mg/kg/day). The rats were divided into three groups. Groups II and III were also treated daily with either the vehicle for L640,035 or L640,035 respectively. Syngeneic indium-111-labeled platelet deposition was determined in the allograft and the native heart at 6, 9, and 13 days after transplantation; group III was studied on the sixth and ninth day only. A rapidly increasing platelet deposition was seen in allografts from rats given azathioprine; whereas the thromboxane antagonist prevented the increase in platelet deposition on the ninth day.