Materials and methods
- Reference values and physiological characterization of a specific isolated pig kidney perfusion model
Animals and experimental groups
After approval of the local official veterinarian institutions, German landrace female pigs (age six months) were used. Six differently treated groups (table 1) were analyzed for reference values. Kidneys from four groups were collected from laboratory animals in an operating theatre (A-D), kidneys of group E originated from slaughterhouse animals at an abattoir. Whereas in group (A) no preservation at all took place, the organs of the groups B-E were preserved before hemoperfusion (B, C, : 2 hrs, D 24 hrs, E about 5 hours due to the process of slaughtering and transport). In group C, albumin was added to the perfusate to approximate physiological colloid osmotic pressure with the two aims: 1.) to normalize effective filtration pressure relations in the glomerula of the kidney and 2.) to reduce the danger of edema.
The control group (CON) originated from 8 living laboratory animals, kept under controlled conditions for 1 week in the stables of the facility, inhouse with the laboratories and the operation room. The animals were provided with blood access via a cannulated external jugular vein for three days. On the second day of this period the individual animals were hold in a metabolic cage for the purpose of 24 hour urine collection. The individual three day mean values of the blood samples and the 24 hour urine values were used as basic data for the CON group.
Selected results from group CON had already been presented in part in a previously published methodological study  to demonstrate a new graphical depiction method.
For the collection of blood of the slaughterhouse animals, as previously described in detail [19,36]., the cervical vessels (Venae jugularis dex.et sin., V. cava cranialis) were punctured and the collected blood was anticoagulated with sodium citrate (18 ml/l) and heparine (5.000 IE/l). The blood was then filtered (Biotest TNSB-3 transfusion device, 200 μm) and stored in sterile blood bags . Alternatively, in the laboratory animals group, blood was collected under sterile conditions via the cannulated external jugular vein.
The pigs of the slaughterhouse groups were electrically stunned and then exsanguinated. Then the organs were removed by en bloc technique, arterially cannulated and flushed with preservation solution (4°C) containing 5.000 IE/L heparine (Liquemin N, Roche). 500 ml of preservation solution (see table 2 for B2-solution pursuant to von Baeyer ) was then applicated into the artery and the kidneys were transferred under sterile, hypothermic (4°C) conditions from the abattoir to the laboratory.
Kidneys from laboratory animals were handled in the same way after being removed surgically. For organ harvesting by surgery, pigs were set under general anesthesia undergoing median laparotomy. The right external jugular vein was cannulated and the animal was heparinized (300 IE/kg body weight). Kidneys were removed and cannulated one by one before the animal was exsanguinated. Normally one kidney was perfused immediately and the other underwent the preservation procedure before the reperfusion.
Perfusion procedures were carried out as previously established for kidneys and other organs [19,37,38]. Ureteral and vascular catheters were implanted and a period of warm rinsing with 500 ml of preservation solution was performed before hemoperfusion with autologous blood was conducted. The hemoperfusion started with an arterial flow of 50–100 ml/min and a mean arterial pressure never allowed to exceed 100 mmHg to ensure an optimal organ warming up and the beginning of renal autoregulation under reperfusion. Blood and urine samples for assessment of parameters were collected after entering a steady state usually after 20–30 min. Then, within clearance periods of 30 min, urine collection and blood sampling was performed and immediately followed by blood gas analysis using an automated blood gas analysator (Radiometer Copenhagen, ABL) to assess pH- and electrolyte status. Further sample fractions were stored for a later transfer to the labotaratory for analysis of multiple other parameters as listed below. Also, venous and arterial pressures and arterial flow were recorded online using ultrasonic flow transducers (Transonic Systems Inc., T206). Organ weight was also assessed directly after surgical resection (prior to eventual cold storage) and before and after reperfusion.
The perfusion system consisted of separated blood and dialysis circuits as described , that may also be used for the perfusion of other organs and tissues, like the liver [39,40], the heart  or the skin . The volume of heparinized (20.000 IE/l) blood was 600 ml, added with standard electrolyte solution (modified Tyrode's solution) to adjust pressures and hemoglobin concentration and to replace urine fluid loss.
The blood was pumped from the reservoir to a low-flux polysulfon dialysis system (model F7, Fresenius, Bad Homburg). Next to dialysis processes, the blood was also oxygenized in this module and then transported to the organ with a second roller-pump. After passage through the organ, the blood reached the reservoir due to hydrostatic pressure differences.
The dialysis circuit containing 10 000 ml of dialysate medium (modified Tyrode's solution) was driven by a roller pump. The dialysate circuit meets the metabolic demands of the organ and, therefore, is permanently oxygenated and nutritional substrates are added as well as creatinine for the determination of the exogenic creatinine-clearance. The substrates are periodically controlled for a steady state in the composition of the dialysate. The temperature was adjusted to 38°C. Controlling of ultrafiltration und thus the perfusate dilution was maintained by continuously weighing the blood reservoir and balancing the afferent and efferent blood roller pumps. The kidneys were kept in a body warm plexi-glass chamber. Urine was collected by way of a ureteral catheter in calibrated glass cylinders.
Apart from basic experimental data (table 3: weight parameters, ischemia time, perfusion time), hemodynamics and blood gases, hemoglobin, blood and urine pH and different electrolytes, the following parameters were measured: free hemoglobin (mg/dl), total blood protein (g/dl), creatinine-concentration in blood (mg/dl) and urine (g/l), urine flow (ml * min1 * 100 g-1). By use of the described formulae (see appendix) the following parameters were determined: creatinine clearance (Clcrea, ml * min1 * 100 g-1), fractional water reabsorption (RFH2O, %), fractional sodium reabsorption (RFNa, %), tubular sodium transport (TNa, mmol * min-1 * 100 g-1). Results are presented for the steady state of the model as 60 min values (hematology: table 4; blood, urine laboratory: table 5) and additionally with the 3 hour state for hemodynamics and renal functional parameters (table 6).
To analyze the influence of multiple determands on complex kidney function parameters, a grapho-analytical method was used, which is described in detail in a previously published article for analyzing nephrological parameters . This nomogram-like method is applied here to examine the creatinine clearance used as approximation of the glomerular filtration rate (GFR).
The creatinine clearance represents the mathematical product of the U/Pcrea quotient and the urine-flow VU. Directly displaying these two terms in a x-y diagram leads to certain curves for similar Clcrea. values in each experimental group, which are difficult to be distinguished from each other. Therefore the x, y data are transformed into logarithmic scaling and linear lines instead of curves are resulting for constant values of the creatinine clearance. In that way figure 1 was constructed and the interrelation of the following parameters can be analyzed: creatinine U/P quotient (U/Pcrea), urine-flow (VU), creatinine-clearance (Clcrea). As a fourth parameter, the fractional reabsorption of water RFH2O(see appendix for the formula) can be displayed, since the reciprocal expression of the U/Pcrea quotient, arranged as (1- P/Ucrea), represents the water reabsorption along the tubular system which is numerically present in the second scale of the y-axis in figure 1.
All assessed data are expressed as mean ± standard deviation (SD). Statistical significance (p
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