2.1. Single-pass perfusion (Variant 1)

Oxygenation of the perfusate is carried out by aerating through sintered glass. This is possible when gelatin-based colloids (Haemaccel), hydroxyethyl-starch (HAES), dextran or pluronic (84) are used, but results in extreme foaming with BSA, which may also promote denaturation of the protein. We have used a combined system of recirculation, which is readily convertible to the single pass mode to enable analysis of metabolism and clearance of uremic middle molecules (233, 234). A membrane oxygenator is used to equilibrate the albumin-containing perfusate. Smyth et al. used a single-pass system with albumin either alone (280) or combined with Ficoll (279) and equilibrated the perfusate at a high flow-rate (2 L gas/min) by means of a capillary dialyzer. These authors provided no information on fluid loss by the hollow fibers. The dialyzer and other parts of the system were immersed in a water bath. This is critical for hygienic reasons. This has been practiced historically in peritoneal dialysis of man, but was later abandoned due to problems with contamination and replaced by dry temperature conditioning. It is better to use water bath jacketed glass coils upstream and downstream of the dialyzer.

Perfusion without colloids is possible, but requires a different procedure compared to an isolated Langendorff preparation of a rat heart (66); it is essential to decapsulate the kidney and not to use ureteral catheters made of PE/PP10, which are too narrow. Cortell (59) points out that PE10 ureteral catheters are particularly unsuitable for the use especially at high diuresis rates (see Fig. 4.7.1.), as in the case of colloid-free perfusate (250, 251). A major advantage of the single pass mode is the constant supply of substrates, such as arginine as a constant source of NO, which is otherwise rapidly depleted in a pure recirculation system especially at small volumes, if it is not replaced continuously (206, 207). If erythrocytes are added, an artificial lung, such as a glass-bulb (film-) oxygenator, membrane oxygenator or a dialyzer (dialung) as in mode 3, must be used for oxygenation where the aerated dialysate is used to equilibrate and regenerate the perfusate. In addition, the effects of the infusion/omission of peptide hormones such as arginine-vasopressin (by proportional infusion or withdrawal of hormones) can be analyzed more easily (305) than when Variant 2 is used.

2.2. Recirculation (Variant 2)

Variant 2 is the technique of choice if one is interested in following cumulative biochemical or pharmacological effects over time. Examples include the study of metabolic processes such as gluconeogenesis, the elimination or breakdown of drugs or the impact of hormones (23, 32, 58, 107, 108, 162, 164, 177, 185, 223, 225, 227, 237, 294). Membrane- or glass-bulb oxygenators were initially used as artificial lungs, but nowadays special capillary dialyzers are preferred. Functionally the best colloid is albumin - bovine serum albumin (BSA) has the lowest viscosity at a given colloid osmotic pressure (COP), and therefore provides the highest perfusion flow rates at any defined perfusion pressure. Compared to other colloids albumin has a well-defined molecular weight. Bowman and Maack have best exploited the advantages of the recirculation method, as their apparatus is easy to set up and the functional results are comparable to those achieved with the method established by the Oxford Group of BD Ross and his followers. Urine was mostly reinfused, except in the case of clearance studies, and the withdrawal of perfusate and urine must be compensated for if the recirculating volume is very small. Pegg, however, has rightly pointed out that the urine contains particles that can block capillaries and enhance resistance to flow (196). Within 30 min after initiation of cell-free perfusion, detached fragments of tubular epithelia appear in the urine sediment (218), and this desquamation process has been verified by morphological studies (7, 263), thus confirming Pegg´s observations. These findings led to the inclusion of a filter between the perfusion pump and the kidney, which also removes particulates originating from the surgical procedure used to isolate the kidney from the animal.

The recirculation mode is particularly suitable for biochemical and pharmacological studies because it facilitates the analysis of cumulative effects. Authors differ on whether BSA can be used in form of the crude Cohn fraction V (23, 33, 162, 163) or should be defatted and dialyzed before use (55, 57). The former group finds the albumin-bound substances present in the Cohn V fraction useful, while the other group prefers tighter control over the composition of the perfusion medium. Moreover, the latter approach is generally thought to provide better functional stability of the preparation, as becomes apparent from continuous monitoring of multiple parameters such as flow, pressure, pO₂ and temperature. Indeed, it was the observation of a rapidly evolving acidosis in the isolated rat liver – perhaps due to the HCO₃ consumption for urea synthesis and/or a lactic acidosis due to hypoxia – in small volumes of perfusate that led to the first use of dialysis (20).

Fig. 2.1.

Urinary sediment from an IPRK perfused with washed human erythrocytes. The micrograph shows several human erythrocytes, one cell ghost and a larger tubular cell, which may be big enough to clog capillaries within the vascular bed.

2.3. Recirculation & regeneration of perfusate by dialysis (Variant 3)

This technique combines the advantages of the single-pass perfusion and recirculation modes, and my colleagues and I have used it particularly for physiological studies. In this case, the dialyzer also serves as the oxygenator, as the dialysate is aerated continuously via sintered glass frits in the mode of the single-pass perfusion. Cumulative effects reflected in changing concentrations of small metabolites, as seen in a pure recirculation system, are avoided. One disadvantage of this method is the greater technical effort required to compensate for the ultrafiltration implemented by the dialyzer. We used a level control of perfusate and automatic supply of dialysate that compensates for ultrafiltration and the removal of perfusate and urine samples. We routinely use a dialysate-to-perfusate ratio of ≥20, i.e., 200-250ml perfusate to 5000 ml of dialysate. This provides a quite stable composition of the perfusate as in a single pass perfusion mode and prevents the rapid loss of arginine as a source of nitric oxide (NO) (206, 207). Thus, the method is best suited for time-consuming micropuncture experiments (182, 184, 206, 207, 245, 248, 251, 266, 267, 291, 292). It also proved to be effective in other respects (106, 135, 136, 241, 242, 263, 267). So-called low-flux dialyzers with smaller pore size, which prevent the passage of albumin into the dialysate (unlike high-flux dialyzers which, however do not play a role in vivo due to the sealing capability of whole blood), are most appropriate in this context. Over the past 12 years, this technique has been applied to larger mammals like rabbits (3, 315), dogs (110) and pigs (89, 90). When Baumung and Peterlik (20) first used dialysis in liver perfusion they had to build their own dialyzer using cellophane tubes. But today´s mass produced dialyzers are affordable and durable, provided appropriate cleaning procedures are followed before reuse. After the first publication of this manual in 2017, I found the publication by Shatkin et al., who was the first to use a dialyzer in a bypass when perfusing an isolated dog kidney (275b).

2.4. Reperfusion of an anatomically perfusion-fixed kidney (Var. 4)

By reperfusion of anatomically fixed kidneys, we were able to analyze the glomerular filter apparatus under conditions that are not applicable in vivo. For example, it was possible to analyze the permeability of albumin at acid pH near the protein´s isoelectric point or below, when the surface charge of albumin switches from negative to positive. The idea was triggered in response to the workload involved in the large numbers of micropuncture experiments required to analyze glomerular permeability for albumin in the isolated perfused rat kidney (245, 248, 266, 292). The electric charge within the glomerular filter apparatus is preserved even in the perfusion fixed kidney. Reale et al. demonstrated the conservation of negative charge at the glomerular capillary membrane histochemically after perfusion fixation using glutaraldehyde (210-215). The aim was to obtain a preparation where GFR is high enough and identical to the urinary output. Therefore, we perfused kidneys for 15min at 100mmHg (effective perfusion pressure) with the addition of verapamil (4.4 µmole/l); beginning 2 min before fixation we increased the perfusion pressure to 150mmHg and maintained it at that level during the fixation period. The fixation solution was a 1.25% glutaraldehyde, phosphate-buffered salt solution at pH 7.1 with 6% HAES as a colloid. Why the colloid additive? Our first fixation done without colloid but under normothermia, revealed that the abrupt drop in the colloid-osmotic pressure to zero leads to a drastic increase of the perfusion resistance, thereby delaying and hampering the fixation. The addition of HAES (hydroxyethyl starch) improved fixation quality significantly and thus allowed us to establish this model (s.a. the morphological study: (263)). The kidneys were stored together with their double-barreled aortic cannula at 4°C in a special designed custom-built acrylic glass box, until used for reperfusion days or even weeks later, s.a. Fig 5.4.1.- 5.4.4. (50-53, 78, 266, 317).