JOURNAL

OF SURGICAL

RESEARCH

22, 128-142

(1977)

An Isolated Rabbit Kidney Preparation for Use in Organ Preservation Research Division

B.J. FULLER,~ D.E. of Cryobiology and Animal

PEGG,~. Division,

Submitted

A. WALTER,

Clinical

Research

for publication

Isolated organ perfusion techniques are playing an increasingly important part in many aspects of experimental biology: in biochemistry, physiology, and pharmacology and in organ preservation and transplantation [25]. We have been particularly concerned with renal preservation [18, 20, 211 and, in common with other workers, have relied on transplantation to assess viability after different preservation procedures. Transplantation must remain the final test of function, but as a tool for comparing experimental variations in the preservation procedure it has serious disadvantages. Transplantation is expensive in both time and resources, and it is often difficult to obtain reproducible quantitative data. Many simple viability tests have been advocated, but none has been entirely satisfactory [7, 111, although a combination of several such tests may be more reliable [32]. We felt that a normothermic perfusion technique measuring the principal functions that the kidney performs in vivo might be a more suitable basis for a quantitative laboratory viability assay. The choice of experimental animal was crucial. The traditional surgical model is the dog, but animals of good quality are expensive while cheaper animals are often unhealthy and tend to give variable results. Rabbits proved to be a convenient and reliable model in earlier renal preservation studies reported from this laboratory [20, 211; healthy animals are inexpensive, and

July

AND C.J. Centre,

Harrow,

GREEN United

Kingdom

1, 1976

rabbit kidney is a convenient size (-15 g) both for biochemical studies and for transplantation. Most of the recently published studies of normothermic renal perfusion have been on dog or rat kidneys, and the results obtained with well-controlled bloodless perfusates, particularly in rats, are now beginning to approach in vivo physiological function [S, 261. Since very little work on rabbit kidneys has been published, we decided to study a considerable range of possible perfusates and to investigate the physiology of isolated rabbit kidneys in some detail. Accordingly, five different perfusates were tested, and glomerular filtration rate, protein leakage, and ultrastructural appearances were used as criteria for the selection of the most suitable perfusate. Studies were then made of structural, circulatory, and metabolic effects during a 2-hr perfusion period. Finally, the glomerular and tubular function of the preparation was defined in the necessary detail for its use as a functional assay in organ preservation research. MATERIALS

AND METHODS

I. Animals. New Zealand albino rabbits, of either sex, weighing 2.5-3.5 kg were used. For in vivo control observations light anesthesia was produced with nitrous oxide, oxygen, and halothane. An anterior midline incision was made, the ureter was severed at the bladder, and timed urine collections from the left kidney were made. Ten microcuries of [14C]hydroxymethyl inulin with 50 mg of carrier inulin, and 20

’ Present address: Academic Department of Surgery, Royal Free Hospital, Pond Street, London NW3 2QG, United Kingdom. 128 Copyright @ 1977 by Academic F’rebs, Inc. All rights of reproduction in any form reserved

ISSN

00224304

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&i of 1311-labeled bovine serum albumin (BSA) with 500 mg of carrier albumin were injected intravenously, and after a lo-min equilibration period, clearance values for inulin and BSA were determined during two consecutive 20-min periods. Renal tissue for estimations of cortical and medullary sodium and potassium content was obtained from similarly anesthetised animals. Fresh tissue and ischemic tissue for adenine nucleotide measurements were obtained as follows: While blood was still flowing normally, one pole of the kidney was excised and allowed to fall into a bath of acetone/ solid CO, to effect rapid freezing. In those experiments in which the effect of warm ischemia was studied, a period of 2 min was allowed to pass between clamping the renal artery and taking the tissue sample. For the perfusion experiments, anesthesia was induced by the intravenous injection of 50 mg/kg of sodium pentobarbitone, and 5000 IU of heparin were injected. A loose ligature was placed around the left renal artery, which was then clamped near the aorta; as soon as the arterial cannula (polished stainless steel, 1.2-mm o.d.) had been introduced, perfusate flow was started. This procedure gave a warm ischemia time of not more than 2 min. The cannula was fixed in position

KIDNEY

129

PREPARATION

by tightening the loose ligature, and the renal vein and ureter were severed. Blood in the kidney was washed out using 200 ml of perfusate at a pressure of 110 mm Hg, and the kidney was then transferred to the perfusion cabinet. The right kidney was removed and weighed, and this weight was used as the reference value for the perfused kidney. 2. Perfusion apparatus. The perfusion apparatus is shown diagrammatically in Fig. 1. Fluid was aspirated from the reservoir by a peristaltic pump (Watson Marlow Type MHRE) and passed through a filter of 142mm diameter and 0.22~pm pore size (Millipore) and a bubble trap to the arterial cannula. The venous effluent was allowed to return by gravity to the reservoir, where it was continuously bubbled with 95% 0,: 5% CO, gas. Polyamide (nylon) tubing (Portex Ltd.) with a diameter of 3.5 mm was used for all connections because it is less permeable to oxygen and carbon dioxide and absorbs less fatty acid than silicone rubber 1191. The whole system was enclosed in a humidified, thermostatically controlled cabinet at 37°C. During perfusion, arterial pressure was monitored from a T-connection on the arterial cannula using a strain gauge pressure transducer (Ether UP4) and was main-

Bubble trap Sample point

Urine collection

Flow measurement 95% 02,5% co2

FIG.

1. The perfusion

apparatus

contained

in a thermostatically

controlled

chamber.

P = pressure

transducer.

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tamed at 110 mm Hg by automatic control of the pump speed [21]. Perfusate flow was measured by timed collections of the venous effluent. Timed urine collections were made from the cut end of the ureter, and, at the midpoint of each urine collection, a perfusate sample was taken from a tap close to the arterial cannula. 3. Perfusates. Details of the basic perfusate are shown in Table 1. The ionic composition was similar to that of rabbit plasma. Metabolic substrates included glucose and octanoate, and the antibacterial agent gentamicin was also added. Inulin and albumin were included as carriers for the labeled materials used to determine glomerular filtration rate and protein retention respectively; these were 10 &i of [i4C]hydroxymethyl inulin and 20 PCi of 1311-labeled BSA/liter of perfusate. To this basic perfusate the colloids listed in Table 2 were added. BSA was obtained from the Sigma Chemical Co., and dextran T70 (dextran) was obtained from Pharmacia Ltd. Hydroxyethyl starch (HES) was obtained from McGaw Laboratories; the preparation used is designated cryoHES. Pluronic F108 (pluronic) was supplied by the BASF Wyandotte Corporation. The total volume of the circuit was 750 ml; the pH of the perfusate was 7.45; and the ~0, was 600-650 mm Hg at the arterial cannula. 4. Physical and chemical methods. Viscosity was measured at 37°C using a cone and plate viscometer (Wells-Brookfield). Surface tension was measured by the maximum pull exerted on a partially immersed copper wire loop attached to the beam of an analytical balance. Plasma, perfusate, and urine levels of 14C-labeled inulin were measured by liquid scintillation spectrometry (Packard Model 3375 counter), and 1311labeled BSA was estimated by gamma spectrometry (Packard Model 3002 counter). Oxygen tension was measured in arterial and venous samples by using a Clark cell (Beckman macroelectrode). Samples of arterial perfusate were taken into a glass syringe and measured immediately to pre-

VOL. 22, NO. 2, FEBRUARY

1977

vent loss of gases by diffusion. Venous samples were taken by directing the venous outflow into a vial containing mineral oil and then sampling with a glass syringe. It was shown that, in the absence of a kidney, there was no difference in oxygen tension between samples taken from a threeway tap close to the arterial cannula and samples collected under oil as the perfusate issued from the cannula. Intrarenal oxygen tension was measured using a solid platinum wire electrode similar to that described by Cater et al. [4]. A silver/silver chloride reference electrode was used with a polarizing voltage of 600 mV, and the system was calibrated at +37”C using perfusate equilibrated with 02, Nz, or air. The response of the electrode was found to be linear. The calibration of the electrode was checked between readings. The sodium and potassium contents of the cortex and the medulla were measured by the method of Flear and Florence [6]. The commercially available reagent kits for ATP, ADP, and AMP (Boehringer Mannheim Ltd.) were used to measure adenine nucleotides. The glucose oxidase method was used for glucose estimations, and urea measurements were made by the urease method: Again Boehringer Mannheim kits were used. Osmolality was estimated with a Fiske osmometer, Model G-66 (Fiske Associates). TABLE COMPOSITION

Na+ K+ Ca*+ Mgz+ ClHCO,H,PO,HPO,*Glucose Octanoate Urea Gentamicin Albumin Inulin

1

OF THE PERFUSATE”

147.8 mh4 5.2 mM 1.0 mM 0.3 mM 131.5 mM 25.0 mM 0.28 mM 0.84 mM 5.0 mM 2.0 mM 0.7 mM 0.1 g/liter 1.O g/liter 0.1 g/liter

o Plus colloids, as shown in Table 2.

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PHYSICAL

PROPERTIES

AND

CLEARANCE

Solution Blood

Perfusates 5% BSA 5% Dextran 3% Dextran and 0.7% BSA 3% HES 3% Pluronic

PREPARATION

131

2 WITH BLOOD AND SYNTHETIC PERFUSATES

MEASUREMENTS

MW of colloid

KIDNEY

Viscosity (CP)

Surface tension (dynes cm-‘)

Colloid osmotic pressure (cm H,O)

Inulin clearance at 2 hr (ml mini g-i)

Albumin clearance at 2 hr (ml min-’ g-i)

69,000 for albumin

2.09”

58.4”

29b

0.43 + 0.06’

5.4 + 0.1 x lo-”

69,000 70,000 42,500 70,000 42,500 and 69,000 120,000 40,000 14,000

I .05 2.86

65.9 71.6

24d 66d

0.118 2 0.015 0.119 + 0.020

0.071 t 0.009 0.036 2 0.004

1.81

50.3

34d

0.112 + 0.014

0.040 2 0.006

1.30 1.26

73.6 38.0

32d 69”

0.787 k 0.090 0.461 k 0.063

0.059 t 0.006 0.053 k 0.006

Weight average

Number average

n Mean value for man. * Mean value for rabbits. c Values determined in viva in lightly anesthetized rabbits. d Colloid osmotic pressure was calculated according to the equation of Hint (12).

5. Measurement of perfusate distribution. The regional distribution of perfusate flow was measured by labeling the vascular space with an impermeant marker (5 mg/ml of blue dextran; MW, 2 x 106; Pharmacia Ltd.) [5] and then carrying out an efflux with fresh perfusate. The perfusion circuit was modified so that the kidney could be perfused, first, from a reservoir which contained the dye and, then, from a second reservoir which contained oxygenated prefiltered pet&sate; timed serial collections of the venous effluent were made. The concentration of dye in each sample of collected fluid was estimated by determining the optical density at 540 nm (Gilford, Model 300-N spectrophotometer). A graph of effluent dye concentration against time was plotted and resolved into three exponential components: The intercept of each component on they axis was proportional to the flow through that compartment [51. 6. Preparation for electron microscopy. Two kidneys were prepared for ultrastructural examination after perfusion for 2 hr with each of the five different perfusates. Twenty-five milliliters of 1% glutaraldehyde in 0.1 M phosphate buffer (pH

7.2-7.4 and osmolality 380 mosmol/kg) were perfused through each kidney at 5 ml/ min. The kidney was then immersed in fixative, and pieces of cortex were diced into l-mm cubes; fixation was continued for 24 hr. The cubes were then washed and postfixed in 2% osmium tetroxide in Millonig’s buffer. The fixed kidney tissue was dehydrated, infiltrated in a 1: 1 mixture of Epon: acetone overnight, and, finally, embedded in Epikote 812. Ultrathin sections were cut on an LKB Ultrotome I using glass knives and were collected on 200-mesh copper grids. The sections were stained in 1% alcoholic uranyl acetate followed by lead citrate and were examined in a Zeiss EM 9s 2 electron microscope. 7. Calculations. Glomerular filtration rate (GFR) was assumed to be equal to the clearance of inulin (C,,). From the GFR and the perfusate concentration of each solute, the quantity filtered per minute was calculated, and by subtracting the excreted load (urine concentration x urine volume) reabsorption of each solute was determined and expressed either as a percentage of the quantity filtered or in absolute terms per gram of fresh kidney weight.

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Protein leakage was measured by determining the clearance rate of BSA (CA& and calculating the quotient CALB/CIN. The ratios of the clearances of urea and potassium to the inulin clearance were also calculated. Oxygen consumption was calculated from the arteriovenous difference in oxygen tension and perfusion flow rate. RESULTS

1. The Effect of Different Colloids merular Function and Structure

on Glo-

The viscosity, surface tension, and colloid osmotic pressure of normal blood and the perfusates are compared in Table 2. None of the perfusates was really close to whole blood in all the properties measured, although the dextran/albumin mixture was closest. Five percent dextran gave higher values than blood in each case, and the pluronic perfusate gave lower values for viscosity and surface tension; because of the low mean molecular weight of this colloid, it is unlikely that the calculated colloid osmotic pressure reflects the oncotic pressure exerted in vivo. The HES solution had a low viscosity and a high surface tension, but the colloid osmotic pressure was physiological. Table 2 also shows the control determinations of inulin and albumin clearance (CiN and CA& obtained in lightly’ anesthetized rabbits. The in vivo ratio CALBICIN was 1.25 x 10m3; if albumin were totally retained the ratio would be 0; if it were freely filtered it would be 1. The clearance values obtained in vitro after 2 hr of perfusion are also shown. Perfusions using albumin, dextran, and dextramalbumin mixtures at physiological pressures did not allow normal rates of glomerular filtration in isolated rabbit kidneys as measured by GIN. However, with the perfusate containing pluronic, CiN was not significantly different from that found in vivo, and, with HES, the measured GFR was somewhat higher. In all experiments the clearance of albumin was significantly

VOL. 22, NO. 2, FEBRUARY

1977

higher than that seen in the rabbit in vivo. The ratio of CALB to GIN is plotted as a function of time in Fig. 2, which shows that the pluronic- and HES-perfused kidneys had the lowest albumin leakage and were stable in this respect throughout a 3hr perfusion period. Electron microscopic examinations were made of lo-20 glomeruli from different regions of two kidneys perfused with each perfusate studied. In every case the basement membrane of the glomerular capillaries was intact and showed no evidence of morphological alteration, but structural changes were evident in the capillary endothelial lining. In kidneys perfused with 5% BSA and 3% dextran + 0.7% BSA there was a distinctive swelling and coalescence of the endothelium, with a consequent partial occlusion of the endothelial fenestrae in most of the capillary loops observed. Extensive sections of endothelial lining were missing in the kidneys perfused with dextran alone and HES. Loss of endothelium was also a feature of the pluronicperfused kidneys but was less severe. Moderate fusion of the epithelial cell foot processes was common in all the kidneys examined, and large balloon-like vacuoles in the visceral epithelial cells, which encroached extensively into the urinary space 1.0 r 0.9 0.8 0. 7 L :I$

+

0.2 t I

rI

1

2

0.1 t 0.0 ' 0

I 3

Time (hours) FIG. 2. The ratio of albumin clearance to inulin clearance (C,/C,,) as a function of time in each group of perfusions. (O), 5% (w/v) BSA; (0), 5% (w/v) dextran; (A), 3% dextran +0.7% BSA; (W), 3% pluronic; (O), 3% HES.

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between the capillary loops, were a distinctive structural alteration in the kidneys perfused with dextran alone and HES. Glomerular morphology was best preserved in the kidneys perfused with pluronic or the dextran-albumin mixture. These observations were used to select one colloid for further study: Clearly the choice lay between pluronic and HES since these were the only compounds studied that yielded acceptable value for GFR. HES gave somewhat better protein retention, but pluronic gave very much better structural preservation. Pluronic was chosen. 2. Structural, Circulatory, and Metabolic Effects of Perfusion with Pluronic Solution Electron micrographs prepared from kidneys perfused for 2 hr with pluronic perfusate are shown in Plate 1. Some of the glomerular capillaries were devoid of endothelium, but in many others it was intact (Plate la) and the only structural change seen in the visceral epithelial cells was some mild fusion of podocyte foot processes. The proximal tubules were well preserved, showing intact basement membranes, well-ordered basal cell interdigitation, and normal mitochondria (Plate lb). There was no evidence of damage to the brush border, and the nuclear chromatin had a completely normal appearance. The only unusual feature was the presence of numerous small vacuoles in the basal and midregions of the cells (Plate lc), some of which appeared to be releasing dense membranous material into the tubular lumens. Many of these vacuoles were almost certainly lysosomal in origin and contained dense material adhering to one side (Plate lc). The peritubular capillaries were generally preserved intact (Plate Id). The lumens of the distal tubules were very distended, but the tubular cells themselves were of completely normal appearance. The perfusion pressure of 110 mm Hg produced a flow rate of 13.3 t 0.5 ml/mm/g

KIDNEY

PREPARATION

133

at 15 min, which increased to 15.9 + 0.4 ml/mm/g at 2 hr (mean + SEM; n = 18). The intrarenal distribution of flow was studied by blue dextran washout, and a typical efflux curve is shown in Fig. 3. Curve stripping yielded three components accounting for 85.0 + 1.7, 7.8 * 1.2, and 7.1 ? 0.8% of the total flow, respectively (mean ? SEM; n = 6). In an additional series of four experiments, perfusion was stopped 10 set after starting the washout. In all cases the cortex was found to be cleared of blue color when the kidney was sectioned, while medullary and corticomedullary regions each retained a blue coloration. It was concluded that the high flow rate compartment in isolated rabbit kidneys is the outer cortex and that the other two compartments are probably the corticomedullary region and the medulla. Isolated perfusion had a profound effect on the renal concentration gradient between the cortex and the medulla. The Na+ and K+ contents of cortical (C) and inner medullary (M) tissue were measured after 2 hr of perfusion and in the contralateral unperfused kidneys. The CM ratios for potassium concentration were 1.08 + 0.09 and 0.93 ? 0.05 for unperfused and perfused kidneys, respectively, (mean ? SEM; n = 6) whereas the C:M sodium ratios were 1.74 + 0.13 for unperfused kidneys and 1.02 2 0.04 for perfused kidneys. It seems probable that the high perfusate flow rate was responsible for washing out the sodium gradient. On the other hand, the high perfusate flow rate improved the rate of delivery of oxygen to the perfused organ. The oxygen consumption was 3.93 2 0.3 1 pmol/min/g at 15 min and decreased slightly throughout perfusion, reaching 3.04 ? 0.18 pmol/min/g at 2 hr (mean + SEM; n = 6). Measurements of tissue oxygen tension were made at the end of each perfusion; readings were made at five depths during insertion of the electrode and again on removal and averaged. Figure 4 shows the results. At 2 mm (cortex) and 5 mm (cortico-

PLATE 1. Electron micrographs showing representative areas from rabbit kidneys perfused for 2 hr at 37°C with the perfusate containing 3% (w/v) pluronic. Although the endothelium was absent from many of the glomerular capillary loops observed, micrograph (a) shows several loops in which the endothelium was intact. Mild fusion of the podocyte foot processes was the only structural change seen in the visceral epithelial cells. Proximal convoluted tubules were well preserved, showing intact basement membranes, well-ordered arrangements of basal cell interdigitations, and longitudinally orientated mitochondria (b). The brush border has a normal appearance, but there are numerous small vacuoles, many of which are certainly lysosomal in origin and contain discrete dense material adherent to one side (c). The peritubular capillaries are mostly intact (d). The distal tubules were distended but otherwise of normal morphology. Scale bar represents 5 pm in (a) and (d) and 2 pm in (b) and (c). 134

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KIDNEY

PREPARATION

13.5

60

0

20

40

60

80

100

120

140

160 180 Time (secondsI

FIG. 3. Graph showing the concentration of blue dextran in the venous effluent from a kidney perfused with dye-free solution after loading with dye-containing solution. The washout curves of six kidneys yielded similar curves which stripped into three exponentials having intercepts of 85.0 t 1.7, 7.8 * 1.2, and 7.1 2 0.8%, respectively.

medullary zone) the tension was close to the arterial value (615 mm Hg). Deeper insertion of the electrode revealed a zone of rapidly falling oxygen tension (outer medulla at 8 mm and inner medulla at 14 mm), and, in the deepest regions of the kidney, oxygen tensions below 100 mm Hg were recorded. The venous oxygen tension was approximately 280 mm Hg. It was important to know whether the supply of oxygen and other substrates was adequate for the energy requirements of the perfused kidney. Groups of six kidneys were used to estimate the adenine nucleotide content of normal control kidneys, kidneys subjected to 2 min of warm ischemia, and kidneys perfused for 2 hr after

2 min of warm ischemia (Fig. 5). Control, ischemic, and perfused kidneys all had similar total nucleotide contents. However, the ATP/ADP ratio, which was 1.71 in the fresh kidneys, fell to 0.51 in kidneys subjected to a 2-min period of warm ischemia and increased to 1.06 after perfusion for 2 hr. These studies showed that, in spite of the abnormally high flow rates obtained and the abnormal intrarenal distribution of flow, the metabolic state and structure of the perfused kidneys were reasonably well preserved. However, the corticomedullary concentration gradient, which plays an important part in urine concentration, was lost.

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3. Functional Characteristics of Kidneys Perfused with Pluronic Solution Six kidneys were studied. Figure 6 shows the GFRs and the u/p (urine/perfusate) inulin ratios obtained during the 2-hr perfusions. The GFR increased during the first 90 min of perfusion and then remained constant, but the highest u/p inulin ratio, reflecting the most efficient water reabsorption, occurred at 1 hr and then declined. Figure 7 shows the percentage of glucose reabsorbed from the filtered load and the total quantity reabsorbed per gram of fresh kidney weight. It can be seen that, although the kidneys took up glucose actively, complete reabsorption was not achieved. The maximum percentage of reabsorption (80%) occurred after 1 hr of perfusion and thereafter remained constant, whereas the reabsorbed load increased for 90 min in parallel with the GFR. Sodium reabsorption (Fig. 8) also reached its maximum at 1 hr (67%); the reabsorbed load remained constant during the second hour, but the percentage uptake declined slightly but significantly. The mean ratio of urine to perfusate osmolality ranged from 0.96 to 1.05 during the 2-hr period, showing that at no stage was osmolar concentration of the filtrate effected. The mean ratio of the potassium clearance to the inulin clearance ranged from 1.26 at the beginning to 1.10 at the end of perfusion and was always significantly >l, indicating that net potassium secretion had occurred. The urea clearance values were always less than the simultaneous inulin clearances (mean ratios lay between 0.6 and 0.7), implying tubular reabsorption of urea. DISCUSSION

Composition

of the Perfusate

The selection of glucose and octanoate as the only metabolic substrates and the inclusion of gentamicin require some justification. In pilot experiments a comparison was made between a perfusate containing

VOL. 22, NO. 2, FEBRUARY

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5 mkl glucose as the sole energy source and perfusates to which either pyruvate, glutamate, acetate and propionate or octanoate, or palmitate had been added. With the basic perfusate there was a tailing off in the fractional reabsorption of water, glucose, and sodium during the second hour, but this was largely prevented by the addition of octanoate or palmitate. None of the other supplemented perfusates brought about a significant improvement, and omitting the gentamicin had no detectable effect on function. Considerable attention was given to the selection of a colloid. Using dextran, bovine serum albumin, a mixture of these compounds, hydroxyethyl starch, or Pluronic F108, we found it impossible to match the colloid osmotic pressure of syn-

I OO

4

8

12

16

Depth of electrode insertion (mm) FIG. 4. Oxygen tension measured in renal tissue at increasing depths from the kidney surface in perfused kidneys (mean values of four readings at each depth are shown for each of six perfusions). The mean arterial oxygen tension recorded at the time the intrarenal measurements were taken was 615 mm Hg. The oxygen tension in the tissue was seen to fall rapidly as the electrode was inserted more deeply intc the kidney. The zone with the most rapid fall was the juxta-medullary region, and deeper in the medulls the oxygen tensions were much lower than the venom value (280 mm Hg).

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KIDNEY

PREPARATION

137

10 -

9-

ADP

lO-

A. Fresh Kidneys.

a. 2 Mins. warm ischaemia.

2L’Hours isolated perfusion.

FIG. 5. The adenine nucleotide concentration of kidney tissue and the relative proportions of AMP, ADP, and ATP in three groups of experiments (mean ? SEM for six observations in each group). A = freshly excised kidneys; B = kidneys subjected to 2 min of warm ischemia; C = kidneys perfused for 2 hr. There were no significant differences in total adenine nucleotide content between the three groups, and thus perfusion had not resulted in loss of tissue nucleotides. The proportion of ATP was slightly lower in the perfused group than in the freshly excised organs, but, as expected, the ratio was much reduced in the ischemic kidneys.

thetic perfusates with that of blood, while keeping other physical properties, such as viscosity and surface tension, similar. The most successful imitation was obtained with the dextran/albumin mixture that was originally designed to produce normal capillary fluid retention [18], but the GFR was low. Likewise, dextran and albumin separately produced low GFRs. Dextran has also been shown by other workers [17] to have this effect and, although albumin has yielded almost normal filtration rates in rats [3, 261 and did so in these animals in our laboratory [9], low values were obtained in rabbits. In our hands, only hydroxyethyl starch and

Pluronic F 108 gave acceptable glomerular filtration rates in the rabbit. Franke et al. [8] obtained similar results with Pluronic F108 in rats. The mechanisms responsible for these differences remain obscure. Viscosity of the filtrate probably is not a factor: Dextran might have produced a viscous filtrate but albumin would not, and yet both gave equally low GFRs. The electron microscopic studies gave little help: Both dextran and HES produced severe vacuolation of the epithelial cells, and the endothelium was extensively desquamated with both compounds, and yet HES gave a high GFR

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0.0

FIG. 6. The glomerular filtration rate (GFR) (m) and u/p inulin ratio (0) measured during isolated perfusion for 2 hr at 37°C (mean + SEM for six nerfusions). The GFR increased during the first 90 mitt, but maximum water reabsorption was seen at 1 hr. _

and dextran gave a low GFR. Glomerular structure was best preserved with the dextramalbumin mixture and Pluronic F 108, and yet the former gave a low GFR and the latter gave a high GFR. It is generally agreed that the basement membrane is the actual filter membrane [31], and this appeared to be morphologically intact with all the perfusates tested: Nevertheless, it probably was damaged since considerable proteinuria was seen, and the fact that protein leakage was greatest when the GFR was least suggests that varying degrees of damage to the membrane were responsible for the differences observed. From the functional point of view there was little to choose between HES and pluronic, but structure was better preserved with pluronic, and pluronic is cheaper and more readily available.

isolated rat kidneys [8, 261. There appear to be two factors responsible for the increased flow: The viscosity of the perfusate (1.26 cP) is approximately 60% of that of whole blood, which would increase the flow rate by 66%; in addition, therefore, there must be a decrease in vascular resistance. The intrarenal distribution of perfusate flow was also very different from that reported for the organ in vivo [29]. Three distinct compartments were identified: One compartment, shown to be the outer cortex,

Flow Dynamics During the first hour of perfusion the vascular flow rate increased 20% and then stabilized for the second hour. This is approximately three times the blood flow in normal rabbit kidneys [24], a finding which is in agreement with observations made on

Time (minutesl

FIG. 7. Glucose reabsorption in perfused kidneys as a percentage of the filtered load (0) and as total glucose uptake (0) during assessment for 2 hr (mean 2 SEM for six perfusions). The ability of isolated kidneys to reabsorb glucose increased during the first hour of perfusion.

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received the majority of the perfusate flow; it was impossible to distinguish between the two remaining compartments by dye washout, but they were assumed to be the corticomedullary region and the deep medulla. The theoretical validity of this efflux method is well established [5], and, although it is technically difficult to make efflux collections at such high flow rates, the good agreement between replicate determinations suggests that the data obtained in these experiments are reliable. When the absolute regional flows are compared with the published data for normal kidneys in vivo [24, 29, 301 it is found that the medullary flow during perfusion is approximately six times the normal flow, whereas the factor for the cortex is only three times. This absolute and relative increase in medullary flow seems to be an important characteristic of the isolated perfused kidney since it has also been observed in the rat [5] and in hypothermically perfused dog kidneys [33]. It seems likely that the greatly increased medullary flow is responsible for the observed removal of the corticomedullary concentration gradient and the production of isosmotic urine. The same effect is produced by frusemide [28]. Metabolic State

The mean oxygen consumption reported here (approximately 3.6 ~mollminlg kidney weight) agrees with data published elsewhere, both for kidneys in vivo and for the organ in isolated perfusion [15, 271. The slight decrease in oxygen consumption during perfusion was probably due to the repayment of an oxygen debt incurred during the ischemic period; whatever the mechanism, it was sufficient to swamp the expected increase in oxygen consumption as the GFR and sodium reabsorption increased during the first hour. The pattern of intrarenal oxygen tensions closely resembled that seen in vivo and in the isolated rat kidney 1151. It is perhaps surprising that such low medullary oxygen tensions

KIDNEY

PREPARATION

100 -0 a E z0 ” c ;” g

ao-

60 40-

f : x

zo-

OLI 0

/

I

I

1.

40

60

90

120

Time iminutes) FIG. 8. Sodium reabsorption in perfused kidneys as a percentage of the filtered load (O), and as total sodium uptake (0) during assessment for 2 hr (mean 2 SEM for six perfusions). As with glucose reabsorption, the ability of isolated kidneys to reclaim sodium from the filtrate increased in the first hour of perfusion.

should be found in isolated kidneys which had a very high perfusate flow rate and a venous oxygen tension commonly as high as 300-400 mm Hg. This can be explained by the dilution of the effluent from the medulla (which presumably does have a low oxygen tension) with the much greater volume of effluent from the cortex, and the occurrence of shunt-diffusion of oxygen from arterial to venous vessels. The ratio of ATP to ADP that we obtained for fresh rabbit kidneys was somewhat lower than that reported for rats [ 161. It is possible that anesthesia or operative trauma were partly responsible or that our technique of freezing was less effective than Wollenberger’s freeze-clamping method; since our technique was consistent, it would have affected both control and experimental kidneys equally. We confirmed that very short periods of interrupted blood flow result in a rapid utilization of high energy phosphate compounds [I]. However, perfused rabbit kidneys were able to repair this deficit to a considerable degree. It might have been possible to prevent the fall in adenine nucleotide levels by isolating the kidneys using a cannulation method that involved no interruption of perfusate flow, but when this was done there was no significant improvement in initial or subsequent

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function [9]. Thus it would seem that adequate total quantities of oxygen and energy substrates were provided during perfusion. Function

of Isolated

Rabbit Kidney

There is some doubt as to the normal GFR in rabbits. Our in vivo value was obtained in anesthetized animals by using a single-injection technique and may be a little low, although it falls within the range (0.38-0.94 ml/min/g) given by Smith [27] and is similar to Korner’s value (0.41 ml/min/g) for both anesthetized and unanesthetized rabbit kidneys [ 141. Rosenfeld and Sellers [23] obtained GFRs of 0.12-0.61 ml/min/g in blood-perfused rabbit kidneys, with a mean of 0.3 mYmin/g. It is reasonable therefore to claim that the pluronic perfusate gives a physiological GFR at physiological arterial pressure. The gradual increase in GFR during the first 90 min of perfusion was striking and quite different from the reported behavior of perfused rat kidneys [8, 261. It was not related to warm ischemic injury, since kidneys cannulated without interruption of blood flow showed a similar pattern [9]. There is some evidence that in rabbits a considerable proportion of the glomeruli are inactive at any given time [27], and it is certainly clear that these animals are particularly subject to renal vasoconstriction following arterial occlusion [13]. It seems likely that the reduction in vascular resistance during the first hour and the increase in GFR were due to a progressive opening up of more glomeruli. This phenomenon should not affect the usefulness of the preparation as long as its existence is recognized. The fractional reabsorption of water, glucose, and sodium were all subphysiological, reaching maxima of 63, 80, and 67%, respectively, and the urine was isosmotic. The reasons for these defects are probably complex and have not been investigated in detail in the present study. Metabolic deprivation seems unlikely, although it cannot be ruled out completely: The lack of hormonal control and disturbance of regional

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perfusate flow are probably more important. Potassium was secreted both by our preparations and by perfused rat kidneys [8], a phenomenon which may be linked to the high rate of sodium excretion [2]. The handling of urea was similar to that of the organ in vivo, in that the urea clearance ranged from 60 to 70% of the inulin clearance. All perfused kidney preparations so far described have shown important defects of tubular function, although isolated rat kidneys are undoubtedly more efficient than rabbit kidneys [8, 261, and we have confirmed this in our laboratory [9]. The principal advantage of rabbit kidneys is their size and ease of handling, and, for certain purposes, especially for organ preservation, this is useful. CONCLUSIONS

The perfused rabbit kidney preparation developed in this paper is relatively simple to set up, and it assumes stable function during the second hour of perfusion. The GFR is close to the physiological normal, and although the reabsorption of water, sodium, and glucose is impaired and there is significant protein leakage, the performance is reproducible. The preparation is in fact superior to other perfused rabbit kidney preparations. The preparations’ defects as a physiological model do not, we believe, interfere with its use as an assay for screening organ preservation methods, and in fact it has already been used in studies of hypothermic renal preservation for transplantation [lo] and in studies of cryopreservation [22]. Although perfused rat kidneys would probably be preferred for physiological studies, rabbit kidneys are currently being used to study the metabolism of thyroid hormones, and further improvements may render them suitable for wider use. SUMMARY

An isolated rabbit kidney preparation has been developed principally for use as

FULLER

ETAL.:

ISOLATED

RABBIT

a laboratory assay to compare experimental renal preservation techniques. Perfusion was carried out at 37°C and an arterial pressure of 110 mm Hg using bloodless perfusates containing bovine serum albumin, dextran, hydroxyethyl starch, or Pluronic F 108. Glomerular filtration rate and protein leakage were determined with each perfusate. It was found that low filtration rates and high degrees of protein leakage were obtained with perfusates containing either albumin, dextran, or a mixture of these colloids, but both pluronic and hydroxyethyl starch gave acceptable glomerular filtration rates and relatively low protein leakage. The pluronic perfusate was selected for further study. Isolated rabbit kidneys had a greatly reduced vascular resistance, and medullary flow was increased to a proportionally greater degree than cortical flow. The concentration gradient of Na+ normally found between cortex and medulla was lost, and this was associated with an inability of perfused kidneys to effect osmolal concentration of urine. Oxygen consumption was within the limits published for the organ in vim, and during perfusion the total nucleotide content of renal tissue was well maintained, but there was a small decrease in the ATP/ADP ratio. Function improved steadily during the first hour of perfusion to give a GFR during the second hour that was close to the physiological norm. The fractional reabsorption of water, glucose, and sodium reached maxima of 63, 80, and 67%, respectively. Although the performance of the preparation is subphysiological, it is stable during the time required for the comparison of preserved kidneys and is reproducible. The preparation has already yielded valuable data in this field. REFERENCES 1. Bergstrom, J., Collste, H., Groth, C., H&man, E., and Melin, B. Water, electrolyte and metabolite content in cortical tissue from dog kidneys preserved by hypothermia. Proc. E.D.T.A. 8: 313, 1971.

KIDNEY

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PREPARATION

2. Berliner, R. W. Renal mechanism for potassium excretion. Harvey Leer. 55: 141, 1961. 3. Bowman, R. H., and Maack, T. Effect of albumin concentration and ADH on H,O and electrolyte transport in perfused rat kidneys. Amer. J. Physiol. 226: 426, 1974. 4. Cater, D. B., Silver, I. A., and Wilson, G. M. Apparatus and technique for the quantitative measurement of oxygen tension in living tissues. Proc. R. Sot. Lond. B 151: 256, 1959. 5. Chapman, B. J. An isolated Mammalian Kidney Preparation. Thesis, Southampton University, Southampton, 1970. 6. Flear, T. C., and Florence, G. A rapid and micro method for the analysis of skeletal muscle for water, sodium, potassium chloride and fat. Clin. Chim. Acta 6: 129, 1961. 7. Foreman, J. Prediction of viability of rabbit kidneys preserved by hypothermic perfusion. Cryobiofogy

12: 231, 1975.

Franke, H., Huland, H., Weiss, Ch., and Unsicker, K. Improved net sodium transport of the isolated rat kidney. Z. Gesamte Exp. Med. 156: 266, 1971. 9. Fuller, B. J. The Functional Assessment of Fresh 8.

and Preserved less Perfusion.

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Kidneys

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Ph. D. Thesis, CNAA,

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London,

1974. 10. Fuller, B. J., and Pegg, D. E. The assessment of renal preservation by normothermic bloodless perfusion. Cryobiology 13: 177, 1976. 11. Hardie, I. R., Clunie, G. J. A., and Collins, C. M. Evaluation of simple methods for assessing ischaemic injury. Surgery 136: 43, 1973. 12. Hint, H. C. Reports of Symposia Rheomacrodex 1: 2, Pharmacia (G.B.) Ltd., London 1964. 13. Honda, N.. Morikawa, A., Nihei, H. Aizawa, C., and Yoshitoshi, Y. Postocclusive vascular responses in isolated perfused rabbit kidneys. Amer. J. Physiol. 222: 1581, 1972. 14. Korner, P. I. Renal blood flow, glomerular filtration rate, renal PAH extraction ratio, and the role of vasomotor nerves in the unanesthetized rabbit. Circ. Res. 12: 353, 1963. 15. Leichtweiss, H. P., Lubbers, D. W., Weiss, Ch., Baumgartl, H., and Reschke, W. The oxygen supply of the rat kidney: Measurements of intrarenal PO,. Pjluegers Arch. 309: 328, 1969. 16. Nishiitsutuji-Owo, J. M., Ross, B. D., and Krebs, H. A. Metabolic activities of the isolated perfused rat kidney. Biochem. J. 103: 852, 1967. 17. Nizet, A. Influence of serum albumin and dextran on sodium and water excretion by the isolated dog kidney. PJl’uegers Arch. 301: 7, 1968. 18. Pegg, D. E. Some effects of dextran and of bovine serum albumin on the isolated perfused rabbit kidney. Cryobiology 6: 419, 1970. 19. Pegg, D. E., Fuller, B. J., Foreman, J., and Green, C. J. The choice of plastic tubing for

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organ perfusion experiments. Cryobiology

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1972.

Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion using a defined perfusion fluid. Cryobiology 9: 420, 1972. 21. Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion. 1. The importance of pressure-control. Cryobiology 10: 56, 1973. 22. Pegg, D. E., and Wusteman, M. C. The function of rabbit kidneys following perfusion with 2 molar glycerol at 5°C. In Proceedings of 14th International Congress of Refrigeration, Moscow, 20.

1975. 23.

Rosenfeld, S., and Sellers, A. L. Pressureflow studies in the isolated artificial heart-lung perfused mammalian kidney. Amer. J. Physiol. 199: 499, 1960.

Rosenfeld, S., Sellers, A. L., and Katz, J. Development of an isolated perfused mammalian kidney. Amer. J. Physiol. 196: 1155, 1959. 2.5. Ross, B. D. Perfusion Techniques in Biochemistry, pp. 221-257, Clarendon Press, Oxford. 1972. 26. Ross, B. D., Epstein, A., and Leaf, E. P. Sodium resorption in the perfused rat kidney. Amer. J. Physiol. 225: 1165, 1973. 24.

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Smith, H. W. The Kidney. Oxford University Press, Oxford, 1951. Stowe, N. T., Wolterink, L. F., Lewis, A. E., and Hook, J. B. Diuretic and hemodynamic effects of frusemide in the isolated dog kidney. Arch. Pharmacol. 277: 13, 1973. Thorburn, D., Kopald, H. H., Herd, A., Hollenberg, M., O’Morchoe, C. C., and Barger, A. C. Intrarenal distribution of nutrient blood flow determined with krypton 85 in the unanesthetized dog. Circ. Res. 13: 290, 1963. Thurau, K., Renal haemodynamics. Amer. J. Med. 36: 698, 1964.

Vernier, R. L. Ultrastructure of the glomerulus and changes in fine structure associated with increased permeability of the glomerulus to protein. In C. E. W. Wolstenholme, and M. P. Cameron, (Eds.) Renal Biopsy, Clinical and Pathologica/ Significance. Churchill, London, 1961. 32. Weinerth, J. L., and Abbott, W. M. Analysis of injury in complex organ preservation. Ann. Surg. 31.

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33. Withey, W. R., Chapman, B. J., and Munday, K. A. Renal blood flow in the intact hypothermic canine kidney. Cryobiology 11: 583, 1974.