Kidney International, Vol. 66 (2004), pp. 77–90

A pathophysiologic study of the kidney tubule to optimizeorgan preservation solutions

NIAZ AHMAD, LUTZ HOSTERT, JULIAN R. PRATT, KATHARINE J. BILLAR, DAVID J. POTTS,and J. PETER A. LODGE

Department of Transplant Surgery, St. James’s University Hospital, Leeds, United Kingdom; Molecular Medicine Unit, St. James’sUniversity Hospital, Leeds, United Kingdom; and School of Biomedical Sciences, University of Leeds, United Kingdom

A pathophysiologic study of the kidney tubule to optimize organpreservation solutions.

Background. Tissue damage at the time of organ transplan-tation has a negative impact on the subsequent success of theprocedure, both in the immediate and longer term. Hypother-mia is the principal element used to prolong organ viabilityex vivo, but paradoxically also induces cellular edema throughinhibition of energy-dependent adenosine triphosphatases(ATPases). This induces an electrolyte imbalance that leads tofluid influx and cell swelling. It is important, therefore, that im-provements are made in the preservation of ischemic organs toreduce this injury.

Methods. This study has applied a novel in vitro system tomodel cold and warm ischemic-induced renal tubule swellingthat characterizes tissue damage in ischemia/reperfusion injury.Biochemical blockade of ATPases in this system using stro-phanthidin modeled the effects of energy depletion and inducedcell swelling. By measuring such tubule swelling and changes totubular cell volume in isolated rabbit renal proximal tubules, ananalysis was made that defined the basis on which an optimalpreservation solution may be developed.

Results. The data show that our model could reproduceischemically induced cell swelling and characterized the re-sponse at the cellular level of tubules to different componentsof preservation solutions. The data indicate that an isosmolar,phosphate-buffered, sucrose solution prevented tubule swellingmore effectively than Euro-Collins, hyperosmolar citrate, orUniversity of Wisconsin solutions that are in routine clinicaluse.

Conclusion. Future developments in organ preservation maysignificantly improve transplant outcomes. Our novel analysisforms the basis of future whole-organ studies that ultimatelymay allow us to propose an optimum platform for improvedpreservation solutions.

The process of organ procurement and implantationconstitutes a severe physical stress on solid organs used

Key words: ischemia/reperfusion, preservation, transplantation.

Received for publication October 7, 2003and in revised form December 15, 2003Accepted for publication February 3, 2004

C© 2004 by the International Society of Nephrology

in transplantation. Despite major advances in organtransplantation, surgical technique, transplant immunol-ogy, preservation techniques, and preservation solutions,every transplant organ faces two inevitable insults—ischemia and subsequent reperfusion. A period of coldischemia extends from the time of organ procurement tothe time when it is removed from cold storage just beforeimplantation. This is followed by a shorter but potentiallymore hazardous period of warm ischemia during implan-tation. The final physical stress is that of reperfusion ofthe organ with recipient blood, the so-called reperfusioninjury.

To minimize the effects of organ damage and is-chemia/reperfusion (I/R) injury, but also to allow time fororgan allocation, organ transportation and surgery, organprocurement requires both in situ flush with a cold preser-vation solution, and hypothermic storage at 0 to 4◦C.During ischemia, active transport mechanisms involvingNa+/K+ and Ca2+/Mg2+ adenosine triphosphatase (AT-Pase) are inhibited [1], which leads to a steady influx ofNa+, Cl−, and Ca2+ into the cell with subsequent osmoticinflux of water causing cellular swelling. Hypothermiareduces the rate of tissue damage by depressing the cel-lular metabolism, but although it forms the central com-ponent of any organ preservation strategy, hypothermiaalso directly contributes to cellular edema by direct de-pressant effects on ATPase-dependent active transportmechanisms [1].

Ischemia also results in lowered pH and an accumu-lation of toxic products of anaerobic respiration (e.g.,lactate and hypoxanthine) that contribute to free radi-cal damage upon reperfusion of the organ with recipientblood [2, 3]. It is well known that severe I/R injury leads todelayed graft function (DGF) posttransplant [4]. Theseauthors have quoted a 5% incidence of DGF for liver,and a 20% to 30% DGF for renal transplantation, whileUnited Kingdom Transplant reported a 19% incidence ofDGF in UK adult cadaveric renal transplantation nation-wide over 5 years to 2002 (UKT, Bristol, UK, personalcommunication). Such DGF has been linked to poorer

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78 Ahmad et al: Ischemia-induced renal tubule cell edema

Table 1. Constituents of preservation solutions used in this study

Hyperosmolar Phosphate-buffered University ofComponents mmol/L Euro-Collins citrate sucrose 140 Wisconsin

Glucose 194Mannitol 186Sucrose 140Raffinose 30Lactobionic acid 105HES % 5Phosphate

Na2HPO4 42.3NaH2PO4.2H2O 26.7K2HPO4 32.2KH2PO4 15 25

NaHCO3 10NaOH 27KOH 100Citrate 54

Na+3-citrate 28

K+3-citrate 26

KCl 15MgSO4 41 5Glutathione 3Allopurinol 1Adenosine 5Insulin IU 40Penicillin G IU 200,000Dexamethasone mg 16Osmolality mOsm.kg−1 355 400 300 325pH 7.0 7.1 7.0 7.4

prognosis [5, 6]. In a clinical study, one- and five-yeargraft survival was significantly reduced from 95% and81% in early functioning grafts to 87% and 70% in de-layed functioning grafts, respectively. In addition, a linkwas established between DGF and the incidence of acuterejection that negatively impacted upon graft half-life [7].The effects of I/R injury and the subsequent tissue dam-age that manifests as DGF may render the organ morevisible to the immune system of the recipient and promoteactivation of both innate and adaptive immunity againstthe donor organ [8, 9]. In particular, lipid peroxidationwill lead not only to membrane damage and activationof arachidonic acid pathways with subsequent release ofprostaglandins and leukotrienes, but may also increasetissue susceptibility to complement activation throughthe alternative pathway [10]. Such proinflammatory me-diators will stimulate not only the release of cytokinesand chemokines by damaged tissue, but also contributeto an acute-phase response that further promotes inflam-mation. Recent experimental studies have demonstratedthat long-term outcomes may well be affected by tissuedamage induced as a result of less than optimal ischemiaand that modifications to preservation solutions can sig-nificantly improve organ function [11].

The core components of any preservation solution areimpermeant agents that prevent fluid entry into cells,buffers that maintain pH, and ions (Table 1). Secondarycomponents may confer additional benefits but are not

essential for organ preservation. These include free radi-cal scavengers, calcium antagonists, colloids, complementregulators, and antiplatelet agents. Solutions such as hy-perosmolar citrate (HOC) also use a hypertonicity of 400mOsm in a bid to prevent fluid entry into cells whoseisotonic environment is approximately 285 to 300 mOsm.

Currently used buffer systems include phosphate, cit-rate, histidine, and bicarbonate. Preservation solutions,with the exception of University of Wisconsin (UW;pH 7.4), commonly have an acidic pH (6.9 to 7.1) inrelation to plasma. This followed work which indicatedthat an acidic pH may actually protect against hypoxicinjury [12–14]. The inclusion of high potassium in thepreservation media has been thought to be important inpreventing potassium loss during cold storage [15, 16].It is also thought that a high potassium concentrationhelps to prevent the build up of intracellular calciumduring ischemia. Euro-Collins (EC), hyperosmolar cit-rate (HOC), and UW solutions have a high potassiumcontent and are thus classed as “intracellular” solutions.Phosphate-buffered sucrose (PBS140), developed byAndrews and Coffey [17] for morphologic preservationof kidney tubules, however, contains only sodium andno potassium and is thus classed as an “extracellular”solution.

The cellular edema caused by the influx of water is theprimary pathophysiologic event that affects ischemic or-gans. In the kidney, it is the corticomedullary segments

Ahmad et al: Ischemia-induced renal tubule cell edema 79

of renal tubules that appear most vulnerable to I/R in-jury [18]. This compartmental susceptibility is possiblyexplained by the no-reflow phenomenon whereby severeedema constricts blood vessels and limits delivery of oxy-gen on reperfusion of affected tissues [19]. Tubule cellvolume is actively regulated in vivo but this regulationis lost in the ischemic tissue since the process is energydependent and ATP is almost totally depleted in an is-chemic organ within 4 hours of ischemia [20]. The aims ofthis study were to develop a new in vitro system to modelthe effect of ischemia on tubule cell swelling, and to an-alyze the effect of preservation solutions and their corecomponents in preventing this tissue damage. Followingfrom this study, new recommendations are made for thebasic constitution of an optimized preservation solution,specifically to address tubule cell edema. This forms theprincipal pathologic insult to whole organs, and preven-tion of such cell swelling could have significant benefit fortransplant outcome if improved solutions are adopted bytransplant centers.

METHODS

Animals and anesthesia

Specific pathogen-free (SPF), New Zealand White(NZW) rabbits 1.5 to 2.5 kg in weight were used through-out the experiments (Charles River, Surrey, UK). UnitedKingdom Home Office Guidelines (Scientific ProceduresAct, 1986) were strictly adhered to throughout this work.Terminal anesthesia was induced by intravenous pento-barbitone 60 mg·kg−1 (Sagattal, RMB Animal HealthLimited, Dagenham, UK).

Experimental plan

In brief, isolated rabbit kidneys were perfused in situwith a preservation fluid under test and maintained on icefor varied cold ischemic times. Following this, single prox-imal tubules were micro-dissected from the renal cortexand assessed for tubule swelling as a result of cold is-chemia, and during a period of warm ischemia using theprotocol described below.

Perfusion of kidneys

A 50 mL syringe reservoir was set at a height of 50 cmabove the table and was filled with 40 mL of cold preser-vation solution (0 to 4◦C) just prior to the flush. A widebore polyvinyl tubing (2 mm internal diameter) was con-nected to the reservoir. The draining end was taperedfor cannulation of the aorta or the renal artery. The tipof the catheter was also barbed with a scalpel blade toavoid the ligature slipping. A midline incision was madeand a laparotomy performed. The lower abdominal aortawas ligated above the iliac bifurcation with a 3/0 silk tie(Ethicon Limited, Edinburgh, UK) followed by ligation

of the aorta below the origin of the celiac trunk but abovethe origin of both renal arteries. An incision was made inthe aorta above the lower tie, and with the flush solu-tion running a catheter was introduced through this in-cision. The catheter was then secured with a 3/0 silk tie.Blood was drained by venting the inferior vena cava inthe chest. The warm ischemia time of the whole proce-dure was less than 1 minute. After 25 mL flush solutionhad passed through, the kidneys were excised and placedin glass beakers containing 100 mL of the preservationfluid under test on ice. These were then stored in the re-frigerator at 4◦C for a period dictated by the protocol ofthe experiment.

Dissection of proximal tubules

After a defined period of storage (0, 24, 48, or 72 hours,according to the experimental protocol) the kidney wasremoved and the capsule was carefully stripped using fineforceps and dissection scissors. A thin (approximately0.5 mm thick) cross-section slice was cut with a scalpelblade and placed in a Petri dish half filled with the preser-vation solution under test. The Petri dish was placedon a refrigerated metal dissecting surface of a purposebuilt dissecting table (Conair Refrigerator Circulator,Warrington, UK) under a stereoscopic zoom microscope(Nikon SMZ-2) mounted directly above the Petri dish. Aservo-driven focusing mount controlled by a foot pedalleft both hands free for dissection. At ×800 magnifica-tion various segments of nephron can be identified. Theyappear translucent-gold in color against the black back-ground. Both proximal and distal tubules have a typicalconvoluted appearance and are readily identifiable withinthe outer cortex. The early S1 and S2 segments of the prox-imal tubules can be identified due to their convolutednature and the attachment of the S1 segment to theglomerulus by a narrow neck. In contrast, the proximalstraight tubules have no convolutions and have a typical“ground glass” appearance. The cortical collecting ductis also straight and has a small diameter and an indistinctimage. The thin loop of Henle is the smallest in diam-eter and is mostly confined to the medulla, as are themedullary collecting ducts, which are easily identified astightly packed straight tubules in the medulla. The thickascending limb of loop of Henle is difficult to differenti-ate from the cortical collecting ducts (CCD). However, itdoes not have the less defined appearance of CCD underhigh magnification. Cortical S1 and S2 segments of proxi-mal tubules were used for these experiments. Two pairs ofmicro-fine forceps (Thackray, Leeds, UK) and a 23 G nee-dle mounted on a 1 mL syringe were used for dissection.The cortical tissue was gently teased apart until a tubulewas seen bridging the gap or hanging freely at the edgebetween two tissue pieces. Finally, the ends were cleanlycut by the sharp edge of the mounted needle acting as a

80 Ahmad et al: Ischemia-induced renal tubule cell edema

micro-blade. A transfer pipette was used to transfer thesingle dissected tubule to the perfusion bath. The transferpipette consisted of a Pasteur pipette with the distal 1.5to 2.0 cm bent at right angle. The bulb was controlled byan adjustable screw which allowed fluid to be drawn upand ejected. Before transferring the tubules, the inside ofthe pipette tip was coated with dilute albumin solution(5 mL of bovine 25% albumin solution in Tyrode’s bufferdissolved in 25 mL of saline) to prevent the tubules fromsticking to the pipette glass.

The microperfusion rig

A standard inverted microscope (e.g., ×2.7, ×4, ×10,and ×20 objectives and a ×20 eyepiece) was used in theseexperiments. The perfusion bath (J. White, MD, Wash-ington, USA) consisted of a circular 0.5 cm thick acrylicplate. A longitudinal 2.0 × 0.5 cm full thickness slot wascut in the center and formed the walls of the perfusionbath while a glass cover slip stuck to the base with meltedwax formed the floor of the bath. The chamber held 1 cmdepth of bathing fluid. There were multiple inlets into thechamber to allow experimental manipulations and mon-itoring (temperature, pH, etc.).

Experimental reperfusion protocol

Tubules were then set up on an inverted microscopeand the tubule diameter was measured at 5-minuteintervals using an eyepiece graticule (Graticules Ltd.,Tonbridge, Kent, UK). The graticule was calibratedagainst a standard scale under various magnifications.This form of analysis was validated against later measure-ments using a photodiode array [21] and found to be ac-curate and reproducible. For the period of measurement,the tubules were initially bathed in oxygenated physio-logic saline for 10 minutes, during which time the cell sizeequilibrated and reached a steady volume. The bathingfluid was then replaced by either physiologic saline or atest preservation solution with or without strophanthidin.This test period lasted for 35 minutes and was related tothe warm ischemia of transplantation. Finally, the bathingfluid was again replaced by oxygenated physiologic saline,allowing the cells to recover. This second period in salinelasted for 25 minutes and paralleled reperfusion in trans-plantation while also acting as a second control period.

Strophanthidin treatment of isolated tubules

Strophanthidin (5b ,20[22]-Cardenoline-19-one-3b ,5,14-triol) is a 405 kD cardiac glycoside derivative of digi-talis and is related to digoxin and ouabain in its properties.Strophanthidin is a selective inhibitor of Na+/K+ ATPase,and competes with K+ for Na+/K+ ATPase receptors[22]. In cardiac myocytes, strophanthidin exerts a posi-tive inotropic effect similar to that of digoxin. The rise inintracellular sodium with strophanthidin treatment is due

to suppressed sodium extrusion via the Na+/K+ ATPasepump, and activated sodium influx via Na+/H+ exchange[23]. Specific sodium channel blockers (e.g., SUN1165),therefore, partly abolish the rise in intracellular sodiumresulting from use of strophanthidin and digoxin [24].Tubules prepared as described above were incubated withdoses of strophanthidin as indicated. At concentrationsgreater than 10−3 mol/L, the solubility of strophanthidinwas a limiting factor. For all test experiments a dose of10−3 mol/L was applied.

Calculation of tubule cell volume

At 37◦C fluids and solutes in the tubular lumen arepumped out across the basolateral membrane causingthe tubular lumen to collapse. The outside diameter ofa collapsed tubule (two rows of cells) can thus be usedto calculate cell volume mathematically (ql.mm−1) withthe equation p r2 × l, or 3.1416 × (Tubule diameter/4)2 ×1000.

Statistics

Data were analyzed using Student t test or repeatedmeasures analysis of variance as appropriate. P < 0.05was considered to be significant. For each solution un-der test, tubules from 6 separate kidneys from 6 differ-ent animals were used for each time point of analysis(N = 6).

RESULTS

Isolated tubule segments were prepared and exposedto different preservation solutions in order to examinecell swelling in a situation that paralleled ischemia. In allexperiments tubule diameter was measured across themidpoint of the tubule segment (Fig. 1).

Evaluation of impermeants in organ preservation

Proximal tubules were prepared as per protocol and,following an initial equilibration period in physiologicsaline, maintained in preservation solutions at 37◦Cthat contained different impermeants (Table 2). Resultswere grouped into three categories: monosaccharides(Fig. 2A), disaccharides (Fig. 2B), and trisaccharides andanions (Fig. 2C). Impermeants currently used in preser-vation solutions were then compared as a group (Fig. 2D).This comparison showed that mannitol performed betterin preventing ischemic induced cell swelling than eitherglucose, fructose or mannose (P < 0.001). Although thedisaccharides and trisaccharide prevented cell swellingto some degree in comparison to the monosaccharides,within this group sucrose and raffinose were most ef-fective. Addition of anions in the form of gluconateor lactobionate (at concentrations given in Table 2)demonstrated that lactobionate was more effective as an

Ahmad et al: Ischemia-induced renal tubule cell edema 81

A

B

C D

Fig. 1. Photograph of a normal unperfused rabbit proximal tubule (A) (×200) and a swollen tubule (B) (×200) after prolonged ischemia. (C andD) illustrate the midpoint measurement at higher magnification (×700) of the normal (C) and swollen tubules (D) shown in the inserts.

impermeant (P < 0.001) than gluconate. By direct com-parison of the effect of impermeants used in standardsolutions (Fig. 2D), sucrose performed as well as raffi-nose (UW) with no significant difference between thetwo in this analysis (P > 0.05), and significantly betterthan lactobionate, glucose (EC) or mannitol (HOC) (P <

0.005). From these experiments it appeared that the dis-accharide sucrose was as effective in the prevention ofcell swelling as the trisaccharide raffinose and the anionlactobionate used in UW. Sucrose was significantly more

effective an impermeant than glucose or mannitol, usedin EC and HOC, respectively.

The use of strophanthidin to mimic warmischemic-induced cell swelling

To investigate the effect of strophanthidin on cell vol-ume a dose titration of this agent was applied to isolatedrabbit proximal convoluted renal tubules and demon-strated increased cell swelling as a result of Na+/K+

pump blockade in a dose and time-dependent manner

82 Ahmad et al: Ischemia-induced renal tubule cell edema

Table 2. Test preservation solutions using various “impermeant” molecules

Phosphate-bufferedImpermeant concentration mmol/L−1

concentration OsmolalitySolutions Impermeant mmol/L−1 Na2 HPO4 NaH2PO4 pH mOsm·L−1

PBG Glucose 140 42.3 26.7 7.0 300PBF Fructose 140 42.3 26.7 7.0 300PBMntl Mannitol 140 42.3 26.7 7.0 300PBMnse Mannose 140 42.3 26.7 7.0 300PBS140 Sucrose 140 42.3 26.7 7.0 300PBT Trehalose 140 42.3 26.7 7.0 300PBMlts Maltose 140 42.3 26.7 7.0 300PBR Raffinose 140 42.3 26.7 7.0 300PBL Lactobionatea 140 9.26 5.83 7.0 300PBGcnt Gluconatea 140 9.26 5.83 7.0 300

Abbreviations are: PBG, phosphate-buffered glucose; PBF, phosphate-buffered fructose; PBMntl, phosphate buffered mannitol; PBMnse, phosphate-bufferedmannose, PBS140, phosphate-buffered sucrose; PBT, phosphate-buffered trehalose; PBMlts, phosphate-buffered maltose; PBR, phosphate-buffered raffinose; PBL,phosphate-buffered lactobionate; PBGcnt, phosphate-buffered gluconate.

Postfix after phosphate buffer indicates the sugar/anion molecules.aSodium salt used, phosphate concentration reduced.

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Fig. 2. Comparison of impermeants showed that of the monosacharrides (A), mannitol prevented ischemic induced cell swelling significantly betterthan either glucose, fructose or mannose (P < 0.001). Within the disaccharides and trisaccharides (B), sucrose and raffinose were most effective. Ofthe anions (C), lactobionate was more effective as an impermeant than gluconate (P < 0.001). By direct comparison of the effect of impermeantsused in standard solutions (D), sucrose performed as well as raffinose [used in University of Wisconsin solution (UW)] with no significant differencebetween the two in this analysis (P > 0.05).

(Fig. 3A). Such cell swelling was shown to be rapidlyreversible by the flushed removal of strophanthidin(Fig. 3B) such that tubules returned to their normal di-ameter pre-strophanthidin. This protocol therefore ap-peared to mimic the warm ischemic-induced tubule cellswelling that was more severe after 24 hours cold is-

chemia. In vitro blocking of the Na+/K+ pump appearedto create a similar situation that was alleviated by removalof the agent. Therefore, strophanthidin was used to in-duce biochemical cell swelling of rabbit proximal convo-luted renal tubules to model warm ischemia. This modelwas used to test the effectiveness of various preservation

Ahmad et al: Ischemia-induced renal tubule cell edema 83

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No strophanthidin10–5 M strophanthidin10–4 M strophanthidin10–3 M strophanthidin

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Fig. 3. A dose titration of strophanthidin demonstrated dose-dependent increase in cell swelling with increasing pump blockade (A). To test thereversibility of strophanthidin-induced cell swelling, tubules were exposed to various periods of cold ischemia, which resulted in an increase in theresponse to strophanthidin-induced warm ischemic pump blockade (B). The influence of cold ischemia also manifested as an increased cell sizeeven after strophanthidin was washed out of the system.

solutions against the hypothesis that such solutions pre-vent cell swelling that occurs under ischemic conditionsbecause of metabolic inhibition of the Na+/K+ pumptransporter.

Assessment of impermeants in HOC and EC

The data showed that sucrose acted as the most ef-fective impermeant during experimental ischemia. Inorder to establish the efficacy of the impermeants ofthe most commonly used renal preservation solutionsEC and HOC, glucose and mannitol, respectively, wereexchanged for sucrose. Strophanthidin blockade wasapplied in order to assess cell swelling during the warmischemic phase (Fig. 4A to D). The data showed that cellswelling was significantly reduced in tubules preservedin EC containing sucrose instead of glucose following 0(Fig. 4A, P < 0.001) or 24 hours (Fig. 4B, P < 0.001) of coldischemia. However, improved preservation of HOC byusing sucrose instead of mannitol was only evident after24 hours of cold storage (Fig. 4C and D, P < 0.05). Fromour data it was apparent that sucrose provides superiorprotection from cell swelling over glucose or mannitol.

Further assessment of modifications to HOC

Since HOC is the most commonly used kidney preser-vation solution in the United Kingdom, a further analysisof HOC was made. Osmolality has a critical impact uponfluid dynamics within cells and tissues. When the osmo-lality of HOC was reduced from 400 to 300 mOsm·kg−1

[isosmolar citrate (IOC)] our data showed minimal im-pact at time 0 (P > 0.05), but significant improvement(P < 0.05) after 24 hours cold ischemia as compared withtubules exposed to normally constituted HOC (Fig. 5Aand B). The addition of MgSO4 to Collins original preser-

vation solution was a beneficial development. To establishthe requirement for MgSO4 in HOC a comparison wasmade using HOC in the experimental analysis of tubulecell edema (Fig. 5C and D). The data showed that therewas no difference in cell swelling between these groups.In the third modification, sodium and potassium citratebuffer of HOC was replaced by a sodium and potassiumphosphate, while maintaining the pH at 7.1 and the osmo-lality at 400 mOsm·kg−1. MgSO4 was also replaced withan equivalent amount of mannitol as it tends to forma precipitate with phosphate. The data showed that re-placement of a citrate-based buffer in HOC with a phos-phate buffer had no immediate effects on the ischemic-induced cell swelling at time 0 (Fig. 5E, P > 0.05), but after24 hours cold ischemic time, significant improvement wasnoted (Fig. 5F, P < 0.05).

Comparison of an optimized solution with commerciallyavailable preservation solutions

The data derived from the above experiments sug-gested that a phosphate-buffered, isosmolar, sucrosesolution would combine the best characteristics for a re-nal preservation solution that would prevent proximaltubular cell swelling over prolonged periods of cold is-chemia. Such a solution (PBS140) was applied to thestrophanthidin-based isolated proximal tubule experi-ments, and compared against the ability of EC, HOC,and UW solutions to control cell volume during warm is-chemia. The data showed that PBS140 was as good as UWin preventing cell swelling (Fig. 6A). The experiment wasrepeated on tubules rendered cold ischemic for 24, 48,and 72 hours in order to establish effects of solutions onincreasingly severe ischemic injury. Here results showedthat PBS140 was comparable if not better in reducing cell

84 Ahmad et al: Ischemia-induced renal tubule cell edema

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Fig. 4. Sucrose was added to Euro-Collins (EC) as a replacement for glucose (A) and showed a highly significant improvement in tubule preservation(P < 0.001). After 24 hours of cold ischemia (B), replacing glucose with sucrose in EC further demonstrated the efficacy of sucrose as an impermeant(P < 0.001). Without any prolonged cold ischemia, replacing the impermeant mannitol in hyperosmolar citrate (HOC) with sucrose (C) had nostatistically significant effect on cell swelling after strophanthidin-induced pump blockade. After 24 hours of cold ischemia (D), sucrose providedsignificant protection when it replaced mannitol in HOC (P < 0.05).

swelling than any of the commercially available solutions,including UW (Fig. 6B to D).

Cell volume changes following cold and warm ischemiawith current preservation solutions

By analyzing pre- and post-warm ischemic cell volumes(pl.mm−1) over 0, 24, 48, and 72 hours of cold ischemia,it was evident that at the earliest time points EC failedto prevent cell swelling that was further increased afterwarm ischemia (Fig. 7A). Similar analysis showed that us-ing HOC (Fig. 7B) prevented the early cell swelling thatwas exhibited by tubules preserved in EC but did littleto reduce warm ischemic cell swelling. After prolongedperiods of cold ischemia a time dependent increase incell volume was shown and susceptibility to warm is-chemia was maintained in the presence of HOC. The useof UW (Fig. 7C) prevented to some degree the cold in-duced cell swelling but an increase in the response oftubules to warm ischemia was seen with increasing dura-

tion of cold ischemia. However, using PBS140 (Fig. 7D)effectively reduced the susceptibility of tubules to coldischemia over 72 hours and reduced the subsequent cellvolume response upon exposure to warm ischemia evenafter 72 hours of cold ischemia. In this analysis PBS140demonstrated its capacity to prevent cell swelling moreeffectively than UW.

From these data it is evident that, following strophan-thidin blockade, both PBS140 and UW show the leastpercentage increase in cell volume after CIT 0 (+10 and+8% respectively), when compared to the 30% increaseof EC and 36% increase of HOC. It also appears that thestarting cell volume after cold ischemia was lowest forPBS140 and highest for EC (Table 3). After a prolongedcold ischemic insult of 72 hours, PBS140 proved to bethe most effective of the preservation solutions in pre-venting warm ischemic tubular edema. Here a minimalincrease in cell volume of 7% was observed compared to23%, 38%, and 25% for UW, EC, and HOC, respectively(Table 4).

Ahmad et al: Ischemia-induced renal tubule cell edema 85

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Fig. 5. Comparison of hyperosmolar citrate (HOC) against isomolar citrate (IOC) demonstrated only marginal protection when tissues had notendured any prolonged cold ischemia (A). After 24 hours of cold ischemia (B), a significant reduction in cell swelling was seen if an isosmolar solutionwas used (P < 0.05). An analysis of the requirement for MgSO4 in the solution showed that its exclusion had no effect on strophanthidin-inducedcell swelling after 0 hours of cold ischemia (C, P > 0.05). Similarly, after 24 hours of cold ischemia (D), the exclusion of MgSO4 in the solutionhad no effect on strophanthidin-induced cell swelling (P > 0.05). The exchange of the citrate buffer for a phosphate buffer (E) did not show animprovement after minimal cold ischemia, while replacing citrate with a phosphate buffer in HOC showed significant improvement if tubules hadbeen rendered cold ischemic for 24 hours (F, P < 0.05).

DISCUSSION

This study analyzed the effect of preservation solu-tions and their core components on preventing cellularedema. An isolated renal proximal tubule system was de-

veloped that reproducibly demonstrated the edematousresponse of single tubules to cold and warm ischemia. Al-though this model examined only one aspect of ischemicinjury, it is cellular edema that forms the principal lesion

86 Ahmad et al: Ischemia-induced renal tubule cell edema

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Fig. 6. By using the strophanthidin-based model, a comparison (A) of the commercially available solutions with a phosphate buffered sucrosesolution (PBS140) showed that cell swelling was minimized by either University of Wisconsin (UW) or PBS140 compared to Euro-Collins (EC)and hyperosmolar citrate (HOC) at time 0 (P < 0.01). The effect of 24 hours of cold ischemia (B) showed that cell swelling was most effectivelyprevented during a warm ischemic episode if PBS140 was used (P < 0.01). After 48 hours of cold ischemia (C), our data show prolonged protectionwith PBS140 comparable with UW. Prolonged protection with PBS140 comparable with UW was also seen at 72 hours of cold ischemia (D).

leading to pathologic features of ischemically damagedorgans. For this reason the prime component of a preser-vation solution is its impermeant, whose function is theprevention of cell swelling. The nature of cell swellingin this model was believed to be due to abrogation ofcell volume regulation. Maintenance of a constant cellvolume is dependent on the rate of solute influx beingequal to solute efflux. Biologic membranes are highlypermeable to water and so ionic disturbance in the in-tracellular and extracellular fluid leads to osmotic move-ment of water across the plasma membrane. In the con-text of ischemic depletion of ATP, energy dependentsolute transporters such as the Na+/K+ATPase are nolonger able to regulate cell volume. An accumulationof intracellular Na+ is followed by influx of anions tomaintain electroneutrality [25–27] and water follows os-motically into the cell. The use of strophanthidin in oursystem to block the Na+/K+ pump modeled the phys-iologic response of tubules to starvation of ATP withregard to cell volume control. The cell swelling that

was detected was reversible once strophanthidin was re-moved from the perfusion solution, as tubules attemptedto regain cell volume control. The isolated tubule experi-ments therefore putatively reflected cellular edema, andsince the experiments were conducted at 37◦C followingperiods of cold preservation, the model system allowedan analysis of tubule responses to both cold and warmischemia.

The usefulness of the isolated tubule experiments wasvalidated by the experimental data in this study thatshowed that UW solution was more effective in prevent-ing cellular edema than EC or HOC. UW solution isregarded as the “gold standard” in organ preservationsolutions so these observations widely reflected otherwork using whole organs [28], but this study provideda novel physiologic analysis of the edema that forms theprincipal source of preservation injury. The isolated per-fused tubule system permitted a direct investigation ofthe effects of alterations to preservation solutions thatmight predict outcomes in whole organ transplants, and

Ahmad et al: Ischemia-induced renal tubule cell edema 87

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Fig. 7. Tubule cell volume after 0 to 72 hours of CIT and subsequent increases following warm ischemia are shown for tubules preserved in EC (A),HOC (B), UW (C), and PBS140 (D). The data showed that PBS140 minimized tubule volume changes after both cold and warm ischemic periods,providing evidence of reduced edema compared to the other solutions.

Table 3. Percentage change in cell volume during pump blockade with strophanthidin and upon reversal of pump blockade compared to the firstcontrol period in physiologic saline (0 hour cold ischemic time)

Cell volume ql·mm−1

Test solution Physiologic salinePhysiologic salineTest solutions Cell volume Cell volume % change Cell volume % change

PBS140 221 ± 16 196 ± 18 −11 243 ± 22 +10UW 225 ± 16 185 ± 17 −18 244 ± 30 + 8EC 282 ± 35 455 ± 18 +62 367 ± 46 +30HOC 244 ± 14 257 ± 15 +5 332 ± 16 +36

Abbreviations are: PBS140, phosphate-buffered sucrose; UW, University of Wisconsin; EC, Euro-Collins; HOC, hyperosmolar citrate.

Table 4. Percentage change in cell volume during pump blockade with strophanthidin and upon reversal of pump blockade compared to the firstcontrol period in physiological saline (72 hours cold ischemic time)

Cell volume ql·mm−1

Test solution Physiologic salinePhysiologic salineTest solutions Cell volume Cell volume % change Cell volume % change

PBS140 323 ± 37 254 ± 22 −21 344 ± 45 +7UW 325 ± 27 269 ± 16 −17 398 ± 29 +23EC 291 ± 16 468 ± 28 +61 402 ± 18 +38HOC 427 ± 45 465 ± 42 +9 534 ± 48 +25

Abbreviations are: PBS140, phosphate-buffered sucrose; UW, University of Wisconsin; EC, Euro-Collins; HOC, hyperosmolar citrate.

88 Ahmad et al: Ischemia-induced renal tubule cell edema

provided an elegant method to potentially design the op-timal platform for a preservation solution to reduce cel-lular edema.

For a variety of reasons, transplant centers worldwidehave adopted alternatives to UW for renal preservationthat are accepted as less than optimal because of the“gold standard” status ascribed to UW. By implication,therefore, a higher degree of tissue injury possibly oc-curs at the time of transplantation where alternatives toUW are used. Delayed graft function and acute tubularnecrosis due to I/R injury have been linked to subsequentacute rejection episodes [4, 5], the incidence of which is amajor predictor of chronic allograft nephropathy—itselfthe most common cause of allograft failure. It is cleartherefore that improvements in preservation solutionsmay have significant benefits for clinical transplantationworldwide.

The specific analysis of the core components of preser-vation solutions in this study highlighted not only the dif-ferences between various solutions in current clinical use,but also could be used to establish the constituents of anoptimized solution for improved organ preservation. Thisstudy confirmed the beneficial effects of impermeants inpreventing cell swelling, and appeared to show that su-crose could be as effective as the raffinose that is usedin UW. Indeed, cell swelling was reduced in the presenceof EC or HOC if their impermeants were changed tosucrose from mannitol or glucose, respectively, demon-strating that tubule cell impermeability to monosaccha-rides is at best only partial, and is not sustained throughprolonged ischemia. The data in this study, therefore,showed that the renal tubules were more impermeant tosucrose. Unlike glucose, sucrose is not transported acrosscell membranes, nor broken down by sucrase activity inthe nephron, and so remains in the extracellular spaceunder both hypothermic and normothermic conditions.Therefore, sucrose is a more suitable impermeant agentfor use in organ preservation than glucose, which oncetransported into cells by the Na+/glucose transporter maybe used as a substrate for anaerobic metabolism and fur-ther contribute to the accumulation of toxic metabolitesduring ischemia. In addition, our observations also sug-gested that tubular cell membranes may become morepermeable to mannitol over prolonged periods of cold is-chemia. The data demonstrated that the benefits in usingsucrose were even more apparent the longer the periodof cold ischemia.

Cell swelling was also reduced after a prolonged coldand warm ischemic period if an isosmolar solution wasused instead of a hyperosmolar solution. In a previousstudy, a comparison of hyperosmolar and isosmolar formsof HOC showed better preservation of isolated perfusedrat kidneys using isosmolar solution [29]. In the currentstudy of isolated tubules, on return to physiologic saline,an irreversible increase in tubule diameter was seen in

the 0 hour HOC group. A similar effect has been ob-served during the investigation of cell volume regulationduring hypertonic shock [30]. During volume regulationin hypertonic medium, intracellular Na+ and Cl− con-centrations increase with no concurrent alteration in K+

concentration. This imbalance drives an influx of waterupon rapid exchange of medium. The data in this studywould suggest that cell volume after exposure to HOCremains at this increased level, though the reason for thisis unclear. However, this could have important implica-tions for the clinical use of hypertonic preservation solu-tions such as HOC and EC; cell swelling triggered byreturn to isotonic conditions from hypertonic storagecould be responsible for some post-ischemic tissuedamage.

This study also investigated the effect of exchangingthe buffer from citrate (HOC) to phosphate (HOPBM).After 0 hours of preservation there was no significantdifference in tubule diameter. However, after 72 hoursof cold ischemia, tubule diameter in the HOC group wasgreater than the HOPBM group. This parameter was notsignificantly different when comparing the IOC group at72 hours. Taken together, these findings suggest that cit-rate is more permeable than phosphate, at least underhypertonic conditions.

The direct comparison of solutions over 72 hours in-dicated that cell volume during warm ischemia was es-sentially uncontrolled in tubules preserved in EC, andworsened with prolonged CIT in HOC. Upon reperfu-sion with saline at the end of the experiment to removethe preservation fluid under test, regulatory volume de-crease (RVD) was repeatedly seen in the tubules exposedto EC as they presumably recovered from the more se-vere warm ischemic swelling induced in the presence ofglucose. Such RVD was not seen with the other solutionsthat did not induce the same degree of swelling. How-ever, the data shows that tubule diameter was lowest inthe PBS140 group at the end of the experimental proto-col after 24, 48 or 72 hours of CIT, even when comparedto the performance of UW.

Our data clearly demonstrate the shortcomings ofHOC, as well as solutions that used a less effective im-permeant, such as EC. These solutions have been usedwidely in clinical transplantation but clearly there mayhave been increased posttransplant complications as a re-sult. Direct comparisons with the common preservationsolutions have been made in clinical and experimentaltransplant studies and these have revealed that UW isbetter than either EC or HOC.

However, the experimental analyses presented herewould suggest that an isosmolar, phosphate-buffered su-crose solution such as PBS140 could form the basic plat-form for an optimized renal preservation solution. Earlierstudies have hinted that this may be the case [31–33], butthis study is the first to definitively examine at the tubular

Ahmad et al: Ischemia-induced renal tubule cell edema 89

level, the physiologic response of renal tissue to preser-vation solutions. The data here suggest that phosphate-buffered sucrose solutions have the potential to offerbetter conditions for organ preservation than UW. In-deed, in a previous trial of PBS140 in clinical renal trans-plantation, improved results posttransplant were seen[34]. The data presented here revealed that PBS140 pre-vented cell swelling more effectively than UW in this ex-perimental setting, and that warm ischemic cell swellingafter 72 hours of cold ischemia was reduced threefold byPBS140, compared to UW-preserved tissue.

These observations could therefore have significant im-plications for the clinical management of ischemic dam-age. If PBS140 could reduce cell swelling more effectivelythan UW, then by implication less tissue damage occurs inPBS140 preserved tissues. This can be interpreted in twoways: first, less tissue damage occurs in short (<24 hours)cold ischemic times, and second, tissues remain more vi-able for longer ischemic times. In addition, PBS140, beinga relatively simple solution, overcomes the shortcomingsof using UW. PBS140 has a lower viscosity, making its useeasier and permitting more rapid and efficient perfusionof the donor organ. These factors all have the potentialto significantly improve transplant outcomes by reducingthe initial tissue damage at the time of transplantation.

This study, therefore, dissected the core componentsof clinical preservation solutions and has proposed thebasis of an optimized preservation solution that may per-form better than all other solutions currently in commonclinical practice. Further studies in whole organ and intransplant systems are already underway. Any possiblebenefit in switching from EC or HOC-based solutionswould probably be seen in reduced rates of acute tubularnephropathy or in the incidence of DGF, but longer termanalysis of transplant recipients is required to demon-strate the effectiveness of better organ preservation ontransplant outcome. The potential exists to reduce thenon-specific tissue injury that occurs in all donor organsand to extend the possible pool of available organs.

ACKNOWLEDGMENT

We would like to thank The Combined Transplant Fund, St. James’sUniversity Hospital, Leeds, for supporting this research.

Reprint requests to Dr. Julian R. Pratt, Ph.D., Molecular MedicineUnit, Clinical Sciences Building, St James’s University Hospital, Leeds,LS9 7TF, UK.E-mail: j.r.pratt@leeds.ac.uk

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