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Scandinavian Journal of Clinical and LaboratoryInvestigation

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Isolated kidney perfusion: the influence ofpulsatile flow

Charlotte von Horn & Thomas Minor

To cite this article: Charlotte von Horn & Thomas Minor (2018) Isolated kidney perfusion: theinfluence of pulsatile flow, Scandinavian Journal of Clinical and Laboratory Investigation, 78:1-2,131-135, DOI: 10.1080/00365513.2017.1422539

To link to this article: https://doi.org/10.1080/00365513.2017.1422539

© 2018 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.

Published online: 04 Jan 2018.

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ORIGINAL ARTICLE

Isolated kidney perfusion: the influence of pulsatile flow

Charlotte von Horn and Thomas Minor

Department of Surgical Research General, Visceral and Transplantation Surgery, University Hospital Essen, University Duisburg-Essen, Essen,Germany

ABSTRACTWithin the scope of transplantation research, ex vivo kidney perfusion has been proven an attractivemodel to study ischemia-reperfusion and preservation injury. Renal perfusion techniques also occupyscientists with the aim to optimize organ reconditioning and preparation prior to transplantation. Thisstudy investigated the influence of a pulsatile perfusion pattern that brings flow conditions closer tophysiological situations, on renal perfusion characteristic and kidney function in the isolated perfusedkidney. Kidneys were perfused via a roller pump at constant pressure set to 90mmHg, or with additionof pulsatile pressure peaks (90/70mmHg; 60/min) using an adjustable positive displacement pump. Itwas found that pulsatile pressure significantly enhanced renal flow rates as compared to non-pulsatileperfusion mode, especially after preceding preservation of the kidney by static cold storage. Theimproved vascular conductivity went along with a notable improvement of clearance of creatinine,sodium reabsorption and reduced tubular cell injury (Loss of fatty acid binding protein). The better vas-cular conductance upon pulsatile perfusion could be attributed to improved endothelial release of niticoxide and reduced secretion of endothelin-1 into the perfusate. It is concluded, that pulsatile perfusionmode should be preferred in isolated kidney perfusion as resulting in better preservation/recovery ofrenal perfusion and function.

ARTICLE HISTORYReceived 18 July 2017Revised 7 November 2017Accepted 13 December 2017

KEYWORDSKidney; isolated perfusion;pulsatile flow; in vitro; pig;porcine; pulsatility

Introduction

The ex vivo perfusion of the isolated kidney has been usedsince decades as a useful tool for the study of renal physi-ology and as valuable model in the evaluation of variouspathophysiologic conditions [1–3].

In the field of transplantation research, the isolated per-fused kidney serves as a surrogate model for assessment ofischemia reperfusion or preservation injury and supplantsactual in vivo transplantation in experimental animals [2].The widespread use of isolated kidney perfusion in experi-mental screening studies thus contributes to reduced straininflicted to recipient animals suffering from dys- or non-functioning kidneys in transplantation studies.

Moreover, the isolated kidney preparation is technicallyless demanding and therefore more reproducible than actualtransplantation in vivo. Pathophysiological studies take bene-fit from the isolated approach, allowing for variations in theexperimental setting that might be difficult or impossible inthe living animal.

Recently, translational as well as clinical research hasfocussed on renal machine perfusion as alternative preserva-tion method in competition with static cold storage.

This interest has fuelled a variety of refinements concern-ing the optimal physical perfusion conditions. Novel perfu-sates have been developed for better preservation of the

kidney during hypothermic perfusion [1,4–6]. Systematicinvestigations have specified adequate perfusion pressuresunder hypothermic conditions [7] as well as at normothermia[8].

Less data are available concerning the mode of renalperfusion, e.g. if arterial pressure should be kept at a con-tinuous level or if a pulsatile pattern, mimicking the physio-logical situation, should be preferred.

Recent upsurge in research on mechanistic background ofperfusion preservation suggests pulsatile shear stress at thevascular endothelium to be a major effector for improvedpreservation of kidneys by hypothermic machine perfusion(HMP) [9,10] and several reports on HMP suggest pulsatileperfusion to be superior to constant pressure modes[9,11,12].

However, no systematic investigation has been carriedout so far focussing on the use of the isolated kidney asreperfusion model or in physiological experiments.

Although pulsatile perfusion pattern is sometimes advo-cated [13], the use of a roller pump instead of non-pulsatilepumping systems is often thought to suffice for pulsatilestimulation without control of the actual pressure curve atthe renal artery.

This study thus was designed in order to investigatethe respective role of vascular pulsatility for isolated kidney

CONTACT Thomas Minor chirfor@uk-essen.de Department for Surgical Research General, Visceral and Transplantation Surgery, University Hospital Essen,University Duisburg-Essen, Hufelandstr. 55, D-45147 Essen, Germany� 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/Licenses/by-nc-nd/4.0/),which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION, 2018VOL. 78, NO. 1-2, 131–135https://doi.org/10.1080/00365513.2017.1422539

perfusion in healthy (freshly excised) kidneys as well as inpreparations that had suffered a longer period of static pres-ervation as usually encountered in transplantation practiceand organ preservation research.

Materials and methods

All experiments were performed in accordance with the fed-eral law regarding the protection of animals. The principlesof laboratory animal care (NIH publication no. 85-23,revised 1985) were followed.

Kidneys were removed from anesthetized, non-survivingfemale German Landrace pigs weighing between 25 and30 kg by trans-peritoneal access through midline abdominalincision. The renal artery was cannulated on the back-tableand the kidneys flushed by 100 cm gravity with 100ml ofHistidine–Tryptophan–Ketoglutarate (HTK) solution (Dr. F.K€ohler Chemie, Bensheim, Germany) at 4 �C.

The isolated kidneys were put on an ex vivo perfusioncircuit for normothermic perfusion as detailed below eitherimmediately or after 20 hours of static cold storageimmerged in the preservation solution at 4 �C for 20 h.

Isolated kidney perfusion

Prior to isolated perfusion, the ureter was cannulated withPE-tubing for collection of urine during the experiment.

Isolated normothermic organ perfusion was done in anestablished set up [12] with some modifications.

The system contained a thermostatically controlled(37 �C) moist chamber and perfusate reservoir, a precisionroller pump, a hollow fibre oxygenator (Hilite LT 1000,Medos, Stolberg, Germany) with heat exchanger also con-nected to the thermostat, a transit time flow meter with flowthrough probe interposed in the perfusion line (Hugo Sachselectronic, March-Hugstetten, FRG) and a pressure trans-ducer connected to the arterial inflow line immediately atthe renal artery.

The perfusate consisted of 1000ml Krebs–Henseleit buffersupplemented with 2.2% bovine serum albumin and 20mlof concentrated amino acid solution (RPMI 1640-50x).Creatinine (0.01 g/l) and urea (1 g/l) were also added toallow for calculation of renal clearances.

The perfusate was oxygenated with 95% oxygen and 5%carbon dioxide and flow through the kidneys in the moistchamber was adjusted to achieve a mean perfusion pressureof 90mmHg by manual control of the roller pump speed.

In half of the cases, arterial inflow was subjected to anadditional pulsatile pattern, generated by means of anadjustable positive displacement pump, which alternately (60times/min) added or withdraw an adjusted pressure to/froma fluid column connected to the vent of the oxygenator. Theactual driving pressure at the renal artery thus variedbetween approx. 70mmHg and 90mmHg during one cycle.

Urine was collected outside of the perfusion chamberand urinary fluid loss was replaced every 30min by addingequal amounts of balanced salt solution to the perfusionmedium.

Renal functionPerfusate and urine samples were analysed for concentra-tions of urea and creatinine in a routine fashion at theLaboratory centre of the University Hospital.

From these data and the volume of the produced urine,the respective clearances were calculated as urinary creatin-ine/urea� urine flow/perfusate creatinine/urea in ml/min.

Tubular reabsorption of sodium was approximated bycalculating the fractional excretion of sodium (FENa) accord-ing to:

FENa = 100� urinary sodium� urinary creatinine/(per-fusate sodium� perfusate creatinine). Oxygen consumptionwas calculated from the pO2 differences between arterial andvenous sites, measured in a pH-blood gas analyser (ABL815flex acid-base laboratory, Radiometer, Copenhagen) andexpressed as mlmin1 g1 according to trans-renal flow andkidney mass.

Tubular cell injuryStructural injury to renal tubular cells was analyzed usingthe release of the intracellular carrier protein L-type fattyacid binding protein (LFABP), as a compound predomin-antly expressed in liver tissue as well as in proximal tubularcells of kidney [14].

Measurements were done with a commercial ELISA kit(USCN life science, Wuhan, China) according to the instruc-tions of the manufacturer on a fluorescence micro platereader (Tecan, Grailsheim, Germany).

Vascular mediatorsConcentrations of major mediators, involved in the controlof vascular tone were determined in the perfusate using ana-lytic kits from the following companies according to theinstructions of the manufacturers to analyse perfusate levelsof endothelin 1 (USCN life science, Wuhan, China) andtotal nitric oxide (R&D Systems, Wiesbaden, Germany)

StatisticsAll values were expressed as means ± SEM. After proving theassumption of normality, differences between the pulsatileand non-pulsatile perfusion were tested by parametric com-parison of the means using Student’s t-test or one-way ana-lysis of variance and post hoc comparison with TukeyKramer multiple comparison test, where appropriate (Instat3.01, Graph Pad software Inc, San Diego, CA). Statistical sig-nificance was set at p< .05.

Results

Pulsatile perfusion pattern

The kinetics of the arterial perfusion pressure with or with-out proactive induction of pulsatile pressure peaks isdepicted in Figure 1. It is seen that little fluctuations in per-fusion pressure are induced by the rotations of the roller

132 C. VON HORN AND T. MINOR

pump, but that these do actually induce a pulse wave patternduring isolated kidney perfusion. By contrast, implementa-tion of alternating pressure peaks in the arterial line resultedin a net fluctuation of perfusion pressure and a real pulsatileperfusion profile.

Vascular integrity during perfusion

In sham preparations that were perfused immediately afterorgan retrieval, renal perfusate flow was only moderatelyaffected by pulsed vs non-pulsatile perfusion mode, althoughpulsed perfusion resulted in tendentially higher flow rates(cf. Figure 2).

After cold storage, however, these differences becamemuch more evident and significantly higher flow rates wereobserved upon pulsed perfusion. Of note that in the lattercase near normal values of renal perfusate flow could beobtained while vascular conductivity was found notablyimpaired upon non-pulsatile perfusion after ischemicpreservation.

Molecular triggers of vascular conductivity

Vascular tone is dominantly influenced by the balancedinterplay of the two major mediators of vascular relaxation(NO) and vasoconstriction (endothelin) at the luminalendothelium.

In our model, we could substantiate a significant increasein endothelin-1 activity after ischemic preservation in theperfusate of non-pulsatile perfused kidneys, which was notdisclosed after pulsed perfusion (cf. Figure 3).

Similarly, a reduction of the endogenous vasodilator NOwas apparent upon non-pulsatile perfusion as compared topulsatile perfusion, which was most pronounced after pre-ceding ischemic preservation, although these differences didnot reach statistical significance.

Figure 3. Perfusate levels of the vasoconstrictor endothelin-1 (ET-1) andendogenous vasodilator NO, as well as their ratio upon isolated perfusion offreshly excised (sham) or previously cold stored (CS) kidneys upon non-pulsatileor pulsatile (puls.) perfusion conditions. (�: p< .05 vs respective non-pulsatileperfusion).

Figure 1. arterial pressure record upon non-pulsatile and pulsed perfusion ofthe isolated kidney as described in material and methods.

Figure 2. Renal perfusate flow (RPF) upon isolated perfusion of freshly excised(sham) or previously cold stored (CS) kidneys upon non-pulsatile or pulsatile(puls.) perfusion conditions. (�: p< .05 vs respective non-pulsatile perfusion).

SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION 133

The difference between pulsatile and non-pulsatile perfu-sion conditions became more evident when plotting theindividual ratios of ET and NO as integral denominator ofvascular tone. Pulsed perfusion consistently resulted in a sig-nificant reduction of this ratio and hence promoted a morevasoconductive phenotype.

Renal function

Glomerular function of the preserved kidneys was estimatedby the calculation of renal clearances for creatinine(Figure 4). This was found to be notably impaired in bothgroups after ischemic preservation as compared to non-ischemic sham conditions. However, pulsatile perfusion con-ditions led to a more than three-fold and significantimprovement of post-preservation function of the kidneys incomparison to non-pulsatile reperfusion.

Likewise, fractional excretion of sodium as a readout ofrenal tubular function was massively deteriorated after ische-mic preservation and non-pulsatile perfusion, while onlymoderate alterations could be evidenced upon pulsed perfu-sion even after renal ischemia(FE Na, Figure 4).

Discussion

The main finding of the present study points out thatsignificant impairments of vascular function andconductivity are observed upon renal perfusion in vitro afterstatic preservation and this vascular dysfunction is in largeparts mitigated by the implementation of a pulsatile perfu-sion pattern in the experimental set-up. The dependency ofvascular conductivity on pulsatility of perfusion pressurewas less pronounced but also noticeable in freshpreparations.

Of note, the effects of pulsatile pressure were not limitedto mere vascular phenomenae but also have significantimplications on renal function during isolated perfusion, e.g.clearance of creatinine and tubular cell integrity.

Renal tubular cells are strongly dependent on adequateoxygenation, as transmembranous transport systems are

major energy demanding processes [15,16]. Therefore, main-tenance of sufficient vascular perfusion of tubular epithelialcells in the kidney is considered to be pivotal to safeguardphysiological cell function.

Likewise, impaired vascular perfusion also favours areduction of glomerular filtration by simple reduction oftranscapillar hydraulic pressure difference [17].

Under physiological conditions in vivo, endothelial cellsare constantly exposed to mechanical stimuli exerted by pul-satile blood flow and pressure. This mechanical stimulationtranslates into a strong induction of endothelial nitric oxidesynthase (eNOS) as well as inhibition of endothelin (ET-1)[18] as to guarantee for optimized vascular conductivity andtissue perfusion.

Experimental studies have further disclosed a subcellularnetwork, ochestrated by the flow dependent activationof thetranscription factor Kr€uppel-like factor 2 (KLF-2) and trig-gering the transcription of a variety of anti-inflammatoryand anti-thrombogenic genes while depressing the upregula-tion of cellular adhesion molecules on endothelial cells[10,19,20].

To this regard, pulsatile flow has been shown to be muchmore effective than steady shear stress [12,20] or oscillatoryflow [21].

The effects of endothelial stimulation are lasting for sev-eral minutes up to hours after cessation of vascular perfu-sion. Thus, they are already declining during earlynonpulsatile perfusion after retrieval, but are still operative.The lack of mechano-stimulation to the endothelium during(prolonged) static preservation in contrast notably aggravatesthe endothelial dysfunction upon reperfusion [19].

Vascular shear stress directly stimulates the phosphoryl-ation of eNOS in an endothelial protein kinase A (PKA)dependent manner resulting in the production of theendogenous vasodilator nitric oxide (NO) [22].

NO is known to maintain medullary blood flow by pro-moting vasodilation of descending vasa recta and juxtaglo-merular arterioles [23], while oxygen free radicals, putativelyarising after ischemic preservation, may reduce local bioa-vailbility of NO.

Pulsatile endothelial stimulation has been shown to resultin elevated activation of eNOS already within 20min in a

Figure 4. Glomerular (clearance of creatinine) and tubular (fractional excretion of sodium- FE-Na) renal function upon isolated perfusion of freshly excised (sham)or previously cold stored (CS) kidneys upon non-pulsatile or pulsatile (puls.) perfusion conditions. (�: p< .05 vs respective non-pulsatile perfusion).

134 C. VON HORN AND T. MINOR

cell culture model [24]. This indicates a swift effect of pulsa-tile perfusion even before the transcriptional machinery willbe reactivated after static storage condition in favor of a vas-opretective endothelial phenotype.

In line with previous results, the present study discloseda notable dysbalance of endogenous mediators that regulatevascular tone (NO and ET-1) upon nonpulsatile perfusionto the effect of a more vasoconstrictive reaction, which wasattenuated by using the pulsatile perfusion mode.

Being closer to physiological conditions upon post-trans-plant reperfusion in vivo, we would thus recommend pulsa-tile perfusion also for use in ischemia reperfusion modelsaiming to supplant actual transplantation. As pulsatile perfu-sion actually also affected renal filtration and tubular cellfunction it is conjectured that in vitro experiments will bet-ter mimick renal physiology if performed in a pulsatile per-fusion mode.

Acknowledgements

The authors gratefully acknowledge the valuable technical help of B.L€uer in executing the experiments and performing the molecularanalyses

Disclosure statement

The authors report no conflicts of interest.

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