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Effect of remote ischaemic preconditioning onrenal protection in patients undergoinglaparoscopic partial nephrectomy: a blinded

randomised controlled trialJiwei Huang, YongHui Chen, Baijun Dong, Wen Kong, Jin Zhang, Wei Xue,DongMing Liu and Yiran Huang

Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Objective To evaluate whether remote ischaemic

preconditioning (RIPC) reduces renal injury inpatients undergoing laparoscopic partial nephrectomy(LPN).

Patients and methods In all, 82 patients undergoing LPN were randomly

assigned to either the RIPC or control group, with 40 and38 patients, respectively completing 6-months follow-up.

RIPC was conducted after induction of anaesthesia,which consisted of three 5-min cycles of right lowerlimb ischaemia and 5 min of reperfusion during eachcycle.

The primary outcome was the absolute change in

glomerular filtration rate (GFR) of the affected kidney byrenal scintigraphy from baseline to 6 months.

The secondary outcomes included urinaryretinol-binding protein (RBP) levels measured at 24 and48 h, serum creatinine, and estimated GFR (eGFR) at 1and 6 months, and changes in GFR by renalscintigraphy.

Results There were no differences in the change of GFR of the

affected kidney at 6 months, while it was significantlydecreased by 15.0% in the control group vs 8.8% in theRIPC group at 1 month (P= 0.034).

The urinary RBP levels increased 8.4-fold at 24 h in thecontrol group compared with a lower increase of 3.9-foldin the RIPC group (P< 0.001).

There were no differences in the serum creatinine levelor eGFR at 1 and 6 months between the two groups.

Conclusions In patients undergoing LPN, RIPC using transient lower

limb ischaemia may reduce renal impairment in theshort term, but failed in the longer term despite anon-significant trend in favour of RIPC.

These novel data support the need for a larger study ofRIPC during LPN surgery.

Keywords

remote ischaemic preconditioning, laparoscopic partialnephrectomy, warm ischaemia, urinary retinol-bindingprotein, radionuclide imaging

Introduction

Partial nephrectomy (PN) is now the standard surgicaloption for localised small renal tumours instead of radicalnephrectomy [1]. The renal vascular pedicle usually needsto be temporarily occluded during PN, which can lead torenal damage. Several studies have previously shown thatprolonged warm ischaemia time (WIT) leads to somedegree of renal function loss [2,3]. Lane et al. [2] reportedon 30 patients that underwent laparoscopic PN (LPN) in asolitary kidney and showed that a WIT of>20 min wasassociated with significantly decreased renal function.

Several methods have been developed to reduce this

functional damage during LPN [47]. However, mosttechniques are complicated to manage and limited toexperienced surgeons. So there is an urgent need for areliable, simple, and quick technique to reduce renaldamage in LPN and robotic surgery.

One potential strategy for reduction of renal injurysustained during LPN is ischaemic preconditioning, whichrefers to application of a brief episode of ischaemia andreperfusion, which results in tolerance to subsequent severerenal ischaemic injury. Ischaemic preconditioning has been

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shown have promising results in preventing renal injury inanimal studies [8]. However, an ischaemic preconditioningprotocol of temporarily occluding the renal artery wouldnot only lengthen the operation but also be impractical toapply.

A more practical and harmless approach may be achievedusing remote ischaemic preconditioning (RIPC), whichmeans that brief ischaemia of an organ or tissue alsoprotects remote organs from a sustained interval ofischaemia. In rats, RIPC by brief hind limb occlusion hasbeen reported to improve renal function by 3060%, andreduce renal tubule damage and kidney injury molecule-1expression after 25-min ischaemia in the rat kidney [9].Several encouraging trials of RIPC have suggested clinicalbenefit in heart and spinal cord surgeries [1018], but as faras we know, no kidney surgeries have applied the RIPCmethod to reduce renal ischaemic insults. Thus, the aim ofthe present study was to assess whether RIPC is effective in

reducing renal injury in patients undergoing LPN.

Patients and MethodsAt our institution, consecutive eligible patients diagnosedwith renal tumours who underwent LPN were recruited forthis randomised, prospective, clinical trial from January2010 to August 2011. The criteria for inclusion in this studyincluded: tumours of40%

as determined by radionuclide scintigraphy. We excludedpatients aged >80 years, those with heart, hepatic,pulmonary or other renal diseases, and those withperipheral vascular disease affecting the lower limbs. Thestudy protocol was approved by the Institutional EthicsCommittee. Written, voluntary, informed consent was takenfrom all patients.

All preoperative images of the patients were reviewed bythree urological surgeons according to the PreoperativeAspects and Dimensions Used for an Anatomical (PADUA)classification [19], to reflect tumour complexity, and aconsensus was reached by open discussion in cases of

dissension. All three observers were blinded to the clinicaloutcomes.

Patients were randomly assigned to one of two groups:control or RIPC before LPN surgery. Randomisation wascarried out using a computer-generated, random-sequencegrid maintained by the principal investigator (Y.H.) in a 1:1ratio. In all, 91 patients were assessed for eligibility, ofwhom 82 were actually recruited and randomly assigned tothe RIPC group (41 patients) or control group (41). Onepatient in RIPC group was lost to follow-up at 1 month

and two in the control group were lost to follow-up at 6months. In all, 78 patients completed the 6-monthfollow-up (Fig. 1).

Procedure

RIPC (by W.K.) consisted of three 5-min cycles of rightlower limb ischaemia, which was induced by an automatedcuff-inflator placed on the right thigh and inflated to200 mmHg, with an intervening 5 min of reperfusionduring which the cuff was deflated. An audio Doppler wasplaced on the pedal artery to ensure lower limb ischaemia.Control patients underwent the same placement of theblood pressure cuff around the right thigh but withoutinflation. The RIPC protocol was applied after anaesthesiainduction and before renal pedicle occlusion started.Patients and the surgeons (Y.H.C and D.M.L.) wereblinded to treatment allocation.

The LPN technique has been described in detail previously[20]. The renal hilum was accurately isolated and then theartery only was clamped, without cooling, in all cases.

Outcome Measures

Urine for retinol-binding protein (RBP) estimation wascollected in sterile bottles from the patients catheter at

three time points: immediately after insertion before thestart of surgery (time 0), and at 24 and 48 h after LPN.Urine was transported to the laboratory and stored at-20 C for subsequent analysis.

Serum creatinine levels and the technetium (99mTc)-diethylene triamine pentacetic acid (DTPA) GFR wereevaluated preoperatively, and at 1 and 6 months after LPN.Total renal function was calculated based on the GFRestimation using the abbreviated equation developed by theModification of Diet in Renal Disease (MDRD) Study

Fig. 1 Consolidated Standards of Reporting Trials (CONSORT)

diagram.

Assessed for eligibility (n =91)

Enrollment

Randomized

Allocated to RIPC (n =41)

Received RIPC intervention (n =41)

Allocated to control (n =41)

Received control intervention (n =41)

Required open conversion (n =1)

Lost follow up at 1 month (n =0)

Lost follow up at 6 months (n =2)

40 analysed at 1 month38 analysed at 6 months

Lost follow up at 1 month (n =1)

Lost follow up at 6 months (n =0)

40 analysed at 1 month40 analysed at 6 months

Excluded (n =9)

Not meeting inclusion criteria (n =3)

Refused to participate (n =6)

Other reasons (n =0)

Allocation

Follow-Up

Analysis

Remote ischaemic preconditioning and renal protection in patients undergoing LPN

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Group, as follows: GFR (mL/min/1.73 m2) = 186 (serumcreatinine) -1.154 (age) -0.203 0.742 (if female) 1.210 (ifblack) [21].99mTc-DTPA renal scintigraphy was used toevaluate the GFR of each kidney and both kidneys together.GFR values were calculated from the 99mTc-DTPA renalscintigraphy data bygcamera estimation using the Gates

method and were normalised by body surface area(1.73 m2).

The primary outcome was the change in GFR of theaffected kidney by renal scintigraphy from baseline to 6months. The secondary outcomes included the urinary RBPlevel measured at 24 and 48 h after LPN, serum creatinine,eGFR at 1 and 6 months and changes in total GFR andGFR of the affected kidney by renal scintigraphy frombaseline to 1 month, and the change in total DTPA GFRfrom baseline to 6 months.

Statistical Analysis

The study had a power of 90% to detect abetween-treatment difference of 3.2 mL/min/1.73 m2 (8%)

in the absolute change in GFR of the affected kidney frombaseline to 6 months after LPN, assuming a SD of 4.25 andwith the use of a two-sidedt-test with an a level of 0.05.On the assumption of a 10% rate of loss to follow-up orother reasons leading to data loss, we established a targetsample size of 82.

For continuous variables, the Students t-test was used forthose variables reported as the mean (SD) and the Wilcoxonrank-sum test for the variables reported as the median withinterquartile range (IQR). For categorical variables, thechi-square test was used. Differences were considered to bestatistically significant atP< 0.05.

ResultsDemographic and Tumour Characteristics

Table 1 shows the baseline characteristics of the groups.There was no difference in the demographic andperioperative data between the patients in the twotreatment groups. The median WIT was 25.0 min in the

Table 1 Baseline patient demographics, tumour and operative characteristics.

Variables RIPC group Control group

Mean (SD):

Age, years 49.8 (11.7) 49.3 (13.9)

BMI, kg/m2 24.2 (2.5) 24.1 (2.8)

N (%):

Male patients 29 (70.7) 30 (73.2)

ASA score:

12 39 (95.1) 40 (97.6)

34 2 (4.9) 1 (2.4)Patients with hypertension 8 (19.5) 9 (22.0)

Patients with diabetes 5 (12.2) 6 (14.6)

Mean (SD):

Preoperative GFR of affected kidney, mL/min/1.73 m2,

(scintigraphy)

39.3 (6.5) 38.6 (4.8)

Preoperative GFR of contralateral kidney, mL/min/1.73m2,

(scintigraphy)

38.3 (6.3) 39.3 (5.3)

Tumour size, cm 3.0 (1.4) 3.1 (1.1)

N (%):

Right-side tumour 25 (61.0) 26 (63.4)

Pathological diagnosis:

RCC 35 (85.4) 33 (80.5)

Benign 6 (14.6) 8 (19.5)

Stage:

T1a 36(87.8) 34 (82.9)

T1b 5(12.2) 7 (17.1)

PADUA score:6 6 (14.6) 5 (12.2)

7 14 (34.1) 15 (36.6)

8 14 (34.1) 11 (26.8)

9 4 (9.8) 6 (14.6)

10 3 (7.3) 3 (7.3)

11 0 1 (2.4)

12 0 (0.0) 0 (0.0)

Median (IQR):

WIT, min 25.0 (20.030.0) 25.0 (22.329.8)

Operative duration, min 120.0 (95.0160.0) 132.5 (106.3180.0)

ASA, American Society of Anesthesiologists.

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both groups. The time taken from the termination of theRIPC protocol to the renal artery cross-clamping was not>30 min in all patients. There were no adverse events of theRIPC observed in the study.

Evaluation of Kidney Damage

There were no statistically significant differences betweenthe groups for baseline concentrations of urinary RBPbefore LPN (Table 2). Control patients had a highlysignificant 8.4-fold increase in urinary RBP levels over thefirst 24 h after LPN, from a median (IQR) level of 0.08(0.060.15) mg/L at baseline to 0.67 (0.560.80) mg/L at24 h. In the RIPC group, the median (IQR) urinary RBPlevels were 0.09 (0.070.11) mg/L preoperatively vs 0.35(0.200.52) mg/L at 24 h after LPN, a 3.9-fold increase.

The serum creatinine levels and eGFRs are given inTables 3 and 4. There was no statistical difference in the

values between the groups at the various time points.

When comparing the percentage change in 99mTc-DTPA

GFR between the groups, RIPC significantly reduced theGFR of the affected kidney at 1 month after LPN (Table 5).The corresponding changes in the scintigraphy of theaffected kidney treated with RIPC was significant at 1month, at a mean (SD) of-8.8 (12.1)% in the RIPC group

vs -15.0 (13.4)% in the control group (P= 0034), but notsignificant vs the control group at 6 months, at -6.1(12.2)% in the RIPC group vs-10.5 (12.4)% in the controlgroup (P= 0123). There were no significant differences inGFR changes in the contralateral kidney at 1 and 6 monthsbetween the two groups.

Table 2Assessment of renal damage by Urinary RBP.

Time point RIPC group(n =41)

Control group(n =40)

P

Median (IQR) urinary RBP level, mg/L:

Preoperative 0.09 (0.070.11) 0.08 (0.060.15) 0.636

24 h after LPN 0.35 (0.200.52) 0.67 (0.560.80)

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As far as the contribution of the operated kidney to overallrenal function (evaluated by nuclear medicine) wasconcerned, the mean (SD, range) preoperative value was50.7 (5.0; 40.159.9)% in the RIPC group and 49.6 (4.0;43.057.8)% in the control group (P= 0.275); at 1 monthafter LPN it was 46.5 (5.7; 31.358.1)% in the RIPC groupand 43.3 (5.2, 33.255.1)% in the control group (P =0.015);and at 6 months after LPN it was 48.0 (5.9, 34.259.9)% inthe RIPC group and 45.3 (4.5, 34.952.3)% in the controlgroup (P= 0.023) (Fig. 2).

All 78 patients survived the median follow-up phase of 18months which ranged from 11 to 31 months. One patient

was found with lung metastatic disease within 18 months ofLPN and received sunitinib treatment.

DiscussionSeveral methods have been developed to protect renalfunction during LPN, e.g. hilar unclamping [4], renalhypothermia [5], segmental renal artery clamping [6] andeven zero ischaemia anatomical PN [7]. The renal hilarunclamping technique is associated with significantly moreblood loss and longer surgery than that with renal hilarclamping. Cooling methods are commonly used during

open PN, while there is no consistent and reliable meansof cooling the kidney yet during LPN. Segmental renalartery clamping and anatomical renal artery branchmicrodissection techniques are promising, but limited tosome experienced surgeons and have not been widelyapplied yet. RIPC is free of material cost, easy toimplement, and harmless. In animal models, several studieshave shown that RIPC is an effective tool to protect thekidney [9,22]. In clinical trials, RIPC has shown promisingresults in randomised controlled trials as a means formyocardial protection and neuroprotection during

coronary bypass surgery [10], surgical repair of congenitalheart defects [11], percutaneous coronary interventions[12,13], and spinal surgery [14]. Several studies havereported a protective effect of RIPC on renal impairment[1517]. Ali et al. [15] reported a relative risk ofpostoperative renal impairment (serum creatinine level of

>2.0 mg/dL) of 0.25 in patients who had undergoneabdominal aortic aneurysm repair and received RIPCbefore surgery in a randomised controlled trial.Zimmerman et al. [16] reported that RIPC significantlyreduced the relative risk of acute kidney injury by 0.43 inpatients undergoing cardiopulmonary bypass-assistedcardiac surgery. In contrast, Choi et al. [18] failed to showany renal protective effect of RIPC in 76 patientsundergoing complex valvular heart surgery. Although easyto manipulate, the effect of RIPC on renal protection isdisputed in heart surgery and based on a rather short-termobservations. The present study is the first randomisedclinical trial to translate RIPC to urological surgery. Theresults showed that RIPC, mediated by transient lower limbischaemia, can reduce the urinary RBP increase and GFRdecline of the affected kidney in the short-term in patientsundergoing LPN, but failed in the longer term.

RBP is produced by the liver and has a short plasmahalf-life of only 12 h. Unbound serum RBP is freely filteredat the glomerulus and then actively reabsorbed in theproximal tubule. Therefore, an increase in the urinary RBPlevel is a specific sign of proximal tubular damage. Thepresent results show that the active transport of RBP waspartially conserved on the first postoperative day in theRIPC group. However, on the second postoperative day,urinary RBP levels were not significantly different and bothgroups showed some recovery. The rapid recovery of theproximal tubule from ischaemic injury may be the mainreason obscuring the effect of RIPC.

For renal function, serum creatinine, eGFR (evaluated usingthe MDRD equation) and total GFR (evaluated by nuclearmedicine) at 1 and 6 months after LPN were notsignificantly different between the groups. These valuesreflecting total renal function did not change significantlyand may be largely due to prompt compensation by thenormal contralateral kidney. Therefore, to evaluate whether

individual function of the operated kidney had beenprotected by RIPC, we used radionuclide scintigraphy with99mTc-DTPA. We found that the percentage decline in the99mTc-DTPA GFR of the operated kidney at 1 month afterLPN was significantly slowed by RIPC compared with thatof the control group. At 6 months after LPN, the percentagechanges in GFR of the operated kidney were notsignificantly different between the groups, this is perhapspartly due to the gradually recovery of the kidney at thelong-term observation, or to some degree because of nottaking renal volume loss into consideration [23]. However,

Fig. 2Contributional change of the affected kidney to total renal

function by differential function evaluated by 99mTc-DTPA renal

scintigraphy. Values are presented as the mean SEM.

RIPC48.0%

45.3%46.5%

43.5%

50.7%49.6%

55.0%

50.0%

45.0%

40.0%

35.0%baseline 6 months1 month

control

P =0.015 between groups# P =0.023 between groups

#

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Correspondence:Yiran Huang, Department of Urology,Renji Hospital Affiliated to Shanghai Jiao Tong UniversitySchool of Medicine, 1630 Dongfang Road, Pudong District,Shanghai 200127, China.

e-mail:yrhuangrenji@163.com

Abbreviations: DTPA, diethylene triamine pentacetic acid;IQR, interquartile range; MDRD, Modification of Diet inRenal Disease; PADUA, Preoperative Aspects andDimensions Used for an Anatomical (classification); (L)PN,laparoscopic partial nephrectomy; RBP, retinol-bindingprotein; RIPC, remote ischaemic preconditioning; WIT,warm ischaemia time.

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