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Acute Effects of Calcineurin Inhibitors on Kidney Allograft Microperfusion Visualized by Contrast-Enhanced Sonography

Kihm, Lars P.; Blume, Carsten; Seckinger, Jörg; Hankel, Vinzent; Stoffler, Dominik; Morath, Christian; Zeier, Martin; Schwenger, Vedat

doi: 10.1097/TP.0b013e31824f3dae
Clinical and Translational Research
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Background Calcineurin inhibitors induce detrimental vascular remodeling, which may be one cause of chronic allograft failure. Real-time contrast-enhanced sonography (CES) is a relatively new technique in providing quantitative information on microvascular tissue perfusion in kidney allografts in more detail. The purpose of the study was to explore whether acute changes of kidney allograft microperfusion due to the administration of cyclosporine A (CsA) and tacrolimus (Tac) can be evidenced using real-time CES.

Methods In an explorative single-center clinical trial, renal parenchymal tissue perfusion of 32 stable kidney allograft recipients was evaluated with CES before and 2 hr after the intake of CsA or Tac. In addition to laboratory and clinical parameters, Doppler indices and estimated glomerular filtration rate were measured.

Results Although systolic and diastolic blood pressure and color Doppler indices did not significantly differ, there was a significant decrease of renal blood flow 2 hr after the intake of CsA compared with baseline (4.78±2.31 dB/s, 49%, respectively). In contrast, kidney allograft microperfusion was neither significantly reduced in patients receiving CsA paralleled by calcium channel blockers nor significantly reduced in patients receiving Tac. Furthermore, there was a significant correlation between renal blood flow obtained before drug administration and kidney function.

Conclusions CES revealed a 49% reduction of kidney allograft microperfusion 2 hr after the intake of CsA, which might be abrogated by calcium channel blockers. In comparison to CsA, Tac did not result in a significant decrease of kidney blood flow.

Department of Nephrology, University Hospital, Heidelberg, Germany.

The authors declare no funding or conflicts of interest.

Address correspondence to: Lars P. Kihm, M.D., Department of Nephrology, University Hospital, Im Neuenheimer Feld 162, 69120 Heidelberg, Germany.

E-mail: lars.kihm@med.uni-heidelberg.de

L.P.K., C.M., M.Z., and V.S. participated in the research design, performance of the research, data analysis, and writing of the manuscript; and C.B., J.S., V.H., and D.S. participated in the research design and performance of the research.

Received 7 June 2011. Revision requested 30 June 2011.

Accepted 7 February 2012.

Since the introduction of cyclosporine A (CsA) in the 1980s, the incidence of acute rejection episodes has decreased impressively (1). However, overall long-term graft survival has remained constant over the past decades, indicating a shift in the causes of mortality from acute allograft rejection toward the consequences of long-term immunosuppression (2). Despite the evidence of calcineurin inhibitor (CNI) toxicity, which is involved in chronic allograft failure (3–5), CNIs remain a fundamental part of immunosuppressive protocols after kidney transplantation (6). In addition to chronic vascular remodeling, acute changes of renal blood flow caused by CsA intake have been described (7, 8). Tacrolimus (Tac) is suspected to induce less pronounced renal vasoconstriction compared with CsA (9, 10). At present, routine kidney allograft sonography is performed by measuring resistance and pulsatility indices in the larger intrarenal arteries only (11–13). In contrast to color Doppler sonography, contrast-enhanced sonography (CES) allows microvascular kidney perfusion imaging, with the use of gas-filled microbubbles, and is able to detect early stages of chronic renal allograft nephropathy (14). Using CES, we previously demonstrated a superior microvascular allograft perfusion 1 year after a switch from CsA to everolimus (15). The purpose of this study was to quantify acute changes of kidney allograft microperfusion caused by the administration of the CNI CsA and Tac using real-time CES.

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RESULTS

Patients’ Characteristics

Characteristics of the 32 patients are given in Table 1. The median age was 49.3 years, and 34.4% of the patients were women. Systolic and diastolic blood pressure did not significantly differ before and after the administration of CsA or Tac. Heart rate and color Doppler indices, resistance index (RI), and pulsatility index (PI) were not significantly different before and after CsA or Tac intake (Tables 2 and 3). CsA and Tac levels significantly increased after CsA administration (Tables 2 and 3, Fig. 1).

TABLE 1

TABLE 1

TABLE 2

TABLE 2

TABLE 3

TABLE 3

FIGURE 1

FIGURE 1

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Renal Blood Flow at Baseline and Kidney Function

There was a significant correlation between renal blood flow obtained before CNI administration and kidney function (estimated glomerular filtration rate [eGFR]: r=0.60, P<0.001 and serum creatinine: r=−0.54, P<0.01; Fig. 2). No correlation could be determined between kidney function and color Doppler indices, transplant age, or recipient age.

FIGURE 2

FIGURE 2

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Change of Renal Blood Flow After Drug Administration

There was a significant decrease of renal blood flow 2 hr after the intake of CsA (Table 2, Figs. 1A and 3A). Kidney allograft microperfusion was reduced by 4.78±2.31 dB/s, 49%, respectively (Fig. 3A). No significant decrease of allograft microperfusion could be obtained after the intake of Tac (reduction of microperfusion by 0.79±1.64 dB/s, 5.9%, respectively; Table 3, Figs. 1B and 3A). The detailed analysis of patients receiving CsA revealed a reduced impairment of kidney allograft perfusion in patients receiving calcium channel blockers (CCB) (CsA+CCB: −2.53±4.56 dB/s vs. CsA−CCB: 7.00±5.12 dB/s, 23% vs. 74%, respectively, P<0.01; Fig. 3B). There was no difference in the decrease of renal blood flow after CsA intake with respect to kidney allograft function (CsA+eGFR >30 mL/min: 54% vs. CsA+eGFR <30 mL/min: 39%, P=n.s.) or angiotensin-converting enzyme inhibitor (ACEI) medication (CsA+ACEI: 55% vs. CsA−ACEI: 45%, P=n.s.; Fig. 3C and D). The decrease of renal blood flow did not correlate with the absolute (r=0.13, P=n.s.) or relative (r=0.09, P=n.s.) increase of the CsA concentration.

FIGURE 3

FIGURE 3

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DISCUSSION

The main finding of this study was that acute effects of CsA on the microperfusion of the kidney allograft could be visualized by CES. Quantifying to what extent microperfusion was impaired by CsA, we observed a marked reduction of renal blood flow of 49% 2 hr after the administration of CsA. In contrast, the intake of Tac did not result in a significant reduction of kidney allograft perfusion. Different influences of CsA and Tac on kidney allograft perfusion have been evaluated earlier (8, 9, 16). Whether acute changes of microperfusion might reflect prognostic impact can only be speculated on.

The decrease of renal blood flow and eGFR related to CsA administration was evaluated earlier, using different invasive methods in healthy volunteers (17) and kidney transplant recipients (7). A decrease of renal blood flow assessed by sonography was also shown by Nankivell et al. (8) using a quantitative Doppler ultrasound technique. However, this method relies on the quantification of the vascularization assessed by Doppler sonography, which is limited by the approximation of the mean velocity and the differentiation of both high-flow and low-flow pixels (18). Therefore, a diagnostic tool to obtain renal blood flow noninvasively in real time is desirable.

CES is an easy-to-perform and noninvasive cost-effective technique, which can be performed at bedside within minutes, providing information about kidney microperfusion in real time. The reference standard in the assessment of tissue perfusion (19) is achieved by continuous infusion of the microbubble agent, followed by high mechanical index (MI) destructive pulse, as performed here. The microbubbles consist of a central sulfur hexafluoride core with a surrounding phospholipid monolayer, remaining in the systemic circulation for approximately 11 min before spontaneous degradation with absorption of the gaseous component by the lungs and the phospholipid shell by the liver. Thus, new-generation ultrasound contrast media are well tolerated and allow repeated measurements without major concerns.

The visualization of an impaired renal microperfusion due to CsA administration with CES here is in line with previous data demonstrating a decrease in renal plasma flow in experimental animals (20), organ transplant recipients (21, 22), and healthy controls (23). Several factors are involved in direct CsA-induced vasoconstriction (24). Accompanying an intrinsic vasoconstrictive activity of CsA, an increased endothelin production (25), and an activation of the renin-angiotensin system have been shown. In addition, stimulating effects of CsA on sympathetic nerve activity play a role in the acute effects of CsA by increased renal vascular resistance and secondarily decreased GFR (26, 27). Furthermore, CsA decreases endothelial nitric oxide synthase-mediated NO production through various mechanisms (28, 29), leading to decreased vasodilatation (30). One has to keep in mind that such a drastic reduction of kidney allograft perfusion occurs twice daily in patients on an immunosuppressive regimen containing CsA. In contrast, the administration of Tac was accompanied by a minor reduction of kidney allograft perfusion compared with CsA. This finding is in line with others pointing out different effects on the microvasculature of the CNI CsA and Tac (8, 16). The mechanisms of this difference are not fully understood; however, different endothelial release of endothelin may be involved (31). This might at least in part explain the positive effect of switching patients with chronic allograft dysfunction from CsA to Tac (32).

It is noteworthy that the reduction of kidney allograft perfusion due to CsA intake was independent of kidney function and ACEI medication, which might reflect data demonstrating no differences in kidney allograft function with respect to the administration of ACEI (33). While the parallel administration of CCB almost abrogated the decrease of perfusion, which has been proposed earlier (8, 34) and might lead to better graft survival in the long term (35, 36).

In addition to the ability to visualize the decrease of renal blood flow due to CsA intake with an easy-to-perform and safe ultrasound technique, the study promises an additional value of CES in the care of kidney transplant recipients. We previously demonstrated a better preservation of kidney allograft microperfusion obtained with CES in patients 1 year after they were switched from CsA to the mammalian target of rapamycin inhibitor everolimus (15). Taking into consideration the described acute CsA-induced effects on vasoconstriction, it would be of great clinical interest as to whether this might result in long-term consequences, as demonstrated with our previously published data regarding the ability of CES to predict chronic allograft nephropathy (14). In this study, kidney allograft microperfusion before drug administration correlates with kidney allograft function. This is in line with data recently published from Kay et al. (37), demonstrating a correlation between microbubble perfusion early after transplantation and kidney function at 3 months after transplantation.

Although promising, investigators still hesitate to implement CES in the clinical routine setting, mostly because of a lack of knowledge and experience. CES has been successfully evaluated in the diagnosis of acute pyelonephritis in native kidneys (38) and kidney allografts (39), expanding the indication for its use. Hitherto, CES can be a valuable additional tool in the early and long-term care of transplant recipients if used in a professional and scientific manner.

CES allows the visualization of deleterious acute effects of CsA on kidney allograft microperfusion. A reduction of kidney allograft perfusion due to CsA might be abrogated by CCB and is not obtainable in patients receiving Tac. Whether the degree of the acute reduction of allograft microperfusion predicts the susceptibility to CsA has to be evaluated in further studies.

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MATERIALS AND METHODS

Patients

This study was an explorative single-center clinical trial. Men and women aged 18 to 65 years with a stable primary kidney allograft and on an immunosuppressive drug regimen consisting of CsA or Tac, mycophenolic acid, and low-dose steroids were eligible for the study. The local ethical committee approved the study protocol, and all patients gave written informed consent. Exclusion criteria were defined as renal arterial stenosis or arteriovenous fistula of the allograft, screened by color-coded Doppler sonography, the presence of synthetic vascular grafts, severe liver disease, congestive heart failure, a lymphocele more than 150 mL, proteinuria more than 1 g/day, leukocytes less than 2.5/nL, or hemoglobin less than 8 g/dL. Patients were evaluated at baseline, followed by drug intake, and 2 hr thereafter. We chose the time point of 2 hr after drug ingestion, because it has been demonstrated that the 2 hr concentration of CsA correlates closely with the area under the curve (40) and is recommended in a consensus statement of the CONCERT group (41).

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Assessment of Resistance and Pulsatility Indices by Color Doppler Ultrasonography

Sonography was performed by an ATL HDI 5000 (Philips Medical Systems, Bothell, WA), using a 2.5-MHz convex array transducer. Intrarenal Doppler signals were obtained from three representative arcuate and interlobar arteries. The peak systolic velocity (Vmax), the minimal diastolic velocity (Vmin), and the mean velocity (Vmean) were determined; the renal segment arterial RI was calculated as 100×(1−[Vmin/Vmax]) and the renal segment PI was calculated as 100×([Vmax−Vmin]/Vmean). The median RI and PI were assessed using six different measurements, performed by a single investigator.

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Real-Time CES

Baseline investigation of the allograft was performed in B-mode. After identification of the optimal long-axis view of the allograft, the transducer was kept in a stable position, whereas the imaging mode was changed to low MI contrast-specific imaging according to the international guidelines for the use of contrast agents in ultrasound (19, 42). After adjustment of MI settings and reduction of B-mode gain, major allograft structures were barely visible. Five milliliters of SonoVue, a second-generation contrast agent, was applied by a perfusor (VueJect, syringe pump for ultrasound contrast agent; Bracco Research SA, Geneva, Switzerland) over a time period of 90 sec, for constant microvascular perfusion. Gain settings were optimized for each study; imaging was performed with low MI (MI=0.1) in color-coded harmonic power pulse inversion mode, with a low frame rate (7–10 images/sec), color gain 70%, and dynamic range with a maximum of 170 dB. Initial visualization of the contrast agent occurred after 15 to 25 sec. When optimal renal contrast amplification (visualization) was achieved (after 35–45 sec), a 300-msec pulse, with a high MI (MI=1.0) (burst imaging), was transmitted. Thereafter, the imaging was reset to low power, and replenishment was visualized for 10 to 15 sec. For quantitative analysis of renal tissue perfusion, a region of interest was placed in the renal cortex. Mean volume of region of interest was 380 mm2 (range 310–460 mm2). Interlobar and arcuate arteries were carefully excluded from regions of interest. Contrast intensity was measured after flash and spline curves of contrast intensity versus time were calculated according to an exponential function y=A×(1 − e−βt), as described previously (14, 15). The plateau of signal intensity (A) and the slope of maximal signal intensity rise (β) were measured, and the product of A×β was calculated in the renal cortex to estimate the blood flow. Images and perfusion were analyzed quantitatively, using a commercially available software tool (Q-Lab, Release 4.1, Philips, Bothell, WA).

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Laboratory Measurements

CsA concentration was analyzed by means of the Dimension Xpand enzyme immunoassay (Dade Behring, Deerfield, IL). Tac concentration was analyzed by means of the EMIT Dimension EXL (Siemens, Munich, Germany). The GFR was estimated according to the modification of diet in renal disease formula (43). All other laboratory measurements were carried out using standard methods in a certified laboratory.

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Statistical Analysis

For all statistical analysis, Prism Version 5 (GraphPad Software, La Jolla, CA) was used. Demographic and clinical variables are presented as mean±standard deviation. Correlations were analyzed using the Pearson correlation coefficient. For investigation of changes within the group, one-tailed Wilcoxon matched-pairs signed-rank test was used. For investigation of changes between groups, the Mann-Whitney U test was used. Differences were considered significant at P less than 0.05.

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Keywords:

Cyclosporine; Kidney transplantation; Contrast sonography

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