Time course of low molecular weight proteins in the early kidney transplantation period—influence of corticosteroids

Uwe Pöge1, Thomas Gerhardt1, Arend Bökenkamp2, Birgit Stoffel-Wagner3, Hans-Ulrich Klehr1, Tilman Sauerbruch1 and Rainer P. Woitas1

1 Department of Internal Medicine I, University of Bonn, Germany, 2 Department of Pediatrics, VU University Medical Center, Amsterdam, The Netherlands and 3 Department of Clinical Biochemistry, University of Bonn, Germany

Correspondence and offprint requests to: Dr Uwe Pöge, Medizinische Klinik u. Poliklinik I, -Allgemeine Innere Medizin, Universitätsklinikum Bonn, Sigmund-Freud-Straße 25, D 53105 Bonn, Germany. Email: u.poege{at}uni-bonn.de



   Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Background. Cystatin C (Cys C) is an established new marker of renal function in patients with various renal diseases and in kidney transplantation. However, few data are available for the early post-transplantation period.

Methods. Twenty-two patients who underwent renal transplantation (RTx) were evaluated for the kinetics of Cys C from day 0 to 14 in relation to creatinine and beta-2 microglobulin (B2MG). Blood samples were obtained immediately before and after transplantation and on a daily basis thereafter. Serum levels before transplantation (100%) were used to calculate reduction ratios.

Results. The decrease of the analytes differed considerably: immediately after RTx Cys C declined by 27.3% (P<0.01). However, after 3 days, on average, all patients showed a significant increase in Cys C levels (15±2.5%; P<0.01). B2MG levels fell quickly by 55.4 and 73.8% after days 1 and 7, respectively, and remained stable thereafter. In contrast, creatinine did not decease immediately after RTx but fell slowly by 67.5% at the end of the study. Prior to rejection, all analytes showed a similar behaviour. Rejection treatment with high-dose methylprednisolone induced a significant increase in Cys C (+22.8±7.9%, P<0.05), while in parallel, creatinine and B2MG decreased (–12.9±3.4 and –8.4±6.89%).

Conclusions. Corticosteroid treatment for induction of immunosuppression or rejection therapy significantly induces Cys C, but decreases B2MG. Cys C and B2MG are not helpful in establishing the diagnosis of rejection earlier. Thus, our data indicate that Cys C and B2MG testing does not accurately reflect changes in the glomerular filtration rate early after transplantation.

Keywords: beta-2 microglobulin; creatinine; cystatin C; kidney transplantation



   Introduction
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Creatinine is the most commonly used marker of excretory renal function in patients after renal transplantation (RTx). However, it has limitations due to its dependence on muscle metabolism, protein intake and physical activity [1]. When renal function changes rapidly, e.g. after ischaemic acute renal failure, especially creatinine shows a poor correlation with glomerular filtration rate (GFR) [2]. Day to day changes in body volume, rate of production and tubular leakage may contribute to this observation. Furthermore, mild to moderate reductions in GFR cannot be detected and, due to tubular secretion, it will overestimate GFR in patients with moderately to severely reduced renal function. Since correct estimation of GFR is mandatory to detect graft failure or rejection immediately in the early post-transplantation phase, alternative parameters to assess kidney function may be useful [3].

Cystatin C (Cys C), a cationic 13 kDa protein, is produced by nucleated cells at a constant production rate, freely filtered without tubular secretion or reabsorption and catabolized in the proximal tubule. Therefore, it is considered to be an excellent marker of GFR in stable renal allograft recipients even in the creatinine blind range [4–8].

Beta-2 microglobulin (B2MG) is another endogenous marker of renal function. Although B2MG serum levels may be influenced by immune reactions, recent reports indicated that it may be superior to creatinine for estimation of GFR [9,10].

So far, no data regarding Cys C in comparison to B2MG and creatinine are available for the immediate post-transplantation period in adults. We, therefore, analysed the time course of these analytes in the very early post-transplantation phase.



   Materials and methods
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 Materials and methods
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Twenty-two consecutive patients (seven female, 15 male) with end-stage renal disease who underwent RTx at our institution were included in the study. All patients were of Caucasian origin and their mean age was 52.1±2.7 years (mean±SEM).

Renal failure was related to glomerulonephritis (n = 8), autosomal dominant polycystic kidney disease (n = 5), diabetic glomerulopathy (n = 3), reflux nephropathy (n = 1) and unknown reasons (n = 4).

Two patients were on peritoneal dialysis, 19 on haemodialysis and one patient received a pre-emptive transplantation. The mean duration of renal replacement therapy had been 55.7±7.6 months in patients who received a cadaveric kidney. Four patients received an allograft from a living donor and had been on dialysis for 4.5±0.9 months. Three patients received a kidney while participating in the Eurotransplant Senior Program. Patients characteristics are presented in Table 1.


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Table 1. Demographic characteristics of the study cohort

 
The study was approved by the local Ethics Committee and informed consent was obtained from all patients enrolled into this study.

The immunosuppressive regimen consisted of the microemulsion formulation of cyclosporine A, mycophenolate mofetil and prednisone. All patients received an induction treatment with interleukin-2 receptor monoclonal antibodies (basiliximab) on the day of transplantation and 4 days later. Additionally, 500 mg of methylprednisolone was administered at the onset of transplantation.

Starting on the day of surgery, allograft function was evaluated on a daily basis for the following 2 weeks. The first blood samples after surgery were drawn immediately when the patients arrived in the intermediate care unit, i.e. an average delay of 1 h 52 min±6 min after the end of surgery. Further blood samples were always drawn at 08:00 every day for the next 14 days. Serum creatinine, Cys C and B2MG were analysed in the same blood sample.

Acute rejection was defined as a presumed or histologically proven rejection with subsequent rejection treatment. Delayed graft function (DGF) was defined as the necessity of dialysis during the first week after transplantation. All haemodialysis sessions were performed with high flux membranes. The surface area of the membranes ranged between 1.4 and 1.8 m2.

Serum creatinine was determined on the DimensionTM clinical chemistry system (Dade Behring, Marburg, Germany) with a commercially available assay based on a modification of the Jaffé method according to the manufacturer's instructions. Serum levels of Cys C and B2MG were analysed by fully automated, latex-enhanced immunonephelometric methods (N Latex cystatin C and N Latex beta-2 microglobulin, respectively, on a Behring Nephelometer II (Dade-Behring).

Ninety-five per cent reference intervals established from a representative cohort of 100 female and 100 male healthy blood donors (median age, 31 years; 2.5–97.5 percentiles, 19.0–60.5 years) were 0.475–0.820 mg/l for Cys C, 53–106 µmol/l for creatinine and 1.085–2.015 mg/l for B2MG. The intra assay coefficient of variation (CV) was 1% for creatinine (mean 61 µmol/l; n = 20), and the inter assay CV was 2.5% (79.6 µmol/l; n = 20). The intra assay CV for Cys C was 1.9% (mean 0.6 mg/l; n = 20), the inter assay CV was 3.6% (mean 0.6 mg/l; n = 20). The intra assay CV of B2MG was 2.5% (mean 1.5 mg/l; n = 20), and the inter assay CV was 2.6% (1.4 mg/l; n = 20) [10].

Statistics
Values are given as mean±SEM. Statistical analysis was performed using the t-test or the Wilcoxon signed-rank test where appropriate (StatView® version 5.0 for Windows; SAS Institute Inc., Cary, NC). P-values <0.05 were considered significant.



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Ischaemia time was 14.2±4.6 h (n = 18) for the cadaveric RTx and 1.0±0.3 h (n = 4) for living-donor RTx. The donor age was 47.3±2.6 years (range 19–68) for the total cohort. Living donors tended to be older than the donors of the cadaveric group (53.5±2.4 vs 45.0±3.1 years; P = 0.2). Mean creatinine, Cys C and B2MG levels before transplantation were 763±39.8 µmol/l, 6.5±0.36 mg/l and 28.7±2.5 mg/l, respectively.

Time course of the analytes after transplantation
The reduction differed considerably between the analytes (Figures 1a–c and Table 2): immediately after RTx, creatinine showed no reduction (–0.15%) but decreased slowly by 18.0 (day 1), 56.7 (day 7) and 67.5% (day 14). In contrast, B2MG and Cys C declined immediately after RTx by 28.4 (P<0.01 vs creatinine) and 27.3% (P<0.01 vs creatinine), respectively. On day 1, the reduction of B2MG and Cys C was more than twice as high than for creatinine (55.4 and 46.4%; P<0.01 vs creatinine for each). A maximal decline by 73.8 and 79.4% was seen for B2MG on days 7 and 14, respectively. Interestingly, even patients with DGF showed a remarkable decrease of B2MG levels prior to the onset of haemodialysis treatment (Table 2).





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Fig. 1. Time course of the analytes after RTx. Data are given as mean±95% confidence interval separately for DGF (n = 7) and PGF (n = 15). (a) Creatinine (µmol/l), (b) Cys C (mg/l), (c) B2MG (mg/l).

 

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Table 2. Reduction ratios (percentage) of the analytes

 
After the initial decline in Cys C levels, all patients showed an increase in Cys C concentrations between days 1 and 8, irrespective of the primary graft function (PGF). In the majority of patients, Cys C increased from days 1 to 2 (n = 13, 15.9±3.5%) and during days 2 to 4 (n = 5, 16.7±6.0%), whereas in another four patients, an increase took place between days 5 and 8 (11.7±3.8%). The maximum overall increase was 15.3±2.5% (P<0.01).

Delayed graft function
After transplantation DGF was diagnosed in seven patients. Immediately after transplantation, Cys C levels in patients with primary renal function showed a more pronounced decrease than in patients with DGF (29.3±1.7 vs 22.3±1.4%, P = 0.029). In comparison, neither reduction in creatinine nor reduction in B2MG serum concentrations differed significantly between patients with PGF and DGF (creatinine: 1.7±2.5 vs –3.1 ±4.9%, NS; B2MG: 28.0±2.4 vs 29.4±2.6%, NS).

Time course of the analytes prior to rejection
Throughout the study period, 10 rejection episodes in 10 different patients took place and were treated. All analytes showed a similar behaviour in the days prior to rejection treatment. Cys C serum levels paralleled the time course of creatinine in six out of eight patients who did not require dialysis. In one case, Cys C increased the day before creatinine rose and, in one further case, Cys C remained stable. The time course of B2MG was similar.

Rejection therapy
The 10 rejection therapies were performed via daily administration of 500 mg of methylprednisolone intravenously for 3 days. On average, rejection treatment was initiated 8.7±0.7 days after transplantation.

Rejection therapy resulted in a significant decrease (12.9±3.4%) of creatinine in all eight patients who were not dialysed. B2MG levels were not significantly altered by the treatment and decreased slightly (–8.4±6.89%; 5.0±1.2 vs 4.3±0.81 mg/l, NS). In contrast, seven out of eight patients who did not require dialysis showed an increase in Cys C levels (22.8±7.9%; P<0.05) (Figure 2).



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Fig. 2. Changes of creatinine, Cys C and B2MG during rejection treatment of eight patients who were not on dialysis. Data are presented as relative concentrations based on the serum concentrations 3 days prior to rejection treatment. Arrows indicate rejection treatment with high-dose methylprednisolone (500 mg each).

 
Although, high flux dialysis reduced Cys C concentrations, the two patients on dialysis also showed an increase in Cys C levels (7.2±3.3%). Following the steroid bolus, the highest Cys C levels occurred on day 2 (2.1±0.3 days) of the treatment. To exemplify the time course of the analytes more clearly a representative example of patient no. 12 is shown in Figure 3.



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Fig. 3. Time course of serum creatinine, Cys C and B2MG in patient no. 12 during the study period. Renal biopsy, which was obtained during revision surgery due to urine leak of the distal ureter, showed acute rejection. Creatinine concentration is given in mg/dl to achieve an improved clarity of the graph (use multiplication factor 88.4 for conversion to µmol/l). Arrows indicate rejection treatment with high-dose methylprednisolone.

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study illustrates three important differences in the time course of creatinine, Cys C and B2MG in the first 2 weeks after transplantation: first, Cys C decreased more rapidly than the other markers of renal function; secondly, on average, a marked increase of Cys C concentration occurred on the third day after transplantation; thirdly, high-dose methylprednisolone application for rejection therapy led to a significantly increased Cys C serum concentration.

The slope of the initial Cys C decline was more pronounced in patients with PGF than in patients with DGF and comprised ~30% of the initial concentration. In contrast, in both groups, creatinine did not decrease immediately after transplantation. Several hypotheses may explain these findings: in post-ischaemic acute renal failure, a situation comparable to transplantation, a considerable proportion of filtered creatinine leaks back across damaged tubular epithelium and enters the interstitium. Furthermore, Myers et al. [11] reported a relative tubular impermeability for intraluminal macromolecules of a size >3 nm in acute renal failure. Thus, we may speculate that Cys C and B2MG (3–4 and 2.6 nm in diameter, respectively), in contrast to creatinine (0.3 nm diameter), cannot cross the tubular epithelium and return into blood circulation. This could explain the delayed reduction of creatinine in comparison to the rapid fall of Cys C and B2MG levels during the first days after transplantation.

However, also in patients with DGF a 20% decrease in Cys C levels on average was noted immediately after transplantation. Two considerations based on the data of Tenstad et al. [12] and Herget-Rosenthal et al. [13] may explain our finding: a study on 125I-labelled Cys C in rats showed a very rapid Cys C breakdown and found 70% of the radioactive Cys C degradation products within 20 min in the blood circulation [12]. This suggests that the initial decline of Cys C may be explained with the primary reperfusion of the kidney graft and concomitant rapid metabolism by (still intact) tubular cells. On the other hand, urinary Cys C excretion of the intact molecule was found to be increased in non-anuric patients with acute tubular necrosis [13]. Similarly, this may also occur after transplantation as the majority of our patients (five out of seven) categorized into the DGF group was non-anuric with a diuresis ranging from 300 to 1350 ml per day.

Previously, in vitro studies showed a dose-dependent increase of up to 80% of Cys C secretion by HeLa cells following dexamethasone exposure [14]. This results from a steroid-dependent induction of promoter activity and transcription of the Cys C gene. Consequently, we previously observed an increase of Cys C starting on day 3 after kidney transplantation in paediatric patients [15]. Our current results confirm this finding in adults. Thus, in our opinion the marked re-increase of Cys C concentration was due to our immunosuppression protocol which included 500 mg of methylprednisolone prior to transplantation. Therefore, despite clinical improvement of the graft function, Cys C increased 3 days after transplantation on average. However, further parameters that could affect Cys C concentrations and that have to be taken into account are differences in the rate of accumulation and intra-individual variations of Cys C [3].

As a component of the major histocompatibility antigen (MHC) class I, B2MG is produced by all nucleated cells. Bacterial and viral infections increase the B2MG levels due to stimulated MHC expression [16]. As corticosteroids exert antilymphoproliferative effects, this may lead to the observed decline in the B2MG levels after corticosteroid comprising immunosuppression [17,18]. According to this observation, a notable reduction of B2MG was seen even in patients who fulfilled the criteria of DGF later in the time course.

Cys C has been proposed to detect rejection episodes earlier than creatinine [3]. However, as in transplanted children [15], we did not find any significant differences between Cys C and creatinine in adults in the days prior to when a rejection was anticipated.

On the contrary, our data indicate a steroid-dependent Cys C increase after high-dose methylprednisolone therapy which occurred earlier than previously published. Thus, the steroid-induced Cys C increase did occur already after 1 day of therapy and peaked after 2 days. Simultaneously, both the serum creatinine and the B2MG serum concentrations decreased.

This observation may be explained by the effects of glucocorticoids on B2MG expression as mentioned above, as well as by improved renal function. Thus, we also may speculate that the observed decrease of Cys C 1 day after the last steroid pulse is due to better renal elimination. A similar steroid-dependent increase of Cys C has also been described in 13 patients with deteriorating renal function who received a 3 day course of high-dose methylprednisolone years after transplantation [6]. However, a slight, albeit insignificant, increase in creatinine was also observed. Therefore, from that study it remains unclear whether the increase in Cys C reflected a more accurate detection of impaired renal function or a steroid-dependent effect.

Controversial data have been published with respect to the effect of continuous low-dose steroid therapies on Cys C. In paediatric patients suffering from nephrotic syndrome, continuous steroid treatment did not affect Cys C levels [19]. In contrast, adults on low-dose steroids tend to have somewhat higher Cys C levels, albeit not significantly. However, Cys C reciprocals in the low-dose steroid group were significantly lower than in the steroid-free groups [6]. Likewise, higher Cys C levels were found in asthmatics who were on low-dose steroid regimens compared to steroid-independent asthmatics [20]. These discrepancies may result from differences in the patient cohorts and kidney function.

The described Cys C elevations and an accurate diagnosis of rejection may be confounded by a high within-subject variation of Cys C. This phenomenon has been described in several small studies mainly with healthy individuals [21,22]. In contrast, in an investigation on 29 diabetics and 11 healthy persons, Tan et al. [23] described a higher biological variation for creatinine than for Cys C. Hence, a recent survey concluded that for serial monitoring of renal function, Cys C may be at least as reliable as creatinine [24].

In conclusion, each of the analytes, creatinine, Cys C and B2MG, displays some advantages as well as some drawbacks in monitoring renal function precisely. However, due to modulation of Cys C and B2MG concentrations by immunosuppressive therapy both analytes are of limited value for assessing renal function in the early post-transplant period.



   Acknowledgments
 
We thank Ms C. Blasius and Ms A. Carstensen for excellent technical assistance in performing the creatinine, Cys C and B2MG assays.

Conflict of interest statement. None declared.



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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 31.12.03
Accepted in revised form: 6. 5.04