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Abstract

Introduction:

This study explores the effect of different corticosteroid administration timings on the incidence of slow/delayed graft function.

Methods:

One hundred and twelve kidney transplants from January 2011 to March 2014 were retrospectively analysed. Thirty-six cases were excluded because they were donor-specific antibody positive (n=16), received thymoglobulin/plasma exchange (n=11), were ABO-incompatible (n=6) or suffered graft loss from vascular thrombosis within the first week post-transplant (n=3). The study period straddled three eras of corticosteroid administration, from intra-operative intravenous (IV) hydrocortisone (Era 1; n=26), to intra-operative IV methylprednisolone (Era 2; n=38) and pre-operative IV methylprednisolone (Era 3; n=12). The primary endpoint was the incidence of slow/delayed graft function. Secondary outcomes included estimated glomerular filtration rate at discharge and 120 and 365 days, rejection (acute and one-year), wound complications, post-transplant diabetes, increase in low-density lipoprotein or body mass index, and cytomegalovirus or BK viraemia within one year.

Results:

On univariate analysis, pre-operative methylprednisolone was associated with lower incidence of slow/delayed graft function (17%, 55%, 58% in Eras 3, 2, 1 respectively; p=0.041), superior estimated glomerular filtration rate at discharge (median 56, 37 and 43 ml/min for Eras 3, 2, 1 respectively; p=0.033) and at 120 days (median 60, 52, and 46 ml/min for Eras 3, 2, 1 respectively; p=0.017). On multivariate analysis, pre-operative IV methylprednisolone (vs. Eras 1 and 2 combined; odds ratio 4.79 (90% confidence interval 1.16–19.80); p=0.07) and living donor type (vs. deceased; odds ratio 5.56 (90% confidence interval 2.25–13.77); p=0.002) were associated with lower incidence of slow/delayed graft function.

Conclusion:

Pre-operative methylprednisolone was associated with reduced slow/delayed graft function and improved early estimated glomerular filtration.

Keywords

Renal transplantation induction of immunosuppression corticosteroids timing delayed graft function eGFR

Introduction

Corticosteroids are a mainstay in renal transplantation, exerting an immunosuppressive effect largely by binding nuclear receptors to alter the balance of transcribed pro- and anti-inflammatory factors.¹ As this takes time, steroids have a clinical latency of hours before attaining maximum immunosuppressive effect. It has been the practice that steroids are administered intra-operatively to induce immunosuppression in renal transplantation. However, this may not provide sufficient time for steroids to modulate gene expression and exert their immunosuppressive effect before the antigenic stimulus of graft implantation occurs.

There is a paucity of literature on this topic. A single randomized controlled trial (RCT) involving 111 transplant recipients was conducted in 1996. This found a lower incidence of acute rejection in patients receiving pre-operative methylprednisolone, as compared with patients receiving intra-operative methylprednisolone (22% vs. 47%; p < 0.05), but no significant difference in estimated glomerular filtration rate (eGFR) at discharge or one-year graft survival.² However, the incidence of delayed graft function was not reported. This is a dated study with high acute rejection rates and newer immunosuppressant regimens have since reduced the rate of acute rejection to 10% in most transplant centres.^3,4 There is no further study exploring the best timing of corticosteroid induction. Studies on other immunosuppressants have found no significant difference between pre- and intra-operative cyclosporin/azathioprine (2004), but superior outcomes were observed with intra-operative thymoglobulin over post-operative administration (2003).^5,6

The Singapore General Hospital (SGH) renal transplant protocol underwent three eras of corticosteroid administration. Longstanding use of intra-operative intravenous (IV) hydrocortisone 500mg (Era 1) ceased in December 2011. From January 2012 to September 2013, intra-operative IV methylprednisolone 500mg (Era 2) was given. This was subsequently changed to pre-operative IV methylprednisolone 500mg (Era 3) from October 2013. This study explores the effect of different timings of corticosteroid administration on the primary end-point of delayed graft function (DGF), defined as serum creatinine > 250µmol/l at one week post-transplant, or the need for dialysis in the first week post-transplant.

Methods

A retrospective analysis of all patients receiving renal transplantation in SGH from January 2011 to March 2014 was performed with Institutional Review Board approval. Certain high-risk groups were excluded, including patients with donor-specific antibodies, or who received ABO-incompatible transplants, thymoglobulin or plasma exchange. Patients who suffer graft loss within one week from vascular thrombosis were also excluded. All cases were followed up for one year from the date of transplant.

Intra-operative steroid (Eras 1 and 2) was administered at induction of anaesthesia, while pre-operative steroid (Era 3) was scheduled for administration 2 h prior to operation time. No patient in our cohort received pre-operative oral corticosteroids but all living kidney donor transplant recipients would have received 2–3 days of calcineurin inhibitors before the scheduled transplant operation, which was a practice across all eras. There was otherwise no change to transplant protocols across the three eras.

The primary endpoint was the incidence of DGF. Secondary outcomes included eGFR at discharge as determined using the Modification of Diet in Renal Disease (MDRD) equation, eGFR at 120 and 365 days, acute rejection (within 30 days post-transplant), one-year rejection, wound complications, post-transplant diabetes, increase in low-density lipoprotein (LDL) cholesterol or body mass index (BMI), and cytomegalovirus (CMV) or BK viraemia within one year.

Statistical analysis was performed using SPSS version 22 (SPSS, Chicago, Illinois, USA). Data was expressed as median (interquartile range) or mean ± standard deviation as appropriate. Proportions were expressed as percentages. Chi-squared tests were used for categorical variables and the Kruskal–Wallis test for continuous variables. Multivariate analysis using binominal logistic regression was also performed. A p value of less than 0.05 was considered to be statistically significant.

Results

One hundred and twelve renal transplants were performed in SGH from January 2011 to March 2014. Thirty-six cases were excluded because they were donor-specific antibody positive (n=16), received thymoglobulin or plasma exchange (n=11), were ABO-incompatible (n=6) or suffered graft loss from vascular thrombosis within the first week of transplantation (n=3). Therefore 76 patients were included, of which 26 were from Era 1, 38 from Era 2 and 12 from Era 3.

The three steroid eras had no statistically significant difference in demographics, immunological risk profile and immunosuppressive regimen at induction (Table 1), except for higher panel reactive antibody levels in Era 3 (median 5%, 0% and 0% in Eras 3, 2 and 1 respectively; p=0.036) and fewer male recipients in Era 3 (25%, 42% and 65% male in Eras 3, 2 and 1; p=0.045). In addition there was a trend towards more living donors in Era 3 (67%, 32% and 35% in Eras 3, 2 and 1 respectively; p = 0.08) and fewer expanded criteria donors in Era 3 (17%, 26%, and 50% in Eras 3, 2 and 1 respectively; p = 0.09). One patient from each steroid era suffered acute rejection within 30 days post-transplant, giving an overall rate of acute rejection of 4%. Four patients from Era 1 (15.4%), five from Era 2 (13.2%) and one from Era 3 (8.4%) suffered rejection within one year post-transplant. One-year patient and graft survival were all 100%.

Table 1.

Baseline patient characteristics.

	Era 1	Era 2	Era 3	p value
	Intra-operative hydrocort.	Intra- operative methylpred.	Pre- operative methylpred.
n	26	38	12
Recipient age, years ± SD	47.1 ± 2.0	47.0 ± 1.5	46.7 ± 3.1	0.94
Recipient male gender (%)	18 (69)	22 (58)	8 (67)	0.62
HLA mismatch, alleles (interquartile range)	3 (2–3)	3 (2–5)	3 (1.5–4.5)	0.72
Positive crossmatch (%)	0 (0)	3 (8)	2 (17)	0.14
Recipient PRAB, % (interquartile range)	0 (0–3.5)	0 (0–17)	5 (0–59)	0.04
Donor type, living (%)	9 (35)	12 (32)	8 (67)	0.08
Donor type, expanded criteria deceased donor (%)	13 (50)	10 (26)	2 (17)	0.09
Donor gender, male (%)	17 (65)	16 (42)	3 (25)	0.04
Cold ischaemia time, h (interquartile range)	6 (1–12)	8 (1–13)	2 (1–13)	0.40
Second warm ischaemia time, min (interquartile range)	35 (32–44)	35 (29–40)	32 (29–34)	0.19
Immunosuppression at day 0 (%)
Cyclosporin + CellCept + prednisolone	11 (42)	25 (66)	8 (67)
Tacrolimus + CellCept + Prednisolone	11 (42)	11 (29)	3 (25)	0.34
Other combinations	4 (15)	2 (5)	1 (8)
Basiliximab induction at day 0 (%)	25 (96)	35 (92)	12 (100)	0.52

HLA: human leukocyte antigen; PRAB: panel reactive antibody; hydrocort.: hydrocortisone; methylpred.: methylprednisolone; SD: standard deviation.

Analysis of the outcomes across three eras of perioperative steroid administration (Table 2) found that pre-operative methylprednisolone (Era 3) was associated with a reduced incidence of DGF (17%, 55% and 58% in Eras 3, 2 and 1 respectively; p=0.041), superior eGFR at discharge (median 56, 37 and 43 ml/min for Eras 3, 2 and 1 respectively; p=0.033) and at 120 days (median 60, 52 and 46 ml/min for Eras 3, 2 and 1 respectively; p=0.017). There was a trend towards reduced one-year rejection in Era 3, but this did not reach statistical significance. There was no significant disparity between the three eras in wound complications, post-transplant diabetes, increase in LDL cholesterol or BMI, and CMV or BK viraemia within one year.

Table 2.

Clinical outcomes across three eras of steroid administration.

	Era 1	Era 2	Era 3	p value
	Intra- operative hydrocort.	Intra- operative methylpred.	Pre- operative methylpred.
Rejection within 30 days (%)	1 (3.8)	1 (2.6)	1 (8.3)	0.68
Rejection within one year (%)	4 (15.4)	5 (13.2)	1 (8.3)	0.84
eGFR at discharge, ml/min per 1.73m² (IQ)	43 (31–55)	37 (25–55)	56 (45–77)	0.03
eGFR at 120 days, ml/min per 1.73m² (IQ)	46 (37–54)	52 (40–66)	60 (53–76)	0.02
eGFR at 1 year, ml/min per 1.73m² (IQ)	50 (39–59)	51 (38–63)	61 (48–74)	0.14
Delayed graft function (%)	15 (58)	21 (55)	2 (17)	0.04
Wound complications (%)	4 (15)	5 (13)	0 (0)	0.37
CMV viraemia (%)	14 (54)	15 (40)	4 (33)	0.39
BK viraemia (%)	7 (27)	12 (32)	5 (42)	0.66
Post-transplant diabetes (%)	5 (19)	2 (5)	1 (8)	0.19
Post-transplant LDL increase^a, mmol/l (IQ)	0.95 (−0.7, 1.5)	0.74 (−0.5, 1.4)	0.35 (−1.1, 0.8)	0.31
Post-transplant BMI increase^a, mmol/l (IQ)	0.2 (−1.2, 1.5)	0.3 (−0.7, 1.8)	0.6 (−0.9, 2.5)	0.68

Calculated as the difference between one-year post-transplant LDL or BMI versus pre-transplant LDL or BMI respectively.

BMI: body mass index; CMV: cytomegalovirus; eGFR: estimated glomerular filtration rate; hydrocort.: hydrocortisone; IQ: interquartile range; LDL: low-density lipoprotein; methylpred.: methylprednisolone.

Analysis of the factors affecting incidence of DGF (Table 3) found that steroid era (as above), donor type (living 24% DGF, deceased 66% DGF; p=<0.001), second warm ischaemia time (<35min: 32% DGF, ⩾35min: 75% DGF; p=0.002), cold ischaemia time (<8h: 38% DGF, ⩾8h: 64% DGF; p=0.022) and recipient race (Chinese 36% DGF, Malay 74% DGF, Indian 75% DGF; p=0.020) significantly affected the incidence of DGF.

Table 3.

Incidence of DGF according to recipient and donor characteristics.

	Incidence of DGF			p value
Recipient age, years	<50: 55%	⩾ 50: 44%		0.353
Recipient race	Chinese: 36%	Malay: 74%	Indian: 75%	0.020
Donor type	Living: 24%	Dead: 66%		<0.001
Donor gender	Male: 61%	Female: 40%		0.066
Cold ischaemia time	<8h: 38%	⩾8h: 64%		0.022
Second warm ischaemia time	<35min: 32%	⩾35min: 75%		0.002
Steroid era	Era 1: 58%	Era 2: 55%	Era 3: 17%	0.041
Day 0 immunosuppression	CsA+MMF: 48%	Tac+MMF: 60%	Others: 29%	0.305

DGF: delayed graft function; CsA: cyclosporin; MMF: mycophenolate; Tac: tacrolimus.

Reduction in DGF was important as patients without DGF, versus those with DGF, had lower one-year rejection rates (5% vs. 21%; p=0.001) and superior eGFR at discharge (median 52 vs. 34 ml/min; p=0.001), 120 days (median 58 vs. 46 ml/min; p=0.015) and one year (median 57 vs. 47 ml/min; p=0.007).

Discussion

Delayed graft function can be thought of as acute kidney injury manifesting in the transplant kidney; this may lead to chronic allograft nephropathy and inferior long-term graft survival.^7–11 Indeed, in our cohort, immediate graft function and higher eGFR at discharge is associated with reduced one-year rejection and improved eGFR at later time points.

The pathophysiology of DGF is multifactorial, including injury from the recipient’s immune response, as well as ischaemic and reperfusion injury to the graft.¹² Given these mechanisms, the role of inductive immunosuppression in minimizing DGF and improving transplant outcomes is clear. While this is well accepted, no recent literature explores the optimal timing of corticosteroid administration.

We demonstrate that pre-operative methylprednisolone was associated with reduced incidence of DGF and improved early eGFR, despite higher panel reactive antibody (PRAB) levels in the pre-operative steroid group, which one would expect to negatively affect outcomes.^13,14 Therefore pre-operative methylprednisolone may be a potential strategy to decrease the incidence of DGF. This finding concurs with that of the 1996 RCT in spite of two decades of progress in immunosuppression in renal transplantation.³ While DGF is associated with increased acute rejection,¹² the incidence of acute rejection in our cohort was only 4%, such that this study was underpowered to detect a statistically significant decrease in one-year rejection with pre-operative methylprednisolone, despite a trend to this effect.

We postulate several mechanisms by which a change in steroid protocols may have led to these outcomes. Going from Era 1 to Era 2, there was a change in glucocorticoid used (hydrocortisone to methylprednisolone) but no change in timing of administration (intra-operative in both eras). The effect of this change may relate to both pharmacodynamics and pharmacokinetics. In terms of pharmacodynamics, methylprednisolone has five times the anti-inflammatory potency of hydrocortisone due to its increased affinity for glucocorticoid receptors,¹ therefore a switch from 500mg hydrocortisone to the same dose of methylprednisolone would have resulted in a relative increase in anti-inflammatory effect. In terms of pharmacokinetics, alterations in the chemical structure of the steroid backbone (e.g. unsaturation of the ∆1-2 bond and methylation) give synthetic glucocorticoids like methylprednisolone longer half-lives than naturally-occurring hydrocortisone, which may also contribute to the former’s superior immunosuppression.^1,15

Notably, the use of a more potent steroid did not significantly increase steroid side effects such as post-transplant diabetes, or the complications of increased immunosuppression such as CMV or BK viraemia. This is no surprise given that these complications are associated with chronic use of high-dose steroids, and the impact of a single glucocorticoid dose in the peri-transplant period is probably negligible. In fact, a lower incidence of DGF may provide the opportunity to taper immunosuppression earlier and more confidently.

Going from Era 2 to Era 3, there was no change in glucocorticoid administered (methylprednisolone), but a substantive change in timing of glucocorticoid administration (intra-operative to pre-operative). The molecular effects of glucocorticoids shed light on how a change in timing of administration may result in reduced DGF and superior eGFR. Glucocorticoids act via altering DNA transcription, as well as by non-genomic mechanisms. The alteration in DNA transcription occurs via various pathways, including binding to glucocorticoid response elements to induce transcription of anti-inflammatory molecules (e.g. p11, which alters arachidonic acid release), and interacting with transcriptional activators like NF-κB to repress transcription of the pro-inflammatory genes they regulate.^16,17 Non-genomic mechanisms include various post-transcriptional and translational targets, such as binding membrane receptors to alter cellular signalling.

In this the relative importance of genomic versus non-genomic mechanisms is contested. If the mechanism of steroid action is predominantly genomic, then there will be a significant time lag from steroid administration to the onset of immunosuppression, in the range of 2–4 h;^18,19 on the other hand, if the steroid action is predominantly non-genomic, there is the potential for rapid onset of immunosuppression, which has been shown to occur within minutes in vitro.^20,21 The evidence for non-genomic rapid effects, at present, comes mainly from in-vitro studies, but may yet be further characterized by RNA expression analysis and newer technologies.²² On the other hand, the evidence for genomic mechanisms is well established. For instance, the knowledge that glucocorticoids trigger T-cell apoptosis – an active process requiring protein synthesis – argues strongly for the importance of genomic mechanisms, even if these may be complemented by novel non-genomic mechanisms.¹⁸ Our finding, that earlier administration of glucocorticoids before graft implantation is associated with superior outcomes, further points to the significance of genomic mechanisms.

This study has several limitations. Sample size was small, especially the number of Era 3 patients. There was a trend towards more living donors and fewer expanded criteria donors in Era 3; although this did not reach statistical significance, it remains a potential confounder. While there was no change to official transplant protocols, a retrospective observational study design cannot exclude potential differences in transplant practices between the three steroid eras. Further studies may explore the exact optimal time interval between steroid administration and graft implantation, as well as the effect of timing of steroid administration on long-term graft survival.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Declaration of Conflicting Interest

The authors declare that there is no conflict of interest.

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