1. ACTIVATION OF MITOCHONDRIAL APOPTOTIC PATHWAY IN CADAVER KIDNEY
TRANSPLANTATION LEADS TO THE SLOW GRAFT FUNCTION Olexander S. Nikonenko, MD, DSc, Andriy V. Trailin, MD, PhD, Tamara M. Nikonenko, MD, PhD Medical Academy Of Postgraduate Education, Zaporizhzhia, Ukraine
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Introduction
Results
Immediate graft function (IGF) occurred in 55 patients, SGF - in 20 patients.
Disturbance of the initial function of kidney allograft (KAG) has an adverse effect on its survival. The initial function can range from slowing the rate of decrease of serum creatinine to oligo-anuria. In most cases, the criterion for the impairment of KAG initial function is defined as the need for dialysis in the first week after transplantation, that is delayed graft function1,2.
Recently a new category has been introduced - the slow graft function (SGF), or the slow decline of serum creatinine, which also has a negative effect on subsequent function and survival of KAG2.
SGF is caused by interplay of ischemic and immunological factors. Ischemia-reperfusion injury (IRI) in cadaveric KAG is associated with necrotic and apoptotic cells death1,3. The restitution of blood supply serves to end the ischemic period and begin the immunological allograft response.
Since early rejection episodes are an independent predictor of long-term graft loss, part of the deleterious effects of SGF could be explained by undiagnosed rejection associated with SGF due to acute tubular necrosis or apoptosis. We have previously shown that the extent of the acute tubular necrosis (ATN) in postperfusion biopsies does not differ in patients with immediate graft function (IGF) and SGF4.
We, therefore, tested the hypothesis that activation of specific apoptotic pathways represents a mechanism for tubular cell death around cadaveric kidney transplantation which result in SGF .
The degree of ATN
IGF
SGF
The degree of ATN does not differ between groups.
The number of apoptotic cells was higher in SGF-group: 8 (3-10) vs. 5 (2-7), Р<0,05. % %
Percentage of positive
tubular cells
Percentage of positive
tubular cells
Objectives
In SGF-group the percentage of bax-positive tubular cells was higher than in IGF-group: 17,4% vs. 9,9% (Р<0,0001). The percentage of bcl-2-positive tubular cells was less than in IGF-group: 27,9% vs. 42,1% (Р<0,0001).
In this study, we compared postperfusion kidney biopsies from patients with IGF and SGF. We aimed to study the extent and mechanisms of apoptotic cell death during IRI in cadaver kidney transplantation and gain an insight into those factors linking reperfusion to alloimmunity and development of SGF. We also studied whether the performance of a kidney in the early posttransplant period can be predicted by postperfusion biopsies investigation.
Mitochondrial pro-apoptotic processes were more active in proximal tubules, whereas anti-apoptotic ones – were in distal tubules.
Methods
The study was conducted on the 75 recipients of cadaveric kidneys transplanted in our centre during Biopsy was taken in 30 minutes after reperfusion and sections were stained with H&E and immunoperoxidase labeling with antibodies to leukocyte markers: CD45R0 (T-lymphocytes), CD68 (macrophages), and key regulators of mitochondrial apoptosis: bax and bcl-2. Severity of tubular ischemic injury up to ATN was quantified in cortical tubules. Apoptotic cells number per HPF were counted amongst tubular cells in H&E-stained sections. The number of T-lymphocytes and macrophages in interstitium per HPF, the percentage of bcl-2- and bax-positive cells amongst tubular cells were also scored. Image analysis was used to quantitatively evaluate interstitial T-lymphocytes and macrophages infiltration with ImageJ 1.34s software. Criterion for SGF was serum creatinine level on the 3d day ≥ 600 µmol/l and/or ≥ 300 µmol/l on the 7th day post-transplant.
IGF
SGF
IGF
SGF
Cells death stimulated higher graft infiltration by macrophages in SGF-group : 13 (8-26) vs. 5 (2-12) in IGF-group, Р<0,0001, and T-lymphocytes 19 (3-36) vs. 8 (4-21) in IGF-group, Р<0,0001.
Conclusions
1. The frequency of SGF in our patients was 26,7%.
2. The percentage of apoptotic cells was significantly higher in SGF-group. The degree of ATN was not significantly different between these groups. The present findings indicate that apoptosis is the main mean of cells death under IRI.
3. In SGF-group the percentage of bax-positive tubular cells was significantly higher and the percentage of bcl-2-positive tubular cells was significantly less than in IGF-group. Our data suggest that renal tubular epithelial cells apoptosis during IRI mediated at least in part by activation of mitochondrial pathway.
4. SGF is caused by interplay of ischemic and immunological factors. We have shown higher graft infiltration by macrophages and T-lymphocytes in postperfusion biopsies of SGF-group. Early allograft rejection might be triggered by intragraft mononuclear cells infiltrate induced by IRI and the appearance of apoptotic cells. Undiagnosed rejection may represent one of the causes of SGF, and this may explain deleterious effects of slow graft function to KAG subsequent function and survival.
5. The higher apoptotic cells number may be predictive of cadaver graft immunogenicity and provoke SGF after reperfusion.
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Count: 489
Total Area: pixel2
Average Size: pixel2
Area Fraction: 2.545%
Image analysis
Apoptotic cell
CD68
References
Delayed graft function in kidney transplantation // Lancet 2004.
Delayed Graft Function: State of the Art // American Journal of Transplantation 2001.
Apoptosis and cell desquamation in repair process of ischemic tubular necrosis // Virchows Arch
Donor kidney histological changes as risk factors for delayed and slow kidney allograft (KAG) function // Transplantation. – 2008.
bax
bcl-2
CD45R0
Presented at the 15th Congress of the European Society for Organ Transplantation, 4-7 September 2011, Glasgow, Scotland