ASSESSMENT OF RENAL GRAFT FUNCTION DEPENDING ON PRE-TRANSPLANT MORPHOLOGY

a Department of Intensive Care in Surgery Branches, Palacký University, University Hospital, Olomouc, Czech Republic b 2 Department of Surgery, Palacký University, University Hospital, Olomouc c Department of Pathology, Palacký University, Olomouc d Department of Obstetrics and Gynecology, Palacký University, University Hospital, Olomouc e 1 Department of Surgery, Palacký University, University Hospital, Olomouc f Department of Preventive Medicine, Palacký University, Olomouc e-mail: lubuskal@fnol.cz


INTRODUCTION
Over the past decades, transplant medicine as a multi-disciplinary branch has undergone many significant changes.Satisfactory results are being achieved nowadays with both short-and long-term success.90% of renal gafts 1 survive one year after kidney transplantation.Mortality of patients with kidneys form ideal donors has dropped from 56 cases per 1000 patients in 1998 to 48 deaths per 1000 patients in 2002 2 .However, the scarcity of kidneys suitable for transplantation persists.This situation is particularly apparent abroad due to the higher incidence of patients with chronic renal failure waiting for transplantation and lack of donors.In quest to increase the number of available organs, so called expanded criteria for cadaveric donors are being put into practice enabling transplantation of kidneys that would have never been considered before.Detailed information on donated organ can be obtained from pre-transplant biopsy.This can reveal even those kidney disorders that remained latent during the donor's life.The aim is to assess the kidney condition in relation to the donor's age, associated diseases and to evaluate damage related to kidney procurement.Both acute and chronic changes are being monitored.The performance of pre-transplant biopsy also enabled us to recognize changes that occur in the graft only after transplantation, e.g.chronic nephropathy, cyclosporine nephrotoxicity, etc. 3,4 .The outcome of transplantation can be influenced by other risk factors as well.There are always numerous immune and non-immune effects that may impact on the graft even after successfully performed transplantation.These factors can subsequently lead to graft damage and impair its function and survival [4][5][6][7][8] .Among risk factors on the donor's side belongs kidney from a non-heartbeating donor, inotropic support of the donor, use of cold storage preservation instead of pulsatil perfusion, extended cold ischemia time, age over 55, diabetes mellitus or hypertensive disease in anamnesis.Unfavourable effects on the graft function may be caused by hypovolaemia on recipient's side, by perioperative administration of albumin, haemodialysis with ultrafiltration performed 24 h prior transplantation, the number of previous transplantations, patient's weight, thrombophilia condition, OKT3 therapy (orthoclon), presence of antiphospholipid antibodies, performed cytotoxic antibodies, acute tubular necrosis (ATN), cyclosporine nephrotoxicity, urinal fistula or obstruction 8 .
Pre-transplant biopsy enables histopathological assessment of some morphological changes that have significant prognostic effects on the function and survival of the graft 9 .An array of factors has been examined for prognostic purposes, particularly conditions affecting glomeruli (glomerulosclerosis), interstitium (fibrosis), arteries, arterioles (intimal fibrosis and hyalinization) and tubules (vacuolar dystrophy, brush border, desquamation of epithelium, tubular dilatation, presence of haemoglobin cylinders).
In our study we evaluated the above specified morphological parameters and degree of their histopathological changes from the viewpoint of their influence on the graft function in post-transplant period.

Objectives of the work -Prognostic factor of the graft function
• Examination of histopathological changes of glomeruli, arteries, arterioles, interstitium and tubules of transplanted cadaveric kidneys and subsequent evaluation of impact of these morphological changes on immediate and long-term function of the graft in posttransplant period.• Determination of relation between degree of morphological histopathological changes of the graft and the donor's age, presence of hypertension on donor's side and nontraumatic cerebrovascular accident as the cause of donor's death.

MATERIALS AND METHODS
During 1994-1997 a total of 117 cadaveric grafts were examined and transplanted.The age of donors ranged from 6 to 77 years (average -37.6; median -39.0).All transplantations were performed at the Transplant Centre in Olomouc Teaching Hospital and the grafts were histopathologically examined at the Institute of Pathological Anatomy of Palacký University Faculty of Medicine in Olomouc.The graft function was monitored for a period of five years after transplantation.Immediate graft function was evaluated in the first week after transplantation as a primary function, delayed function and afunction.Long-term function was evaluated after 1, 2, 3, 4 and 5

Tubular dilatation (TD) 0 -absent 1 -present
Haemoglobin cylinders in distal tubules (HC) 0 -absent 1 -present years, and over all those periods the number of functional and afunctional grafts was monitored.The serum creatinine level determined by Jaffe reaction without proteinace assessed the quality aspect of the graft function within all monitored periods.
Biopsy sample in a form of wedge excision was obtained at the time of organ procurement from the pole of cadaveric graft after in situ kidney perfusion and its Assessment of renal graft function depending on pre-transplant morphology The statistic result showed significantly higher number of grafts with delayed function at the end of week 1 after transplantation with histopathological finding of epithe-lial desquamation (P 0.02; OR 5.42; 95 % CI 0.91-41.18),presence of haemoglobin cylinders (P < 0.001; OR 4.78; 95 % CI 1.92-12.02)and loss of brush border (P 0.006; OR 3.08, 95 % CI 1. 26-7.67).Differences between the number of functional and afunctional grafts with these morphological changes had no statistical significance.No statistically significant difference in the number of grafts with primary function, delayed function or afunction were found at the end of week 1 with histopathological lesions of glomeruli, interstitium, arteries, arterioles, presence of tubular dilatation and vacuolar dystrophy.
When evaluating the number of afunctional grafts in 1, 2, 3, 4 and 5 years after transplantation, we were able to prove a statistically significant difference only with histopathological finding of a brush border loss 1 year after transplantation (P 0.02; OR 0.12; 95 % CI 0.01-1.05)and presence of vacuolar dystrophy after 2 years (P 0.03; OR 0.25; 95 % CI 0.05-1.12)and after 3 years (P 0.03; OR 0.26; 95 % CI 0.06-1.17).Both morphological changes did however prove a protective effect on the graft function.The serum creatinine levels rose with the degree of lesion and lapse of time after the transplantation.At the end of year 5, the grafts with higher degree of histopathological damage demonstrated the highest levels of serum creatinine.The only exemption of levels was found with mild degree of vacuolar dystrophy in the 1 st , 3 rd , and 5 th year and with epithelial desquamation in the 1 st year.The study

DISCUSSION
Performance of pre-transplant biopsy followed by histopathological evaluation of morphological finding in kidney provides more comprehensive information on transplanted organ.The professional literature published many studies focussing on the influence of detected degree of lesion in individual morphological parameters on immediate and long-term kidney function after transplantation.The majority of these studies analysed the impact of lesion in glomeruli, arteries or interstitial lesions, some also reflect tubular lesions.In our work, we observed all four prime morphological units.
The percentage of defective glomeruli in the case of glomerulosclerosis (GS) in our patient group had no influence on immediate or long-term function of transplanted kidney.We noticed no negative impact on graft function in patients with glomerulosclerosis ≥ 20 %.Other works describe the same results 5,10 .Some studies on the contrary quote impaired graft function with this degree of glomerulosclerosis 11,12 .Gaber et al. 11 states that delayed graft function occurred in 87 % with glomeruli affection ≥ 20 %.Escofet et al. 12 demonstrates the five-year survival in 35 %.In our group of patients, we recorded delayed function only in 17 % with the same degree of glomeruli affection and five-year graft survival in 57 %.In harmony with the literature we confirmed the correlation between glomerulosclerosis degree and the hypertension of the donor and nontraumatic cerebrovascular accident as cause of death 10,11 .However we did not confirm any relation between donor's age and degree of glomeruli impairment also described in the literature 5,10,11,[13][14][15] .
The degree of arterial and arteriolar lesions in our group of patients had no significant influence on immediate or long-term graft function.The significance of arterial lesions is disputed and questioned by other studies as well 10,14,16,17 .Nevertheless, other authors showed the influence of arterial changes on the post-transplant kidney function 4,16,[18][19][20][21][22][23][24] .In our group only mild arterial lesions were noticed, no moderate or severe lesions.As for arterioles, we evaluated mild and moderate changes, no severe changes were recorded.In harmony with the literature we confirmed the correlation between the degree of arterial and arteriolar lesions and donor's age, hypertension and nontraumatic cerebrovascular accident as cause of death 4,14,[21][22][23][25][26][27] .
The degree of graft lesion caused by interstitial fibrosis did not influence the immediate or long-term function of the graft.There was no case demonstrating severe changes.Nonetheless, there are cases in the literature which report the influence of this parameter on graft function 14,16,28,29 .Other authors, in contrast, validate our results 16,17,24,30 .In the case of interstitial fibrosis we found a link between degree of lesions and donor's age, hypertension and nontraumatic cerebrovascular accident as the cause of death.The literature describes only the correlation between the donor's age and the degree of interstitial fibrosis 14,21,27,31,32 .
In the case of tubules we evaluated the degree of vacuolar dystrophy, presence of epithelial desquamation, loss of brush border, presence of tubular dilatation and presence of haemoglobin cylinders in distal tubules.A negative impact on immediate function of the graft was recorded for epithelial desquamation, presence of haemoglobin cylinders and loss of brush border.The cases described in the literature deal only with the epithelial desquamation and vacuolization.A negative impact of these morphological changes on delayed graft function has been proved 3,5,16,17,30,33 .Influence on the long-term function was observed only in the case of vacuolar dystrophy and loss of brush border, but both morphological changes, on the contrary, showed protective effects on graft function.We found nothing in the literature describing similar findings.Pokorná et al. 5 analyses the negative impact of vacuolization and desquamation presence on immediate and late level of graft function.

CONCLUSION
It is possible to transplant kidneys and attain both short-and long-term satisfactory results even with a mild degree of arterial lesion, moderate degree of arteriolar lesion, moderate degree of interstitial fibrosis, glumerulosclersis greater or equal to 20 % and tubular cells lesions.The donor's age, hypertension and nontraumatic cerebrovascular accident as the cause of death demonstrate a direct correlation with the degree of arterial and arteriolar lesions and with degree of interstitial fibrosis.The same outcomes have also been confirmed with glomerulosclerosis, except for the influence of donor's age.It is advisable to perform biopsy of the kidney on donors over 40 years of age with hypertension and nontraumatic cerobrovascular accident as the cause of death.However, the pretransplant biopsy is just one of many factors contributing to the outcome of transplantation.

Table 1 .
Classification of histopathological evaluation of biopsy samples.

Table 3b .
Graft function 1 week after transplantation.removalfrom the donor's body.Each sample was fixed by 10% formalin, and then processed and embedded in paraffin.Histological slices of 3-4 micrometers were stained with haematoxylin-eosin.The brush border of proximal tubules was evaluated in PAS (Periodic Acid Schiff) staining.Trichrome staining according to van Gieson was applied to evaluate fibrosis, and basal membranes of glomeruli and tubules were visualized by staining according to Jones.All samples were evaluated by means of light microscopy in harmony with the classification stated in Tab.1.Statistical analysis was performed by software Statistica Version 6 and Epi Info Version 6.All values with p < 0.01 were considered statistically significant.RESULTSProportional representation of individual types of morphological changes according to classification inTable No. 1 is stated in percentage in Table No. 2. The connection between histopathological findings and graft function in a time-dependent manner from the date of transplantation are shown in detail in Tables No. 3-9.

Table 9 .
Odds ratio -afunction / function.between degree of histopathological changes in arteries, arterioles, interstitial fibrosis and the donor's age, hypertensive disease and nontraumatic cerebrovascular accident as cause of death.Same outcomes were confirmed with glomerulosclerosis, except for the influence of donor's age.