Kidney transplantation is recognized as treatment of choice for most patients with end stage kidney disease. However, despite substantial progress during last few decades in field of transplantology, long term graft outcome is still unsatisfactory (1). Kidney allograft outcome is influenced by the quality of transplanted organ as well as multiple donor and recipient interactions which are non-immune and immune in their nature. Seron et al. (2) and Isoniemi et al. (3) have postulated that histological changes in posttransplantation protocol biopsies can be useful in predicting long term graft outcome. Other studies (4, 5) suggested that particular morphological changes detectable at the very early stages after transplantation or even in preimplantation biopsies, before chronic changes superimpose could even be more useful to assess organ quality and predict long term graft outcome. Donor biopsies usually do not reveal acute inflammatory lesions and the range of chronic lesions is even narrower than in protocol ones (6). Therefore some authors have proposed to evaluate donor biopsies with a very detailed semiquantitative scale in order to differentiate groups in this range of lesions (7, 8), while others have proposed to employ a morphometric analyses (9, 10). Although more laborious and time consuming than conventional histologic evaluation, morphometric methods reduces the subjectivity and variability in interpretation.

Results of some quantitative histopathological studies underscore the role of nephron number and glomerular size/volume as well as its variability in the pathogenesis of arterial hypertension and kidney failure (11, 12). It has been found that patients with essential hypertension are characterized by lower nephron number and higher glomerular volume (GV) in comparison to normotensive patients (13). Counting of total glomerular (nephron) number in the kidney is very laborious and time consuming method not useful in every day clinical practice. However, results of autopsy studies (13, 14) revealed an inverse relationships between kidney mass and glomerular volume as well as between glomerular volume and glomerular (nephron) number, so the GV could serve as surrogate of total glomerular number in clinical studies (14, 15). Calculation of glomerular number from biopsy material is not possible. However, it is thought that in these specimens estimation of glomerular density is a marker of glomerular number which determine the adaptive capacity of grafted kidney to its new conditions after transplantation. An inverse correlation between GV and total glomerular number noted in autopsied kidneys of people without evidence of renal disease (13, 14) and between GV and glomerular density (GD) in IgA Nephropathy (IgAN) (16), Idiopathic Membranous Nephropathy (IMN) (17) and Minimal Change Disease Nephrotic Syndrome (MCDNS) (18) patients represent structural adaptation of glomeruli and their decreased functional reserve, susceptibility for subsequent renal injury and faster loss of function as well as worse response to therapy. Fogo et al. (19) documented in children with MCDNS, that the presence of glomerular hypertrophy in a biopsy is an indicator of an increased risk of progression to focal segmental glomerulosclerosis (FSGS). Koike et al. (18) showed that MCDNS adult patients with low GD (< 3.4/mm²) had not only larger GV but also characterized by higher degrees of chronic histopathological changes, such as global glomerulosclerosis and interstitial fibrosis indices in comparison to high GD group (> 3.4/mm²). Alperovich et al. (20) has been found that transplanted kidneys with higher GV characterized worse long term outcome in comparison to kidneys with lower GV. Therefore it was found, that variations in glomerular volume may influence the clinical course of various forms of primary glomerulonephritis as well as outcome of transplanted kidneys. Recent studies have underscore the importance of GD (the number of non-sclerotic glomeruli per renal cortical area of biopsy specimen) on kidney outcome in patients with different forms of primary glomerulonephritis. An individual value of GD in a diagnostic biopsy (≥ 10 glomeruli) showed approximately a 7-fold variation, even in patients with relatively well preserved kidney function (eGFR > 60 ml/min). Patients with IMN, IgAN and MCDNS and low GD characterized more rapid progression in comparison to patients with high (16-18). These results identified GD as an important histological predictor of kidney diseases progression. Summarizing the presented data, the value of pre-implantation kidney biopsy seems to be unquestionable. However, there is still no full agreement, whether histological changes available at transplantation provide only information concerning donor-derived organ injury or which lesions in pre-implantation biopsies allow the best prediction on outcome, and whether it is necessary to perform pre-implantation biopsies in both kidneys of the same donor (paired kidneys), or it will be representative to biopsy only one kidney.

Relationship between duration of cold ischemia time (CIT) and the incidence of DGF has been well established (21-24). However, there is still no clear consensus concerning the influence of CIT-induced DGF on long-term graft function and outcome (21, 22, 25-27). Recent studies of Kayler et al. (22) and summary of data by Terasaki (28) on paired kidneys have shown, that if no longer than 15h, CIT has no influence on long- term graft outcome between longer- and shorter-CIT groups. They concluded that the CIT--caused DGF was only the result of ischemic acute tubular injury (ATI), which as fully reversible state has no impact on long term graft outcome. In their study Louvar et al. (29) have been found a significant correlation within paired kidneys for the occurrence of DGF and kidney allograft failure. They noted, that within pairs of recipients from a common donor, when DGF occurred in one recipient, the adjusted odds for DGF in the recipient of the contralateral kidney was > 200% higher, suggesting that unmeasured donor genetic and biochemical factors, so called “nature” contribute more than CIT to a recipient risk for DGF and late graft outcome. On the contrary, results of the Collaborative Transplant Study (30) and analysis of US Renal Data System (31) have found greater risk of graft failure in recipients with CITs > 18h and continuous worsening of graft outcome associated with each 6h CIT increase, respectively. Notably, most of these studies did not included into the analysis kidneys weight and pre-transplant histology of the paired kidneys. Therefore, it is suspected, that CIT induced injury superimposed on already existing chronic changes are responsible for observed relationship between CIT and long term graft outcome and may be partially explain the differences in published results (22, 28). Taking into account the above mentioned facts, the integrated analysis which include not only pre-procurement kidney function but also its mass as well as histologic changes and morphometric characteristics in pre- (4, 5) and posttransplantat biopsies (2, 3) is useful to predict graft outcome.

The aim of this study was: 1. to perform a preimplantation histomorphometric analysis of glomeruli obtained by needle biopsies of paired kidneys harvested from adult deceased donors, and 2. to assess the relationships between kidney weight, glomerular volume (GV) and glomerular density (GD) as well as 3. 1- and 3-years outcome of paired kidneys transplanted into different recipients in context histomorphometric studies, influence of CIT and donor-recipient HLA mismatch.

To minimize most transplant related differences influencing graft function and arising from local practices, only paired kidneys without any gross abnormalities (congenital or acquired), harvested from adult deceased donors and transplanted in a single center between January 1, 2005 and December 31, 2007 were included into the analysis. After procurement, all the kidneys were stored in “Viaspam” solution until implantation, without machine perfusion. In 28 deceased donors with adequate biopsy material (according to Banff criteria – at least 7 glomeruli and 1 artery section), weight of both kidneys were obtained. Kidney biopsies were performed on the back table after surgical preparation using semiautomatic needle “PRECISA” 16 G x 200 mm (Italy). For measurement of kidney weight an electronic scale was used. Kidney weight measurements were performed with precision of 1 g.

Kidney biopsies were fixed in 4% buffered formalin, processed and embedded in paraffin, then cut on 2-3 μm thick sections, and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), Masson’s trichrome, periodic acid silver methenamine and Congo red. Immunohistochemistry for IgA, IgG and IgM was performed in each case. Only cases containing at least 7 glomeruli and 1 arterial section (measurement of six glomeruli provides a reliable estimate of glomerular size by the MPA method (32)) as well as corticomedullary junction and negative or unspecific immunostaining underwent subsequent histomorphometric analyses using “OLYMPUS BX51” microscope connected with “OLYMPUS CX50” camera (Olympus, Japan) and “cellSense Standard” softwere (Olympus, Japan). Total number of complete (being encircled by non-interrupted Bowman capsule except vascular and tubular poles) glomeruli, as well as numbers of normal (Norm), globally sclerosed (GS), segmentally sclerosed (SS), hyperperfused (Hyp) and ischemic (Ish) glomeruli were counted in the section containing the highest number of glomeruli. Glomeruli with a completely patent capillary network, relatively uniform capillary diameters, no excess of mesangium (no more than three nuclei per mesangial stalk), and no areas of sclerosis, adhesions to Bowman’s capsule or podocyte alterations were defined as normal. Hypertrophic glomeruli were characterized by combinations of two or more of the following histologic features: a) evident increase in size in comparison to most surrounding glomeruli, b) a dilated hilar capillary region in comparison to most surrounding glomeruli, substantially wider than the lumen of it feeding afferent arteriole, usually with evident dilatation of the primary branches, c) widely patent, often distended capillary loops, in the absence of increase in mesangium or any focal adhesions, sclerosis or podocyte alterations, d) marked disparity in capillary loops. Mesangial increase and sclerosis with narrowing of glomerular capillaries and tuft adhesions to Bowman’s capsule defined glomeruli of FSGS type. As ischemic were classified glomeruli which show progressive shrinkage of the tuft, compared with most surrounding glomeruli, with wrinkled capillary walls surrounding narrowed but still patent capillary loops in most areas. Globally sclerotic glomeruli characterized with no patent capillary loops (33). Results were expressed as the percentage of complete glomeruli.

Morphometric analysis of complete, nonsclerosed glomeruli was performed on PAS, Masson’s trichrome and periodic acid silver methenamine stained sections at 200 x magnification. Glomerular diameters and maximal profile areas were measured. Glomerulus in the present study was defined as an area inside a Bowman’s capsule containing tuft. Maximal glomerular volumes (maxGV) were estimated from the maximal glomerular profile area according to the formula: GV (μm³) = 4/3 p r³ where:

GV – glomerular volume (assuming that its 3-daimentional shape is a sphere),

r = GPA μm²/p where:

r – glomerular radius (calculated from the glomerular area, assuming that its shape is a circle),

GPA – glomerular profile area.

Glomerular density (GD) was calculated in the section containing the highest number of complete glomeruli and defined as the number of non-globally sclerotic glomeruli per mm² of renal cortical area of needle biopsy.