Living donor kidney transplants: a biopsy study 1 year after transplantation, compared with baseline changes and correlation to kidney function at 1 and 3 years

Ståle Sund1, Anna V. Reisæter2, Per Fauchald2, Øystein Bentdal3, Kirsten Sundby Hall4 and Torstein Hovig1

1 Institute of Pathology, 2 Department of Medicine, 3 Department of Surgery and 4 Department of Clinical Chemistry, The National Hospital, Rikshospitalet, University of Oslo, Oslo, Norway

Correspondence and offprint requests to: Dr S. Sund, Institute of Pathology, The National Hospital, Rikshospitalet, N-0027 Oslo, Norway.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Introduction. Chronic changes in biopsies from long-term stable kidney allografts have been reported to correlate with graft prognosis. Morphological changes in baseline (`zero-hour') biopsies have been described as well, but their importance for long-term prognosis have been less clear. The aim of the present study was to evaluate biopsy changes from baseline to 1 year after transplantation in patients receiving kidneys from living donors, and to assess the possible prognostic implications of these findings.

Methods. Light microscopical changes in 18 gauge full-core biopsies were scored semi-quantitatively in 33 patients 1 year after transplantation, and compared to baseline changes previously reported [1]. All cases were also examined with transmission electron microscopy. The semi-quantitative data from baseline and at 1 year were correlated with kidney function 1 and 3 years after transplantation. The reproducibility of baseline findings regarding arteriosclerosis and arteriolar hyalinosis was tested by comparison with biopsies 1 week after transplantation (n=43).

Results. We found a significant increase in mesangial glomerular sclerosis (P<0.001), interstitial fibrosis/tubular atrophy (if/ta) (P=0.002), and mononuclear cell interstitial infiltration (P=0.003) after 1 year, compared to baseline changes. There was an increase of arteriosclerosis (P=0.028) and arteriolar hyalinosis (P=0.006) when compared to biopsies taken 1 week after transplantation, but not when compared to the `zero-hour' findings. Electron microscopy revealed one case of recurrent immune-complex glomerulonephritis and another case of recurrent light chain deposition kidney disease. Comparing 1-week vascular findings with baseline gave a low level of reproducibility, probably due to sampling error. Baseline biopsy findings could not predict long-term kidney function. In the 1-year biopsy, if/ta was significantly correlated with serum creatinine (P=0.007) and glomerular filtration rate (GFR) (P<0.001) at 1 year, with serum creatinine at 3 years (P=0.011), and with the first-year cumulative dose of methylprednisolone (P=0.004). Serum creatinine at 1 year, however, was found to be the most accurate predictor of 3-year kidney function (P<0.001). Donor age was correlated to kidney function at 3 years (P=0.013) but not at 1 year after transplantation.

Conclusion. Morphological changes in baseline biopsies of living donor kidneys tend to become more pronounced in well-functioning allografts during the first year after transplantation. In the 1 year biopsy, if/ta seems to be the most reliable variate for grading of chronic changes. However, 1-year serum creatinine predicted long-term kidney function more precisely than did the biopsy scores. Based on the results of the present study, a protocol 1-year biopsy does not seem warranted in the management of the graft recipient with a stable kidney function.

Keywords: kidney transplant biopsy; baseline; prognosis; transplant outcome; reproducibility



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
In a previous study, we described the morphological changes in kidney baseline (`zero-hour') biopsies from living donors [1], and found that significant alterations, especially arteriosclerosis and arteriolar hyalinosis, were prevalent even in normally functioning kidneys. The possible impact of such baseline changes on kidney graft function have been reported in several studies, but the results have been somewhat conflicting [27].

Morphological changes in arterial vessels are part of the chronic transplant nephropathy, together with interstitial fibrosis, tubular atrophy and glomerulopathy [8,9]. Similar changes in kidney transplants with a stable function have been shown to predict long-term renal function [10].

The impact of morphological changes in cadaveric donor kidneys may be difficult to evaluate because of the confounding effects of donor factors, such as pre-existing disease, and primary renal dysfunction related to pre-transplantation shock-induced kidney damage and prolonged `cold' ischaemia [11,12]. We therefore wanted to study changes in grafts from living donors, in whom a pre-operative screening has confirmed the absence of kidney disease or hypertension, and in whom the pre-operative regimen as well as the surgical procedure are optimally standardized. Furthermore, our material offered the possibility to compare the developing changes with those of the baseline biopsies.

In the current study, our main purpose was to answer the following questions: (i) what is the evolvement of the histopathological changes in follow-up full core biopsies up to 1-year after transplantation; in particular, what happens to the arterial and arteriolar changes? (ii) Do the graft biopsy findings predict graft outcome? (iii) What is the effect of sampling error in studying these questions? (iv) Should a protocol 1-year biopsy be recommended in the management of the kidney graft recipient?



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Patients
Thirty-three patients who were transplanted with a kidney from a living donor from March 1992 to January 1994 at our centre were included in this study. Patients were included consecutively and exclusion criteria were as follows: recipient aged less than 18 years, HLA-A, -B and -DR identity between donor and recipient, anticoagulation treatment with warfarin or known bleeding diathesis, and intra-abdominal placement of the kidney graft.

A baseline biopsy was obtained from 57 patients [1], and 33 of these were available for a biopsy 1 year after transplantation. Twenty-four patients withdrew from the longitudinal study for the following reasons: 14 patients declined further protocol biopsies, three patients were withdrawn for medical reasons, six patients lost their grafts within the first 3 months because of rejection, and one patient died after 3 months from a cerebrovascular insult. From 1 to 3 years after transplantation, three patients lost their grafts because of chronic rejection and two patients died with functioning grafts (one with pneumonia and one with diabetes mellitus suffering sudden death).

There were 20 males and 13 females among the 33 kidney graft recipients and the mean age was 41.9 years (range 20.4–74.4). Thirty-two patients were transplanted for the first time and one patient received a second graft. Among the donors there were 14 males and 19 females, and the mean age was 51.3 years (range 20.7–71.5). The donors were screened pre-operatively according to standard procedure, and were confirmed to have normal kidney function, normal urine and normal peripheral blood pressure [1].

The recipients suffered from chronic glomerulonephritis (n=14), focal segmental glomerulosclerosis (n=3), chronic pyelonephritis (n=4), diabetic nephropathy (n=3), plasma cell dyscrasia (n=3), autosomal dominant polycystic kidney disease (n=2), nephrosclerosis (n=2), interstitial nephritis (n=1) and nephronophtisis (n=1).

At 1 year, approximately 50% of the patients were treated with one or two antihypertensive drugs, while one patient received antihypertensive treatment with three drugs. Nearly one third of the patients were also treated with furosemide. The follow-up time was 3 years.

The project was approved by the regional ethical committee and all patients gave their written consent to participate in the study.

HLA typing, antibody screening and cross-match tests
Prospective serological HLA typing of recipients and donors was performed twice on peripheral blood mononuclear cells, isolated by the immunomagnetic method [13]. Patient sera were screened for lymphocytotoxic panel reactive antibodies (PRA) by the immunomagnetic method [13], each time using T lymphocytes from 15 random blood donors. Patients whose sera reacted against one or more of the panel cell donors on more than one occasion during the last year, in the absence of autoreactive antibodies, were considered PRA positive. Immunomagnetic lymphocytotoxic B and T cell cross-matches [13] were negative in all cases. One patient had PRA against 5–20% of the panel of T lymphocytes and was treated with prednisolone and cyclophosphamide for 4 weeks prior to transplantation.

Thirty donors were first degree relatives and three were spouses. Twenty-eight recipient–donor pairs were one haplo-type mismatched and two pairs were two haplo-types mismatched. Between the spouses the number of HLA-A and -B mismatches were three and of HLA-DR mismatches zero, one and two, respectively.

Immunosuppressive treatment
The post-transplant immunosuppression consisted of a standard triple therapy regimen: cyclosporin A, started at 10 mg/kg/day tapered to 2–5 mg/kg/day, aiming at a trough value of 75–175 µg/l at 3 months and 75–125 µg/l 1 year after transplantation; prednisolone 80 mg/day tapered to 10 mg/day after 8 weeks, and azathioprine 2.0 mg/kg/day tapered to 1.0 mg/kg/day after 7 days.

Patients with an increase of serum creatinine of 20% or more, when other causes of renal dysfunction were excluded, were treated for acute rejection. Pulses of methylprednisolone were given for 4 days, to a total of 1.25 g. Steroid resistant rejections were verified by biopsy and treated with antithymocyte globuline (ATG Fresenius, Bad Homburg, Germany) a total of 1–2 g in doses of 200 mg or OKT3 (Ortho Pharmaceutical Corp, New Jersey, USA) 2.5 or 5 mg/day for 10 days [14]. Repeated episodes of acute rejection were primarily treated with methylprednisolone and if steroid resistant, with ATG or OKT3. Twenty-seven out of 33 patients (82%) experienced one or more episodes of acute rejection during the first 3 months. Cumulative i.v. doses of methylprednisolone were 1.0–3.875 g during the first 3 months, and 1.25–4.125 g during the first year after transplantation. Fourteen patients needed additional treatment with ATG and/or OKT3. Six patients had also one or more episodes of late (>3 months) rejections. All patients receiving antibody treatment had at least one rejection episode verified by biopsy (n=14); in the group of patients treated with bolus of methylprednisolon only (n=13), nine had at least one biopsy-verified rejection (including `borderline' biopsy changes).

Kidney biopsies
Full-core biopsies were obtained with a biopticut biopsy gun (BioptiCut, Radioplast, Bromma, Sweden) with a needle size of 18 gauge. The baseline biopsies were taken during donor nephrectomy, shortly before the kidneys were removed from the donors [1]. Follow-up biopsies were taken at 1 week, 8–12 weeks (not reported here), and 1 year, guided by ultrasonography. Specimens for light and electron microscopy were processed in the same manner as previously described [1]. Most biopsies were representative for renal cortical tissue according to the Banff-93 criteria [15], containing at least seven glomeruli and one artery (Table 1Go).


View this table:
[in this window]
[in a new window]
 
Table 1. Representativity of 18-gauge graft biopsies: no. of structures (mean and range) per biopsy
 
Scoring of biopsies
Light microscopical changes were estimated and scored semi-quantitatively (0–3) according to a scoring system partly based on the Banff schema—93/95 [15,16], as previously described [1]. The following variables were graded: arteriosclerosis (as), arteriolar hyalinosis (ah), glomerular mesangial sclerosis (gs), interstitial fibrosis and tubular atrophy (if/ta), and mononuclear cell interstitial infiltration (mi). The evaluation of glomerular changes included estimation of the fraction of globally sclerosed glomeruli (gsg) and a `combined' glomerulosclerosis score (gsc), combining the gs score and the gsg as described in a preceding publication [1]. The sum of scores in each biopsy was recorded as the chronicity index (CI) (CI=as+ah+gsc+if/ta+mi) [1]. Lastly, the differences between the 1-year and baseline biopsy scores were recorded in each case. Biopsies obtained 1 week after transplantation were graded for as and ah, in order to analyse the reproducibility of the vascular scores at baseline (n=43).

Kidney function
Renal function was measured as serum creatinine at 1 and 3 years after transplantation. In addition, GFR was measured at 1 year as 99mTc-DPTA clearance [17].

Statistical methods
The data were entered on SPSS for Windows Release 7.5.1 [18] and partly on S-PLUS version 3.4 Release 1 [19]. For analyses of changes in morphology scores we used the Wilcoxon one-sample test. Relationships between morphological changes and renal function were calculated with Spearman's rank correlation, supplemented by testing of group differences with the ANOVA F-test. Relationships between data of renal function were analysed with Pearson's correlation and linear regression. Group differences with respect to rejection data (rejection vs non-rejection, different subsets of rejection) were analysed with the Mann–Whitney U test. The predictive power of 1-year functional and histopathological variates with respect to 3-year kidney function (serum creatinine more or less than 130 µmol/l) was assessed by cross validation. A logistic model was used for the prediction. For each of the patients a logistic model was fitted using the remaining variates. All tests were two-tailed. Cohen's kappa analysis was used for testing reproducibility of early vascular scores in kidney biopsies.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 References
 
Vascular changes in 1-week biopsies compared to baseline: as and ah
There was no significant difference between the mean arteriosclerosis scores at baseline (1.40) and at 1 week (1.28) (Table 2aGo). When comparing these two scores in each patient, however, they often differed from each other. A Cohen's kappa [18] of 0.046 for arteriosclerosis grading confirmed a poor reproducibility of the baseline score.


View this table:
[in this window]
[in a new window]
 
Table 2.
 
The baseline ah could often not be reproduced in the 1-week sample. Thus, the mean ah score at baseline was significantly higher (1.19) than the 1-week score (mean 0.56) (P<0.001) (Table 2aGo). When comparing the ah scores in each individual, the reproducibility of the baseline grading was again found to be low (Cohen's kappa 0.122). When recording only absence or presence of hyalinosis, however, the discrepancy was less: out of 33 ah positive cases at baseline, 22 (67%) remained positive at 1 week. And among 10/43 cases without hyalinosis at baseline, all but one showed absence of ah at 1 week as well. The intra-observer reliability of the scoring both with respect to arteriosclerosis and arteriolar hyalinosis was previously found to be satisfactory [1].

The number of glomeruli, arteries and arterioles in general did not differ in baseline [1] vs the 1 week biopsies (Table 1Go). The fraction of biopsies containing renal capsular tissue was 21/43 (48.8%) in baseline, vs 27/43 (62.8%) at 1 week (NS). Medullary tissue or tissue from the corticomedullary junction was found in 33/43 (76.7%) and 20/43 (46.5%) at baseline and at 1 week, respectively (P=0.002).

One-year biopsy findings compared to baseline
Arteriosclerosis (as)
The arterial changes in the 1-year biopsies consisted mainly of fibrointimal thickening of the vessel wall. In three biopsies, a small number of intimal lymphocytes were demonstrated (Fig. 1Go). No case was interpreted as acute vascular rejection. Foam cells were not observed.



View larger version (219K):
[in this window]
[in a new window]
 
Fig. 1. Graft biopsy 1 year after transplantation: Arcuate artery with intimal lymphocytes (arrow). Serum creatinine at 1 year, 96; at 3 years 89 µmol/l. (PAS stain, original magnification x400.).

 
The as scores are given in Table 2bGo, compared with baseline data. In the 1-year biopsies, as well as in baseline, more than 50% of the specimen showed as grade 2–3, and the as scores in the two groups were not significantly different. Although 10 patients showed an increase in as score during the first year, 14 patients demonstrated the same score and seven cases even a lower score in the 1-year biopsy. When comparing the 1-year changes with the biopsies taken 1 week after transplantation, we found an increase of arteriosclerosis (Table 2cGo). Comparing the two biopsies from each patient, 11 cases showed an increase and seven a decrease of as score, while 10 patients showed identical scores at 1 week and 1 year. The 1-year arteriosclerosis was also compared with the mean score of baseline and 1-week as. The difference between the two sets of scores was then not significant (P=0.12).

Arteriolar hyalinosis (ah)
Approximately 2/3 of the 1-year biopsies showed ah (Table 2bGo). Compared to baseline changes, 9/31 (29%) patients showed an increase of hyalinosis, 10 patients remained unchanged, and 13 biopsies showed a reduced score.

There was, on the other hand, a significant increase in ah from 1 week to 1 year (Table 2cGo). Thus, 40% of the patients (12/30) showed an increase of ah, while most of the remaining biopsies showed the same score and only two patients a reduced score at 1 year. An increase was seen both with respect to high-grade (2–3) changes, and when recording all cases including mild (grade 1) changes.

Glomerulosclerosis (gs)
The glomerular changes are summarized in Table 3Go. The mean score of mesangial sclerosis increased from baseline to 1 year. There was a similar increase of the glomerulosclerosis score gsc, combining mesangial sclerosis with the fraction of globally sclerosed glomeruli [1]. Thus, an increase of mesangial sclerosis by one grade in preserved glomeruli was present in 19 cases; no change was found in 12 biopsies, while one single case showed reduction of mesangial sclerosis by one grade. An increase of the combined score gsc was found at 1 year in 14/32 biopsies, whereas no change was found in the remaining samples.


View this table:
[in this window]
[in a new window]
 
Table 3. Glomerular and tubulointerstitial changes: baseline vs 1-year biopsy (n=32)
 
Thickening of capillary walls was not observed, except for occasionally short capillary segments in continuity with areas of mesangial sclerosis. No case with double contours of basement membranes was found.

The fraction of globally sclerosed glomeruli was found to increase from a mean value of 5.25% in baseline to 8.59% in the corresponding 1-year biopsies (NS). In 22/32 (69%) of the 1-year biopsies, 10% or less of the glomeruli were sclerosed, while two cases showed more than 25% sclerosed glomeruli.

Electron microscopical examination confirmed mesangial sclerosis to be the most consistent morphological feature. One case revealed recurrence of immune-complex glomerulonephritis, and one showed recurrence of light chain deposition in the graft. Another case with dense deposits and glomerular staining of IgA in the baseline biopsy [1], did not show deposits at 1 year. No case of typical chronic transplant glomerulopathy [9] was found.

Interstitial fibrosis/tubular atrophy (if/ta)
There was a significant increase compared to baseline (Table 3Go). Thus, grade 3 if/ta was found in nine cases vs none at baseline. Altogether 18 patients (56%) showed an increased score, whereas if/ta grade remained unchanged in nine biopsies and reduced by one grade in five cases.

Mononuclear cell interstitial infiltration (mi)
No case of unequivocal acute cellular rejection was found. Two cases were, however, consistent with Banff borderline and interpreted as probable acute-on-chronic rejection. One of these biopsies revealed a cortical stenosing arteriolopathy as well as signs of `arteriolitis' in the vasa recta. A few mononuclear cells lying within tubular epithelium was a common finding in atrophic tubuli.

A mononuclear cell infiltration grade 1–2 was found in approximately 40% of the biopsies (Table 3Go). In 12/32 cases, there was an increase in mi score compared to baseline; 19 patients showed an unchanged grade 0 score (not reaching the 5% limit for a grade 1 score [1]), and one single case a reduction of infiltrating cells by one grade.

Chronicity index (CI)
CI was significantly increased compared to baseline (Table 4Go). While two biopsies had the same CI at baseline and at 1 year, 17 biopsies showed an increase of CI by 1–3 grades and six biopsies by 4–6 grades. On the other hand, six biopsies showed a reduction of CI by 1–5 grades compared to baseline.


View this table:
[in this window]
[in a new window]
 
Table 4. Chronicity index (CI): baseline vs 1-year biopsy (n=31)
 
Correlation of biopsy findings to renal function
Functional significance of 1-year biopsy findings
As shown in Table 5Go, serum creatinine showed a very slight increasing tendency only from 1 to 3 years after transplantation (P=0.06). Thirteen out of 33 patients (39%) at 1 year, and 10 out of 28 patients (36%) at 3 years had excellent kidney function with serum creatinine of 130 µmol/l or less. Four out of 28 patients (14%) showed an increase of more than 20% in serum creatinine between 1 and 3 years; one of these suffered from recurrence of light change deposition in the graft, and another had a recurrence of immune-complex glomerulonephritis.


View this table:
[in this window]
[in a new window]
 
Table 5. Renal function data 1 and 3 years after transplantation
 
The correlations of the various histopathological parameters with kidney function at 1 and 3 years are shown in Table 6Go and Figure 2Go. If/ta correlated well with kidney function at 1 and 3 years. The extent of mi also correlated with renal function. One of the two mi-grade 2 biopsies showed recurrence of light chain deposition, and the other (patient no. 2) had a failing graft function; when excluding these from the analysis, no significant correlation between the mi score and serum creatinine at 1 or 3 years was found.


View this table:
[in this window]
[in a new window]
 
Table 6. One-year variates: correlation with kidney function 1 and 3 years after transplantation
 


View larger version (6K):
[in this window]
[in a new window]
 
Fig. 2. One-year variates correlated with serum creatinine (µmol/l) 3 years after transplantation (n=28). (a) Interstitial fibrosis/tubular atrophy (if/ta) of graft biopsy. Spearman's rank correlation 0.472, P=0.011. (b) Combination of the following variates in graft biopsy: Arteriosclerosis (as)+Glomerulosclerosis (gsc)+Interstitial fibrosis/tubular atrophy (if/ta)+Mononuclear cell interstitial infiltration (mi). Spearman's rank correlation 0.401, P=0.037. (c) Serum creatinine. Pearson's correlation 0.762, P<0.001.

 
The occurrence of a few arterial intimal lymphocytes (Figure 1Go) did not seem to influence kidney function. In three patients with this finding, serum creatinine at 1 and 3 years was 96–89, 122–116, and 126–147 µmol/l, respectively.

Kidney function at 1 and 3 years did not correlate with the 1-year biopsy scores of as, ah, or gs. The fraction of globally sclerosed glomeruli correlated with kidney function at 3 years (P=0.009), but not at 1 year. Estimates of the respective differences between the 1-year and baseline scores did not improve the correlation between morphological and functional parameters (data not shown).

The CI tended to be higher in those patients with less well preserved kidney function; the correlation was significant, however, only to GFR at 1 year (Table 6Go). Subtracting the ah score from the CI improved the correlation to kidney function both at 1 and 3 years (Figure 2bGo). By combining the chronicity scores in different ways, we found the combination of if/ta and mi to give the best correlation to kidney function, but not clearly better than if/ta alone (Table 6Go).

One-year serum creatinine and GFR both correlated significantly with 3-year serum creatinine (Table 6Go, Figure 2cGo). By cross validation analysis, the 1-year serum creatinine was found a better predictor of 3-year serum creatinine than GFR and each of the chronic biopsy scores at 1 year, or any combination of these scores, predicting the value correctly for 22 out of 27 patients. Furthermore, given the creatinine value, including any one of the other variates did not improve the prediction, except for the case where as was added, predicting correctly for 23 out of 27 patients.

Functional significance of baseline biopsy findings
We found no significant correlation between kidney function at 1 or 3 years with the baseline scores of as, ah, gs, if/ta, CI, or fraction of globally sclerosed glomeruli.

Furthermore, there was no correlation between as/ah in the 1-week biopsies and renal function at 1 and 3 years.

Impact of donor age
Donor age correlated significantly with kidney function at 3 years (P=0.013), but not with 1-year kidney function. The fraction of globally sclerosed glomeruli at 1 year correlated with donor age (P=0.011) but not the 1-year if/ta.

Impact of rejection
In patients with one or more episodes of acute rejection (n=27) the mean score of if/ta was 1.9 vs 1.0 in patients with no rejection episodes (n=6; P=0.052). There was no difference in the score for if/ta in patients with steroid sensitive rejections and patients receiving additional T cell antibody treatment. There was, however, a correlation between the first year cumulative doses of methylprednisolone in the individual patient and the 1-year if/ta (P=0.004).

Serum creatinine at 1 year tended to be higher in patients suffering acute rejection vs those without rejection (P=0.05), while there was no significant difference in kidney function at 3 years between the two groups. Cumulative dosage of methylprednisolone correlated to reduced kidney function both at 1 year (n=33, P<0.001) and at 3 years (n=28; P=0.037).

We found no correlation between 1-year ah and the first year cumulative methylprednisolone doses. Neither occurrence of acute rejection nor cumulative dosage of methylprednisolone correlated to donor age.

Discussion
In the present study on living donor kidneys, baseline (`zero-hour') biopsy changes tended to become more pronounced during the first year after transplantation. This could be shown for if/ta, gs, and mi but not for the vascular changes. When comparing the 1-year samples with biopsies taken 1 week after transplantation, however, an increase in ah and as was found.

In the follow-up of the individual patient, however, the 1-year chronicity scores were less predictable. Thus, even though if/ta was increased in the 1-year biopsy group, 14/32 biopsies showed unchanged or decreased if/ta score from baseline to 1 year. CI was unchanged or in fact reduced from baseline to 1 year in 8/31 cases.

These data may seem paradoxical, but are most likely explained by sampling error. Mild-to-moderate morphological changes may be seen at baseline as an expression of benign (partly age-related) nephrosclerosis [1]. These changes may be essentially similar to those of chronic transplant nephropathy [8,9]. A difference in morphological score between baseline and a follow-up graft biopsy may therefore merely reflect the focal nature of the processes involved. We have shown that even high-grade vascular changes may be seen in `zero-hour' biopsies from living donors [1], adding to the uncertainty in comparing with follow-up samples.

We assume this point has been generally underestimated in studying chronic allograft changes. In our opinion, the value of the allograft biopsy in diagnosing low-grade chronic transplant nephropathy may be questioned. On the other hand, severe (grade 3) interstitial fibrosis/tubular atrophy is restricted to the 1-year biopsies in our study and seems a reliable marker of chronic post-transplantation parenchymal damage.

Sampling error may be especially relevant in the study of early vascular changes, as shown by our comparison between baseline and biopsies taken 1 week after transplantation. Thus, we found the reproducibility of baseline as to be only slightly better than random. This may be explained by the sample size: the single 18-gauge biopsy, with an average of four arterial profiles, is too small for a reproducible scoring of this lesion.

The scoring of baseline ah seemed even more unreliable compared to the 1-week biopsy. The difference in the biopsy procedures may contribute to this result: at our centre, the baseline biopsies were obtained with the biopsy gun positioned directly onto the surface of the kidney. These biopsies would therefore be expected to contain tissue from a scarcely deeper level of the renal parenchyma. This is supported by our finding that the baseline samples more often included tissue from the corticomedullary junction, and may indicate that the baseline ah is more pronounced in a juxtamedullary location. Reporting only presence or absence of hyalinosis improved the reliability of the baseline finding: we then found a 67% reproducibility of baseline hyalinosis, which is exactly the same figure as presented by Wang et al. [6] in comparing baseline with subsequent graft biopsies.

Because the biopsy procedure at baseline is different from those at follow-up, it might be that the 1-week sample represent a more `correct' early reference point than the actual `zero-hour' biopsy. Thus, we found a highly significant increase in biopsy ah 1 year after transplantation when compared to the 1-week finding, but not so when compared to baseline. The findings in as were similar, but less convincing with respect to statistical significance. Otherwise, the 1-week biopsies seem less ideal as a baseline, because of frequently occurring interstitial oedema, new-onset interstitial cellular infiltrates, and glomerular hypercellularity (data not shown).

The clinical significance of the ah at 1 year is uncertain. We found no correlation between the hyalinosis and kidney function at 1 or 3 years. Nor was there any correlation to acute rejection, as measured by cumulative 1-year methylprednisolone dose. On the other hand, long-term cyclosporin treatment—even in low doses—has been shown to induce ah in native kidneys as well as in allografts [2022]. Our patients were treated with a standard triple regimen, aiming at low trough values of CsA; consequently our study was not designed to reveal possible morphological effects of differences in CsA doses. Because of individual differences in susceptibility to CsA nephrotoxicity, such morphological effects are, anyway, unpredictable [20].

Gs was described by Isoniemi et al. [10,23] as part of their CADI (Chronic Allograft Damage Index) score. Our 1-year findings confirm the evolvement of such mesangial alterations in the graft with stable function. This mesangial sclerosis may reflect a subclinical or `silent' transplant glomerulopathy [23], and seems much more common than the more specific `chronic transplant glomerulopathy' [15]. Quantitative analyses of mesangial sclerosis of various aetiologies have recently been shown to correlate with renal function [24]. We did not find any significant impact on renal function after 1 or 3 years, possibly because the changes tended to be mild or mild-to-moderate only. In our study, electron microscopy was implemented mainly to find or exclude specific causes of glomerular changes, such as recurrence of kidney disease or de novo glomerulonephritis. The findings of recurrent disease in two patients (vide supra) underlines the importance of ultrastructural examination.

Allograft inflammatory infiltrates are known to occur—apart from in acute rejection—in `well-to-do' grafts. Our finding of leukocyte infiltration in many biopsies, as well as frequently occurring mild tubulitis in atrophic tubuli [25] is in conjunction with other studies [10,26,27]. The pathological significance of such infiltrates in the single case is often difficult to assess. Croker et al. [28] studied the effect of macrophage infiltration in graft biopsies and found correlation between their Macrophage index (MI) and graft outcome. We found an uncertain correlation to long-term renal function, but the majority of biopsies showed mild (grade 1) changes only.

A few of our biopsies showed a scarce arterial lymphocytic infiltration within a fibrous intima, that might be interpreted as `chronic vascular rejection'. This finding did not, however, have any obvious impact on kidney function, and its relevance seems therefore doubtful.

If/ta was the histopathological variate that most closely correlated to long-term kidney function. This finding confirms studies previously presented by several investigators [10,23,29,30]. Nicholson et al. [31] studied the development of allograft fibrosis by histomorphometric assessment and found that changes at 6 months after transplantation can be used as a surrogate endpoint marker of chronic graft damage. Furthermore, our data support the proposal by the Banff Working Classification, that chronic allograft changes be graded essentially by the extent of if/ta [15,16].

Serum creatinine 1 year after transplantation correlated highly significantly with creatinine at 3 years, and proved to be the single most reliable predictor of long-term kidney function. Adding any of the histopathological scores, or any combinations thereof, did not at all, or only marginally, increase the predictive value of 1-year creatinine in cross validation analysis. The reason for this result is probably a lack of precision in the semi-quantitative scoring system, together with the effect of sampling error already discussed.

Serum creatinine is not, either, an ideal measure of kidney function. It cannot be ruled out that the biopsy findings might have shown a better correlation to a more precise measure of kidney function like GFR at 3 years. The 1-year data might indicate such a closer correlation between the biopsy findings and GFR (Table 6Go). Recording of GFR 3 years after transplantation was not, however, part of our protocol.

The frequency of cases with one or more acute rejection episodes in this study was 82%. This figure corresponds to that reported in another study from our centre including a higher number of patients [32] and to data published by Olbricht [33]. The frequency with which acute rejection occurs depends on several factors, such as HLA mismatch [32,34], immunosuppressive protocol [35,36], and the current definition of acute rejection at the actual centre. In the present study, HLA identical siblings were excluded, and the majority of transplantations were one HLA haplo-type mismatched. All patients were treated with cyclosporin A, prednisolone, and azathioprine, with no induction therapy. At the time of patient inclusion in the study, the protocol CsA dosage was, in fact, lower than what is currently recommended (vide supra). In the majority of the patients, acute rejection was verified by graft biopsy. Our graft survival is, in general, at the same level as that of other centres [32,37]. As reported by Opelz [38], successfully treated episodes of acute rejection had a negligible influence on long-term graft function.

None of our baseline biopsy variates correlated significantly with graft outcome. This is in agreement with the reports by Nyberg et al. [2] and Curschellas et al. [3], who studied `zero-hour' biopsies from living and cadaveric donors, respectively. Minakawa et al. [4], on the other hand, observed their baseline `vasculopathy' to correlate with short-term but not with long-term prognosis; similar observations were made by Taub et al. [5], who found reduced graft survival in patients with donor baseline as at 2 years, relating however mainly to increased early graft loss during the first 3 months after transplantation. Wang et al. [6] found that absence of ah was correlated with a better graft prognosis, and have recently reported [39] that fibrous intimal thickening in arteries in cadeveric donor baseline biopsies was correlated with occurrence of graft loss and delayed graft function.

We could not show any prognostic significance of our vascular baseline changes. Our analysis is, however, based mainly on patients with long-functioning grafts. The number of early graft losses were too small for a meaningful correlation with `zero-hour' changes. One might speculate, furthermore, that the significance of vascular changes is less in living than in cadaveric donors; in the latter, a potentially reduced renal reserve capacity might be challenged by additional stress such as prolonged ischaemia.

In conclusion, we have found that if/ta in the well-functioning allograft 1-year after transplantation is the most reliable histopathological parameter in predicting kidney function at 3 years. Serum creatinine at 1 year seems, however, an even better predictor of long-term prognosis. In fact, none of the chronic biopsy scores in our study added substantially to the predictive value of the 1-year serum creatinine. It might be that increasing the amount of tissue, by taking larger biopsies or more than one biopsy at the time, could improve the reliability of the histopathological findings [29,40].

According to our experience, the value of the morphological examination of the long-term allograft is first and foremost in the case of declining kidney function; the graft biopsy is the superior tool in diagnosing recurrent disease vs chronic transplant glomerulopathy or de novo nephritis. Recent data [41] suggest that protocol biopsies may be helpful to optimize the level of immunosuppression in patients with `subclinical' rejection, perhaps as a step towards a more individualized approach in patient follow-up. We do not at our centre, however, recommend a routine 1-year biopsy in the management of the patient with a well-functioning transplant, at least not so in the case of the living donor and with the current biopsy procedures.



   Acknowledgments
 
The authors want to thank Odd Kolbjørnsen, Institute of Mathematical Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, for his performance of statistical analyses. We thank our Dept. of Radiology, especially Dr Jarl Jakobsen for performance of the kidney biopsies.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 References
 

  1. Sund S, Reisæter AV, Scott H et al. Morphological studies of baseline needle biopsies from living donor kidneys: Light microscopical, immunohistochemical and ultrasstructural findings. APMIS 1998; 106: 1017–1034[ISI][Medline]
  2. Nyberg G, Hedman L, Blohmé I, Svalander C. Morphologic findings in baseline kidney biopsies from living related donors. Transplant Proc 1992; 24: 355–356[ISI][Medline]
  3. Curschellas E, Landmann J, Dürig M et al. Morphologic findings in `zero-hour' biopsies of renal transplants. Clin Nephrol 1991; 36: 215–222[ISI][Medline]
  4. Minakawa R, Tydén G, Lindholm B, Reinholt FP. Donor kidney vasculopathy: impact on outcome in kidney transplantation. Transplant Immunol 1996; 4: 309–312[Medline]
  5. Taub HC, Greenstein SM, Lerner SE, Schechner R, Tellis VA. Reassessment of the value of post-vascularization biopsy performed at renal transplantation: the effects of arteriosclerosis. J Urol 1994; 151: 575–577[ISI][Medline]
  6. Wang HJ, Kjellstrand CM, Cockfield SM, Solez K. On the influence of sample size on the prognostic accuracy and reproducibility of renal transplant biopsy. Nephrol Dial Transplant 1998; 13: 165–172[Abstract]
  7. Cosyns J-P, Malaise J, Hanique G et al. Lesions in donor kidneys: nature, incidence, and influence on graft function. Transplant Int 1998; 11: 22–27[ISI][Medline]
  8. Paul LC, Solez K. Chronic rejection of vascularized organ allografts. In: Paul LC, Solez K (eds). Organ Transplantation. Long-Term Results. Marcel Dekker, Inc. New York, Basel, Hong Kong: 1992: 99–134
  9. Mihatsch MJ, Ryffel B, Gudat F. Morphological criteria of chronic rejection: differential diagnosis, including cyclosporine nephropathy. Transplant Proc 1993; 25: 2031–2037[ISI][Medline]
  10. Isoniemi HM, Krogerus L, von Willebrand E, Taskinen E, Ahonen J, Häyry P. Histopathological findings in well-functioning, long-term renal allografts. Kidney Int 1992; 41: 155–160[ISI][Medline]
  11. Ojo AO, Wolfe RA, Held PJ, Port FK, Schmouder RL. Delayed graft function: risk factors and implications for renal allograft survival. Transplantation 1997; 63: 968–974[ISI][Medline]
  12. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival rates of kidney transplants from spousal and living unrelated donors. New Engl J Med 1995; 333: 333–336[Abstract/Free Full Text]
  13. Vartdal F, Gaudernack G, Funderud S et al. HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation—a fast and reliable technique. Tiss Antigens 1986; 28: 301–312[ISI]
  14. Midtvedt K, Tafjord AB, Hartmann A et al. Half dose of OKT3 is efficient in treatment of steroid-resistant renal allograft rejection. Transplantation 1996; 62: 38–42[ISI][Medline]
  15. Solez K, Axelsen RA, Benediktsson et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993; 44: 411–422[ISI][Medline]
  16. Solez K, Benediktsson H, Cavallo T et al. Report of the third Banff conference on allograft pathology (July 20–24, 1995) on classification and lesion scoring in renal allograft pathology. Transplant Proc 1996; 28: 441–444[ISI][Medline]
  17. Sapirstein LA, Vidt DG, Mandel MJ, Hanusik G. Volumes of distribution and clearances of intravenously injected creatinine in dog. Am J Physiol 1955; 181: 330–336[Free Full Text]
  18. Norusis MI. SPSS for Windows. SPSS Inc., Chicago 1993
  19. Venables WN, Ripley BD. Modern Applied Statistics with S-PLUS, (Second Edn). Springer New York 1997
  20. Sund S, Førre Ø, Berg KJ, Kvien TK, Hovig T. Morphological and functional renal effects of long-term low-dose cyclosporin A treatment in patients with rheumatoid arthritis. Clin Nephrol 1994; 41: 33–40[ISI][Medline]
  21. Zachariae H, Kragballe K, Hansen HE, Marcussen N, Olsen S. Renal biopsy findings in long-term cyclosporin treatment of psoriasis. Br J Dermatol 1997; 136: 531–535[ISI][Medline]
  22. Solez K, Racusen LC, Marcussen N et al. Morphology of ischemic acute renal failure, normal function, and cyclosporine toxicity in cyclosporine-treated renal allograft recipients. Kidney Int 1993; 43: 1058–1067[ISI][Medline]
  23. Isoniemi H, Taskinen E, Häyry P. Histological chronic allograft damage index accurately predicts chronic allograft rejection. Transplantation 1994; 58: 1195–1198[ISI][Medline]
  24. Vleming L-J, Baelde JJ, Westendorp RGJ, Daha MR, van Es LA, Bruijn JA. The glomerular deposition of PAS positive material correlates with renal function in human kidney diseases. Clin Nephrol 1997; 47: 158–167[ISI][Medline]
  25. Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55: 713–723[ISI][Medline]
  26. Rush DN, Henry SF, Jeffery JR, Schroeder TJ, Gough J. Histological findings in early routine allograft recipients. Transplantation 1994; 57: 208–211[ISI][Medline]
  27. Rush D, Jeffery J, Trpkov K, Solez K, Gough J. Effect of subclinical rejection on renal allograft histology and function at 6 months. Transplant Proc 1996; 28: 494–495[ISI][Medline]
  28. Croker BP, Clapp WL, Abu Shamat AR, Kone BC, Peterson JC. Macrophages and chronic renal allograft nephropathy. Kidney Int [Suppl] 1996; 57: S42–S49[Medline]
  29. Nickerson P, Jeffery J, Gough J et al. Identification of clinical and histopathologic risk factors for diminished renal function 2 years posttransplant. J Am Soc Nephrol 1998; 9: 482–487[Abstract]
  30. Dimény E, Wahlberg J, Larsson E, Fellström B: Can histopathological findings in early renal allograft biopsies identify patients at risk for chronic vascular rejection? Clin Transplant 1995; 9: 79–84[ISI][Medline]
  31. Nicholson ML, Harper SJ, Wheatley TJ, McCulloch TA, Feehally J, Furness PN. Renal transplant fibrosis: Histomorphometric assessment of early renal transplant biopsies for markers of chronic rejection. Transplant Proc 1997; 29: 2793–2794[ISI][Medline]
  32. Foss A, Leivestad T, Brekke IB et al. Unrelated living donors in 141 kidney transplantations. Transplantation 1998; 66: 49–52[ISI][Medline]
  33. Olbricht CJ. Living-donor kidney transplantation with tacrolimus. New Horizons Kid Transplant 1998; 2: 21
  34. Reisæter AV, Leivestad T, Vartdal F et al. A strong impact of matching for a limited number of HLA-DR antigens on graft survival and rejection episodes. Transplantation 1998; 66: 523–528[ISI][Medline]
  35. The tricontinental mycophenolate mofetil renal transplantation study group. A blinded randomized clinical trial of mycophenolate mofetil for the prevention of acute rejection in cadaveric renal transplantation. Transplantation 1996; 61: 1029–1037[ISI][Medline]
  36. Opelz G. Efficacy of rejection prophylaxis with OKT3 in renal transplantation. Transplantation 1995; 60: 1220–1224[ISI][Medline]
  37. Terasaki PI, Cecka JM, Gjertson DW, Cho YW. Spousal and other living renal donor transplants. In: Cecka JM, Terasaki PI (eds) Clinical Transplants 1997. UCLA Tissue Typing Laboratory, Los Angeles: 1998: 269–273
  38. Opelz G. Critical evaluation of the association of acute with chronic graft rejection in kidney and heart transplant recipients. Transplant Proc 1997; 29: 73–76[ISI][Medline]
  39. Wang H, Solez K, Kjellstrand CM, Cockfield SM. The importance of donor vascular pathology in determining renal allograft outcome. J Am Soc Nephrol 1998; 9: 702A
  40. Colvin RB, Cohen AH, Saiontz C et al. Evaluation of pathologic criteria for acute renal allograft rejection: Reproducibility, sensitivity, and clinical correlation. J Am Soc Nephrol 1997; 8: 1930–1941[Abstract]
  41. Rush D, Nickerson P, Gough J et al. Beneficial effects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol 1998; 9: 2129–2134[Abstract]
Received for publication: 2.12.98
Accepted in revised form: 8. 6.99