Low recipient age is still a major risk factor for graft failure after renal allograft transplantation (Tx) (1, 2). Graft thrombosis, acute tubular necrosis, and primary nonfunction are major risk factors in the youngest recipients, particularly in the early postTx period (2, 3). Younger recipients also have more acute rejections and a poorer outcome thereafter (1, 2). Acute rejection is the major risk factor for chronic rejection (4, 5). In the long term, chronic rejection is the leading cause of graft loss in children (1, 6). In the 1997 North American Pediatric Renal Transplant Cooperative Study report, 5-year graft survival for cadaveric donor (CAD) recipients was only 61% compared with 77% for living related donor (LRD) recipients. Results in CAD recipients <2 years of age have been especially discouraging (1). The importance of using histopathological criteria in defining chronic rejection has been emphasized (7). There are nevertheless few reports on long-term allograft histology in children in the literature.
We have previously reported good long-term graft survival in children transplanted when under the age of 5 years, mainly with adult cadaveric kidneys. Seven-year graft survival was 94% for LRD recipients and 79% for recipients of a CAD kidney. However, long-term graft function slowly deteriorated with time and, in patients transplanted at <2 years of age, was inferior to grafts in patients transplanted between 2 and 5 years of age (8).
In the present study, we report a long-term prospective follow-up of renal histology in small children with functioning allografts. Our aim was to ascertain whether there were any histological changes with time and, if so, what risk factors related to these changes. In addition, our aim was to analyze what risk factors contributed to the inferior graft function in children who received transplants at <2 years of age.
PATIENTS AND METHODS
Fifty-one kidney allograft recipients who were under the age of 5 years when they received their first graft have received 55 grafts between 1987 and 1997, at our center. The most common disease leading to Tx was congenital nephrosis with mutations in the nephrin gene (NPHS1) in 43 patients (84%) (9). Other diagnoses were a urethral valve in three patients and polycystic kidney disease, prune-belly syndrome, neuroblastoma, congenital nephritis, and congenital nephrotic syndrome in one patient each. Other patient characteristics are listed in Table 1. Forty-three patients were followed for at least 1.5 years. Prospective follow-up of renal histology was started in 1990; therefore, a biopsy specimen was not available from the first five patients at 18 months. Biopsies were not taken (either for clinical reasons or refusal to participate) from three additional patients at 1.5 years, from one patient at 3 years, from three patients at 5 years, and from one patient at 7 years.
Immunosuppression and acute rejection.
Our pretransplantation protocol and management of NPHS1 patients have been reported previously (10, 11). Triple immunosuppression with a pharmacokinetically determined individualized starting dose of cyclosporine (CsA) was used, and given to preschool children in three daily doses because of their faster CsA metabolism (10, 12). CsA doses were calculated and CsA blood trough levels measured at the time when the biopsies were taken and functional studies were made. Fine-needle aspiration biopsies (FNAB) were taken routinely, and the diagnosis of acute rejection was based on FNAB, as well as clinical and laboratory data (8, 13).
Renal function and histology.
The patients were followed prospectively and studied for renal function and histopathology 1.5, 3, 5, and 7 years after Tx. Only protocol biopsies were included in this study. Functional investigations and clinical data taken at the same time as the biopsies were used for the analysis. Glomerular filtration rate (GFR) was measured by 51Cr-EDTA clearance. Effective renal plasma flow was estimated by para-aminohippuric acid clearance (8). Percutaneous renal core needle biopsies were performed under ultrasound guidance, using an automated punch device (Biopty-Cut®; Radiplast, Bromma, Sweden). A needle with an outside diameter of 1.2 or 1.6 mm was used to obtain biopsies sized 0.9×20 mm. The specimens were fixed in 4% formaldehyde, embedded in paraffin, and cut into serial 4-μ m sections. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and methenamine silver periodic acid-Schiff for light microscopy. The biopsies were coded and examined by two investigators independently (E.Q. and L.K.) without knowledge of kidney function or time of Tx. Biopsies with a minimum of five glomeruli and an artery were considered representative and accepted for analysis. On these criteria, 29 (83%) of 35 biopsies were suitable for analysis at 1.5 years after Tx. The corresponding figures were 30/35 (86%), 25/26 (96%), and 13/14 (93%), at 3, 5, and 7 years after Tx, respectively. The mean GFR of all the patients excluded was 76.0±21.9 ml/min/1.73 m2 at 1.5 years, and 82.0±31.8, 59.7±19.1, and 49.1±15.4 ml/min/1.73 m2 at 3, 5, and 7 years, respectively. GFR of these patients did not differ significantly from those in the study (unpaired t test). The mean number of glomeruli in the analyzed biopsies ranged from 12 to 18, at different time points. Adequate clinical data were not available for one patient at 18 months and one patient at 7 years at the time when the biopsies were taken, and those two patients were therefore excluded from the functional and clinical correlations.
The Banff classification (BC, 1991) was used for diagnosis and grading of chronic allograft nephropathy and any signs of acute rejection and CsA-associated changes (14). In addition to our preliminary report (15), the histological findings were also scored semiquantitatively, using our more extended scoring table (Table 2) (16, 17). The interstitial changes were classified as focal (less than two of four visual fields) or diffuse. The glomerular changes were scored separately for either segmental or global changes. Global glomerular sclerosis was also noted separately as a percentage of the total number of glomeruli examined in each biopsy slide (percentage of sclerotic glomeruli [PSG]). Changes in arteries, arterioles, and veins were scored separately, as were proximal and distal tubules (Table 2). The “chronic allograft damage index” (CADI) has previously been shown to correlate with decreasing graft function in adult renal transplant recipients (16). The histopathological changes in our patients were mostly very mild. Because of this, we used a scale from 0 to 6 instead of 0 to 3, as in the original paper (16). Thus, 0=no changes, 1=very mild, 2=mild, 3=mild to moderate, 4=moderate, 5=severe, 6=very severe. CADI was calculated as the sum of the scores of diffuse interstitial inflammation and fibrosis (0–6 each), mesangial matrix increase and glomerular sclerosis (0–6 each), intimal proliferation of vessels (each score for arterial, arteriolar, and venous changes is divided by 3; thus, vessels together scored 0–6) and tubular atrophy (each score for proximal and distal tubules is divided by 2; thus, tubules together scored 0–6). With a scale from 0 to 6, the total range for CADI is 0–36.
Nine patients were treated with recombinant human growth hormone (GH). GH was started at earliest 1 year after Tx (mean: 1.9) if height was below −2 standard deviation scores or height velocity was below the 25th centile. A GFR over 20 ml/min/1.73 m2, normal thyroid function, and no malignancy were other prerequisites for starting GH treatment. Mean duration of treatment was 4.6 (range: 1.0–7.0) years.
Nonparametric data of two groups were compared with the Mann-Whitney U test and the Wilcoxon signed rank test was used for testing repeated measures of nonparametric data. Parametric data between two groups were compared with the unpaired t test and parametric repeated measures were tested with the paired t test. The Spearman rank correlation coefficient was used to evaluate correlations between nonparametric data. A P value of less than 0.05 was considered significant.
The study design was approved by the Ethical Committee of the Hospital for Children and Adolescents, University of Helsinki, and informed consent was obtained from parents or patients before their inclusion in the study.
Seven patients (14%) have lost their grafts (one of them twice). Two of the five patients with NPHS1 who lost their grafts after developing nephrosis had a protocol biopsy taken at 1.5 years. One of them had a grade II chronic transplant nephropathy (C2) with an overlying grade I acute rejection. The other patient’s biopsy was regarded as normal. One patient lost her graft early after Tx on account of acute rejection combined with acute tubular necrosis. One patient had glomerular necrosis and arterial thrombosis in addition to C2 in his protocol biopsy at 7 years, because of a hemolytic uremic syndrome at the time of biopsy. This patient subsequently lost his graft. In addition, one patient with a functioning graft died from a lymphoproliferative disease almost 10 years after Tx. This patient had a biopsy regarded as normal at 7 years.
The histological changes were mostly scored as mild or very mild, and most biopsies were regarded as normal according to the BC (Table 3). The percentage of biopsies with chronic allograft nephropathy remained stable. The median CADI score for all patients was 2.5 (score: 0–36) at 1.5 years and 4.0, 4.0, and 3.5, at 3, 5, and 7 years after Tx, respectively (Fig. 1). Thus, as a whole, the histopathological findings did not show signs of deterioration. The incidences of the most frequent individual histopathological findings and their corresponding mean scores are presented in Table 4. Interstitial inflammation with focal lymphocytes tended to decrease with time, whereas the incidence of mild diffuse fibrosis tended to increase with time. A major finding was the increase in global glomerular sclerosis. The proportion of totally sclerotic glomeruli increased markedly with time. At 1.5 years, the mean PSG was only 3%, but at 5 years after Tx it had increased to 36% (P =0.0008, Wilcoxon). The glomerular sclerosis was not reflected in graft function. The most frequent vascular findings were arterial and arteriolar intimal proliferation, encountered in 20% and 24% of the patients at 5 and in 46% and 15% at 7 years, respectively. The most prominent tubular finding was epithelial atrophy in 76% of the patients at 1.5 years and in 54% at 7 years. Tubular casts were found in approximately half of the biopsies. They were almost exclusively hyaline. However, the mean and median scores were low, indicating that the changes encountered were mild.
Acute rejection and cyclosporine toxicity.
Of our patients, 67% had suffered at least one, and 31% two or three rejection episodes verified by FNAB during the first 3 months after Tx. Late acute rejection episodes were seen in only two patients. One of them had a clinically and fine-needle documented late acute rejection at the time of protocol core biopsy. This acute rejection episode was partially treated at the time of biopsy and showed only mild tubulitis (i.e., <4 mononuclear cells/tubular cross-section). The rejection was reversible with steroids. Borderline changes (i.e., very mild acute rejection) were additionally found in two patients without clinical signs of acute rejection.
The number of early acute rejections influenced CADI. Patients with one or more rejection episodes had a higher mean CADI at every follow-up than those without rejection episodes (5.2±6.1 vs. 3.0±2.6 at 1.5 years, 6.8±5.1 vs. 4.3±3.9 at 3 years, 8.9±5.5 vs. 3.5±3.1 at 5 years, and 6.7±6.6 vs. 5.2±4.6 at 7 years). The difference in CADI was not statistically significant at 1.5 and 3 years. The increase in the mean CADI (ΔCADI) between 1.5 and 3 years was similar in the two groups (2.2±7.1 vs. 2.3±6.8). ΔCADI increased to 4.8±8.2 in the group with one or more rejection episodes between 1.5 and 5 years in contrast to a ΔCADI of 0.9±5.6 in the group without rejection episodes. At 5 years, the difference in CADI was significant (3.5±3.1 vs. 8.9±5.5, P =0.001, Mann-Whitney U test). A correlation between acute rejection episodes and CADI at 5 years was also shown with the Spearman test (ρ 0.67, P =0.001).
When patients were divided according to HLA mismatch (0–1 in the AB loci and 0 in the DR loci vs. other), cold ischemia time (<24 vs. >24 hr for CAD recipients), or donor age (<40 vs. >40 years), this did not affect the CADI.
Histopathological findings assumed to be related to CsA toxicity were rarely found. Isometric vacuolization was seen in three patients (in one patient each at 1.5, 3, and 7 years). Their mean CsA doses were moderate, from 2.9 to 6.7 mg/kg. Nodular hyaline afferent arteriolar deposits were seen in six patients (in two at 1.5 and 7 years and in one patient at 3 and 5 years, respectively). Except for one patient with a CsA dose of 10.0 mg/kg, their doses were moderate, ranging from 5.3 to 6.7 mg/kg. Except in one case, the hyaline deposits and vacuolization appeared in different patients. CsA doses or blood levels did not correlate with any of the histological parameters evaluated.
Renal function and histology.
The body surface area-related mean GFR deteriorated slowly with time, although the CADI remained stable and low. In contrast, we recorded an improvement in absolute GFR with time (Fig. 1). Patients classified, according to the BC, as having a normal biopsy, had a better mean GFR (74.6±19.2) 3 years after Tx than the group of patients with some degree of chronic allograft nephropathy (59.4±18.0) (P =0.04, unpaired t test). Patients with C2 had a mean GFR below 60 ml/min/1.73 m2 at every follow up. The CADI index also correlated inversely with graft function (ρ −0.44, P =0.02 and ρ −0.46, P =0.03, at 3 and 5 years, respectively, Spearman). However, individual histological scorings (e.g., diffuse fibrosis) did not correlate with graft function.
The CADI score at 3 years correlated inversely with later function at 5 years after Tx (ρ −0.58, P =0.01, Spearman). The patients with a higher than median CADI score (i.e., >4.5) at 3 years had a lower GFR at 5 years than those with a lower CADI score (50.6±17.4 vs. 70.4±18.6, respectively, P =0.03, unpaired t test).
Histology and donor source.
CAD recipients suffered more early acute rejections and showed more pathological findings in the biopsies than did LRD recipients. However, the differences were slight and the median CADI was significantly higher in CAD recipients only at 3 and 5 years (Table 5). The LRD recipients had a higher CADI at 1.5 years. The majority of the LRD recipients received their graft <2 years of age, and had a higher CADI at 1.5 years compared to recipients >2 years of age (Table 6). Neither difference was, however, statistically significant.
The changes in CADI with time for CAD recipients were not statistically significant; thus, the median CADI remained stable with time for both CAD and LRD recipients. These minor changes did not affect graft function, which was similar in the two groups. The absolute GFR even improved with time, significantly so for LRD recipients. The CsA doses were also closely matched between the groups.
Histology and recipient age.
Patients <2 years of age had a lower absolute GFR (Table 6). The patients transplanted at >2 years of age improved their function with time, in contrast to the group transplanted at <2 years of age. However, no significant difference in the CADI score was found in patients receiving their graft <2 years of age as compared with patients >2 years of age at Tx (Table 6). The CADI also remained stable with time in both groups. Individual histopathological changes did not differ between the groups, except for the incidence of totally sclerotic glomeruli, which increased in both groups with time, but at a different pace. PSG was 3% for both groups at 1.5 years, 21% for recipients <2 years vs. 16% for recipients >2 years, at 3 years and 41% vs. 32%, respectively at 5 years (all P =NS). After 7 years, 45% (SD: 14) of the glomeruli were collapsed in the group transplanted <2 years of age as compared with 24% (SD: 15) in the group transplanted >2 years of age (P =0.04, Mann-Whitney U). There were no differences in the mean number of acute rejections, and the CsA doses were also evenly distributed between the groups. The tubular epithelial atrophy, which can be associated with impairment of renal blood flow (18), did not differ significantly between the age groups (Mann-Whitney U test).
Histology and growth hormone.
Two patients receiving GH lost their grafts (one on account of nephrosis, the other after developing a hemolytic uremic syndrome). The graft survival rate of the GH-treated patients and that of patients not receiving GH did not differ. No late acute rejections occurred in the GH treatment group. The groups did not differ with respect to the number of early acute rejections (1.3±1.1 vs. 0.9±0.8, respectively, P =NS, unpaired t test). GFR did not differ significantly between the groups. The mean CADI was slightly higher in the group receiving GH at 3 (8.3±5.7 vs. 5.1±4.3) and 5 years (9.6±5.4 vs. 5.6±5.0), but the difference was not statistically significant.
Seventy-five percent of the patients were without anti-hypertensive treatment 1.5 years after Tx. At 3 years, this proportion had increased to 87%, and no patient was dependent on anti-hypertensive treatment after 5 or 7 years.
This prospective study is one of the first investigations detailing long-term development of histopathological changes of chronic allograft nephropathy in small children. The histological changes encountered were mild, but were influenced by the number of acute rejections, and these changes 3 years after Tx were predictive of future GFR. In contrast to most other reports, in which chronic rejection is the leading cause of long-term graft loss (1, 6) the majority of the lost grafts (5/7) in our patients were because of posttransplantation nephrosis in NPHS1 patients. In patients who received transplants before the age of 2, graft function was inferior to that seen in patients who received transplants after the age of 2 years, although histopathological changes were evenly distributed.
Glomerular mesangial expansion, tubular atrophy, interstitial fibrosis with moderate inflammation, and intimal hyperplasia have been described previously in connection with chronic allograft nephropathy (19). In our patients, the incidence of interstitial inflammation with focal lymphocytes diminished between 1.5 and 7 years after Tx. This may have reflected a slight decrease in immunoactivation, even though renal function is stable and FNAB were normal or had normalized. Diffuse fibrosis, which is regarded as irreversible, increased with time. The incidence of mild tubular epithelial atrophy seemed to decrease with time. This may reflect an adaptation process of the kidney or merely a beginning of an irreversible process leading to extinction of the tubuli together with the development of fibrosis. The casts in the biopsies were almost exclusively hyaline casts. The diagnostic value of casts in renal biopsies are limited (18). In this context, it is likely that it connects to the tubular atrophy. The incidence of arterial intimal proliferation tends to increase with time and the arteries are more affected than the arterioles. The latter observation is more compatible with immunoactivation than with CsA toxicity (20). Intimal thickening without hypertension is also suggestive of chronic rejection (14). Hypertension was not a problem in our patients; 87% were without anti-hypertensive treatment at 3 years, and none received treatment after 5 or 7 years.
Most of the biopsies (52–69%) were regarded as normal according to the BC (14), and the proportion with chronic allograft nephropathy did not substantially increase with time (Table 3). In a study by Broyer et al. (21), 53% of pediatric CAD recipients with a long-term functioning graft had chronic transplant nephropathy grade I at 12–18 months after Tx. The most prominent histopathological changes in our study were consistent with the report by Isoniemi et al. (16), who studied the histopathological findings in adult long-term renal allografts. In that study, the mean CADI score was 1.5 (scale: 0–18) in adults with triple immunosuppression, 2 years after Tx. In our patients, the median CADI scores ranged from 2.5 to 4.0 (scale: 0–36).
Acute rejection is one of the major risk factors for development of chronic rejection, both in adult studies and among pediatric CsA-treated recipients (4, 5). In our study, patients with one or more rejection episodes, even though mostly slight and steroid-responsive, had higher CADI at every subsequent follow-up compared with those without rejection episodes. The increase in the mean CADI (ΔCADI) between 1.5 and 5 years was also greater in the group with one or more rejection episodes than in the group without rejection episodes. Thus, it seems that the histopathological changes caused by early acute rejection episodes progress with time and that early immunological activation initiates a cascade of events leading to tissue destruction (22). In our patients, graft matching, cold ischemia time (CAD recipients), and donor age had no effect on histopathology.
GH treatment might carry the risk of late acute rejection and influence long-term graft function (23). No late acute rejections were noted in our patients receiving GH. GFR was also similar in the two groups receiving GH and not receiving GH. CADI was slightly higher in the GH treatment group, but not significantly so.
Signs of acute CsA toxicity were rarely seen. This is understandable in view of the moderate CsA doses used. CsA doses or blood levels did not correlate with any histological parameter. The patients’ CsA doses were also continually adjusted according to blood trough levels, functional parameters of toxicity (8, 24, 25), and histology.
It is conceivable that these mild histological findings had a minor effect on graft function. However, when combined in the CADI score, they show a negative correlation with function. Furthermore, patients with more chronic changes according to the BC had inferior function. The CADI score has previously been shown to have a predictive value for later deterioration of graft function (26). In our patients, despite low CADI scorings, 3 years after Tx they still correlated inversely with later graft function and in patients with a higher CADI score graft function was worse at later follow-up.
The difference in histological findings between CAD and LRD recipients was small. CADI was significantly higher in the CAD recipients only at 3 and 5 years. The CAD recipients also had significantly more acute rejections. However, the difference in absolute GFR between the groups was not significantly different. The absolute GFR also remained stable with time.
We have shown earlier that patients under 2 years of age at Tx have a lower GFR than patients transplanted between 2 and 5 years of age (8). Analyses of the different risk factors did not give a clear explanation for this difference. In addition, the younger patients only maintained their absolute GFR during follow-up, in contrast to the older patients, in whom we documented a slight improvement in GFR with time. The CADI is similar in these groups. The number of acute rejections are also equally distributed. The number of patients in the study is unfortunately too small to allow further stratifying of the data, according to both age and recipient group. However, the majority of the patients in the group transplanted at <2 years of age are LRD recipients (73%), who have a slightly better function than the CAD recipients.
It has recently been proposed by Salvatierra et al. (27) that maintenance of high aortic blood flow is critical in avoiding early posttransplantation complications (e.g., acute tubular necrosis, graft thrombosis and primary graft nonfunction), in infants receiving adult-sized kidneys (ASK). In our study, we recognized one graft loss on account of acute tubular necrosis. Acute tubular necrosis was also experienced in five other patients, only one of them under the age of 2 at Tx. It is tempting, however, to speculate that the discrepancy in blood flow in small children and the need of an adult-sized kidney also leads to a greater extent of irreversible long-term functional loss of the transplanted kidney as compared with older children.
Global glomerular sclerosis increased significantly with time. The mean PSG was only 3% at 1.5 years, but increased to 36% at 5 years. The mean age of the donor kidneys was close to 30 years. The normal incidence of PSG up to 40 years of age in native kidneys is close to 2%, and an incidence of PSG greater than 10% in this age group is considered to be disease-related rather than related to normal senescence (28). Allografts from donors >40 years of age did not have significantly more sclerosis than allografts from <40 years of age (data not shown). The effect of donor age is difficult to evaluate, however, as very few marginal donors (i.e., very young or old) were used.
In our patients, the increasing glomerular sclerosis is probably secondary to functional and structural adaptation of the glomeruli after loss of functioning nephrons leading to hyperfiltration (29, 30). It has been shown that the ASK adapts its function to the size of the recipient. The mechanism behind the functional adaptation is, however, not clear (31). Lower perfusion in the very young recipient probably leads to irreversible loss of functioning nephrons in the ASK. Glomerular sclerosis increased markedly with time in all patients, but especially in the youngest recipients. This could be explained by greater hyperfiltration in the remaining nephrons with growth of the child. This process is self-perpetuating, as further glomerulosclerosis leads to additional loss of functioning nephrons. It could be speculated that, in small children, recipient functional and structural adaptation after renal allograft Tx is even more important for long-term success than immunological disparity (32).
Early reversible acute rejections have an impact on later chronic histopathological changes, which in turn have an impact on graft function. It seems important to treat acute rejections effectively, in order to minimize the amount of initial damage done to the graft. On the other hand, we must beware of the nephrotoxicity of immunosuppressive treatment (33). We have used protocol biopsies as a safe complementary tool for detecting possible signs of CsA toxicity and to evaluate the progression of chronic allograft changes. Based on these, and the clinical picture, we have adjusted the CsA medication accordingly. The histopathological changes also have a predictive value for later deterioration in function.
The youngest recipients (<2 years of age) form a special risk group. Our rate of early postoperative complications is low and graft survival is good. The recipients <2 years of age did not have more acute rejection episodes, nor did they have more chronic allograft histopathological changes, than recipients between the ages of 2 and 5 years. Nevertheless, recipients <2 years of age receiving an adult kidney seem to lack the capacity to improve graft function in response to growth, in contrast to older recipients. Further investigations are needed to improve our understanding of the nonimmunological structural, functional, and adaptation problems encountered in small children.
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