ADOLESCE-NT: More rapid deterioration of renal function after renal transplantation in adolescence: effect of the endocrine system on immunological and pharmacokinetic factors

Regarding renal transplantations at childhood age, transplant survival for
transplantation at ages 14-24 years is inferior to that at other ages, as
appears from data of transplantations performed between 1990 and 2000 in the
Eurotransplant region (unpublished data) and other studies. Evaluation of more
recent transplantations in the Collaborative Transplant Study has confirmed
this (http:/www.ctstransplant.org).

In the first months after transplantation the incidence of acute rejections in
the age group 12-16 years is higher than that in the younger recipients.
Furthermore, the percentage of patients with complete reversal of acute
rejection episodes is less in adolescents than in younger age groups.
Transplant survival in the first phase after transplantation does not differ
between age groups; differences develop over the course of the years. Still,
acute rejections in the early phase after transplantation may affect survival
of the transplant kidney on the long term, which suggests there may be a
relationship between the two phenomena.

The psychological changes in adolescence, manifested as desire for liberty and
independence, may well result in poorer therapy compliance, which no doubt may
play a role in the poorer transplant survival. A review on compliance showed a
mean prevalence of non-adherence of 43% in adolescents, a significantly higher
proportion than in younger patients and to mixed pediatric/adolescent
populations (22%). Twenty-three percent of late acute rejection episodes in
pediatric kidney transplants and 14.4% of all graft losses have been reported
to be associated with non-adherence. However, one must take into account that
quantification of non-adherence is difficult since no accepted and reliable
method for assessment is currently available. Assessment of utilization of
medication such as pill counts and assessment of prescription refill rates are
often used in clinical drug trials. MEMS (medication event monitoring system),
another, quite expensive method used, works via electronic monitoring devices
attached to medication container caps and records the time and day the bottle
is opened and closed. All mentioned techniques have in common that they do not
measure the actual drug ingestion.
Despite the shortcomings of subjective assessment, self-reporting represents
the most utilized and probably the most cost-effective way to monitor adherence
in clinical settings. Self-report at a confidential interview has been best
measured for detection of both missed doses and erratic timing of medication.
Unfortunately, for obvious reasons patients may be reluctant to disclose
non-adherence to their physician. Disclosure to an independent researcher seems
to be more accurate than to clinical staff.
Finally, blood drug level monitoring may be helpful when a patient*s *through*
level is either inexplicably low or high or variable.

Nevertheless the higher incidence of acute rejections in the first months
after transplantation can most likely not be ascribed to the poorer therapy
compliance, as this is expected to exert effects on the longer term. There are
no reports describing whether the increased incidence of acute rejections and
the more rapid deterioration of graft function at these ages can be explained
by other factors. The enormous changes in hormonal status in adolescence could
affect the immunological reactivity or the pharmacokinetics of
immunosuppressants. Activation of the immune system occurs in many other
diseases in this period: autoimmune diseases such as SLE and Wegener*s
granulomatosis often start to flare up in this period; the minimal changes
nephrotic syndrome often exacerbates in this period before abating in
adulthood. The starting point of this study is therefore the hypothesis that
the inferior transplant survival in adolescence is caused by altered
immunological reactivity.

Development of the immune system
T-cells play an important role in the acceptation and rejection of a
transplanted kidney. T-cells mature in the thymus, an organ that grows until
pubertal age and then gradually involutes, during which process epithelium is
replaced with fatty tissue. T-cells are released from the thymus as naive
T-cells that have not yet been in contact with alloantigens. In the circulation
they slowly proliferate in the absence of antigenic stimuli, the so-called
homeostatic proliferation. In the presence of antigenic stimuli a proliferation
burst may occur.

Reference values of the most important lymphocyte subpopulations that circulate
in the blood (CD3/CD4/CD8 positive T-cells, B-cells, and NK cells) show only
slight changes after the 10th year of life. Also the levels of circulating
immunoglobulines A, G and M show little variation after this age.3Still there
are indications that also at this stage of life an important development takes
place in the lymphocytic system, notably within the T-cell compartment: the
number of naive T-cells goes down, whereas the number of memory-T-cells goes
up.

The naive T-cells / memory T-cells ratio can be estimated by measuring the
concentration of T-cells that contain TRECs. TRECs, T-cell receptor excision
circles, are DNA molecules generated as a by-product during recombination of
the TCR genes, and which are not duplicated during cell division. As a result,
at mitosis the TREC is passed on to one of the two daughter cells. The TREC
level decreases with ageing, as a result both of the reduced production of
T-cells and the many cell divisions which T-cells in the immune system undergo
during an individual*s life. There is a very good correlation between age and
TREC levels in adults, which is being used in forensic medicine. In certain
circumstances (diseases, persistent viral infections) the immune system will
age (more rapidly). On the basis of the TREC levels an individual*s
immunological age can then be estimated and plotted against the chronological
age. In puberty the TREC level might occasionally be extremely high, among
other things due to the effect of growth hormone on the thymus (see below). The
larger number of naive T-cells could result in a stronger immunological
reactivity to a transplanted kidney.

In the present study we will investigate, apart from the TREC levels, the
distribution and activity of the different T-cell subsets in the circulation.
Activity will be investigated of the memory T cells in particular, i.e. the CD4
T effector memory cells (Tem) and the central memory T cells (Tcm), and of
regulatory T-cells (Tregs). Once Tems have been in contact with a certain
antigen, they will proliferate and produce cytokines at renewed contact. In
this way they activate the immune response. In addition we will study the role
of regulatory T-cells (Tregs). Tregs, CD4+CD25+CD127-FoxP3+, play an important
role in maintaining the equilibrium in the immune response. These cells are
capable of controlling unwanted immune responses, for example in autoimmune
processes, and in rejection of transplanted organ. The question is how the
ratio of memory and regulatory T-cells changes during the different
developmental stages in childhood. It may well be that in adolescence, under
the influence of hormonal changes, the Tem and the Tcm are activated while the
Treg are still in a resting phase, which may result in a fiercer immune
response against the transplanted kidney. We hypothesize that this is the most
active period, compared to the pre-adolescence and the young adult phases.

Hormonal changes in children with chronic kidney disease and after renal
transplantation.
It is well known that adolescence is characterized by major hormonal changes,
regarding both the hypothalamus-gonadal axis and the growth hormone axis.

Puberty of children with chronic kidney disease (CKD) is delayed by about 2
years. Boys reach their final height at a median age of about 20 years, girls
at 18 years, versus median 18 and 16 years, respectively, in healthy children.
The growth spurt is shorter and less pronounced, so that the final height is
clearly shorter than in healthy young people. In 35-60% of the children, final
height is less than 2 standard deviations below average. The delayed puberty
development manifests itself in delayed bone maturation, which after
transplantation is even more delayed with corticosteroids use. This is why most
children after a successful renal transplantation will continue to grow for a
longer period. The hypothalamus-gonadal axis is suppressed notably in the
pre-transplantation phase, and is in the subnormal range in the transplanted
adolescent, especially in the first months after transplantation. This leads to
the delayed sexual maturation in children with CKD. The average age of menarche
in these girls mainly depends on the age at which transplantation is performed
during pubertal development , but is later in comparison with the 13.3 years in
healthy girls. High to very high growth hormone levels are also found during
the pubertal growth spurt in children after transplantation, comparable with
the levels in healthy children during puberty.

Relationship endocrinology - immunology in adolescence
If the sex hormone development should be the cause of the difference in
transplant survival, it is to be expected that a difference between boys and
girls would occur in puberty, and which thereafter would persist. 7-year
transplant survival grouped by age and sex, is in the 12-14 years age group
already decreased in girls, who reach puberty earlier than do boys. In boys the
decline does not occur until age 14-16 years. In this age category there is no
longer a difference between boys and girls. Also in adults there is no
difference in transplant survival between men and women, so we may assume that
sex hormones do not play an important role in the puberty effect.

There are, however, clear indications that the growth hormone axis affects the
immune system, and notably the thymus. Growth hormone (GH) acts through the GH
receptor, which is in the cytokine receptor family. Theoretically it is
possible that the high GH levels during and after the maximum growth spurt
influence the other cytokine receptors and consequently exert a disturbing
effect. To the best of our knowledge this has not been investigated in healthy
adolescents nor adolescents with kidney transplants so far. The substantial
levels of GH and growth factors such as IGF-I and the binding proteins during
puberty possibly influence graft acceptation at pubertal age.

Associations between GH, IGF-1 and the immune system have been reviewed
extensively. GH is produced in the pituitary gland. The major role of GH
obviously is promoting growth, which is achieved by stimulation of the
production of IGF-1 in the liver, which through exerting action on the growth
plates results in increased body height. In addition, however, GH and IGF-1 are
also involved in the immune system. Here, too, GH acts through the GH
receptors, which are present on cells of many types, including lymphocytes in
the thymus and thymus epithelium. Receptor cleavage leads to signal
transduction via JAK2 and subsequently via a number of members of the STAT
family, i.e. 1, 3, 5a and 5b. Activated STAT5 may tone down a large part of the
immunosuppressive effect of corticosteroids on lymphocytes 22. JAK2 is also
stimulated by cytokines such as interleukin (IL)-3, -5, -6, -12, -13,
prolactin, erythropoetin and interferon -γ. If the extracellular domain of the
GH receptor is cleaved, it can circulate in the blood as IGF binding protein
(IGF-BP 1 t/m 6), which competes with the receptors. In humans and experimental
animals with GH-deficiency, GH therapy leads to growth of the thymus and the
cell populations therein. GH stimulates the repopulation of the thymus also
after damage to the thymus by radiation or treatment with cytostatics. The
total number of lymphocytes increases, but the ratio of CD4+ and CD8+ cells
does not alter. Also the number of circulating T-cells which just have been
released from the thymus, assessed from the TREC level, increases.
These considerations support our hypothesis that a strong activity of GH as
occurs in adolescence may increase the risk of acute and chronic transplant
rejection by generalized activation of the T-cell system, although a specific
effect is not recognized.

On the other hand, however, an in vitro study has shown that purified T-cells
under the influence of IGF-1 produced the Th2 cytokines IL10 and IL4, not the
inflammatory cytokines IL-2, IL-5, IL-6, interferon -γ and IL-1β, IL-8, tumor
necrosis factor alpha. This would, contradictory to our hypothesis, precisely
point at an anti-inflammatory role of IGF-1. T-cell subsets are established and
undergo dramatic changes during childhood. Reference values are determined in
children aged 0-18 years, but have not been compared with hormonal status. Some
parameters are highly variable in adolescents (Langerak, pers comm.). A study
in healthy adolescents might provide the answers whether or not there is a
relation between the immune system, specifically T-cells, and hormonal status
of hormones such as GH and sex hormones, independent of factors connected to
kidney disease, that might be of influence on this relation.

Also multicenter clinical research on GH treatment for small body height in
children after renal transplantation has not evidently documented an increase
in the incidence of acute rejections. However, the following prerequisites for
treatment applied: proper suppression of the immune system with daily doses of
prednisolone, rather than every other day; and not any rejection episode in the
year before start of treatment. For that matter, GH treatment is rarely started
earlier than after one year after transplantation.
Of the lymphocyte subsets, only B-cell and CD25+ T-cell levels are lower during
treatment with GH as compared with the preceding period. A mixed lymphocyte
culture (MLC) of pediatric recipients and their living kidney donors showed
hyporesponsivity after transplantation; addition of GH to the medium resulted
in an MLC of healthy adults in strong stimulation of the proliferative and
cytotoxic response and the production of interferon -γ, whereas this effect was
seen in only 3 of the 20 pediatric recipients.

In summary, in vitro and in vivo studies provide arguments pro and con about an
immuno-activating effect of GH. The present study will investigate whether
there is a relationship between hormone development, particularly that of the
GH axis, and the immunological activity in children and young adults.

Pharmacokinetics and -genetics
The concentration-time curves that are yearly prepared for all transplanted
children in Erasmus MC-Sophia, at first sight show with regard to cyclosporine
and MMF no differences with age. Regarding tacrolimus, the eldest children (15
until 18 years) show a higher trough level and a higher curve at a similar
relative starting dose, although the same trough level is aimed at (4-8 mcg/l).

In spite of comparable or even higher blood concentrations, the
pharmacokinetics could be different, e.g. as a result of the effect of the drug
on and in the cells.
Most studies of the age effect on pharmacokinetics and -dynamics describe a
special position of only the very young children, while the adolescents show no
peculiarities in metabolism or bioavailability. One study, in children treated
with TCL and MMF, without steroids, reports that children older than 12 years
require less tacrolimus than do the younger children. This is confirmed by our
own findings. Several factors have been proposed to explain this age effect,
such as difference in distribution volume, in relative liver size and thus rate
of conversion, and in glomerular filtration rate.

Regarding the individual enzymes involved in absorption and metabolisation of
the immunosuppressives: CYP 3A4 reaches adult levels around the end of the 1st
year of life, the glucuronidating enzymes UGT1A9 and UGT2B4 shortly after the
2nd year of life. Contrasting findings have been reported for ABCB1, which is
negatively involved in the absorption of calcineurin inhibitors in the bowel:
on the one hand a persistently low level until the 10th year and next
increasing levels until the 55th year of life; on the other hand a maximum
level at birth and next a decrease until a stable level at the age of 0.5 to 2
years. An age-dependent effect of ABCB1 polymorphisms (ABCB1 c.2677GG genotype,
c.2677GT genotype c.2677TT genotype and ABCB1 c.1236C>T genotype) on the
bioavailability of cyclosporine has been described, with the largest effect in
adolescence. Also the CYP3a4 intron 6 C>T polymorphism and CYP 3a5*3 are
associated with altered tacrolimus and cyclosporine A metabolism. The
literature does not contain reports of evident effects of sex hormone or GH on
bioavailability or metabolism of calcineurin inhibitors or MMF. Our own data
show a slight difference in trough levels versus relative dose of tacrolimus
between adolescents and younger children, and boys and girls in puberty. The
numbers of patients are too small, however, to draw conclusions.

In conclusion, the data collected in our own clinical experience and from
literature indicate that the hormonal changes in adolescence affect
immunological reactivity and pharmacokinetics. Nevertheless, the data are not
unanimous concerning the direction and the size of these effects. Therefore a
study is justified to evaluate the relation of hormonal, immunological and
pharmacokinetic parameters in a group of kidney transplant recipients in
adolescence or the adjacent age groups, with comparison to healthy controls.

Answering the question if there are any associations between hormonal status in
adolescence on the one hand, and immunological reactivity and pharmacodynamics
on the other hand; and if such associations can explain the more rapid
deterioration of renal function and loss of the graft.

Prospective cross-sectional (single center) and longitudinal observational
(multicenter) study.

Burden for the patientpopulation is very low and consist of:
1. a maximum of 3 venipunctures, which are combined with the regular controls.
2. a maximun of 3 X-rays of the hand, of which 2 are usually made within the
regular controls. Therefore at least 1 additional x-ray will be made.

Burden for the controlgroup consists of 1 venapuncture and 1 X-ray of the hand.

Time patients have to spent for participation is little.

The risks of participation are negligible.
Risks of venipuncture are hematoma, pain, bleeding, and in immune compromised
patients there is a very small risk of infection.
Risk of hand X-rays, in particular due to the radiation exposure. This exposure
is very small.

The burden and risks of participation therefore are very small