NEPHROLOGY - REVIEW

What is the impact of immunosuppressive treatmenton the post-transplant renal osteopathy?

Kristina Blaslov • Lea Katalinic • Petar Kes •

Goce Spasovski • Ruzica Smalcelj • Nikolina Basic-Jukic

Received: 14 August 2013 / Accepted: 22 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Although glucocorticoid therapy is considered

to be the main pathogenic factor, a consistent body of

evidence suggests that other immunosuppressants might

also play an important role in the development of the post-

transplant renal osteopathy (PRO) through their pleiotropic

pharmacological effects. Glucocorticoids seem to induce

osteoclasts’ activity suppressing the osteoblasts while data

regarding other immunosuppressive drugs are still contro-

versial. Mycophenolate mofetil and azathioprine appear to

be neutral regarding the bone metabolism. However, the

study analyzing any independent effect of antimetabolites

on bone turnover has not been conducted yet. Calcineurin

inhibitors (CNIs) induce trabecular bone loss in rodent,

with contradictory results in renal transplant recipients.

Suppression of vitamin D receptor is probably the under-

lying mechanism of renal calcium wasting in renal trans-

plant recipients receiving CNI. In spite of an increased

1,25(OH)2 vitamin D level, the kidney is not able to

reserve calcium, suggesting a role of vitamin D resistance

that may be related to bone loss. More efforts should be

invested to determine the role of CNI in PRO. In particular,

data regarding the role of mammalian target of rapamycin

inhibitors (mTORi), such as sirolimus and everolimus, in

the PRO development are still controversial. Rapamycin

markedly decreases bone longitudinal growth as well as

callus formation in experimental models, but also lowers

the rate of bone resorption markers and glomerular filtra-

tion in clinical studies. Everolimus potently inhibits pri-

mary mouse and human osteoclast activity as well as the

osteoclast differentiation. It also prevents the ovariectomy-

induced loss of cancellous bone by 60 %, an effect pre-

dominantly associated with a decreased osteoclast-medi-

ated bone resorption, resulting in a partial preservation of

the cancellous bone. At present, there is no clinical study

analyzing the effect of everolimus on bone turnover in

renal transplant recipients or comparing sirolimus versus

everolimus impact on bone, so only general conclusions

could be drawn. Hence, the use of mTORi might be useful

in patients with PRO due to their possible potential to

inhibit osteoclast activity which might lead to a decreased

rate of bone resorption. In addition, it should be also

emphasized that they might inhibit osteoblast activity

which may lead to a decreased bone formation and ady-

namic bone disease. Further studies are urgently needed to

solve these important clinical dilemmas.

Keywords mTOR � Sirolimus � Everolimus �Glucocorticoids � Calcineurin inhibitors � Post-

transplant osteopathy � Adynamic bone disease

Introduction

Renal transplantation is a treatment of choice for many

patients with chronic kidney disease (CKD) that restores

K. Blaslov � L. Katalinic � R. Smalcelj � N. Basic-Jukic (&)

Department of Nephrology, Arterial Hypertension, Dialysis

and Transplantation, University Hospital Centre Zagreb,

Kispaticeva 12, 10000 Zagreb, Croatia

e-mail: nina_basic@net.hr

P. Kes � N. Basic-Jukic

School of Medicine, University of Zagreb, Zagreb, Croatia

G. Spasovski

University Department of Nephrology, Medical Faculty,

University of Skopje, Skopje, Macedonia

e-mail: spasovski.goce@gmail.com

N. Basic-Jukic

School of Medicine, University of Osijek, Osijek, Croatia

123

Int Urol Nephrol

DOI 10.1007/s11255-013-0596-7

exocrine, endocrine and metabolic kidney function. How-

ever, it is only partially efficacious for renal osteodystrophy

as part of the bone and mineral disorders [1], yet with many

unanswered questions [2]. Thus, after successful kidney

transplantation and a good graft function, up to 60 % of renal

transplant recipients experience rapid bone loss, with a

fracture rate up to 10 % associated with severe morbidity and

mortality [2, 3]. This is mostly due to the development of

post-transplant renal osteopathy (PRO), a condition repre-

sented with several different clinical and histological skel-

eton features characterized by imbalance in bone turnover

and bone resorption [2]. Preexisting bone abnormalities,

parathyroid hormone status, calcium, phosphorous and

magnesium disorders, type, dose and duration of immuno-

suppressive agents required to prevent graft rejection are

considered as most important risk factors for such PRO

development [4]. Although glucocorticoid treatment is the

principal contributor to PRO development, a consistent body

of literature evidence suggests that other immunosuppres-

sants [calcineurin inhibitors (CNIs), mycophenolate mofetil

(MMF) and proliferation signal inhibitors (PSI)] might also

play an important role in PRO pathogenesis through their

pleiotropic pharmacological effects [1, 5–8]. On the other

hand, data regarding the role of mammalian target of rapa-

mycin inhibitors (mTORi), such as sirolimus and everolimus,

in the PRO development is still controversial.

Post-transplant renal osteopathy

Bone turnover is a well-established and balanced interac-

tion cycle between two major bone cells: osteoclasts and

osteoblasts. First, osteoclasts erode the bone, and thereaf-

ter, osteoblasts fill in the resorbed area with osteoid matrix

that eventually becomes mineralized to form a new bone.

Parathyroid hormone (PTH), vitamin D and mineral con-

centrations directly affect bone cells in order to tightly

couple bone removal and bone replacement. Subsequently,

the abnormality of any of these parameters results in

changes in bone turnover, mineralization or growth. Nev-

ertheless, the combination of all these factors can be found

in chronic kidney disease but also in successfully trans-

planted patients with an estimated GFR up to 60 ml/min

[9], resulting in PRO that is manifested through at least

three main components: osteopenia–osteoporosis, osteo-

necrosis and bone pain [8]. Moreover, although the kidney

transplantation is supposed to slow endocrine disturbances,

concurrently, the persistent PTH overproduction and its

high levels may be sometimes a common feature in the first

months after transplantation [8–14]. PTH directly stimu-

lates osteoclasts, and bone resorption with subsequent

hypercalcemia and hypophosphatemia (due to hyperphos-

phaturia) along with the decreased osteoblast activity

related to glucocorticoids may consequently develop oste-

openic-osteoporotic syndrome [15]. Furthermore, several

studies emphasize an important role of magnesium in bone

metabolism, and hypomagnesemia might also be found in

renal transplant recipients [16]; it is involved in bone for-

mation and influences the activities of osteoblasts and

osteoclasts [17]. Magnesium also affects the concentrations

of both PTH and the active form of vitamin D, which are

major regulators of bone homeostasis. Several population-

based studies have found positive associations between

magnesium intake and bone mineral density in both men

and women [18]. Other study research found that women

with osteoporosis have lower serum magnesium levels than

women with osteopenia and those who do not have oste-

oporosis or osteopenia [19]. These and other findings

indicate that magnesium deficiency might be a risk factor

for osteoporosis in general population as well as in renal

transplant recipients [16, 17].

Finally, the unavoidable use of immunosuppressive

agents in order to overcome acute and chronic transplant

rejection does not contribute positively to the bone

metabolism. Glucocorticoids seem to induce osteoclasts

activity suppressing the osteoblasts while data regarding

other immunosuppressive drugs such as cyclosporine and

tacrolimus are still controversial [1, 6–8, 20]. Nevertheless,

mycophenolate mofetil and azathioprine appear to be

neutral regarding the bone metabolism [21]. However, the

study analyzing any independent effect of antimetabolites

on bone turnover has not been conducted yet.

Calcineurin inhibitors in post-transplant renal

osteopathy

Since early reports have linked PRO mainly to glucocorti-

coid excess, spared regiments including calcineurin inhib-

itors such as cyclosporine A (CsA) and tacrolimus were

developed. The introduction of CsA to post-transplantation

regimens was associated with a great reduction in rejection

episodes and improved survival. CsA inhibits calcineurin, a

T cell phosphatase, and reduces T cell function via sup-

pression of regulatory genes expressing products such as

interleukin 2, interleukin receptors and the proto-oncogene,

H-ras, and c-myc [22]. The data of several experimental

studies suggest that CsA inhibits bone resorption in cultured

bone [23, 24]. However, CsA administration in in vivo

rodent caused severe bone loss, particularly in trabecular

bone, that was associated with marked increases in both

bone resorption and formation, with increased levels of

osteocalcin and 1,25-(OH)2D3, suggesting that CsA has

independent adverse effects on bone and mineral metabo-

lism that could contribute to bone loss after renal trans-

plantation [22]. In addition, there are data suggesting that

CsA may cause bone loss by direct effects on calcineurin

Int Urol Nephrol

123

genes expressed in osteoclasts [25] or indirectly via alter-

ations in T cell function [26]. Nevertheless, the effects of

CsA on the human skeleton are still unclear, particularly in

view of reports that kidney transplant patients receiving

CsA in a steroid-free regimen do not appear to lose bone

mineral density [27, 28]. Tacrolimus, another calcineurin

inhibitor that inhibits cytokine gene expression, T cell

activation and T cell proliferation, also causes trabecular

bone loss in the rat [22]. Fewer studies have evaluated the

skeletal effects of tacrolimus in humans. However, liver

transplant recipients taking tacrolimus had significantly

higher femoral neck bone mineral density 2 years after

transplantation than those receiving CsA [29], and addi-

tionally, patients on tacrolimus received lower prednisone

dose that might be beneficial for the post-transplant bone

metabolism. Since hypomagnesemia occurs frequently in

tacrolimus-treated patients, Navaneethan et al. [16] studied

the correlation between renal magnesium wasting and ta-

crolimus blood levels in renal transplant patients. They

measured serum magnesium, fractional excretion of mag-

nesium (FEMg) and 24-hour urinary excretion of magne-

sium in 41 transplant patients and 10 healthy volunteers for

correlation with tacrolimus level and reported that serum

magnesium levels correlate inversely with tacrolimus con-

centrations and creatinine clearance. Additionally, Lee et al.

[30] conducted an animal study to investigate the effects of

CsA and tacrolimus on renal calcium, magnesium and

vitamin D metabolism and reported a two- to threefold and

1.6- to 1.8-fold increase in urinary calcium and magnesium

excretion, respectively. Moreover, they observed elevation

in serum 1,25(OH)2 vitamin D without affecting the PTH

level as well as reduced mRNA of vitamin D receptor

(VDR). They suggested that suppression of VDR by calci-

neurin inhibitors is probably the underlying mechanism of

renal calcium wasting. In spite of an increased 1,25(OH)2

vitamin D level, the kidney is not able to reserve calcium,

suggesting a role of vitamin D resistance that may be related

to bone loss.

Mammalian target of rapamycin inhibitors (mTORi)

in transplantation: What we do/not know?

Mammalian target of rapamycin (mTOR) is a serine/thre-

onine kinase that regulates cell-growth-related processes:

mRNA translation, ribosome biogenesis, autophagy and

metabolism in response to nutrient and energy status [31].

It is composed of two distinct signaling complexes: mTOR

complex 1 and mTOR complex 2 [32]. The structural and

functional difference between them remains unclear [31].

Studying the function of antifungal agent rapamycin (si-

rolimus), it is established that it acts through mTORC1

inhibition so it is a part of mTOR we are referring when

talking about mTOR inhibitors in clinical medicine [33].

Aberrantly elevated mTOR activity is a common molecular

defect detected in majority of human cancers, under the

condition of obesity and in genetic syndromes with high

incidence of cognitive deficits [34–36]. Exploring the

antiproliferative and antimigratory effect of mTORi in

human cancers, it has been noticed that they also have a

potent immunosuppressive effect by regulating the growth

and proliferation of T2 cells, which gives them a poten-

tially protective role in renal graft dysfunction and post-

transplant de novo tumorigenesis [37]. Nowadays, siroli-

mus (SIR) and everolimus (EVE) are anti-mTOR drugs

commonly used in the transplantation medicine.

Sirolimus is the generic name for the natural product

rapamycin. It was the first mTORi introduced in the clin-

ical practice in the late 1990s when global phase III clinical

trial comparing sirolimus and placebo in combination with

cyclosporine and corticosteroids in de novo transplant

recipients has shown both 6 months and 1 year lower rate

of biopsy confirmed acute rejection in the sirolimus-treated

group [38]. Additionally, sirolimus has been demonstrated

to prolong graft survival in various animal models of

transplantation for both heterotopic and an orthotopic

organ grafting [39]. However, despite these beneficial

effects, sirolimus has a synergistic effect with the cyclo-

sporin-induced nephrotoxicity, and a prolonged combina-

tion of these two drugs inevitably leads to progressive and

irreversible renal allograft damage [40]. In order to over-

come these difficulties and to improve the bioavailability,

new sirolimus analog, 40-O-hydroxyethyl-sirolimus,

named everolimus has been developed [41]. Phase III

clinical trials comparing everolimus with MMF in patients

treated with cyclosporine and corticosteroids showed sim-

ilar incidence in acute rejection and graft survival, but

higher creatinine values in the everolimus group [42, 43].

However, after cyclosporine dose adjustment, the renal

function significantly improved [43]. At present, there are

several clinical studies that suggest that everolimus treat-

ment in renal transplant recipients improves renal function

with no differences in treatment failure, i.e., acute or

chronic rejection [44]. On the other hand, mTOR inhibitors

cause numerous side effects [45]. Hence, dyslipidemia,

proteinuria, diabetes, edemas and pneumonitis caused by

the use of SIR and EVE are well-established conditions,

but the drug impact on bone metabolism, i.e., PRO bone

disease, remains as an open issue in the present trans-

plantation medicine evidence [37].

The impact of mTORi on bone metabolism: future

perspectives based on the experimental data

It has been well established that mTORi exhibit both

antiproliferative and antiangiogenic activities; thus, their

Int Urol Nephrol

123

interference with bone metabolism is unavoidable. Alva-

rez-Garcia et al. [46] explored the effect of rapamycin on

growth plate in young rats. Four-week-old male rats were

receiving 2 mg/kg per day of intraperitoneal rapamycin or

vehicle for 14 days. Rapamycin markedly decreased bone

longitudinal growth rate (94 ± 3 vs. 182 ± 3 lm/day),

which indicates that rapamycin can severely impair body

growth in fast-growing rats and distort growth plate

structure and dynamics. In the same year, Holstein et al.

[47] investigated the effect of rapamycin treatment on bone

repair in a murine closed femur fracture model. They

demonstrated that rapamycin treatment inhibits callus for-

mation after 2 weeks of fracture healing. In contrast, the

negative impact of rapamycin on fracture healing was

overcome after 5-week treatment. Thus, it is confirmed that

rapamycin only initially delays fracture healing, most

probably by inhibiting cell proliferation and neovasculari-

zation in the callus, and that it has potential to disrupt

vascular endothelial growth factor (VEGF). Additionally,

there is an assumption that rapamycin may also interfere

with insulin-like growth factor I (IGF-I) signaling [48]. To

further investigate the mechanisms of rapamycin action on

longitudinal growth, another experimental study on

4-week-old rats treated with rapamycin for 2 weeks was

performed by Alvarez-Garcia et al. [49]. Compared with

the control group, the rapamycin group had higher levels of

circulating IGF-I as well as the mRNAs for IGF-I and of

the receptors of IGF-I and growth hormone in the liver, but

not in the growth cartilage. So, it confirms that rapamycin

causes a state of resistance to endogenous IGF-I action, and

it suggests another mechanism of action of adverse effect

of rapamycin on the growth plate dynamics. Kneissel et al.

[50] examined the effect of everolimus on the mouse and

human bone cells in vitro and on bone in an ovariectomized

(OVX) rat model. Everolimus potently inhibited primary

mouse and human osteoclast activity as well as the osteo-

clast differentiation. Moreover, despite the in vitro anti-

proliferative activity of everolimus and the observed

inhibition of osteoblast differentiation, no detrimental

effects were detected at different skeletal sites in mature

OVX rats at doses up to 3 mg/kg/day. In addition, this

everolimus dose also prevented the OVX-induced loss of

cancellous bone by 60 %, an effect predominantly associ-

ated with a decreased osteoclast-mediated bone resorption,

resulting in a partial preservation of the cancellous bone.

Finally, all data presented clearly suggest that mTORi

might have inhibitory impact on bone turnover and that

they should be applied with high precaution in clinical

medicine, especially in patients with a previous high

fracture rate or in process of growth, i.e., in patients with

already impaired bone quality or pediatric population.

However, it has not been clear whether these experimental

Chronic kidney disease and pretransplant HPT

Glucocorticoids

POSTTRANSPLANTATION BONE DISEASE

HYPOPHOSPHATEMIA

CNi

mTORi

HYPOMAGNESEMIA+

+

+

+

+

? -

+

Fig. 1 Current knowledge about pathophysiology and immunosuppressant effect on post-transplantation renal osteopathy. CNI calcineurin

inhibitor, mTORi mammalian target of rapamycin inhibitors

Int Urol Nephrol

123

data could be completely extrapolated as for the kidney

transplant recipients. Hence, the performed clinical studies

with mTORi are partially in contrast to the experimental

data. Campistol et al. [51] analyzed the data of bone

turnover markers of two clinical phase 2 randomized, open-

label, parallel-group trials conducted in 19 centers in

Europe on renal transplant recipients receiving triple

therapy with either CsA or sirolimus in combination with

glucocorticoids and azathioprine/MMF. The two treatment

groups were well matched and did not show any significant

differences in medication that could affect bone metabo-

lism. During the first year after transplantation, mean uri-

nary excretion of N-telopeptides and average serum

osteocalcin were consistently lower in patients receiving

sirolimus compared with those receiving CsA. The lower

bone resorption markers suggest that sirolimus preserves

bone mineral density. However, despite the favorable bone

marker profile for sirolimus-treated patients in the trial, an

abnormally increased bone turnover and loss in both groups

was observed. The authors also highlighted a significantly

higher calculated GFR (9.8–15.1 %) and lower calcium

plasma concentration (2.39 ± 0.02 vs. 2.45 ± 0.02 mmol/L)

in the sirolimus-treated patients. Thus, it could indicate that

sirolimus-treated group had also a lower level of PTH,

which might have contributed to the lower rate of bone

resorption. So far, it could be acknowledged that there is no

clinical study analyzing the effect of everolimus on bone

turnover in renal transplant recipients treated with everol-

imus or sirolimus/everolimus comparison, so only general

conclusions can be made.

The current knowledge about pathophysiology and

immunosuppressant effect on PRO is presented in Fig. 1.

In conclusion, the use of mTORi might be useful in

patients with PRO due to their possible potential to inhibit

osteoclast activity directly or by improving kidney function

and subsequent PTH hormone status which might lead to a

decreased bone resorption rate. On the other hand, it should

be emphasized that they might inhibit osteoblast activity

which could lead to a decreased bone formation and ady-

namic bone disease. The data regarding calcineurin inhib-

itors are also controversial, especially since the studies that

exclude negative effect of corticosteroids are rare, but there

is more evidence for their negative impact on bone turn-

over. There is a need for further clinical studies urgently

evaluating this important clinical dilemma.

Conflict of interest None declared.

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