Evaluation the Risk Factors that are Associated with Osteoporosis in Post Kidney Transplantation in a Sample of Iraqi Patients

* Department of Clinical Pharmacy, College of Pharmacy, University of Baghdad, Baghdad, Iraq. ** Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad, Baghdad, Iraq *** Ministry of Health and Environment , Baghdad, Iraq. Abstract Renal transplantation is a principal treatment option for end-stage kidney failure. Bone loss and fracture are serious complication of kidney transplantation, associated with morbidity and mortality. The pathogenesis of post transplantation bone loss is multifactorial and complex. There are changes in the normal bone remodeling system which will lead to more accelerated osteoporotic changes compared to normal individuals. The current work aimed to investigate the incidence of osteoporosis in post kidney transplant patients when compared to the general population. And study the relationship between post kidney transplant immunosuppression therapy and osteoporosis and determine some biochemical changes. Also to evaluate the bone mass and the possible correlation between demographic data and the development of osteoporosis. A case control study, conducted in the Kidney Transplant Center – Medical City Complex during the period (from October 2018 till April 2019), seventy-five kidneys transplant patients were participated in the present study including (23 females &52 males). Apparently healthy seventy-five subjects were selected to participate as a normal group for comparison (control) including (35 females and 40 males). All participants were examined for their bone density using DEXA scan (T – score) and those with cut– point ≤2.5 were diagnosed as having osteoporosis (lumbar and hip bones were examined). The prevalence of osteoporosis and osteopenia was significantly higher in transplant patients compared to control for lumbar and hip bone (for lumbar bones: 33.3% vs. 2.7% for hip bones: 60% vs. 14.7%). Tscore was significantly lower in the transplant patients compared to control for both lumber (-1.9±0.8 vs. 1.1±0.7) and hip bones (-2.3±0.9 vs. -1.3±0.8).In logistic regression analysis; only gender and BMI were the predictors of osteoporosis for lumbar bone, while; the BMI and serum calcium were the predictors of osteoporosis for hip bones. In conclusion , Osteoporosis in post-renal transplant patients is high when compared to general population, only corticosteroids significantly increase risk of osteoporosis, biochemical marker serum level in post kidney transplant patients are significantly different when compared with the general population but did not increase risk of osteoporosis and Body mass index is a risk factor for both lumbar and hip bones osteoporosis ,while gender and serum calcium are risk factors for osteoporosis in lumbar and hip ;respectively.


Introduction
The primary function of Kidney is to maintain stable internal equilibrium by eliminating excess water, electrolytes, and other byproducts, through the formation of ultrafiltrate from the plasma by the filtration action of glomerulus system, which can serve as a system for reabsorption or section of other materials and byproduct (1) .
The main stay of treatment of end stage renal failure (ESRF) is either dialysis (temporary solution), or kidney transplant. Transplant result in better outcome in comparison with dialysis (2) . However, transplant had its drawbacks, including low availability of kidney from donors, use of chronic immunosuppressant medications and others factors; these factors together with superiors outcome compared to dialysis lead to the development of criteria for selective candidates for the operation to include as many as possible patients to benefit from transplantation (3) .
In order to prevent graft loss that caused by immune reaction, several protocol develop, these protocols will prevent acute rejection. Currently reduction of side effects that caused by these protocols become as important as their role in reducing the incidence of acute rejection. In the present time, intensive immunosuppression therapies in the early stages of the transplant become a paramount, followed by maintenance protocol to reduce the risk of rejection (4) .
However, many of these drugs have side effects that will result in more deterioration of bone density and osteoporosis. Osteoporosisa disease that show reduction in bone mass, micro architectural disruption, and enhanced skeletal fragility, with subsequent low bone strength and high rates of fracture (5) . After renal transplantation, there are changes in the normal bone remodeling system which will lead to more accelerated osteoporotic changes compared to normal individuals, while for transplantation-related bone loss results from both an increase in the rate of resorption and a decrease in the rate of bone formation (6) .The current work aimed to investigate the incidence of osteoporosis in post kidney transplant patients when compared to the general population, and study the relationship between post kidney transplant immunosuppression therapy and osteoporosis and determine some biochemical changes. Also to evaluate the bone mass by using dual X-ray absorptiometry (DEXA) and the possible correlation between demographic data and the development of osteoporosis .

Study design
A case control study applied in Kidney Transplant Center -Medical City Complex during the period from October 2018till April 2019. Bone density assessment DEXA scan was used for the assessment of bone density, with T -score ≤ -2.5 to define osteoporosis and between -1.0 to -2.5 to define osteopenia (7) . Laboratory procedure A 5 ml venous blood sample from each participant was collected and then sent for laboratory analysis in the Medical City Complex campus, serum (calcium, phosphorous (PO4), alkaline phosphatise (ALP), vitamin D3, parathyroid hormone(PTH), and albumin) measurement were recorded.

Statistical analysis
For the assessment of continuous variables, independent ttest was used, while for categorical variables chi square testused, ordinal logistic regression analysis used to examine the risk of osteoporosis (in which the order of category from lowest to highest was normal bone, osteopenia, and osteoporosis). All analysis carried out using SPSS version 22.0.0 (Chicago, IL) and GraphPad Prism version 8.2 ( San Diego, California USA), p value considered when appropriate to be significant if less than 0.05

Results
The study included 150 participants, mean age of patients was not significantly different in the study group compared to control(40.9±12.2 vs. 38.4±11.5 years, respectively), with age range from 15 -65 years,the commonest age group for both study and control group was between 40 -49 years ( The prevalence of osteoporosis and osteopenia was significantly higher in transplant patients compared to control for bone lumbar and hip bones (Figure 1 and 2), also; T-Score was significantly lower in the transplant patients compared to control for both lumbar and hip bones ; as illustrated in table  -2.

Figure 1. Osteoporosis status for spinal (lumbar)
bones . *** Highly significant difference (p-value < 0.001). **** Very highly significant difference (p-value < 0.0001) Figure.2. Osteoporosis status for hip bone *** Highly significant difference (p-value < 0.001) **** Very highly significant difference (p-value < 0.0001) Only gender and BMI were the predictors of osteoporosis for lumbar bone, as illustrated in Table  3, while The BMI and serum calcium were the predictors of osteoporosis for hip bone, as illustrated in table 4 .

Discussion
Disturbances in bone metabolism are common complications that affect patients after successful renal transplantation. The usual method for assessing BMD by DXA scan in which osteoporosis defined as T score ≤ -2.5 standard deviation (one standard deviation represent the average of young adult) (8) . BMD in patients who have undergone renal transplantation has been reported to decrease by a mean of 5.5% to 19.5% during the first 6 months but only 2.6% to 8.2% between months 6 and 12 after surgery (9) .
In the present study, mean age ±SD of transplant patients were 40.9 ± 12.2 years, with 73.3% of them distributed between 30 -59 years, this result is close to Coco et al; study which included 59 kidney transplant patients with mean age of 45.5 ± 13 years (10) , also in agreement with Walsh et al; study which included 93 transplant patients with mean age±SD for treatment was (46.1 ± 12.77 years) and for control (46.1 ± 12.93 years) (11) .In this study patients were younger than patients included in the Smerud et al; study with mean age 51.4 ± 13.8 years for all the 129 transplant patients (12) , also lower than other study with mean age of 50.7 ± 15.5 years for 49 control transplant patients (13) .
In the current study, the prevalence of osteoporosis, osteopenia, and normal bone for spinal bone were 33.3% ,48.0% ,and 18.7% ;while for hip bone it was 60.0% ,30.7% ,and 9.3% .
In Gregorini et al, a retrospective cohort study, 60.3% of the patients had normal bone, while osteopenia and osteoporosis were present in 24.6% and 15.1%, respectively (17) , which is in disagreement with the current study since osteoporosis in the current study is higher. In Marcén et al study, 20% had normal BMD in the lumbar spine; 52.5% had osteopenia and 27.5% had osteoporosis. While for femoral neck, 35.0% had normal BMD; 50.0% had osteopenia; and 15.0%, osteoporosis (which mean lower osteoporosis rate when compared to the present result) 14 . In Durieux et al; studyis agree with current study for the lumbar spine 37% had osteoporosis, and 44% had osteopenia, but disagree for the femoral neck 37% had osteoporosis and 40% had osteopenia 16 . In Marcen et al study, 41.9% had osteopenia and 14% had osteoporosis (18) .
The high rate of osteoporosis that observed in this study can be explained by the long duration of transplantation that can will more progressive bone disease, also all patient received corticosteroids (CS) and for extended period of time, since CS is known to cause bone loss by its inhibitory effect on osteoblast cells, and activation of osteoclastic activity, reduction of Ca absorption from the GIT, enhance renal Ca excretion, and increased section of PTH (19) .
In the present study, 34.7% of the patients received cyclosporine(CsA) (as part of combination therapy), also there is no significant relationship between CyA with osteoporosis, (with OR, 95%CI = 1.286 ,0.516-3.208; for lumbar bone and = 1.321 , 0.511-3.419 for hip bones(, which in agreement withMartín-Fernández (20) . This can be explained by the lack of effect of CsA on bone, or since the decrease in BMD in transplant recipients is difficult to evaluate because CsA is usually administered together with glucocorticoids (20) .
Tacrolimus was received in the present study by 50.7% patients (as part of combination therapy), also there was no significant relationship between tacrolimus and osteoporosis, (with OR, 95%CI =0.850, 0.362-2.0; for lumbar bone and, =1.023, 0.414-2.527 ;for hip bones).FK506-based regimens may benefit the skeleton by lowering steroid exposure in transplant recipients. In a small group of kidney-transplant recipients followed for 1year, Goffin et al. (21) ; noted that those who received tacrolimus had a small net increase in bone density compared to a loss observed in those who received cyclosporine.
Sirolimus might impair bone formation by interfering with the proliferation and differentiation of osteoblasts and might contribute to the impairment of osteoclast-mediated bone resorption (20) . In the present study, only 9.3% of patients received sirolimus, there was no correlation between the use of sirolimus and osteoporosis(with OR, 95%CI =1.036,.249-4.317; for lumbar bone and, =0.524,0.098-2.807 ;for hip bones), it is a more novel immunosuppressive agent that produces lower effects on bone (22) .
In the current study, 98.7% of patient received mycophenolatemofetil (MMF) (as a part of combination therapy), and there was no significant relationship between (MMF) and osteoporosis. Mycophenolatemofetil has no influence on bone formation and mass in clinical observations (23) .
Also, the logistic regression analysis revealed that the BMI (OR, 95%CI = 0.850 ,0.748-0.966) and gender (OR, 95%CI = 3.026 , 1.118-8.188) were risk factors that are associated with osteoporosis risk for lumbar bone.While for hip bone, logistic regression analysis for BMI (OR, 95%CI = 0.870 ,0.760-0.996), and serum calcium (OR, 95%CI = 0.514 ,0.265-0.997) were risk factors that are associated with osteoporosis in hip bone. These results study disagree with the results of another study, where no significant relationship was observed between osteoporosis and gender and body mass index (24) . Also differs from another study, where no significant relationship was observed between osteoporosis and serum levels of calcium (25) but it agrees with the same study, regarding nonsignificant relationship between osteoporosis and serum phosphorous and serum parathyroid hormone level.
Osteoporosis after kidney transplantation is multifactorial, while pathophysiologic mechanisms responsible for this condition is not completely elucidated. Pre-transplantation risk factors include duration of dialysis, high or low parathyroid hormone (PTH) levels and preexisting bone disease. Post-transplantation risk factors associated with bone loss and/or fractures are deceased kidney donor, immunosuppressive regimen choice (glucocorticoids, calcineurin inhibitors), and time since transplantation, hypophosphatemia and graft dysfunction. Additional risk factors such as postmenopausal status for women and presence of diabetes have been considered as possible culprits, in adjunction to the classical osteoporosis risk factors such as age and female gender (26) .

Conclusion
Osteoporosis in post-renal transplant patients is high when compared to general population, T-score was significantly lower in the transplant patients when compared to control for both lumbar and hip bone, immunosuppressant therapy (mycophenolate mofetile, tacrolimus, cyclosporine and sirolimus) did not increase the risk of osteoporosis, but corticosteroids significantly increase risk of osteoporosis, Biochemical marker serum level in post kidney transplant patients are significantly different when compared with general population but did not increase risk of osteoporosis. Body mass index is a risk factor for both lumbar and hip bones osteoporosis, while gender and serum calcium are risk factors for osteoporosis in lumbar and hip; respectively.