In this study, kidney transplant recipients showed an improvement in traditional laboratory markers of bone metabolism and significant lower levels of non-traditional molecules when compared to hemodialysis patients. After kidney transplantation, calcium levels were higher, while phosphorus, ALP, 25(OH)D, and PTH were lower when compared to hemodialysis patients, as already reported[2]. Hypercalcemia was found in 29.8% of transplant patients. Similarly, Evenepoel et al. found that at the 1st year post kidney transplantation, 30.9% of patients had hypercalcemia, although this percentage decreased to 12.4% at the 4th year post-surgery [15].
All non-traditional markers of bone metabolism were significantly reduced in our group of transplant recipients when compared to hemodialysis patients and healthy subjects. On one hand, this may represent an improvement of MBD after transplantation in comparison to patients on hemodialysis. On the other hand, this may also indicate a general reduction of bone turnover when compared to healthy individuals. This latter difference could be attributed not only to immunosuppression [16], but also to an impairment of bone homeostasis after transplantation, as MBD after transplantation is usually characterized by low remodeling [17].
Plasma levels of FGF-23 were significantly lower in the transplant recipients when compared to patients on hemodialysis. In the early post-transplant period, FGF-23 levels decrease dramatically as a consequence of the recovery of renal function [18]. Nevertheless, even after the successful transplantation, plasma levels of FGF-23 can remain higher for a certain period, leading to hyperphosphaturia [11]. Although this study has a cross-sectional design and, therefore, cannot address temporal relationships, the lower levels of FGF-23 found in the transplant recipients may reflect the improvement of the 25(OH)D metabolism after transplantation.
Plasma levels of OC are elevated in patients on hemodialysis due to uremia and hyperparathyroidism, acting as a calcification inducer [19]. The levels are expected to decrease post-transplant, as a consequence of the action of glucocorticoids, improvement of kidney function, and bone metabolism [20]. Glucocorticoids reduce osteoblast proliferation and lifespan, consequently leading to bone loss and reduction of OC [21]. As expected, we found significant lower levels of OC in transplant recipients when compared to hemodialysis patients.
Regarding SOST, Cejka et al. found high levels in dialysis patients, which were associated with low bone turnover and a reduced number of osteoblasts, contributing to osteoporosis [7]. Previous studies have shown increased SOST levels in dialysis patients and non-dialysis CKD when GFR decreases [6]. Araujo et al. found a reduction in plasma levels of SOST to below pre-transplant levels at the first year post-transplant [22]. Bonani et al. also reported reduced SOST levels 15 days post-transplant, but the levels progressively rose in 6 and 12 months after transplantation [23]. The explanation for the reduction of SOST after kidney transplantation is still uncertain, but the use of glucocorticoids and the improvement of GFR with greater clearance of the molecule may contribute. PTH inhibits the expression of SOST and DKK1 [7]. After transplantation, significantly lower levels of PTH and increased levels of SOST are expected. However, steroids, both endogenous and exogenous, seem to reduce plasma SOST production [24]. This reduction could also be explained by the increase in SOST clearance after recovery of renal function. Conversely, a recent study with CKD patients showed that the urinary excretion of SOST increases as the GFR decreases [25]. Therefore, the increase in SOST in CKD patients seems not to be due to a reduction in its clearance as there will be an increase in its tubular excretion [25]. Another recent study, however, showed no relation between GFR and plasma SOST levels in kidney transplant patients [26]. Similarly, we found no association between SOST levels and GFR in transplant recipients. These conflicting results call upon the necessity of more studies to comprehend the role of SOST in CKD.
Hemodialysis patients exhibited higher plasma levels of OPG when compared to kidney transplant recipients, as shown in previous studies [5]. Although OPG is an inhibitor of vascular calcification, its increased levels have been associated with higher coronary artery and aorta calcification and cardiovascular events in CKD and kidney transplant patients [27]. Higher plasma levels of OPG could reflect low bone turnover, leading to more calcium availability in the circulation and, consequently, increasing calcification [20]. Bone vascular smooth muscle cells could also act as osteoblast-like cells, releasing OPG. The OPG binds to the RANKL and avoids the formation of osteoclast-like cells, causing the progression of vascular calcification [5]. OPG levels increase as kidney function decreases and can be altered by post-transplantation medications, including calcineurin inhibitors and glucocorticoids. These medications decreased OPG levels, increased osteoclastogenesis, osteoclastic activity, and diminished osteoblast differentiation and proliferation[28]. Accordingly, our post-transplant patients had significantly lowered OPG levels when compared to hemodialysis patients, reaching values similar to healthy controls. Moreover, B-lineage cells are a major source of OPG [29]. As these patients use corticosteroids and other immunosuppressants that affect B-cell metabolism [30], this mechanism may also reduce OPG levels.
Transplant recipients presented lower levels of DKK1 when compared to hemodialysis patients and healthy controls. It is known that DKK1 and SOST are inhibitors of osteoblastogenesis, while OPG inhibits osteoclastogenesis [31]. After kidney transplantation, the decrease in DKK1, SOST, and OPG levels could reflect improvement of bone mineral density. Furthermore, DKK1 is produced by osteoblasts, osteocytes and in bone marrow cells [32], while SOST is mainly produced by osteocytes [33]. The reduction of both molecules is associated with increased bone formation through activation of WNT–β-catenin signaling in osteoblasts [34, 35]. Thus, it is possible that the necessity for more bone formation and normal serum calcium levels could reduce plasma levels of DKK1 and SOST. However, bone densitometry and/or bone biopsy are required to establish the relationship between these molecules and the bone structure.
Hemodialysis patients showed higher OPN levels, possibly related to the inflammatory environment and vascular calcification. On the other hand, OPN levels were reduced in healthy controls and transplant patients, suggesting an improvement of the inflammatory setting after kidney transplantation. As inhibitors of vascular calcification, OPN and OPG are usually higher in patients with vascular calcification, possibly as a negative feedback [10]. Higher levels of OPN and OPG have been previously associated with all-cause mortality in patients with CKD stage 5 in hemodialysis without diabetes [36]. In the present study, significant lower levels of OC, OPN, and OPG levels were found in transplant patients, which could reflect an improvement in extraosseous calcifications. In addition, glucocorticoids have shown to reduce OPN levels in animal models [37]. The use of glucocorticoids could reduce OPN levels in kidney transplant recipients, similar to what is seen with OPG. Nevertheless, further studies are needed to assess this hypothesis.
Regarding correlations between traditional markers and molecules related to bone metabolism, we found that levels of 25(OH)D and FGF-23 levels negatively correlated in transplant recipients and hemodialysis patients. This can be explained by the inhibitory action of FGF-23 on 1α-hydroxylase, reducing the levels of active vitamin D [38]. Similarly, OPN levels and ALP were positively correlated in the transplant group. As expected, levels of both molecules were lower in kidney transplant patients, reflecting low bone turnover. There was a positive correlation between OPG and ALP in hemodialysis patients, similar to what was reported by Morena et al. [27]. In patients with CKD, PTH stimulates the release of OPG by osteoblasts [27]. OPG prevents the binding of RANKL to RANK in osteoclasts, inhibiting osteoclastogenesis and thereby reducing bone reabsorption [39].
Trans-iliac bone biopsy is still considered the gold standard to assess bone metabolism in CKD patients [40]. However, this method is invasive, poses risks, and demands training physicians [41]. In addition, its use is still poorly standardized across services. This is a problem, especially in kidney transplant recipients, since few studies have evaluated MBD in this population. We showed that the combined bone biomarkers panel, specially in association with serum creatinine, can successfully predict hypercalcemia and dyslipidemia, two outcomes that are intimately related to cardiovascular risk in kidney transplant patients [42]. Similarly, the bone biomarkers panel successfully predicted dyslipidemia in hemodialysis patients, which further suggests the ability of these biomarkers to predict cardiovascular risk factors [43].
The present study has limitations, including the cross-sectional design, the performance in a single center, and the lack of investigation on mechanisms. The causality cannot be established in regard to the changes found in bone metabolism markers. This study was carried out in patients more than one year after kidney transplantation, in whom the dose of prednisone was only 5 mg/day, precluding the evaluation about the influence of high doses of corticosteroids. Serial measurements in the same patient before and after kidney transplant would allow understanding dynamic changes of these molecules. Nevertheless, the persistence of differences in the studied biomarkers, regardless of the gender and transplant time, two possible confounding factors, supports the improvement of MBD after kidney transplantation.
In conclusion, bone metabolism biomarkers levels in kidney transplant recipients were lower than in hemodialysis patients, possibly due to the amelioration of inflammation and uremia after transplantation. Longitudinal studies with additional methods to evaluate bone structure are needed to understand the role of these molecules on bone metabolism after kidney transplant.