1. Introduction
Nephropathic cystinosis (NC; 1/200,000 live births) is a monogenic autosomal recessive lysosomal storage disease caused by a bi-allelic mutation of the
CTNS gene (17p13.2), consisting of 12 exons [
1]. This gene encodes cystinosin, a lysosomal seven-transmembrane domain cystine transporter of 367 amino acids. So far, over 140 pathogenic mutations have been reported in the
CTNS gene [
2], the most frequent one being a large deletion of 57 kb involving the promoter region and the first 9 exons and part of exon 10. This deletion represents approximately 50% of mutant alleles in patients of North European and North American origin [
2].
Cystinosin deficiency causes an accumulation of cystine in all organs and tissues, making cystinosis a systemic disease [
3,
4]. Early clinical manifestations are related to complete proximal tubulopathy and therefore include polyuric-polydipsic syndrome, growth retardation, and hypophosphatemic rickets. The natural history of this disorder is marked by chronic interstitial nephritis, leading to end stage renal disease during the second decade of life. In this regard, the beneficial role of cysteamine therapy in NC has been well known for nearly four decades: Although it does not prevent nor improve tubulopathy, it considerably slows the progression of renal lesions [
4,
5], delays the need for transplantation [
6], and prevents late complications [
7].
Since patient survival has improved considerably with cysteamine therapy, late onset complications have emerged, notably bone involvement. Indeed, the concept of “cystinosis metabolic bone disease” (CMBD) is currently emerging [
8]. We were the first to show in a pilot study on 10 teenagers and young adults, at a median age of 23 (range 10–35) years, that 70% of patients complained of a bone symptom (past of fracture, bone deformations, and/or bone pain), with a tendency toward low PTH and low FGF23 levels [
9]. At the same time, an American study in 30 patients displayed similar results [
10]. Physicians are currently aware of this specific “novel” complication of NC, and international guidelines on the diagnosis and management of CMBD were published in 2019 [
8].
Even though its exact underlying pathophysiology remains unclear, at least five distinct but complementary entities can explain CMBD in addition to the classical mineral and bone disorders associated with CKD and post-transplant [
8,
11]: long-term consequences of hypophosphatemic rickets and renal Fanconi syndrome; deficiency in nutrition and micro-nutrition, and notably copper deficiency; hormonal disturbances such as hypothyroidism, hypogonadism, hypoparathyroidism and resistance to growth hormone and IGF1; myopathy; and intrinsic and iatrogenic bone lesions such as direct bone effects of
CTNS mutation on osteoblasts and osteoclasts, both in murine models of cystinosis and cystinosis patients [
11]. Recent experimental data corroborate clinical observations suggesting a toxicity of high-dose cysteamine on bone cells [
12,
13,
14]. We previously showed that if monocytes derived from NC patients PBMCs were more prone to differentiate into osteoclasts than healthy donor monocytes, they displayed less efficient resorption activity. However, intriguingly enough, cysteamine treatment did not revert this tendency nor did it revert the deficient resorption activity in vitro of NC patients-derived osteoclasts [
14]. These findings suggested that cystinosin might be a negative regulator of osteoclast differentiation but also that cystine efflux is not essential to osteoclastogenesis.
Thus, the objectives of the present study are to determine in vitro the impact of CTNS mutations and cysteamine therapy on human osteoclasts derived from peripheral blood mononuclear cells (PBMCs), and to carry out a genotype-phenotype analysis in terms of osteoclastic differentiation and response to cysteamine therapy, to better decipher the functional role of cystinosin in osteoclasts.
4. Discussion
Mineral and bone homeostasis disorders displayed by CKD patients increase as kidney function declines. It results in a high number fractures and ectopic vascular calcifications as a consequence of impaired mineral metabolism. In nephropathic cystinosis, these aspecific mineral and bone disorders are worsened by what is now called CMBD [
8]. From a clinical point of view, we here confirm that bone involvement is a late complication of cystinosis occurring in teenagers and young adults. Indeed, transplant patients were significantly older than patients under conservative management, and they also presented a higher frequency of bone symptoms. Interestingly, even though the study was not designed for this aim, we here show that the only significant difference between patients receiving SA or DR cysteamine is the proportion of patients within the target for LHL. Additionally, the fact that the ratio DR/SA is 0.62, as opposed to 0.75 in previous publications [
15], likely reflects a better compliance in patients receiving DR cysteamine, as expected [
27]. This is an indirect plea in “real life” for using DR whenever possible, to optimize the control of LHL, even though there were no differences in term of osteoclastic differentiation in these two sub-groups.
The underlying pathophysiology of CMBD nevertheless remains complex and multi-factorial, but cellular defects have been well documented at the cellular level, both in osteoblasts and osteoclasts, in terms of differentiation and specific activity [
12,
13,
14]. However, cystinosin function in bone cells, and particularly in osteoclasts, remains unclear. Here, we focused on osteoclasts in order to better explain the altered bone phenotype of patients with nephropathic cystinosis. The main strength of this study is the protocol implemented to obtain human bone cells directly from patients presenting an orphan disease. It is an innovative, minimally invasive but time-consuming technique that allows direct access to osteoclasts from a small sample of total blood sample.
Thus, we extend the results of our previous work on cystinosin-induced osteoclastic dysfunction, in which we showed that cystinosin is required for proper osteoclastic differentiation with a peak of expression on day 6 of the differentiation process following the same pattern as cathepsin K transcripts [
14]. We also showed that cysteamine has anti-resorptive effects in vitro on osteoclasts derived both from controls and patients [
14].
Herein, we have classified cystinosin-identified mutations in three groups corresponding to its predicted in vivo activity, in an attempt to assign a phenotype to a genotype. The main findings of the present study are therefore the following: cells with residual cystinosin activity generate less osteoclasts as opposed to inactive or absent protein, indicating that cystinosin might be a negative modulator of osteoclast formation; moderate doses of cysteamine have no effect on either of the three groups, that is to say that in the RA, IP, or AP group, cysteamine treatment did not increase nor further reduce the number of osteoclasts; osteoclast formation remained of the same order of magnitude, supporting our previous results showing no evidence of a significant effect of cysteamine on osteoclastic differentiation at low doses [
14]. In contrast, we here demonstrate a significant inhibitory effect of cysteamine on osteoclastic differentiation at higher doses. These apparent discrepancies with our previous results may be explained by an increased number of patients in this study (17 versus 7), but also by the different clinical profiles of the patients, our previous cases being older (median age 31 years), at different stages of kidney disease (transplantation, N = 5, hemodialysis, N = 2, no conservative management) [
14]. Anyway, this inhibitory effect of cysteamine on osteoclastic differentiation appeared to be dose-dependent in the pooled IP and AP groups whereas the response profile to cysteamine appeared to be different in the RA group, as the number of osteoclasts at low doses of cysteamine remained comparable to the number of osteoclasts at baseline.
The genotype/phenotype analysis that we propose hinges on the first rational classification of mutations in the
CTNS gene and is based on the functional consequences of these mutations on the structure of cystinosin. The justification of mutations classification is based on a multi-disciplinary approach taking into account both published experimental data and patients’ clinical phenotype, with a discussion involving physicians, biochemists, geneticists, and basic scientists [
2,
20,
21,
22,
23]. In light of the results, this classification appears relevant since it makes it possible to predict a response profile to cysteamine as a function of the patient’s underlying genotype. These findings may be of clinical interest for the management of cysteamine therapy, which should reconcile, especially in patients most at risk of toxicity, effectiveness in reducing lysosomal cystine concentrations and preservation of bone capital. Without cysteamine, the number of osteoclasts was higher in cultures from subjects in whom cystinosin was inactive or absent, as compared to subjects in whom cystinosin retained residual activity. One may argue that a potential bias in the interpretation of these results may be induced by the different number of osteoclasts obtained depending on age, since younger healthy donors are more prone to produce more cells, but there was no significant association between age and osteoclastic number at the end of the differentiation process in the cells obtained from these peculiar patients. The fact that all but one patient received maintenance cysteamine therapy may also influence the results of subsequent cell culture experiments, but it would not be ethical to propose a wash-out period in these patients.
Mechanistically, this observation of a different profile of osteoclastogenesis depending on the underlying cystinosin functionality may be explained by the role that the mammalian target of rapamycin complex 1 (mTORC1) and its interaction with the Ragulator–Rag complex play during osteoclastogenesis, as discussed thoroughly in a recent review on the topic [
11]. Indeed, it has been established that mTORC1 activity is down-regulated during osteoclastic differentiation through the negative regulator TSC1, whose absence impairs RANKL-dependent osteoclastogenesis. Furthermore, Andrzejewska et al. showed that the mTORC1 pathway is downregulated in proximal tubular cell lines derived from Ctns
−/− mice [
28]. A similar down-regulation in human osteoclastic progenitors might account for the overall increased osteoclastogenesis that we observe in NC patients, as compared to controls. Andrzejewska et al. also demonstrated that cystinosin is a component of the vacuolar H + -ATPase–Ragulator–Rag complex, which controls mTORC1 localization to lysosomes and thus, mTORC1 signaling [
28].
DNA mutations in the
CTNS gene have various functional consequences linked to their structural impact on cystinosin. Extensive deletions (such as the 57-kb deletion) cause the absence of protein, while severe truncating mutations lead to the synthesis of an inactive variant. Both these situations amount to a loss of cystinosin efflux function. In contrast, milder mutations allow the synthesized cystinosin variant to retain residual activity. It is interesting to hypothesize that, as well as canceling cystinosin efflux function, severe
CTNS mutations impair the interaction between the Ragulator–Rag complex (of which cystinosin is a component) and mTORC1, preventing its activation. On the other hand, mutations of more limited structural impact might allow, to some extent, to maintain an efflux activity as well as the interaction between mTORC1 and the lysosomal membrane-attached Ragulator–Rag complex. This hypothesis would explain the correlation we observed at baseline between the severity of the mutation, its impact on cystinosin efflux function, and the outcome in terms of osteoclastogenesis (increased osteoclast number in the AP and IP groups, compared to the RA group). It could be argued that the downregulation of mTORC1 is due to the accumulation of cystine, which would logically be greater in the AP and IP groups. However, as shown in the Andrzejewska et al. study, decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in proximal tubular cells, thus suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine [
28].