Abstract :
[en] Impact of Parathyroid Hormone Fragments on Skeletal Hyporesponsiveness in Chronic Kidney Disease
Project description
According to the World Health Organization (WHO), chronic kidney disease (CKD) affects approximately 10% of the global population and represents a major public health challenge. This is due to its association with increased morbidity and mortality, reduced quality of life, and substantial economic burden.
CKD is a progressive and irreversible disease characterized by a gradual decline in glomerular filtration rate (GFR). As kidney function deteriorates, disturbances in mineral and bone metabolism (CKD-MBD) often arise, increasing the risk of fractures, vascular and soft tissue calcifications, and overall mortality. Fracture incidence in CKD patients is reported to be 2–14 times higher than in the general population.
Given these risks, the KDIGO (Kidney Disease: Improving Global Outcomes) international nephrology guidelines emphasize the importance of monitoring specific biochemical markers to guide the management and follow-up of patients with CKD. Recommended biomarkers include serum concentrations of calcium, phosphate, vitamin D, and parathyroid hormone (PTH).
Among these parameters, PTH is an 84-amino-acid peptide that plays a central role in maintaining calcium and phosphate homeostasis. It is synthesized by parathyroid cells and secreted in response to a decline in extracellular calcium concentration.
Once released, PTH maintains calcium–phosphate homeostasis through its actions on bone, kidney, and indirectly the intestine via vitamin D activation. In CKD, phosphate retention induces hyperphosphatemia and stimulates FGF23 secretion, which suppresses calcitriol production and reduces intestinal calcium absorption, leading to hypocalcemia. This triggers increased PTH secretion, and in some patients, disproportionately high PTH levels develop, indicating skeletal hyporesponsiveness and contributing to bone demineralization and increased fracture risk.
The concept of skeletal resistance to PTH is frequently discussed in the context of chronic kidney disease. Nevertheless, the term “PTH hyporesponsiveness” is more appropriate, as the skeletal response to the hormone is reduced rather than entirely absent. Functionally, this condition requires elevated circulating PTH levels to preserve calcium–phosphate homeostasis, resulting in disproportionately high PTH relative to serum calcium. Consequently, bone remodeling and mineralization are disrupted, compromising skeletal integrity and heightening fracture risk.
Eight distinct PTH fragments have been identified in human serum, primarily generated in the liver during PTH metabolism. They are classified as N-terminal fragments, which are rapidly cleared by the liver, and C-terminal fragments, which are released into the circulation prior to renal excretion. These fragments normally account for 15–30% of total circulating PTH, but their accumulation is markedly increased in patients with chronic kidney disease.
In this project, we hypothesize that these recently identified PTH fragments may contribute to skeletal PTH hyporesponsiveness by interfering with PTH binding to its receptor, PTH1R. This hypothesis is supported by studies on FGF23, whose C-terminal fragments disrupt receptor binding and inhibit biological activity. We propose that a similar interference mechanism could occur with PTH C-terminal fragments, particularly given the evolutionary co-emergence of FGF23 and PTH. Elucidating the mechanisms and inter-individual variability of skeletal PTH resistance in chronic kidney disease is essential for preventing bone demineralization and reducing fracture risk.
During my master’s thesis, I developed an LC-MS/MS quantification method for four of the eight identified PTH fragments. I also observed that the concentrations of C-terminal fragments increased as the eGFR decreased. However, fragments and PTH concentrations vary between patients, even among individuals with similar eGFR values. For instance, patient M51 exhibited a serum PTH34–84 fragment concentration 13 times higher than patient F63, whereas their PTH concentration was only three times higher. These findings suggest that fragment concentrations are not solely dependent on eGFR and that this variability may be associated with differing degrees of PTH resistance.
The aim of this project is thus to investigate the mechanisms underlying PTH resistance observed in CKD patients could involve PTH fragments. For this purpose, we will test whether some of them can interfere with the PTH receptor, namely PTH1R, or modulate its signaling.
To achieve this, we will develop an LC-MS/MS assay method to quantify PTH and its eight fragments in serum. Sample preparation will involve solid-phase extraction, followed by analyte separation using ultra-performance liquid chromatography (UPLC) and detection with a triple quadrupole mass spectrometer. The method will then be validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines.
The final two phases of the project will be conducted in parallel. First, PTH and its eight fragments will be quantified in a well-characterized clinical cohort, the CPH-CKD (Copenhagen Chronic Kidney Disease Cohort). This cohort includes patients aged 30 to 75 years, spanning CKD stages G1 to G5, who were recruited at Herlev Hospital and Rigshospitalet. One major strength of this project is that all 998 participants have already been enrolled, and biological samples are already available. Thus, upon reaching this stage, the only remaining step will be to analyze these samples to determine their concentrations of PTH and its fragments. These measurements will enable the identification of potential associations between elevated levels of specific fragments and various biological or clinical parameters, such as treatment resistance or markers of bone remodeling. Moreover, the 15-year longitudinal follow-up of this cohort will provide the opportunity to investigate associations between high concentrations of selected fragments and subsequent clinical outcomes, including fractures and hospitalizations.
On the other hand, we will investigate the interaction of selected fragments with PTH1R in vitro using cell culture assays to determine whether they interfere with PTH binding, potentially contributing to PTH resistance. For these in vitro experiments, osteoblastic cell lines and primary osteoblasts will be stimulated either with PTH alone or with combinations of PTH and fragment modules, based on their in vivo concentrations. After incubation, cells will be lysed and centrifuged, and the supernatants will be analyzed by LC-MS/MS to assess cyclic AMP (cAMP) levels. cAMP functions as a second messenger generated upon activation of adenylate cyclase by the Gαs subunit following PTH binding to PTH1R. If the fragments interfere with receptor binding, we expect a reduction in cAMP production in cultures treated with PTH-fragment combinations compared to cultures stimulated with PTH alone12.
Successfully completing this project will enhance patient care, prevent potentially fatal fractures, and reduce healthcare costs.
