Mechanisms underpinning chronic kidney disease-mineral and bone disorder

Abstract

Chronic kidney disease (CKD) is an irreversible systemic disease characterised by the gradual loss of kidney function over time. Patients with progressive CKD frequently encounter altered levels of circulating and bone-derived hormones which can dysregulate mineral metabolism leading to ectopic calcification and compromised bone formation. Generally, CKD presents with increased levels of parathyroid hormone (PTH), fibroblastic growth factor-23 (FGF23), and phosphate (Pi), all of which are able to affect bone homeostasis through direct and indirect effects on bone-forming and resorbing cells.

FGF23 is a phosphaturic factor that starts to increase in the early stages of CKD. It controls Pi homeostasis and impacts bone mineralisation by inhibiting tissue nonspecific alkaline phosphatase (TNAP) and stimulating pyrophosphate (PPi) expression in bone. PTH is a key regulator of bone remodelling and has biphasic effects on bone metabolism. While intermittent PTH stimulation facilitates bone formation, chronic exposure to PTH as observed in advanced CKD results in bone loss. Besides, high Pi levels that emerge at the latter stages of CKD may aggravate skeletal mineralisation by increasing bone resorption through the promotion of FGF23 and PTH secretion and their associated transcriptional effectors. These observations led to the hypotheses that FGF23, PTH and Pi may possess a “direct effect” on bone cells that mediate bone remodelling and mineralisation.

Although growing evidence indicates that FGF23, PTH and Pi modulate bone matrix mineralisation, it is also possible however that the altered endocrine milieu directly targets the expression of key phosphatases that are critical for skeletal mineralisation. Two of the most widely studied phosphatases involved in skeletal mineralisation are PHOSPHO1 and TNAP. During mineralisation, PHOSPHO1 liberates Pi to be incorporated into the mineral phase through hydrolysis of its membrane-bound substrates leading to an increase in the Pi/PPi ratio within matrix vesicles (MVs). TNAP, highly expressed on the membranes of MVs hydrolyses PPi to facilitate the propagation of hydroxyapatite (HA) in the extracellular matrix (ECM). A complete absence of ECM mineralisation is observed in PHOSHO1; TNAP double knock-out (Phospho1-/-; Alpl-/-) mice. Despite clear links between TNAP and PHOSPHO1 in the control of skeletal mineralisation, there is a lack of evidence about the expression profiles of PHOSPHO1 and TNAP in mineral and bone disorders (MBD) in CKD. The work described in this thesis characterised the expression of PHOSPHO1, TNAP, and other key genes associated with ECM mineralisation in in vitro models of CKD by continuously exposing murine osteoblasts to FGF23, PTH and Pi and in vivo murine model of CKD-MBD.

In the adenine-induced murine model of CKD, mice presented with modest skeletal complications that were characteristic of renal osteodystrophy (ROD), a feature of CKD-MBD. Notably, BMD of trabecular bone was decreased whereas it was increased in cortical bone of CKD-MBD mice. These changes in CKD bones were accompanied by decreased TNAP and PHOSPHO1 expression. However, cortical bone BMD was unchanged in Phospho1 knockout (P1KO) CKD mice suggesting that the increased cortical BMD noted in CKD was driven by the increased PHOSPHO1 expression. The administration of Pi, PTH, and FGF23 in vitro had various effects on osteoblast ECM mineralisation but all three decreased PHOSPHO1 and TNAP expression in culture. Overall, the in vivo and in vitro experimental models of CKD were the first to implicate PHOSPHO1 function in the altered mineralisation status of CKD bones.

Furthermore, this thesis provides the first detailed transcriptomic analyses (RNA-seq) of bone from a murine CKD-MBD model which will prove invaluable in understanding the altered bone metabolism and mineralisation noted during disease progression. Intriguingly, mitochondrial dysfunction was identified as a possible causative pathological mechanism implicated in altered bone remodelling of CKD-MBD. This is a novel observation. Indoxyl sulfate (IS), is a representative renal toxin and acts as a bone toxin by inducing reactive oxygen species (ROS) overproduction inside the cell. This work revealed an extensive characterisation of the effects of IS on osteoblast mitochondrial mass, ROS production, and energy metabolism in primary osteoblasts. The pharmacological mitophagy activator, rapamycin can restore the PARKIN-associated mitophagy pathways mediating the effects of IS on mitochondria. Finally, in vivo models such as mito-QC CKD reporter mice provide direct evidence of mitophagy abnormalities in the bone of CKD-MBD mice. In addition, the reduction of ATG7 (a protein essential for autophagy) in CKD human femurs supports the hypothesis that defective autophagy in CKD bone contributes to mitophagy dysfunction. Such findings will help to identify promising therapeutic options to manage the skeletal complications of CKD encountered in clinical practice.