Phosphate and vascular calcifications in chronic kidney disease – PARKA

Elevated serum phosphate (Pi) is a life-threatening condition which promotes the development of vascular calcifications (VC) and is associated with increased mortality in patients with chronic kidney disease (CKD). High Pi load triggers calcification of the medial layer of the arterial wall resulting in an inevitably more stiffened arterial bed and stimulates the secretion by bone of the phosphaturic hormone FGF23, whose levels increase dramatically in advanced CKD stages thereby contributing to the worsening of cardiovascular diseases. Despite elevated Pi load resulting from CKD, intestinal Pi absorption remains inappropriately unchanged, and therapeutic strategies to control transcellular intestinal Pi absorption have been unsuccessful.

The PARKA project is designed to provide an integrated approach covering the entire spectrum of Pi regulation, from its intestinal absorption to its local vascular effects and systemic role, in order to decipher its mechanism of action leading to the development of VC in CKD, and to identify therapeutic targets able to counteract its deleterious effects. It is a 42-month project (624k€) based on 4 work packages:

• WP0 Project’s management
• WP1 Local and systemic mechanisms of action of Pi on VC during CKD
• WP2 Paracellular intestinal absorption of Pi by BSP-DMP1 axis during CKD
• WP3 Identification of putative therapeutic targets in mice and humans

We will first focus on the role of tissue-nonspecific alkaline phosphatase (TNAP) and the sodium-Pi cotransporter PiT2. TNAP, the key enzyme for bone mineralization, was recently shown to be an early and obligatory inducer of medial calcification in a mouse model of CKD. PiT2 participates in the local effect of Pi on VCs during CKD and was characterized by Team 1 as a Pi sensor in bone controlling Pi-dependent FGF23 secretion in vivo. To study both the local and systemic effects of Pi, and the role of TNAP and PiT2, we will use conditional KOs of PiT2 in vascular cells (VSMCs) (sm22Cre;PiT2cKO) or osteoblasts (OsxCre;PiT2cKO), as well as the TNAP inhibitor SBI-425. Subtotal 5/6 nephrectomy and high-Pi diet feedings will be used to address the impact of high Pi load on medial VC during CKD. To uncover the role of intestinal absorption of Pi during CKD, we will focus on the regulation of the paracellular transport of Pi by the bone sialoprotein (BSP)-dentin matrix protein 1 (DMP1) pathway, since BSP KO mice have a dramatic reduction of paracellular intestinal absorption of Pi, together with a 10-fold increase in DMP1 intestinal expression.

We expect that inactivation of TNAP and/or PiT2 in VSMCs will provide unique answers on the VC mechanisms and identify putative therapeutic targets. Deletion of PiT2 in osteoblasts will dampen the increase in FGF23 during CKD, resulting in high serum Pi levels and exacerbated VC without possible cardiovascular confounding effects of FGF23. Crossing BSPKO mice with OB-PiT2cKO mice is expected to result in normalized FGF23 and Pi serum levels in CKD conditions, alleviating the appearance of VC.

The strength of PARKA is to bring together 4 teams who will share top notch expertise and specific models that will allow an integrated assessment of Pi regulation and action during CKD. This will be achieved via in vivo experiments using established and readily available in-house relevant mouse models, unbiased technical approaches (scRNA-seq and NanostringTM) and attempts to translational validation of our mouse findings in patients with advanced CKD. In vitro approaches will be used to further investigate the underlying molecular mechanisms. Fall-back solutions have been considered to include these in vitro models or organ cultures from the different animal models.