P0870THE ACTIVATION OF KYNURENIC SYSTEM IN BONE TISSUE AS A NEW REGULATOR OF OSTEOBLASTOGENESIS IN RATS WITH EXPERIMENTAL CHRONIC KIDNEY DISEASE DURING LP533401 THERAPY
Authors: Pawlak Dariusz , Mor Adrian , Kalaska Bartlomiej , Domaniewski Tomasz , Sieklucka Beata , Pawlak Anna , Zieminska Marta , Pawlak Krystyna 
Published: June 2020

Abstract

Background and Aims
Abnormalities in bone metabolism represent the most complex disorder accompanying a chronic kidney disease (CKD) development. Serotonin – one of the tryptophan metabolites, has received intensive attention due to its potential role in bone metabolism. The synthesis of peripheral serotonin is initiated by the tryptophan hydroxylase-1 (TPH-1) in the gut. LP533401 is a small-molecule inhibitor of TPH-1. Previously, we found that inhibition of TPH-1 by LP533401 may improve bone mineralization in rats with experimentally induced CKD (Pawlak et al, Bone, 2018). The aim of this study was to establish whether the inhibition of serotonin synthesis by LP533401 may affect kynurenine pathway activity in bone tissue of nephrectomized rats, and to determine the potential consequence of this process in relation to osteoblastogenesis.

Method
Nephrectomized rats were randomized into: untreated (CKD), treated with vehicle (VEH), treated with LP533401 at a dose of 30 (LP 30) and 100 mg/kg (LP 100) daily for 8 weeks. The shame-operated animals served as controls (CON). Tryptophan (TRP) and kynurenine (KYN) concentrations in trabecular and cortical homogenates of femoral bone were determined using high-performance liquid chromatography (HPLC). The expression of tryptophan 2,3-dioxygenase (TDO) as well as genes associated with osteoblastogenesis: activating transcription factor 4 (ATF4), forkhead box protein O1 (FOXO1), Cyclin D1, osteocalcin (Bglap) and sclerostin (Sost) were assessed using the QRT-PCR method.

Results
We demonstrated a presence of TDO-dependent, paracrine kynurenic system in bone homogenates of rats with CKD. The significant decrease in TDO mRNA level was observed in the bone of all nephrectomized groups in comparison with CON (all p&lt;0.0001).
In LP 100 group, the TDO expression was still lower than in CON, but was significantly higher than in VEH and LP 30-treated groups (both p&lt;0.0001). TDO expression was positively associated with KYN concentration and KYN/TRP ratio (R = 0.426, p = 0.016 and R = 0.559, p = 0.001; respectively) in trabecular bone region. ATF4 gene expression in bone was similar in LP 30 and CON, whereas it was significantly higher in other studied groups compared to healthy animals, and the positive relationship existed between ATF4 and TDO expression (R = 0.372, p = 0.042) as well as between ATF4 gene expression and KYN/TRP ratio both in trabecular and cortical bone tissue (R = 0.485, p = 0.006 and R = 0.421, p = 0.023; respectively) of LP-treated rats. In contrast, cyclin D1 mRNA level was the lowest in LP 100 group compared to other studied groups, and the inverse relationship was between this gene and TDO expression (R = -0.622, p = 0.0002). Bglap mRNA level was higher in CKD and VEH group compared to CON (p&lt;0.05 and p&lt;0.0001; respectively). CKD rats treated with LP 30 had the lowest Bglap mRNA levels among all analyzed uremic groups (all p&lt;0.0001), and the positive relationship was between this gene expression and both TDO expression and KYN/TRP ratio (R = 0.468, p = 0.011 and R = 0.474, p = 0.008; respectively) in trabecular bone of uremic rats treated with LP. Similarly, Sost gene expression was significantly reduced in LP 30 group in comparison with other studied groups (all p&lt;0.0001), and it was positively correlated with TDO expression and KYN/TRP ratio (R = 0.540, p = 0.002 and R = 0.409, p = 0.022; respectively) in trabecular bone tissue.

Conclusion
This study for the first time demonstrated the presence of a TDO-dependent, paracrine kynurenic system in the bone of rats with CKD. The modulation of this system during LP533401 treatment was closely associated with the expression of genes participating in osteoblastogenesis, particularly with osteoblast maturation markers. These results represent the next step in studying the role of tryptophan metabolites in renal osteodystrophy.