Fig 1.
Parathyroid Casr gene expression is significantly rhythmic in secondary hyperparathyroidism.
Expression profile of Casr gene over 24 h in parathyroid glands from 44 rats with secondary hyperparathyroidism. Rhythmicity was examined by cosinor regression analysis and showed significant 24 h rhythmicity of expression (p = 0.006). Dots represent one sample. Data are fitted by cosinor regression (solid line). Gray areas indicate dark period and white areas indicate light period. Zeitgebertime (ZT; “time-giver”) is hours since light onset. Gene expression was normalized to the expression of housekeeping gene Hprt1.
Fig 2.
Schematic timeline of the Chronotherapy study. 60 rats were acclimatized for 14 days and, CKD was induced by 5/6 nephrectomy in 40 rats which were shifted to a high-phosphate diet to induce sHPT at w0. Four weeks later at w4, 20 rats with sHPT were randomized to receive Cinacalcet once daily at either ZT2 (N = 10) or ZT14 (N = 10). After three weeks of treatment at w7, Cinacalcet-treated rats were investigated at either ZT2 or ZT14, 24 h since last medication together with untreated normal and sHPT control rats (N = 10 of each). Gray areas indicate dark period and white areas indicate light period. ZT, Zeitgebertime (“time-giver”) is hours since light onset.
Table 1.
Weight and plasma biochemistry.
Fig 3.
Cinacalcet administered early in the inactive phase markedly decrease parathyroid Ki-67 index.
(A) Ki-67 expression in parathyroid glands of rats with CKD-induced sHPT treated with Cinacalcet either early in the inactive light phase (Cina1; N = 10) or early in the active dark phase (Cina2; N = 9) compared to untreated rats with sHPT investigated at similar time points (PNX1; N = 7 and PNX2; N = 7, respective) and to normal rats investigated at similar time points (ctrl1; N = 9 and ctrl2; N = 7, respective). All groups were compared by Kruskal Wallis test with post hoc test after Dunn with Bonferroni adjustment showing significant decreased Ki-67 labeling index of Cina1 compared to Cina2 (p = 0.006) and the untreated CKD groups (p = 0.0001 and p = 0.0002, respectively). (B) For each group, the median Ki-67 immunostained parathyroid sample closest to the group mean is shown. Each dot represents one sample. Scale bars measures 50μm.
Fig 4.
Transcriptional regulation is downregulated, and oxidative phosphorylation is upregulated in parathyroid glands in sHPT.
The transcriptomes of parathyroid glands from untreated CKD groups PNX1 (N = 7) and PNX2 (N = 7) were compared to glands from normal rats investigated at similar time points: ctrl1 (N = 8) and ctrl2 (N = 4), respectively. (A) Comparing PNX1 to ctrl1 14% of all expressed genes were downregulated and 10% upregulated, which was the case for 10% and 8%, respectively, comparing PNX2 to ctrl2. (B) Downregulated genes at both investigated time points were significantly enriched in terms of mRNA processing and histone modification by gene ontology analysis and upregulated genes of both comparisons were enriched in terms related to ATP synthesis/Oxidative phosphorylation and mitotic nuclear division. Dashed line in (A) indicates adjusted p-value of <0.05. For all p-values and further details, please see S2 Table in S1 File.
Fig 5.
Pathways of mRNA processing are downregulated, and pathways of oxidative phosphorylation are upregulated in parathyroid glands in sHPT.
The transcriptomes of parathyroid glands from untreated CKD groups PNX1 (N = 7) and PNX2 (N = 7) were compared to glands from normal rats investigated at similar time points: ctrl1 (N = 8) and ctrl2 (N = 4), respectively. (A) Gene Set Enrichment Analysis (GSEA) found significant downregulation of one pathway: mRNA processing (WP529) and (B) upregulation of the pathway of Oxidative phosphorylation (WP1283) in both comparisons, whereas in the PNX2 vs. ctrl2 comparison also pathways of TCA cycle (WP347), Beta-oxidation meta-pathway (WP372), Fatty acid biosynthesis (WP504), Relationship between glutathione and NADPH (WP2562) and Inflammatory response pathway (WP40) were upregulated. For all normalized enrichment scores (NES), p-values and further details, please see S2 Table in S1 File.
Fig 6.
Cinacalcet administered before the inactive phase leads to downregulation of genes involved in mitotic nuclear division.
The transcriptomes of parathyroid glands from Cinacalcet treated CKD groups Cina1 (N = 7) and Cina2 (N = 10) were compared to glands from untreated CKD rats investigated at similar time points: PNX1 (N = 6) and PNX2 (N = 7), respectively. (A) Comparing Cina1 to PNX1, 67 genes were downregulated and 23 upregulated. No genes were differentially expressed comparing Cina2 to PNX2. (B) The downregulated genes of Cina1 were significantly enriched in multiple terms involved in chromatin organization, mitotic cell cycle checkpoint transition and mitotic nuclear division by gene ontology analysis, whereas upregulated genes showed no significant enriched terms. For all normalized enrichment scores (NES), p-values and further details, please see S2 Table in S1 File.
Fig 7.
Cinacalcet administered before the inactive phase upregulate pathways of energy metabolism.
The transcriptomes of parathyroid glands from Cinacalcet treated CKD groups Cina1 (N = 7) and Cina2 (N = 10) were compared to glands from untreated CKD rats investigated at similar time points: PNX1 (N = 6) and PNX2 (N = 7), respectively. (A) Gene Set Enrichment Analysis (GSEA) found in both Cinacalcet treated groups a significant downregulation of the pathways: ATM signaling pathway (WP654), p53 pathway (WP655) and G1 to S cell cycle control (WP348) whereas only Cinacalcet administered in the early inactive phase (Cina1) resulted in significant upregulation of the pathways Oxidative phosphorylation (WP1283) and TCA cycle (WP347), mRNA processing (WP529) and Prostaglandin synthesis and regulation (WP303) (B). For all normalized enrichment scores (NES), p-values and further details, please see S2 Table in S1 File.