Novel mediators in phosphate homeostasis: noncoding RNAs and the microbiome

Abstract

Phosphate is an essential nutrient and its level in the blood is tightly regulated by well-studied hormonal and non-hormonal factors. The renal sodium phosphate cotransporter (SLC34A1) is essential to maintaining Pi homeostasis. Recently two novel factors have emerged that potentially modulate phosphate body levels; a non-coding SLC34A1-antisense transcript and the gut microbiome. The first part of this study aimed to understand the coordination between SLC34A1- sense/antisense transcripts in the human renal epithelial cell lines HEK293 and HKC8. In both cell lines SLC34A1-sense/antisense RNAs are expressed at very low levels. Transcription of SLC34A1 was stimulated using the epigenetic modifiers Zebularine (Zeb) and dexamethasone (Dex) and its coordinated expression was assessed by RT-qPCR. HKC8 cells treated with Zeb showed that SLC34A1- sense/antisense transcripts are positively correlated, i.e. concordantly upregulated. A similar expression pattern was recorded in HEK293 cells following Dex treatment. The expression changes were paralleled by a reduction in DNA methylation of the sense/antisense promoter regions. In addition a significant increase in histone acetylation (H3K27Ac) at the sense promoter and a decrease in H3K4Me3 at the antisense promoter were detected after 5 days of Dex treatment. After establishing a system to induce SLC34A1-sense/antisense expression with the aid of Zeb and Dex, we sought to experimentally manipulate SLC34A1 locus expression by introducing polyadenylation signals using CRISPR/Cas9 to silence sense or antisense transcription, respectively. Sense- antisense expression was quantified by RT-qPCR using different primer sites up- and downstream of the insertion site. Unexpectedly, termination was incomplete and read-through transcripts were generated. Again, SLC34A1-sense/antisense transcripts were concordantly upregulated regardless of the orientation of the read-through transcript. From a functional perspective, both strategies -hormonal induction and read-through transcription- led to an increase in SLC34A1 protein expression as determined by western blot and 32Pi uptake experiments. The second part of the thesis aimed to determine the impact of a high/low Pi diet on the human gut microbiome. The experimental strategy included a dietary intervention in two healthy groups of volunteers followed by stool sampling, DNA extraction and sequencing. Alpha and beta diversity examination indicated that both high and low Pi regimens resulted in only minor alterations in bacterial compositions. However, a focus on the relative abundance of bacterial phyla and species demonstrated a reduction in Bacteroidetes among probands with reduced Pi intake suggesting potential involvement of this bacterial species in Pi homeostasis. To summarise, this PhD investigated two novel aspects in the field of microbiology of Pi homeostasis; noncoding RNAs and the microbiome. The concordant expression of SLC34A1-sense/antisense transcripts confirms the impact and biological importance of the antisense transcript on SLC34A1-sense expression, potentially affecting renal Pi reabsorption. On the other hand, consuming a high or low Pi diet may have an impact on the abundance of certain bacterial species, but only insignificant effects on the overall composition of the microbiome. A potential link between the affected bacterial groups and Pi homeostasis remains to be investigated in depth.