| dc.description.abstract | Physiological and biochemical abnormalities present in patients with Chronic Kidney Disease (CKD) have been hypothesised to cause ‘dysbiosis’: pathological alterations to a host organism’s resident populations of bacteria. It has been suggested that these microbiological changes may contribute to the progression of CKD. This thesis explores the nature of such dysbiotic changes in the oral and gut microbiota of animals with experimental uraemia, and considers whether modulation of the gut microbiota might be used therapeutically to improve the health of patients with kidney disease. Part 1 Introduction: There is a high incidence of periodontal disease (PD) in patients with CKD, and it has been claimed that low-grade inflammation from PD may contribute to the progression of CKD. Here, it is hypothesised that actually the relationship may be the other way round, with CKD causing oral dysbiosis that subsequently leads to PD. Results: Using several rodent models, it is demonstrated that experimental uraemia reliably induces loss of periodontal alveolar bone height in both rats (mean -0.113mm, p<0.001) and mice (mean -0.02mm, p<0.001). Uraemic animals have a dysbiotic oral microbiome with increased alpha diversity (Simpson Index 0.82 vs 0.75, p=0.054), reduced total bacterial counts (log10 5.80 vs 6.07 log10 cfu/ml, p=0.034), a decrease in health-associated taxa (phylum Firmicutes, log10 5.43 vs 5.88 log10 cfu/ml, p=0.043, and genera Streptococci and Rothia) and an increase in gram-negative taxa (phylum Proteobacteria comprising 9.53% of isolates in uraemic animals vs 2.99% in controls, p=0.003). Induced saliva from uraemic animals had a higher urea concentration than that from controls (3.73 vs 1.62mmol/L, p=0.007), and bacterial isolates which were under-represented in samples from uraemic animals showed reduced tolerance to higher urea concentrations during in vitro broth culture. Uraemic animals which were co-housed with healthy animals demonstrated significantly less bone loss than those housed with other uraemic animals (-0.109mm vs -0.149mm, p=0.038), and transfer of oral microbiota from uraemic animals induced more periodontal bone loss in healthy germ-free mice than transfer of oral microbiota from health animals (-0.042mm, p<0.001). Conclusion: Experimental uraemia causes loss of periodontal bone height. Although some of this may reflect the systemic effects of uraemia on bone, the demonstration of reproducible dysbiotic effects on the oral microbiome and the effects of co-housing and oral microbial transfer on periodontal phenotype suggest that uraemic dysbiosis plays a key role in the aetiology of PD in the setting of CKD. Part 2 Introduction: Both bacterial generation of uraemic toxins and reduced generation of short-chain fatty acids have been suggested as possible metabolomic mechanisms that would implicate gut dysbiosis in the aetiology of CKD. We sought to characterise gut dysbiosis using rodent models of chronic uraemia. Results: Analysis of the gut microbiota of two identically treated cohorts of rats, obtained from the same supplier just a few weeks apart, revealed that batch effect far outweighed the effect of uraemia on the composition of the gut microbiota (batch effect accounting for 9.7% of variance, p=0.007; compared to 4.8% for uraemic vs control animals, p=0.227). These batch differences proved to be functionally significant, with the urinary metabolome also showing far greater effects of batch than of uraemia (batch accounting for 66% of variance between samples, p=0.001, compared to 48% for uraemic vs control, p=0.007). Further cohorts of animals demonstrated similar large variations compared with previous cohorts, and urinary metabolomes between batches proved equally dissimilar, with no reproducible effect of uraemia. To understand this batch variability in the context of previous published work claiming a demonstrable effect of uraemia on gut bacterial populations, a meta-analysis was carried out of all publicly available NGS sequencing data investigating the effect of experimental uraemia on the gut microbiome of rodents. In this combined dataset, the leading determinants of variation were batch (69% of variance, p<0.001), primer type (23.9% of variation, p<0.001) and host species (rat vs mouse, 13.3% of variance, p<0.001). The presence of uraemia did influence sample clustering, but to a very limited extent (1.9% of variance, p=0.026). Conclusion: The effect of uraemia on the gut microbiome is minor, and is eclipsed by inter-batch variation, which makes it hard to state confidently that ‘uraemic dysbiosis’ occurs in the gut. The degree of variability between animals from different batches poses wider questions about the reproducibility of animal research in other settings. Alternative experimental strategies are discussed, such as longitudinal studies which explore how a given intervention affects the microbiota of the same animals over time, using animals as their own controls. Part 3 Introduction: Fermentable dietary fibre, such as fructo-oligosaccharide (FOS), has been shown to induce significant generation of short-chain fatty acids by the gut microbiota, with a range of beneficial effects on health. We sought to establish whether such effects could be demonstrated in experimental uraemia and might offer a microbiome-mediated therapeutic tool for patients with CKD. Results: FOS-supplemented diet produced similar and substantial effects on the gut microbiota of both control and uraemic animals. Whilst uraemia again accounted for minimal amounts of species-level variation between samples (2.4% of variance, p=0.75), diet was associated with a large degree of variance (46.3%, p<0.001), including large increases of the acetate producing genus Bifidobacterium (27.3% of reads vs 1.7%, p<0.001), and increases in propionate- and butyrate-producing taxa including Bacteroidaceae (23% vs 7.4% of reads, p=0.006), Marvinbryantia (4.5% vs 0.09%, p<0.001) and Blautia (1.8% vs 0.25%, p<0.001). Comparable changes were seen in the microbiota of both uraemic and control animals. Using whole genome sequencing metagenomics, the FOS-supplemented diet was associated with significant increases in the abundance of carbohydrate metabolism pathways (3.37x106 reads/sample in all FOS-treated animals vs 2.51x106 in all CELL-treated, p=0.029), including the bifid shunt and other bacterial glycolytic pathways, and in pathways involved in the initiation (2.26x105 vs 1.12x105 reads/sample, p<0.001) and elongation (4.20x104 vs 8.31x104 reads/sample, p=0.004) of short chain fatty acids. Animals fed the FOS-supplemented diet demonstrated substantial increases in caecal volume, and had significantly lower caecal pH, in keeping with the predicted increase in short chain fatty acid production. FOS administration was associated with beneficial effects on various aspects of the uraemic syndrome including a 51% reduction in serum urea concentrations (p=0.004), a 24% reduction in urine output (p=0.032) and a 0.6mmol/L reduction in serum potassium (p=0.02). Conclusion: Fermentable fibre produces substantial changes in the gut microbiome of both control and uraemic animals, associated with substantial improvements in several aspects of the uraemic syndrome. These results suggest that fermentable fibre supplements may offer benefits to human subjects with CKD if the effects seen in experimental animals can be translated into clinical practice. | en_US |
