By Prof. Juan A. Galán-Llopis (Alicante, ES), General University Hospital
This article reflects the highlights of the lecture Prof. Juan A. Galán-Llopis will be giving during the Meeting of the EAU Section of Urolithiasis (EULIS) at EAU21 Virtual. This meeting will be taking place in Virtual Room 2 on Monday 12 July, from 13:30 – 14:30 CEST.
Human microbiome understanding and its relationship with health has represented a revolution in biomedicine. It was facilitated by the emergence of new molecular microbiology techniques, specifically massive sequencing techniques, focused on the study of the gene content and the mRNA, along with proteomics.
The microbiome not only refers to the microorganisms present in a well-defined habitat but also includes the scenario where their activity takes place, resulting in specific ecosystem. The microbiota consists of the set of microorganisms belonging to different kingdoms, without considering their site of activity. The metagenome refers to the total microbial gene content resulting from the study and analysis of an environmental sample using new-generation sequencing techniques.
The intestinal microbiota presents incremental compositional and quantitative variation throughout the tract. The influence of the intestinal microbiome on digestive functions and nutrient absorption, either by direct action on them or by affecting the state of the mucosa, is well established.
Some conditions that may impact on factors associated with lithiasis are the use of dietary oxalate, inflammatory bowel disease caused or exacerbated by intestinal dysbiosis and derived malabsorption, dehydration due to chronic diarrhea caused by pathogenic bacteria, microbiota alterations associated with bariatric surgery, the influence on the composition of the vaginal and urinary microbiota as a consequence of a gut dysbiosis, and the impact of the intestinal microbiota on chronic low-grade inflammation and associated lesions at the renal tubule.
Other conditions affecting the composition of the microbiota and, therefore, influencing stone disease include the nutritional profile, the immune system, antibiotic consumption, age and gender.
Urobiome: the microbiome of the urinary system
The dogma of bladder urine sterility was broken by Wolfe et al, reporting the existence of microorganisms in the urine of healthy women sampled by suprapubic aspirate, by means of sequencing the 16S rRNA gene. Enhanced quantitative urine culture techniques (EQUC) were required to confirm the viability of the bacteria detected. The urobiome has been reported to be highly dependant on the vaginal microbiome, and determined by partial pressure of oxygen, the solutes dissolved in the urine (influenced by diet and metabolism), the presence of organic molecules (such as glycosaminoglycans (GAGs) of urothelial origin) and the urine pH, as well as by the consumption of antibiotics.
Dietary and behavioural indications, such as increasing water intake, minimise the likelihood of infections. Thus, dilution of both the solute concentration in the urine and the colonizing bacterial load of the bladder prevent this proliferation, in addition to facilitating their expulsion and preventing their ureteral ascent. Prebiotics, defined as molecules that, once ingested, selectively promote the development of beneficial microorganisms, can be useful in this setting when, either directly or their metabolites reach the urine.
Bladder colonisation with microorganisms can facilitate migration to the upper urinary tract, where they act directly as nucleants in lithogenic processes, directly or indirectly as a cause of an increase in mucus production and of the resulting inflammatory reaction. In addition, the increased urinary pH associated with bacterial urease activity can lead to the formation of struvite and associated apatite stones.
Most of the dietary oxalate is potentially metabolized by the intestinal microbiota as an energy source. Ever since Oxalobacter formigenes was demonstrated, the relationship between the intestinal microbiome and oxalocalcic lithiasis has become an important focus of research. Some studies show that there is a significant difference in favour of the presence of O. formigenes in healthy individuals, but results are very heterogeneous. In addition, it has been reported to have the ability to promote active transport of oxalate from the blood to the lumen of the intestine.
Being a strict anaerobic and oxalatrophic microorganism, the dietary restriction of foods by oxalate might not only affect its degree of colonisation but also jeopardise its efficacy in studies with patients who follow a strict oxalate-exclusive diet. Probiotic use of bacterial strains with a facultative oxalotrophic metabolism (with the possibility of using other energy sources) such as Lactobacillus sp., or Bifidobacterium sp could be a potential therapeutic alternative. The existence of research projects involving animal models in which the transplantation of faecal material clearly and consistently modified urinary biochemistry reinforces this hypothesis.
The presence of a stable and diverse microbiota has been correlated with the absence of stone pathology. For this reason, it is advisable to preserve the microbiome of patients, both through dietary and lifestyle advice, as well as through prebiotic and probiotic supplements of proven efficacy. For this same reason, it is also of special importance to minimise the impact associated with the consumption of antibiotics, as they can lead to a dysbiotic microbiota with clinical implications.
Oxalate malabsorption is present with inflammatory bowel disease and bariatric surgery. In both cases, differences have been found in the composition of the microbiota as a result of changes in the physiology and intestinal state. Regarding oxalate, either due to a degradation of the intestinal epithelium or an increase in calcium absorption, the concentration of free luminal oxalate is increased, which, if not used by oxalatotrophic populations, will increase urinary oxalate. Fecal material transplantation from donors with a relevant profile can be a compassionate solution in highly recurrent patients.
Low-grade systemic inflammation and oxidative stress
Low-grade systemic inflammation presents multiple organic and metabolic dysfunctions. Thus, the development of chronic kidney disease, atherosclerosis, diabetes, or autoimmune diseases has been correlated with altered inflammatory markers and mediators. The impact of the microbiota on low-grade systemic inflammation has been intensively studied, given its influence on circulating levels of pro-inflammatory or anti-inflammatory immune factors, and in the specific case of stone disease, on tubular damage associated with promotion of the oxidative state. Even in situations of normal renal function, the toxin fraction absorbed, either due to an excessive presence of protein in the diet or due to a proteolytic microbiota, can increase and damage tubular cells.
High counts of Proteobacteria are associated with states of low-grade systemic inflammation. Maintaining the integrity and barrier function of the intestinal epithelium is of vital importance. The influence of the microbiota lies not only in the groups detected but also in the metabolites that result from interaction with food. Thus, the maintenance of a correct luminal pH (in the acid range) or the correct supply of short-chain fatty acids, especially butyric, will benefit the trophism of the epithelial cells, the presence of intercellular junctions (tight junctions) and a correct structure of the adjacent mucus layer, thereby maximizing the epithelial barrier function.
This scientific evidence reinforces the indication to reduce protein intake in the patient affected by calcium oxalate stones in order to minimise the lesions that act as nucleation spots.
Influence of the intestinal microbiota on the urogenital microbiota
The composition of the urobiota is strongly influenced by the intestinal microbiota, mainly in the form of contaminations of faecal origin through the perianal area, much more relevant in the female gender due to anatomical reasons, and it is easy to correlate a higher concentration of bacteria with uropathogenic potential at the distal areas of the bowel, with a higher probability of developing urinary infections.
Multiple investigations have focused on reducing this pathogenic burden with both probiotics (with direct antagonistic capacity or ecological superiority), or with prebiotics (fibers and oligosaccharides that selectively promote the growth of beneficial organisms). Moreover, promoting a eubiotic vaginal microbiota rich in lactic acid bacteria (which will allow the generation of an acidic environment (pH 4.5) and represent an ecological barrier for pathogens during their migration to the urinary system) will have the potential to reduce the potential negative impact of the urogenital microbiota.
Urobiome and infectious stones
The formation of struvite stones is closely related to the presence of microorganisms with urease capacity in the urobiota. Preserving the structure and composition of the bladder urobiome, reducing the risk of colonization and proliferation of bacterial groups with high urease activity is especially relevant in patients with associated morbidities and those with a tendency for recurrent UTI. It can be expected that relevant alterations in the bladder bacterial ecosystem will translate into changes within the whole urinary system. Conditions that facilitate urinary stasis, as well as the implantation of urological devices, such as ureteral or urethral stents, are independent factors associated with a higher probability of developing infectious stones. Antibiotic-associated diarrhea can also facilitate contamination of the urogenital microbiome by gut microorganisms.
In order to reduce the burden of pathogenic bacteria, consumption of prebiotics and fibers with a high degree of polymerisation can be a very useful tool, as is preserving the structure of the genital microbiota, with special relevance at the vaginal level with vaginal probiotics reinforcing its physiological function both as a direct protection against vaginitis and vaginosis and as a reservoir of positive microorganisms that are indirectly provided to the urobiota. Another strategy is directly acting on the urobiota by instilling attenuated strains of pathogenic species such as E. Coli in what we could define as urobiota transplants. These options could be of interest in people affected with highly recurrent diseases or with permanent catheterization or fecal incontinence.
The urobiome in non-infectious stones
The Urobiome has been linked to non-infectious stones. Some studies confirm the presence of a urobiome in the upper urinary tract, with high similarity to the ecological structure found at the bladder level, and others point to certain microorganisms in the nucleation and growth of oxalocalcic and apatitic phosphate stones. Furthermore, the secretion of inflammatory proteins as a tissue response to the presence of bacteria would act in a similar way to the Tamm-Horsfall protein, with ability to add crystals. Finally, the citrate lyase activity of certain bacterial groups could reduce this stone inhibitor, resulting in the formation of calcium-based stones.
Interventions on the microbiome seem to be an important source of new therapies for prevention and management of the stone disease. Consequently, study and analysis of the microbiome should be a factor to be considered in order to precisely determine the different factors that explain the etiology of nephrolithiasis.
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