Introduction
Kidney stone disease (KSD) is a common disease of urinary tract with increasing prevalence and a high risk of recurrence which poses a substantial healthcare burden [
1,
2]. Although great advances have been achieved in the surgical techniques for stone removal, the development of effective medical drugs for treatment and prevention of KSD remains stagnant, since the detailed mechanisms of stone formation are still unclear. Calcium oxalate (CaOx) is the most dominant type of stones, followed by calcium phosphate (CaP), struvite, uric acid, and cystine [
3]. Hypercalciuria and hyperoxaluria are major risk factors for stone formation, and supersaturation of the urine act as driving forces for crystallization [
4]. However, idiopathic CaOx stone formers shared similarity in urine chemistries with healthy individuals [
5]. Besides, most patients have unilateral stone disease, suggesting that supersaturation alone does not necessarily trigger stone formation. Hence, there must be other factors contributing to KSD.
With advanced techniques in sequencing and culturomics, the urinary tract has been discovered to harbor a large and diverse population of microbes [
6‐
8]. The urinary microbiome (urobiome) was proven to be associated with several urologic diseases, such as urinary incontinence, overactive bladder, and urologic tumors [
9,
10]. Thus, the role of the urobiome in KSD has attracted increasing research interest. Some researchers profiled the urobiome in stone formers by 16S rRNA gene sequencing and observed distinct microbial differences between stone formers and healthy individuals [
11‐
14]. In addition, Dornbier et al. found several bacterial species were dominant in the paired bladder urine and stone homogenate from the same patient using 16S rRNA gene sequencing and enhanced quantitative urine culture (EQUC) [
15]. These studies suggest the urobiome might be implicated in stone formation.
All current urobiome-related studies in KSD performed 16S rRNA gene sequencing to investigate the microbial community composition in the urine. As this sequencing technique only sequences 16S rRNA genes of bacteria, it generally provides taxonomic classification up to the genus level, which is difficult to achieve precise and reliable microbial identification at the species level. Although whole metagenome shotgun sequencing allows to sequence the entire genome from samples to resolve species-level taxonomy, it is much more costly and usually requires a great amount of DNA as starting material. However, the urobiome represents a relatively low biomass compared to the gut microbiome. We therefore sought to apply a method which can produce accurate and species-resolution taxonomic profiles to characterize the low-biomass urobiome in KSD. 2bRAD sequencing for Microbiome (2bRAD-M) is a novel sequencing approach to study the microbiome [
16]. This method digests the genomic DNA of the samples using type IIB restriction enzymes to yield iso-length DNA fragments [
17,
18]. The fragments were amplified for sequencing and mapped into species-specific 2bRAD markers for microbial characterization and quantification. 2bRAD-M has proven its ability to profile the low-biomass microbiome at the species level with high fidelity and low costs [
16].
The present study enrolled patients with unilateral stones and collected renal pelvis urine from both sides. We performed 2bRAD-M to profile the renal pelvis urobiome and aimed to determine if the urobiome in the renal pelvis is different between kidneys with and without stone(s). To our best knowledge, it is the first attempt to explore the pelvis urobiome in unilateral stone formers at the species resolution using 2bRAD-M.
Discussion
KSD is a complex disorder with a multifactorial etiology involving environmental and genetic factors. The application of 16S rRNA gene sequencing and the EQUC has revealed that a complex and diverse community of microbes inhabiting the urinary tract of healthy individuals [
6‐
8]. Since then, the urobiome has been verified to have links with some urologic diseases [
9,
10]. Increasing attention has been directed towards the role of the urobiome in KSD. It is well recognized that struvite stones, namely infective stones, were caused by urea-producing bacteria, such as
Proteus mirabilis [
25]. However, clinical studies found that the majority of bacteria isolated from all types of stones were non-urea-producing bacteria, and the most common bacterium isolated from urine and stone samples of patients was
Escherichia coli, suggesting that metabolic stones (CaOx, CaP, uric acid, etc.) were also related to urinary bacteria [
26,
27]. Recently, a distinct difference in microbial community composition was observed between stone formers and healthy individuals, further hinting at the potential importance of the urobiome in stone formation [
11‐
14]. But all these studies applied 16S rRNA gene sequencing to profile the urobiome, and the results mostly do not resolve taxa below the genus level. In addition, most studies conducted comparative analysis of the bladder urobiome between stone formers and healthy controls. However, renal pelvis urine is more reflective of the microbiota colonizing the kidney than bladder urine in terms of the anatomic location. Some researchers have collected renal pelvis urine of stone formers for sequencing [
11,
13,
28], but there are currently no studies with large sample sizes that compare the renal pelvis urobiome with and without stone(s) in unilateral stone formers. Herein, we aim to characterize the renal pelvis urobiome of unilateral stone formers to explore whether microbial differences existed between the stone side and the non-stone side using 2bRAD-M, a novel sequencing technique that can depict the landscape of microbial community at the species level with high accuracy and low costs.
Both alpha diversity and beta diversity indicated that the overall microbial composition of the renal pelvis urine with stone(s) was similar to that without stone(s). There may be several possibilities for these results. First, the contamination of bladder during urine collection could lead to a high degree of similarity between the stone side and the non-stone side. Second, although we recruited unilateral stone formers with an initial stone episode, we cannot exclude the possibility that the patients might have bilateral stone formation before and the small stones may be eliminated in the urine. Since the non-stone side might develop stone formation before, it is easy to understand that both sides shared similarity with each other. Third, the resemblance between the urobiome in the stone side and the non-stone side was consistent with the previous findings [
13]. Liu et al. collected four types of urine samples in kidney stone patients, including bladder urine aspirated before bladder disinfection, newly formed bladder urine after bladder disinfection, kidney pelvis urine in the stone side after bladder disinfection, and kidney pelvis urine in the non-stone side after bladder disinfection. They have disinfected the bladder carefully to prevent contamination, but they found that four types of urine samples had similar microbial richness and diversity. Thus, we assumed that the diversity of the stone side was similar to that of the non-stone side in nature as they both came from the same subject. There might be some differential taxa between two groups, but the overall microbial communities in both groups did not differ significantly. Although Yang et al. found that the overall microbial composition of the stone side and the non-stone side was different, their study was limited by a very small sample size of just 4 patients [
28].
The abundance profiles showed that the dominant bacterial composition at different taxonomic levels was quite similar between the two groups. Some bacterial genera, including
Acinetobacter,
Pseudomonas,
Sphingomonas, and
Staphylococcus, were also previously reported as dominant genera in renal pelvis urine of stone formers [
13,
28]. Of note,
Acinetobacter,
Pseudomonas, and
Staphylococcus are important opportunistic pathogen that can cause UTIs. Some studies revealed that
Acinetobacter is overrepresented in the bladder urobiome of stone formers compared to controls [
11,
12].
Pseudomonas and
Staphylococcus were found to be dominant in stone homogenate, and
Pseudomonas aeruginosa and
Staphylococcus epidermidis were isolated from these samples via EQUC [
15,
29]. Although the major genera
Cupriavidus has not been reported in any previous study about the urobiome in KSD, it has been associated with other urologic diseases. For example,
Cupriavidus was more abundant in the bladder cancerous tissues than paracancerous tissues [
30]. The present study identified 451 bacterial species, and the top 5 most abundant species were
C. pauculus,
A. junii,
A. sp_CIP_110321,
S. paucimobilis, and
A. ursingii. These species are mainly present mainly in the natural environment that have rarely been reported in human infections.
Through paired comparisons and LEfSe, we found that the genus
Corynebacterium and the species
P. disiens was increased and the genera
Prevotella and
Lactobacillus and the species
P. bivia,
P. timonensis,
L. iners,
C. aurimucosum,
F. magna,
P. sp_286 and
P. sp_S150 were decreased in renal pelvis urobiome of the stone side versus the non-stone side. An increased level of
Corynebacterium in renal pelvis urine with stone(s) was also previously observed [
13]. Besides,
Corynebacterium spp. were cultured and isolated from stone homogenate [
15,
31].
Corynebacterium urealyticum, named for its potent ability to split urea, is confirmed to be related to urinary calculi [
32,
33].
Corynebacterium matruchotii, an inhabitant in the oral cavity, is associated with dental calculi formation and induce calcium precipitation and apatite deposition in vitro [
34]. It is well accepted that most CaOx stones form on a base of interstitial apatite deposits called Randall’s plaque [
35]. These clues have pointed to the potential causative role of
Corynebacterium in KSD. Notably, though,
C. aurimucosum was enriched in the non-stone side.
Lactobacillus and
Prevotella have been previously reported to be underrepresented in the bladder urobiome of stone fomers [
11,
14].
Lactobacillus is the common resident of vagina that can provide a healthy vagina environment and prevent the colonization of pathogens through production of lactic acid and antimicrobial compounds [
36].
Lactobacillus is also the dominant member of healthy bladder urobiome, and it could likewise inhibit the growth of urinary pathogens [
8,
37]. Probiotics belonging to
Lactobacillus spp. have been applied to prevent UTIs through intravaginal treatment, and a robust vaginal colonization with
Lactobacillus with reduced recurrence rate was observed [
38].
L. iners is a common
Lactobacillus spp. in the vaginal microbiome and bladder urobiome, which was found to share 99.99% similarity between the vagina and bladder [
39]. Due to its good adaptability,
L. iners can maintain a relatively constant abundance under fluctuating environmental conditions, while other
Lactobacillus spp. may not survive [
40,
41]. Besides,
L. iners prevents pathogens from obtaining important nutrients and inhibit their growth through activation of the innate immune system in vaginal epithelial cells [
42]. Studies also showed that UTI risk is associated with depletion of
L. iners [
43]. The above evidences suggest
L. iners might play a protective role in KSD.
Prevotella is highly present across different body sites, including vaginal, gastrointestinal and urinary tract. Some
Prevotella spp. are proficient producers of the short-chain fatty acids (SCFAs) [
44]. SCFAs are major metabolites of bacterial fermentation that maintain the intestinal barrier integrity and exert anti-inflammatory effects, and it can regulate oxidative stress to prevent acute and chronic kidney injury through the gut-kidney axis [
45,
46]. Since oxidative stress is closely involved in stone formation, it is no surprise that the intestinal microbiome of stone formers showed decreased levels of
Prevotella [
47,
48].
P. bivia,
P. timonensis, and
P. disiens are highly abundant species in vagina, all of which are associated with vaginal dysbiosis [
49]. However, their role in urinary disease has not been reported and requires further exploration.
Correlation analysis has revealed extensive and close connections among different genera and species. The positively correlations indicate that some bacteria might support each other’s growth and colonization, while the negative correlations suggest that some bacteria might compete with other residents in the same niche to hinder their growth. In addition, 20 species from top 30 most abundant species were selected as the optimal marker set to construct a random forest model, which distinguished the stone side from the non-stone side with accuracies of 71.2%. The POD based on the 20 species markers also achieved a powerful classification potential. Since the species markers could be a potentially effective tool for predicting the stone group, the communities of these species might be implicated in stone formation.
Based on existing research on urobiome, it is unlikely that a single pathogen contributes to KSD. The renal pelvis might harbor diverse communities of microbes that are constantly in interaction with each other. Pathogenic bacteria might change urine chemistries to create a lithogenic environment, promote CaOx crystals growth and aggregation through surface structures, and induce the expression of pro-inflammatory proteins and stone matrix proteins in renal tubular epithelium to exacerbate the progress of stone formation [
29,
50,
51]. Protective bacteria might have anti-inflammatory functions, and inhibit pathogenic bacteria colonization to maintain a balanced microbiome composition, favoring an anti-lithogenic environment. We assumed that a loss of protective bacteria and a consequent increase of pathogenic bacteria in the kidney, referred to as urinary dysbiosis, might be a key step in the process of KSD.
Our study had several limitations. First, sample size was modest, and 40% of samples lacked data on stone composition, which limited generalizability, comparison and subgroup analysis. A larger study population is needed to expand urobiome knowledge across stone types. Second, 2bRAD-M was unable to predict the potential functional pathways of the urobiome. The application of metagenomics will allow an insight into structural and functional information of the microbial communities. Finally, like most urobiome studies, our study was just descriptive in nature, failing to directly explore the functionality of the urobiome. Thus, it is difficult to determine the causal relationship between the urobiome and KSD. Longitudinal follow-up studies and experimental studies are necessary to whether the bacteria are contributors, bystanders, or consequences of stone formation.
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