The Microbiome in Benign Renal Tissue and in Renal Cell Carcinoma

Introduction: The renal bacterial colonization has not been explored so far. Objective: The aim of this study was to describe the renal microbiome and to determine differences of the renal microbiome in healthy and tumor-bearing parenchyma. Methods: Ten biopsies from patients undergoing laparoscopic nephrectomy for renal carcinoma with no history of urinary tract infections within the last 6 months were included in this study. The identification of all microorganisms was done using 16S DNA sequencing. The beta diversity analysis was performed by Bray-Curtis dissimilarity. Results: In all kidney samples, a plethora of microorganisms was found, with significant differences between benign and malignant renal tissue (p < 0.0001). Conclusions: There is evidence that healthy kidney tissue as well as renal cell cancer tissue have a specific microbiome, thus opening new perspectives in renal physiology and tumor pathogenesis.


Introduction
The term microbiome is defined as the totality of all microorganisms of a habitat [1]. Due to recent developments in microgenetic and microbiologic analyses, the understanding of different microbiomes in the human body has increased. The mechanisms of bacterial growth and proliferation of a healthy tissue as compared to a tissue affected by disease, such as cancer, has not been explored so far. Emerging evidence shows that there are multiple microorganisms inhabiting many sites of the body, including the urinary tract [1]. In the past, urine was thought to be sterile in healthy individuals. Recent studies show that there are a great number of microorganisms in the urinary tract [1] and changes in patients with type 2 diabetes mellitus [2], overactive bladder syndrome [3,4], urinary incontinence, interstitial cystitis [5], neuropathic bladder [6,7], sexually transmitted infections [8], and chronic prostatitis/chronic pelvic pain syndrome [9,10].
The aim of this pilot study was to examine the kidney microbiome. The second aim was to describe differences between healthy and tumor-bearing tissue based on the

Materials and Methods
This study was conducted with 10 formalin-fixed paraffin embedded (FFPE) tissue samples of 5 kidneys from patients who underwent laparoscopic nephrectomy because of renal carcinoma with no history of urinary tract infections within the last 6 months. Description of tumor entities and stage are recorded in Table 1. Samples were taken from the center of the malignant tissue and from tumor-free renal cortex. The samples were obtained directly from the operation site and put into sterile containers to avoid contamination. After that, they were taken to the pathology department and were embedded in paraffin without delay. From the paraffin-embedded blocks, each piece measuring 20 × 20 × 3 mm, a slice of 10 μm was cut from each block with the laser capture mi-crodissection. With these slices, the genetic analysis was performed. Every kidney has a different microbiome. For better comparability of the 2 categories (tumor and benign renal tissue), the number of bacterial DNA of all tissue samples in each category was added. The testing of the whole genome with the identification of microorganisms was done using 16S DNA sequencing. DNA isolation was done with the GeneRead DNA FFPE Kit (Qiagen). DNA quantification was performed with the Qubit dsDNA BR assay kit using the Qubit 3.0 instrument (ThermoFisher), and quality control was performed with the standard genomic DNA analysis kit on the Fragment Analyser (Agilent Technologies). The library preparation was done with the QIAseq 16S/ITS Screening Panel. The next-generation sequencing of the libraries was performed on MiSeqDx with V3 chemistry. The data was analyzed with the CLC Genomics Workbench with the CLC Microbial Genomics module. For the supportive identification of the bacteria, the COSMOSID database was utilized. The beta diversity analysis was performed by Bray-Curtis dissimilarity utilizing the principal coordinate analysis plot (PCo). Student´s t test was used to determine the numerical difference of the microbiomes.

Results
Basic patient and tumor characteristics are given in  Table 1. A plethora of microorganisms was found, with significant differences between benign and malignant renal tissue (p < 0.0001) (Fig. 1). We isolated 3 domains, 15 phyla, 16 classes, 19 orders, 27 families, 28 genera, and 30 species of microorganisms. In the domain of the Archeae we found two phyla, in the domain of the Eukaryota 6 phyla, in the domain of the bacteria we found 7 phyla. The distribution of microorganisms shows differences in benign and malign tissues (Fig. 2).
The Bray-Curtis dissimilarity showed a clear cluster of the microbiome of the benign tissue in the PCo diagram (Fig. 3).

Discussion
This is the first attempt to describe the microbiome in renal tissue. Our study demonstrates that there is a microbiome in renal tissue and that there are differences in the microbiome of benign and malignant tissue. The major limitation is the small sample size as it was designed as a pilot study.
For this study, 16S DNA analysis in FFPE tissue was utilized. FFPE tissue blocks maintain cellular morpholo- gy and are a standard storage method for human tissues. It allows histological analysis and is the standard method for DNA analysis. RNA and DNA are presented in degraded, highly fragmented forms, which now can be analyzed with a growing number of modern assays. Storage is uncomplicated, and paraffin blocks can be kept for further analysis and reference. In germ identification, there is always a concern regarding contamination during fixation and embedding of the tissue. To clarify the question, a large retrospective study of heart valve tissue biopsied between 1994 and 2005 was performed. Only paraffin blocks from patients with histologically confirmed infectious endocarditis were used. In 75% of the cases, the results of 16S rRNA sequencing were consistent with those from the valve cultures [11].
So far, healthy renal tissue was thought to be free of bacteria, although bacteria can enter the kidneys via the bloodstream [12][13][14]. The finding of bacteria in the kidney was always thought to be related to an infection, a microbiome of the kidney as such is unknown. The role of bacteria in the pathogenesis of kidney cancer is unclear. However, an association between renal cell cancer and viruses has been published in several studies [15][16][17][18].
In the bladder, the association between Schistosoma haematobium infection and the development of squamous cell carcinoma by the endogenous synthesis of nitrosamines and oxygen radicals has been published [19,20]. Xu et al. [21] reported the association of the urinary microbiome with urothelial cell carcinoma in a small number of patients. Pseudomonas and Anaerococcus were the most abundant genera in cancer urine samples. He suggested that urothelial carcinoma may be associated with altered microbiota of the urinary tract. Another Chinese study by Wu et al. [22] observed enrichment of Acinetobacter, Anaerococcus, and Sphingobacterium, and decrease of Serratia, Proteus, and Roseomonas in the cancer group when compared to the non-cancer group. Cancer patients with high risk of recurrence and progression showed an enrichment of Herbaspirillum, Porphyrobacter, and Bacteroides. Therefore, beta diversity was significantly different in the cancer and non-cancer group [22]. Concerning the influence of bacteria on cancer genesis, three Japanese studies evaluated the prophylactic effects of an oral Lactobacillus casei preparation in patients with superficial transitional cell carcinoma of the bladder. The results indicated that Lactobacillus casei strain Shirota could be effective for prevention and treatment of non-muscleinvasive bladder tumors [23][24][25].
Another recent study demonstrated significant variations in microbial populations in prostatic secretions, voided urine, and seminal fluid from patients diagnosed with prostate cancer or benign prostatic hyperplasia. The group of patients with prostate cancer had a significantly higher number of microorganisms compared to the benign prostate hyperplasia group, which differs from one another [26]. The interaction of microorganisms and their hosts is complex. Molecular mechanisms are thought to be responsible for oncogenesis, tumor progression, and response to anticancer therapy by changing the balance of host cell proliferation and cell death, influencing the immune system function and metabolism of host- produced factors and reaction to pharmaceuticals [27]. Therefore, diagnostic and therapeutic considerations concerning cancer and the microbiome require a multidisciplinary approach.

Conclusion
There is evidence that benign and malignant renal tissue have a specific microbiome, which differs from one another, opening a new field on renal function and tumor genesis.

Statement of Ethics
The Karl-Landsteiner University of Lower Austria states in its policy regarding "Good scientific practice," Version 1 from September 2015, under point 2.3 concerning measures not to be submitted to the ethics committee on humans: No submission to the ethics committee is required for medical actions that are exclusively performed for the health benefits of an individual patient. That means, if the objective is not the gain of knowledge, such medical actions ("therapeutic attempt") are not to be considered research projects, and therefore no "ethics commission" is needed. In our case, nephrectomy was performed to cure a renal cancer in each and every tissue sample utilized. Surgery had to be performed, the tissue was not destroyed postopera-tively, but referred to us for further analysis after tumor histology was finished. This is in accordance with Art. 35, Declaration of Helsinki, 2008.
A written informed consent for participation was given by all patients before nephrectomy during the inpatient admission.

Disclosure Statement
The authors have no conflicts of interest to declare.

Funding Sources
Qiagen Company provided the analysis equipment for DNA isolation of microbes. Qiagen company has not contributed to this study in any other way, such as study design, collection, and interpretation of data or the writing of the manuscript. This was performed solely by the authors.
No further funding or sponsoring was received for this study.

Author Contributions
Stefan Heidler contributed the study concept and design, analyzed and interpreted data, and wrote the manuscript. Stephan Madersbacher and Lukas Lusuardi were major contributors in the writing of the manuscript. Christa Freibauer performed the histological examination of the kidneys. All authors read and approved the final manuscript.