key: cord-329118-0gq5yusk
authors: Xiang, Boyu; Hu, Xiangyu; Li, Haoxuan; Ma, Li; Zhou, Hao; Wei, Ling; Fan, Jue; Zheng, Ji
title: ScRNA-seq discover cell cluster change under OAB: ACE2 expression reveal possible alternation of 2019-nCoV infectious pathway
date: 2020-06-06
journal: bioRxiv
DOI: 10.1101/2020.06.05.137380
sha: 
doc_id: 329118
cord_uid: 0gq5yusk

Objective Previous study indicated that bladder cells which express ACE2 were a potential infection route of 2019-nCov. This study observed some differences of bladder cell cluster and their ACE2 expression between OAB mice and healthy mice, indicating the change of infectious possibility and pathway under overactive bladder (OAB) circumstance. Material and method Pubic dataset acquisition was used to get ACE2 expression in normal human bladder and mice bladder (GSE129845). We built up over OAB model and studied the impact on cell typing and ACE2 expression. By way of using single-cell RNA sequencing (scRNA-seq) technique, bladder cell clustering and ACE2 expression in various cell types were measured respectively. Result In pubic database (healthy human and mice bladder), ACE2 expression in humans and mice is concentrated in bladder epithelial cells. The disappearance of umbrella cells, a component of bladder epithelial, was found in our OAB model. In the two mouse bladder samples, ACE2 expression of epithelial cells is 34.1%, also the highest of all cell types. Conclusion The disappearance of umbrella cell may alternate the infection pathway of 2019-nCov and relate to the onset and progression of OAB.

In December 2019, a novel coronavirus named 2019-nCoV emerged in Wuhan, China, and swept around the world in just one month. Except remarkable fever and respiratory disorder, urinary system damage was also detected in some 2019-nCoV patients 1, 2 .Though there has been no evidence of positive detection of 2019-nCoV in urine so far, its detection in urine samples of SARS patients hinted the importance of virus-testing in urine samples of 2019-nCoV patients, which showed that the urinary system was a potential route of infection 3, 4 The expression and distribution of receptors determine the pathway of viral infection, which is of great significance for understanding the pathogenesis and designing treatment strategies 5 .

2019-nCov was reported to share the same receptor with SAR-Cov, Angiotensin-converting enzyme 2 (ACE2), and previous study has proven that bladder epithelial cells are potential target cells for a novel coronavirus and the concentration of ACE2 seemed to have a special pattern which shows a decreasing trend from the out layer of the bladder epithelium (umbrella cells) to the inner layer (basal cells) with the intermediate cells in-between 6 .

Studies and a lot of data show that older people with basic diseases have a higher chance of infection and higher mortality if they are infected 7 , so it is very urgent and important to figure out how the underlying disease affects the way to change the 2019-nCov infection pattern. The purpose of our study is to explore cell cluster structure and ACE2 expression pattern in the bladder and use scRNA-seq to infer the effects of human senile lesions, OAB, on umbrella cells and intermediate cells in the bladder epithelium from the results of mice. Since OAB is a common senile disease which leads to expensive medical expenses and can be very authentic to simulate the effects of senile diseases on the bladder epithelium 8 and it greatly affects the quality of life of the patients, we construct a model by using OAB mice.

Bioinformatics scRNA-seq has been widely used in 2019-nCoV research because it has the ability to analyze gene expression of all cell types in multiple tissues with unbiased high resolution. Because this potential urinary tract infection pathway may be critical to prevent 2019n-Cov, here we used two scRNA-Seq transcriptomes to analyze bladder tissue from normal and OAB mice and combined human and mouse bladder ACE2 expression data from public data base to analyze the impact of underlying disease on this potential infectious system 7 .

Gene expression matrices of scRNA-Seq data from normal humans and mice were downloaded from the Gene Expression Omnibus (GSE129845).

We replicated the downstream analysis using the code provided by the author in the original paper. We built up over active bladder model (OAB) and studied the impact on cell typing and ACE2 expression. By way of using scRNA-seq technique, bladder cell clustering and ACE2 expression in various cell types were measured respectively.

Bladder tissue samples were gathered and instantly stored in the GEXSCOPE Tissue Preservation Solution (Singleron Biotechnologies) at 2-8°C.

Before dissociating the tissue, the specimens were washed with Hanks Balanced Salt Solution (HBSS) for three times and shred into 1-2 mm pieces.

At 37°C in a 15ml centrifuge tube, the tissue pieces were dissolved in 2ml 

In PBS, the concentration of single-cell suspension was 1 × 105 cells/ml. 

To get access to high quality cells, we discarded cells with less than 200 genes and more than 5000 genes, as well as the ones whose mitochondrial content was higher than 20%. After screening, 7330 cells were kept for the following analysis. The tSNE projection of the data calculation was similar to previously mentioned. Cell clusters with resolution 0.8 were obtained by using Seurat Find clusters function. With cell canonical markers, their types were distributed.

From dataset (GSE129845), cell types annotated were basically the same as the original research 9 , except neuron of mouse ( Figure 1A , 1B, 2A and 2B).

The fibroblasts in the red box were annotated as neurons by GPM6A in the original article, but more credible markers (Col3a1, Col1a1, and Dcn) proved that they were fibroblasts. In human bladder, the expression of ACE2 was mainly concentrated in three subtypes of epithelial cells, with a small amount distributed in fibroblasts and monocytes ( Figure 1C and D) . In mouse bladder, ACE2 also gathered in epithelial cells, but with a higher density than human ( Figure 2C and D) . As shown in Figure 3 , the ACE2 expression in human bladder relatively corresponded to that of mouse bladder.

As presented in our independent data (Figure 4) 

According to the latest scRNA-seq studies, ACE2 proved detectable in respiratory, digestive and urinary systems 6, 10, 11 , indicating these systems were potential targets for 2019-nCoV dissemination. Lin, et al have revealed that in bladder, the expression level of ACE2 was relatively high in epithelial cells, especially umbrella cells 6 , which indicated umbrella cells or epithelial cells were more likely to be infected by 2019-nCoV. As we know, the bladder epithelium is a transitional epithelium with high degeneration and rapid cell regeneration, thus bladder diseases like OAB may have an impact on it. In our study, we sought to figure out the changes of scRNA-Seq transcriptome under OAB circumstance. However, healthy or non-cancerous human bladder samples are usually unobtainable. We compared each cell type across species, we found that most human and mouse bladder cells were relatively well correlated. By comparing ACE2 expression profiles, we also found similarities: The highest ACE2 concentration existed in epithelial cells of both species. As an alternative, Mice model was reliable to evaluate the effects of OAB on cell clustering and 2019-nCoV infection risk.

We identified the cells to construct the cell map, and then further clustered the fibroblasts and epithelial cells. We firstly discovered that OAB Umbrella cells are small in quantity but functionally significant. As known, bladder epithelial cells are continuously exposed to mechanical forces. Yet for all that promise, our method is far from being the whole answers.

The results from mouse scRNA-seq were all side evidence without direct relation with clinical features of 2019-nCoV. In addition, we only detected a limited number of mouse bladder samples, rather than hACE2 transgenic mice or human samples. Li et al has reported that compared to human's ACE2, mice's ACE2 is less efficient in SARS-CoV binding. Due to differences in molecular structure, such a binding efficiency difference may also appear in 2019-nCoV. Beyond that, the pathogenicity of 2019-nCoV was only clarified in hACE2 mice 19 , so normal mouse model may fail to simulate pathogenesis and therapeutics of 2019 novel coronavirus pneumonia.

Further studies should turn to hACE2 mice or humans and increase the sample size. 

The Extent of Transmission of Novel Coronavirus in Wuhan, China, 2020

Novel Coronavirus-Infected Pneumonia

Duration of RT-PCR positivity in severe acute respiratory syndrome

Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov

Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection

Reduced Ca2+ spark activity contributes to detrusor overactivity of rats with partial bladder outlet obstruction

Single-cell transcriptomic map of the human and mouse bladders

Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCov

The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes

Exocytosis/Endocytosis in Bladder Umbrella Cells

Hypercompliant Apical Membranes of Bladder Umbrella Cells

Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding

Acute renal impairment in coronavirus-associated severe acute respiratory syndrome

Persistent shedding of viable SARS-CoV in urine and stool of SARS patients during the convalescent phase

The Pathogenicity of SARS-CoV-2 in hACE2 Transgenic Mice