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ACE2 Expression in Kidney and Testis May Cause Kidney and Testis Damage

After 2019-nCoV Infection

Caibin Fan1, Kai Li1, Yanhong Ding1, Wei Lu2, Jianqing Wang1*

1
Department of Urology, The Affiliated Suzhou Hospital of Nanjing Medical University

2
School of Nursing, Suzhou Vocational Health and Technical College

*Correspondence to:

Jianqing Wang, Department of Urology, The Affiliated Suzhou Hospital of Nanjing Medical

University, 26 Daoqian Rd, Suzhou, Jiangsu 215000, PR China; Tel: +86-512-62362011; E-mail:

[email protected]

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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Abstract

In December 2019 and January 2020, novel coronavirus (2019-nCoV) - infected pneumonia

(NCIP) occurred in Wuhan, and has already posed a serious threat to public health. ACE2

(Angiotensin Converting Enzyme 2) has been shown to be one of the major receptors that mediate

the entry of 2019-nCoV into human cells, which also happens in severe acute respiratory

syndrome coronavirus (SARS). Several researches have indicated that some patients have

abnormal renal function or even kidney damage in addition to injury in respiratory system, and the

related mechanism is unknown. This arouses our interest in whether coronavirus infection will

affect the urinary and male reproductive systems. Here in this study, we used the online datasets to

analyze ACE2 expression in different human organs. The results indicate that ACE2 highly

expresses in renal tubular cells, Leydig cells and cells in seminiferous ducts in testis. Therefore,

virus might directly bind to such ACE2 positive cells and damage the kidney and testicular tissue

of patients. Our results indicate that renal function evaluation and special care should be

performed in 2019-nCoV patients during clinical work, because of the kidney damage caused by

virus and antiviral drugs with certain renal toxicity. In addition, due to the potential pathogenicity

of the virus to testicular tissues, clinicians should pay attention to the risk of testicular lesions in

patients during hospitalization and later clinical follow-up, especially the assessment and

appropriate intervention in young patients' fertility.

Keywords: 2019-nCoV, kidney, testis, ACE2

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Introduction

Since December 2019, a novel coronavirus-induced pneumonia was discovered in Wuhan, Hubei

Province, China [1, 2]. The virus is a previously unknown sub-coronal virus (β-round virus)

named 2019-nCoV by WHO, which forms a branch in the subgenus sarbecvirus, subfamily

Orthocoronavirinae. 2019-nCoV is closely related to SARS- CoV with above 85% identity [3]. In

addition to common respiratory symptoms such as cough and fever, some patients may also

experience other symptoms such as diarrhea and liver damage [4], which brings more challenges

to the patient's recovery. Although the source of the 2019-nCoV is still unknown, previous

research has shown that the receptor binding domain of 2019-nCoV was able to bind ACE2

protein on the surface of human cells, which provide strong evidence for human ACE2 being the

receptor for 2019-nCoV [5, 6].

Angiotensin converting enzyme 2 (ACE2) belongs to the angiotensin-converting enzyme family

of dipeptidyl carboxydipeptidases, which is homologous to human angiotensin 1 converting

enzyme. The expression distribution of ACE2 suggests that it might play critical roles in the

regulation of cardiovascular and renal function, as well as fertility. Since the global outbreak of

SARS in 2003, numerous studies have revealed the role of cell surface ACE2 as the cellular

receptor for SARS-Cov and NL63 [7-9]. ACE2 has also been proven to be a major receptor of the

novel 2019-nCoV because 2019-nCoV is closely related to SARS-CoV. As the virus enters the cell

by binding to cell receptors to complete intracellular replication, virus release, and induce

cytotoxicity, the route of virus infection depends on the expression and distribution of the

corresponding receptor [10-12]. Meanwhile, the damage caused by the virus in different organs is

closely related to clinical manifestations and has a major implication for understanding the
medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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pathogenesis and designing therapeutic strategies in clinical practice.

Here in this study, we analyzed the online datasets to uncover the expression pattern of ACE2 in

urinary and male reproductive systems, which is the potential mechanism of abnormal renal

function or even kidney damage in patients infected with 2019-nCoV. Moreover, we emphasized

high ACE2 expression level in testis because of the potential pathogenicity of the virus to

testicular tissues, especially the potential risks affecting fertility.

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Materials and Methods

Clinical data

We summarized the clinical data of three previous studies to extract the incidence of abnormal

renal function or kidney damage in patients infected with 2019-nCoV [2, 13, 14]. All clinical data

are directly obtained from the articles above.

Publicly available scRNA-seq gene expression data sets

In this article, we made use of some online renal single cell RNA-seq (scRNA-seq) gene

expression data sets that were publicly usable. These contained the data reported in GSE131685

and GSE107585.

We used RNA and protein expression data of ACE2 in different human tissues and cancer cell

lines through The Human Protein Atlas portal (Website: http://www.proteinatlas.org/) [15], GTEx

portal (Website: https://gtexportal.org) and The Cancer Cell Line Encyclopedia (CCLE) [16]. All

data are available directly online.

Analysis of scRNA-seq raw sequencing data

Primary analysis of scRNA-seq raw sequencing data

Raw reads were processed to generate gene expression matrices as described previously. Reads

with the same cell barcode, UMI and gene were grouped together to generate the number of UMIs

per gene per cell. Cell number was then determined based on the inflection point of the number of

UMI versus sorted cell barcode curve.

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Results

To determine whether patients infected with 2019-nCoV have abnormal renal function or kidney

damage, we reviewed the latest 3 studies focused on the clinical features of such patients. In these

3 cohorts, one was a familial cluster of six patients, while other cohorts contained 99 patients and

41 patients, respectively. Outcome in two relatively larger sample studies suggest that about 3% to

10% of patients infected with 2019-nCoV had abnormal renal function, including elevated

creatinine or urea nitrogen. In addition, 7% of patients experienced acute renal impairment (Table

1). Considering the large number of infected patients, it is necessary to explore the mechanisms of

abnormal renal function and to promote to take special care on such patients.

As the virus frequently enters the cell by binding to cell receptors, and ACE2 has been proven to

be one of the major receptors of 2019-nCoV in human body, we explored the online datasets to

find out the expression level of ACE2 in urinary system. As we expected, data from CCLE and

GTEx portal indicated that ACE2 mRNA expression level is relatively higher in kidney cells

(Figure 1).

To further determine the protein expression level of ACE2 in kidney cells, we investigated the

Human Protein Atlas portal to find out some details. Results of immunohistochemistry (IHC) also

indicated that the expression level of ACE2 protein is significantly higher in the kidney, especially

in renal tubular cells, although the mRNA expression level is not such high (Figure 2, Table 2).

Unexpectedly, we also found that ACE2 expresses quite highly in testicular cells. The protein and

mRNA expression of ACE2 in the testes is almost the highest in the body. Moreover, both cells in

seminiferous ducts and Leydig cells showed high ACE2 expression level (Figure 2, Table 2).

These results indicate that testicular cells are the potential targets of 2019-nCoV.
medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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To further assess the cell type specific expression of ACE2 and confirm ACE2 expression level in

kidney, we downloaded the gene expression data of single-cell RNA sequencing of human kidney

from Gene Expression Omnibus (GEO) datasets. We first analyzed GSE131685, a published

dataset containing scRNA-seq data of the normal kidney samples from 3 donors [17]. We divided

single cells into subclusters based on the canonical markers and cell classification in the original

literature (Figure 3A), and found specific ACE2 expressions in tubular cells. In contrast, ACE2

expression was not observed in immune cells and glomerular parietal epithelial cells (Figure 3 B).

Results of GSE132023 confirm the previous results (data not shown).

Therefore, ACE2 expression in renal tubular cells and testicular cells may suggest a potential

mechanism of infection and direct damage of renal tubules and testis by 2019-nCoV binding

ACE2 as host cell receptors.

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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It is made available under a CC-BY-NC-ND 4.0 International license .

Discussion

Novel coronavirus- infected pneumonia outbroke in Wuhan in December 2019 and January 2020,

which has posed a major threat to global public health [18]. The numbers of confirmed cases and

deaths are still rising quickly, posing higher challenges for disease control and patient treatment.

The symptoms of the disease are complex. In addition to common respiratory symptoms such as

cough and fever, some patients may also experience other symptoms such as diarrhea and liver

damage [4], or even asymptomatic, which brings more challenges to the patient's diagnosis and

treatment. Studies on the mechanisms of disease pathogenesis could help us understand the

disease comprehensively.

Here in this study, we first reviewed the latest literatures and found about 10% of the patients

infected with 2019-nCoV had abnormal renal function. This indicates the significance to explore

the mechanisms by which the virus affects renal cells. We used the online datasets and

bioinformatic methods and found out that ACE2, one of the major receptors for 2019-nCoV,

expresses quite highly in renal cells, particularly in tubular cells. IHC results showed no ACE2

expression in cells in glomeruli. Renal tubular cells have reabsorption and excretion functions, and

play a key role in excretion of metabolites, maintenance of body fluid balance and acid-base

balance. Renal tubular cell injury could cause renal tubules atrophy, thereby aggravating renal

interstitial fibrosis, secreting a variety of chemokines and growth factors into the stroma,

promoting interstitial inflammatory cell infiltration, interstitial intrinsic cell proliferation, and

extracellular matrix (ECM) accumulation. Therefore, 2019-nCoV could enter the renal tubular cell

by binding to ACE2, which induces cytotoxicity and abnormal renal function. Examination and

follow-up of the renal function of patients infected with 2019-nCoV is necessary to detect the
medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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impaired renal function in time and provide early intervention.

Another major point in this study is the high expression level of ACE2 in testicular cells. It is well

known that viruses such as HIV, HBV and mumps could enter the testicular cells and cause viral

orchitis. Besides, in some cases, virus-induced testicular tissue damage might result in male

infertility and testicular tumor [19]. SARS-CoV is just like the ‘cousin’ of 2019-nCoV and shares

the receptor ACE2 with 2019-nCoV. Previous research has also investigated the possible damage

of the testis in SARS patients and the effects of SARS on spermatogenesis. Their findings

suggested that orchitis is a complication of SARS and that spermatogenesis could be affected after

infection [20]. Current clinical data show that a large proportion of the novel coronavirus

(2019-nCoV) - infected pneumonia patients are young adults and even children, so the potential

testicular damage caused by the virus may exist as a late complication. However, limited

information is available regarding the involvement of reproductive organs in patients infected with

2019-nCoV. Therefore, our findings suggest that clinicians should take care of the possible

occurrence of orchitis. Following-up and evaluation of the reproductive functions should be done

in recovered male SARS patients, especially the young male patients.

Conclusions

Our study demonstrated the highly expression of ACE2 in kidney and testicular tissue and

facilitated the understanding of the mechanisms of abnormal renal function and kidney damage in

2019-nCoV-infected patients. Our findings also suggest the patient cares regarding the possible

occurrence of orchitis. Following-up and evaluation of the reproductive functions may be

necessary in recovered male SARS patients, especially the young male patients.
medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Figures

Fig. 1 Data of mRNA expression level of ACE2 in different human tissues from online datasets.

A. Data from CCLE showed ACE2 expression level in different tissues, including urinary system

(red frame).

B. GTEx portal showed ACE2 expression level in different tissues.

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Fig.2 Data of ACE2 protein expression level in different human tissues from HPA portal.

A. B. ACE2 protein expression level in different tissues.

C. D. Representative IHC staining of ACE2 in kidney and testis.

medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Fig.3 Single cell analysis of published kidney cell atlas.

A. Kidney cell atlas visualized by UMAP, colored by cluster number. Cluster number information

is provided by the authors.

B. The dotplot showing ACE2 gene expression of all major cell types.
medRxiv preprint doi: https://doi.org/10.1101/2020.02.12.20022418; this version posted February 13, 2020. The copyright holder for this
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Table 1. Summary of the renal function characteristics of patients infected with 2019-nCoV
in 3 cohorts

Characteristics Cohort 1 Cohort 2 Cohort 3

Patients n=99 n=41 n=6

Age, years 55·5 (21-82) 49 (41–58) 10-66

Sex

Female 32 (32%) 11 (27%) 3

Male 67 (68%) 30 (73%) 3

Renal Function

Blood urea nitrogen

Increased 6 (6%) N/A 0

Serum creatinine

Increased 3 (3%) 4/41 (10%) 2

Other renal related affairs

Acute kidney injury N/A 3 (7%) N/A

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Table 2. Summary of the IHC results in The Human Protein Atlas project

Testis (n=6) Kidney (n=6)

Cell type Characteristics IHC results Cell type Characteristics IHC results

Staining: High Staining: Not detected

Cells in
6/6 Cells in 6/6
seminiferous Intensity: Strong Intensity: Negative
(100%) glomeruli (100%)
ducts
Quantity: >75% Quantity: None

Staining: High Staining: High

6/6 Cells in 6/6
Leydig cells Intensity: Strong Intensity: Strong
(100%) tubules (100%)
Quantity: >75% Quantity: 75%-25%
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