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. 2016 Feb 22;1(3):16004.
doi: 10.1038/nmicrobiol.2016.4.

MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2

Man-Lung Yeung  1   2   3   4 , Yanfeng Yao  5 , Lilong Jia  2 , Jasper F W Chan  1   2   3   4 , Kwok-Hung Chan  2 , Kwok-Fan Cheung  6 , Honglin Chen  1   2   3   4 , Vincent K M Poon  2 , Alan K L Tsang  2 , Kelvin K W To  1   2   3   4 , Ming-Kwong Yiu  7 , Jade L L Teng  2 , Hin Chu  2 , Jie Zhou  1   2   3   4 , Qing Zhang  6 , Wei Deng  5 , Susanna K P Lau  1   2   3   4 , Johnson Y N Lau  4 , Patrick C Y Woo  1   2   3   4 , Tak-Mao Chan  6 , Susan Yung  6 , Bo-Jian Zheng  1   2   3   4 , Dong-Yan Jin  8 , Peter W Mathieson  6   9 , Chuan Qin  5 , Kwok-Yung Yuen  1   2   3   4
Affiliations

MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2

Man-Lung Yeung et al. Nat Microbiol. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes sporadic zoonotic disease and healthcare-associated outbreaks in human. MERS is often complicated by acute respiratory distress syndrome (ARDS) and multi-organ failure(1,2). The high incidence of renal failure in MERS is a unique clinical feature not often found in other human coronavirus infections(3,4). Whether MERS-CoV infects the kidney and how it triggers renal failure are not understood(5,6). Here, we demonstrated renal infection and apoptotic induction by MERS-CoV in human ex vivo organ culture and a nonhuman primate model. High-throughput analysis revealed that the cellular genes most significantly perturbed by MERS-CoV have previously been implicated in renal diseases. Furthermore, MERS-CoV induced apoptosis through upregulation of Smad7 and fibroblast growth factor 2 (FGF2) expression in both kidney and lung cells. Conversely, knockdown of Smad7 effectively inhibited MERS-CoV replication and protected cells from virus-induced cytopathic effects. We further demonstrated that hyperexpression of Smad7 or FGF2 induced a strong apoptotic response in kidney cells. Common marmosets infected by MERS-CoV developed ARDS and disseminated infection in kidneys and other organs. Smad7 and FGF2 expression were elevated in the lungs and kidneys of the infected animals. Our results provide insights into the pathogenesis of MERS-CoV and host targets for treatment.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Ex vivo and in vitro experiments demonstrating renal infection by MERS-CoV.
a, Pathway analyses of MERS-CoV-infected (top) and SARS-CoV-infected (bottom) Calu-3 cells based on differential gene expression profiles. Red line: the most significant –log(P value) of the most perturbed biological pathway by SARS-CoV. The –log(P value) is calculated based on the hypergeometric distribution (right-tailed Fisher's exact test) of a gene of interest that can be found in a molecular network over that of a randomly selected gene. Data were derived from two independent experiments. b, Ex vivo human kidney cultures were inoculated with either MERS-CoV or SARS-CoV. The MERS-CoV-inoculated human kidneys were cryosectioned at 24 h post inoculation (top). NP of MERS-CoV was immunostained by specific antibodies (green, left). The kidneys were co-stained with cell-type-specific antibodies against cytokeratin 18 (CK18), CD31 and synaptopodin (red, middle). Co-localization of viral antigens with cellular markers is shown in yellow (right). Nuclei counterstained by DAPI are shown in blue. Representative images from three independent experiments are shown. Total RNAs of MERS-CoV-inoculated (left, lanes 1–5) and SARS-CoV-inoculated (right, lanes 1–5) human kidneys samples were collected at the indicated time points post infection (bottom). Viral RNAs were detected by RT-qPCR as described previously. MERS-CoV-infected HK2 (left, ‘+’, lane 6) and SARS-CoV-infected Calu-3 samples (right, ‘+’, lane 6) were included as positive controls. Mock-treated HK2 (left, ‘–’, lane 7) and Calu-3 (right, ‘–’, lane 7) samples were included as negative controls. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was also detected as a loading control. c, Conditioned medium (left) and cell lysate (right) of MERS-CoV-infected (M) and SARS-CoV-infected (S) HK2 and NHMC cells were collected 24 h post inoculation and the viruses were quantified using TCID50 assays as described. C, mock infected; M, MERS-CoV-infected; S, SARS-CoV-infected. Error bars represent the mean ± s.d. of three independent experiments. Samples from a–c represent biological replicates.
Figure 2
Figure 2. MERS-CoV infection induced the expression of caspase-3, Smad7 and FGF2.
a, Apoptosis of MERS-CoV-infected (M) and SARS-CoV-infected (S) HK2 cells were measured by fluorometric analysis of caspase-3 activity. Mock-infected cells (C) were included as negative control. a.u., arbitrary units. b, Western blot analysis of caspase-3, Smad7, FGF2, MERS-CoV NP and SARS-CoV NP in MERS-CoV-, SARS- and mock-infected HK2 cells. γ-Tubulin was also detected as a loading control. Representative images from three independent experiments are shown. c, Heat map showing the candidate genes in the category of ‘renal necrosis/cell death’ identified in Fig. 1a. Arrows point to SMAD7 and FGF2, which were selected for further study. d, The relative expression of Smad7 (left) and FGF2 (right) mRNA was measured using RT-qPCR. e, Quantitative measurement of secreted FGF2 was performed using an anti-FGF2 enzyme-linked immunosorbent assay kit. Statistical significance was evaluated by Student's t-tests and P values are shown in a,d,e. Except for c, all samples were harvested at 24 h.p.i. for measurements. Error bars in a,d,e represent the mean ± s.d. of three independent experiments. Samples from a–e represent biological replicates.
Figure 3
Figure 3. Suppression of Smad7 and FGF2 expression subverted MERS-CoV-induced apoptosis.
a, MERS-CoV-induced Smad7 expression in HK2 cells was dampened by transfection of siRNAs (Smad7-1 siRNA, lane 1; Smad7-2 siRNA, lane 2). Control siRNA-transfected (lane 3) and mock-infected (lane 4) cells were analysed. Left panels: protein expression of Smad7 and cleaved caspase-3. γ-Tubulin was detected as a loading control. Right panels: mRNA levels of Smad7 normalized to GAPDH and the relative apoptotic activities of the MERS-CoV-infected cells. b, FGF2 inhibition by a small-molecule compound or antibodies. Caspase-3 activities in MERS-CoV-infected HK2 cells were measured when FGF2 signalling was blocked with a small-molecule inhibitor AG1296 (top, lane 1). AG490 (top, lane 2), which blocks epidermal growth factor but not FGF2 signalling, and untreated MERS-CoV-infected cells (top, lane 3) served as negative and mock controls, respectively. Caspase-3 expression was determined in MERS-CoV-infected HK2 cells treated with anti-FGF2 neutralizing antibodies (bottom, lane 1) or irrelevant IgG (bottom, lane 2). Mock-infected cells were assessed (bottom, lane 3). γ-Tubulin was detected as the loading control. c, Cell protection by anti-Smad7 oligonucleotide against MERS-CoV-induced cytotoxicity in HK2 (dark diamonds) and Calu-3 (grey squares) cells when compared with that of untreated MERS-CoV infected cell lines. d, The antiviral activity of anti-Smad7 oligonucleotide was measured by comparing the levels of viral load from the MERS-CoV-infected cell extracts. All statistical significance was evaluated by Student's t-tests (P) and Pearson's correlation analyses (r). Images shown in a and b are representative of three independent experiments. Error bars in a–d represent the mean ± s.d. of three independent experiments. Samples from a–d represent biological replicates.
Figure 4
Figure 4. Viral loads and host gene expression in the lung and kidney of common marmosets inoculated with MERS-CoV on day 3 post infection.
a, Quantitative measurement of MERS-CoV RNA in the lungs of MERS-CoV-inoculated common marmosets. Samples were divided into three categories (1.9 × 105 ± 9.9 × 104; 1.6 × 106 ± 5.2 × 105; 2.6 × 106 ± 1.7 × 106) based on the viral load (top). The relative expression levels of Smad7 (middle) and FGF2 (bottom) are indicated. All values were normalized to GAPDH. Statistical significance was evaluated by Student's t-tests and P values are indicated. Error bars represent the mean ± s.d. of three selected tissue samples. b, Co-immunohistochemical staining of MERS-CoV NP, caspase-3, Smad7 and FGF2 in kidneys of MERS-CoV-inoculated common marmosets. All co-immunohistochemical staining was performed on the same slide, except Smad7 and MERS-CoV NP, which were stained on separate slides, and the overlay view was generated using two neighbouring slides. The specificity of anti-MERS-CoV antibodies was confirmed by overnight pre-incubation with a fivefold more concentrated MERS-CoV NP peptide before their application to the kidney sections. The central image in the bottom panel represents the background signal detected after inoculation of rabbit secondary antibodies. Nuclei counterstained by DAPI are shown in blue. c, Haematoxylin and eosin staining of kidney sections of MERS-CoV-inoculated common marmosets. Interstitial infiltration was observed in the infected kidneys (arrow heads). Characteristic histological features of acute kidney injury, including flattened epithelial cells (arrows) and peritubular capillary congestion (white arrows), were detected in the kidney sections of MERS-CoV-inoculated common marmosets. Images shown in b and c are representatives of the four common marmosets' kidneys in which the MERS-CoV RNAs were detected (Supplementary Fig. 8a). D, dilated renal tubules; PC, protein cast.
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