The study findings were published in the peer reviewed journal: Molecular Therapy.
https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(22)00712-2
In this study, we employed both in vitro and in vivo model systems to uncover a previously unrecognized pathogenic mechanism of kidney injury associated with COVID-19, in which dysregulation of STAT3 signaling plays a central role.
Our recent reports about increased STAT3 phosphorylation in COVID-19 patient kidney biopsy suggests that aberrant STAT3 signaling due to the SARS-CoV-2 infection could be one of the pathomechanisms underlying the renal injury in COVID patients,22,23 given that STAT3 activation in kidney cells has been implicated in various kidney diseases.18
However, how the STAT3 signaling pathway in kidney cells is perturbed by SARS-CoV- 2 infection remains unclear.
A massive secretion of cytokines is elicited by SARS-CoV-2 infection in COVID- 19 patients, including IL-6, a potent activator of STAT3 signaling, so the systematic cytokine response to the viral infection is one of the triggers to activate the STAT3 signaling. However, recent reports about the detection of viral mRNA and proteins in kidney biopsies of COVID-19 patients33,34 and infection of human kidney organoids by SARS-CoV-2 viruses in vitro 6 suggest that kidney cells could be infected directly by the virus.
This opens up a new question whether direct viral infection by SARS-CoV-2 can alter STAT3 signaling and modulate the severity of kidney injury due to the exposure to massive cytokines. To address this question, we found that in vitro cultured HK-2 cells infected by SARS-CoV-2 had an elevated level of phosphorylated STAT3 and KIM-1 expression, demonstrating that direct infection of SARS-COV-2 can disturb the STAT3 signaling and cause renal tubular epithelial cell injury.
In order to understand the mechanism for the renal cell injury caused by direct infection of SARS-CoV-2, we generated a number of transgenic zebrafish to express individual viral proteins of SARS-CoV-2 in the embryonic pronephric tubular epithelial cells and found that the viral protein ORF3A could induce renal tubular injury in zebrafish.
ORF3A, one of the largest accessory proteins of SARS-CoV-2, encodes a viroporin with ion channel activitiesthat is localized to plasma membrane and lysosomes and is involved in viral release.35-39 Like SARS-CoV ORF3A, SARS-CoV-2 ORF3A has been shown to induce apoptosis, necrosis and pyroptosis in HEK293T and Vero E6 cells,40,41 and can activate NF-κB and NLRP3 inflammatory pathways in HEK293T and A549 cells.42
However, the pathogenic role of ORF3A has not been characterized in the context of kidney cells or STAT3 signaling. Here, we presented both in vitro evidence with HK-2 cells and in vivo evidence with transgenic zebrafish that ORF3A in kidney cells is indeed able to modulate the NF-kB pathway and increase cytokine production in kidney cells, including TNF-α and IL-6, which are NF-kB target genes and strongly associated with the severity of COVID-19 patient outcomes.20,43
Multiple molecular mechanisms have been proposed to mediate the dysregulation of STAT3 signaling by SARS-CoV-2, such as the imbalance between STAT1 and STAT3 activation and the crosstalk between plasminogen activator-inhibitor 1 and STAT3 signaling.21
We showed in HK-2 cells, both SARS-CoV-2 infection and expression of SARS-CoV-2 ORF3A alone could upregulate TRIM59, which competitively block the interaction between STAT3 and the phosphatase TCPTP and reduce the dephosphorylation of STAT3, leading to persistent activation of STAT3.
This represents a previously unrecognized mechanism of the regulation of STAT3 signaling by a SARS-CoV-2 viral protein downstream to the JAK-mediated STAT3 phosphorylation. Furthermore, since ORF3A could simultaneous activate NF-kB and STAT3 signaling, our data suggest that direct infection by SARS-CoV-2 may create a positive feedback loop to enhance the cellular response to cytokines such as IL-6 in kidney cells and aggravate the injury.
Such simultaneous activation of NF-kB and STAT3 has been observed in clinical samples from patients with other infalmatory diseases as an evidence of IL-6 signaling amplifier,44,45 so it is worthwile exploring whether this mechanism is applicable to other cell types besides kidney cells, and further investigation is needed to test it in other cell types susceptible to SARS-CoV-2 infection, such as pulmonary alveolar cells and endothelial cells.
We discovered that ORF3A of SARS-CoV-2 could interact with TRIM59 and STAT3, but it is unclear whether these interactions are involved in the regulation of STAT3 activation. Previously ORF3A is found to be localized to the lysosome-endosome membranes and inhibiting autophagy in cultured cells.37,39
We found that ORF3A is also present in the nuclei of HK-2 cells, which is consistent with its interaction with STAT3 that translocates to the nuclei when it is phosphorylated. Whether nuclear ORF3A also affects activity of other transcriptional factors requires further studies.
To corroborate our in vitro findings, we also examined the expression TRIM59 in the kidney autopsies of COVID-19 patients with AKI and found that its expression was significantly increased in the renal tubular cells. However, it is still unclear how TRIM59 is regulated in the COVID-19-associated nephropathy.
As ORF3A has been shown to inhibit autophagosome/lysosome function, it is possible that TRIM59 degradation is affected by ORF3A, which deserves further investigation.
To explore the potential role of ORF3A in COVID-19-associated acute kidney injury, we established a murine model with transient ORF3A expression in kidney and showed that ORF3A exacerbated kidney injury induced by ischemia/reperfusion and inhibition of STAT3 activities could ameliorate this effect.
These results demonstrate the cytotoxicity of ORF3A in kidney cells in a mammalian animal model and suggest that STAT3 is a critical mediator of renal injury in COVID-19-associated kidney injury.
Recently, the N protein of SARS-CoV-2 have been reported to cause renal tubular necrosis in mice by dysregulating the TGF-β-Smad3 pathway.14 Thus, multiple pathogenic pathways may be involved in the COVID-19-related renal injury. An appropriate animal model for kidney injury induced by SARS-CoV-2 infection is needed to further confirm these findings.
Zebrafish is a small vertebrate model organism that has been applied to AKI research.25,26,46-49 Because of the ease of genetic manipulation in zebrafish and its organ similarity to human, we used transgenic zebrafish to perform the functional screening of several viral proteins of SARS-CoV-2 in relation to kidney injury and identified ORF3A as a cytotoxic gene for tubular epithelial cells.
Although we did not identify the N gene of SARS-CoV-2 in our screening, presumably because zebrafish kidney is minimally susceptible to fibrosis and may have a different response to TGF-β signaling, the zebrafish may still be useful to test other viral proteins of SARS-CoV-2 to identify virulent gene affecting different organs or cell types. In addition, the zebrafish models could be used to screen drugs for treatment of COVID-19-induced kidney injury.
In summary, SARS-CoV-2 ORF3A protein can augment both NF-kB and STAT3 signaling and exacerbate the injury of renal tubular cells in the presence of cytokines induced by SARS-CoV-2 infection. In addition, ORF3A modulates the STAT3 signaling via TRIM59-mediated inhibition of dephosphorylation. Therefore, targeting the aberrant STAT3 activation may serves as a potential therapy for acute kidney injury in COVID-19 patients.
