Molecular Basis of Kidney Defects in COVID-19 Patients

Background: Kidney damage is considered to be one of the risk factors for severity and mortality among COVID-19 patients. However, molecular nature of such observations remains unknown. Hypothesis: Altered gene expressions due to infection and in chronic kidney disease could explain severity in COVID-19 with kidney defects. Methods: We collected gene expression data from publicly available resources Gene Expression Omnibus CKD, Enrichr for deregulated genes in SARS-CoV infected cells in vitro, DisGeNET and others and carried out enrichment analysis using Enrichr. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 July 2020 doi:10.20944/preprints202007.0452.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. 2 Result: Number of common genes altered in chronic kidney disease (CKD) and SARS-CoV infected cells was 2834. Enrichment analysis revealed that biological processes related viral life cycle and growth, cytokines, immunity, interferon, inflammation, apoptosis, autophagy, oxidative stress and others were significantly enriched with common deregulated genes. Similarly, significantly enriched pathways related to viral and bacterial infections, immunity and inflammation, cell cycle, ubiquitin mediated proteolysis, signaling pathways like Relaxin signaling pathway, mTOR signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway were enriched with the common deregulated genes. These processes and pathways are known to be related to kidney damage. DisGeNET terms enriched include and related to Dengue fever, chronic Hepatitis, measles, retroviridae infections, respiratory syncytial virus Infections and many others. Kidney dysfunction related terms ischemia of kidney, renal fibrosis and diabetic nephropathy. Conclusion: Common deregulated genes in SARS-CoV infected cells and chronic kidney disease, as well as their enrichment with molecular processes and pathways relevant for viral pathogenesis and renal dysfunctions, could explain the severity of COVID-19 with kidney disease. This observation not only provides molecular relation of severity in COVID-19 with renal dysfunctions but might also help in the management and treatment targets for these cases.


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SARS CoV-2 is mainly observed in type II alveolar epithelial cells in lungs. ACE2 expression is also high in renal epithelial and bladder cells. Podocytes and proximal straight tubule can act as kidney host cells for infection and podocyte injury might be responsible for proteinuria seen in some cases [32]. Viral RNA has also been isolated from urine in COVID-19 patients. This observation supports viral tropism for kidney [25]. Electron microscopic examination further showed clusters of coronavirus particles with distinctive spikes in the tubular epithelium and podocytes. Immunostaining with SARS-CoV-2 nucleoprotein antibody was positive in tubules.
This result shows that SARS-CoV-2 could directly enter into the kidney cells, even though the stage of the disease was unknown [28,33].
Accumulative epidemiological evidences suggest that episodes of AKI and subsequent development or progression of CKD are associated. Severity of AKI and repeated episodes of AKI are associated with increased risk of CKD. Studies with animal models have provided evidence for a biological basis linking episodes of AKI with CKD [34]. Mitochondrial dysfunction, cell death, and inflammation are considered to be the underlying pathogenesis of AKI. Dysfunctions of these processes and pathways may lead to progressive chronic disease.
Complex interconnections of these pathways are likely to cross-talk between the tubular epithelium, endothelium, and interstitial compartments (reviewed in Zuk and Bonventre [35]).
Common changes in cytokine level and inflammation between AKI and CKD have been reviewed [36].This observation indicates that at the molecular level, pathways altered in CKD and AKI could be similar. AKI and CKD observed among COVID-19 patients might thus have similar molecular changes altering different pathways.

Altered expression of host genes in response to infection by SARS-CoV and SARS-CoV-2
Once the virus enters into the host, it manipulates host machinery for replication and its survival against immune response of the host. Altered expression of host genes has been identified in response to diverse viral infections. Analysis of host transcriptional response to SARS-CoV-2 and other respiratory infections through invitro, ex vivo and in vivo model systems reveals that each virus deregulates many common genes for antiviral activity as well as unique transcriptional footprints of individual virus. Compared to the response to influenza A virus and respiratory syncytial virus, SARS-CoV-2 shows a different response that lacks robust induction of a subset of cytokines including the Type I, Type III interferon and numerous chemokine.
Taken together, these data suggest that the unique transcriptional signature of SARS-CoV-2 might be responsible for COVID-19 [37]. In SARS-CoV infected peripheral blood mononuclear cells, increased expression of cytokines and transcription regulators has been identified.
Expressions of genes involve inactivation of macrophages and coagulation pathways were also increased [38]. Differential expression genes in interferon pathway between patients with acute respiratory distress syndrome (ARDS) and without ARDS have been identified [39]. During SARS-CoV infection in humans, deregulated cytokine and inflammatory response have been observed and correlated with pulmonary pathology. Levels of type I interferon and interferon stimulated genes (ISGs) were elevated in patients infected with SARS-CoV.IL-6 and IL-8 genes, as well as TNF-α and IL-1β genes, have also been associated with ARDS [40,41]. In SARS-CoV infected monocyte cells, expression of interferon and related genes was decreased after 24h of infection. Expression of Toll-like genes was increased while expression of MyD88, TRIF-1 and SITPEC was decreased in same conditions. Expression of cytokine was increased and expression of TRAF4, TRAIL, TGFβ2, Endoglin, IL-6R and IL-13 were decreased. Expression of chemokine, complement C3 (C3), PDGFRA and LTA4H was increased. Expression of genes associated with fibrosis was increased and expression of PROS1 (protein S), MMP2, SPON2, PLOD3, ADAMTS4 and TIMP3 was decreased [42]. Altered expression of many host genes has been reported after infection of SARS-CoVat different time points and catalogued in Enrichr at https://amp.pharm.mssm.edu/Enrichr/ [43,44]. To summarize, expression of many genes associated with cytokine, chemokine and inflammation are altered in SARS-CoV infected cells.
Similar result is also reported for cells expressing SARS-CoV-2 infected cells.

Altered expression of genes in chronic kidney disease and acute kidney injury
To identify the markers which potentially contribute to tubular cell damage and tubulointerstitial fibrosis in CKD, altered expressions of hundreds of genes have been identified. Functional classifications of the differentially expressed genes in CKD revealed that deregulated genes were associated with cell cycle, blood vessel morphogenesis, cardiac development, cell adhesion, erythropoietin pathway, inflammation mediated by IL-2 signaling, response to unfolded proteins and others. Expression of hepatitis A virus cellular receptor 1(HAVCR1) gene, also known as KIM-1, LCN2 gene, involves in transport of lipids, steroid hormones and retinoids and plays a role in innate immunity by limiting bacterial growth as a result of sequestering iron-containing siderophores and others were deregulated [49]. Differentially expressed genes from peripheral blood in advanced stage of CKD were enriched and associated with insulin-like growth factor activity, neuroactive receptor interaction, the complement system, lipoprotein metabolism and lipid transport. Genes were also associated ubiquitin pathway, cytoskeletal remodeling, the clathrin-coated endosomal pathway, T-cell receptor signaling and CD28 pathways, and many immune pathways [50]. Various processes and pathways like cytokine and interferon signaling, ubiquitination mediated proteasome degradation, activation of NFkB, cell cycle, infection, apoptosis, Wnt signaling, p53 dependent DNA damage response and others were enriched with deregulated genes in CKD [51].
Differential expressions of many genes like LCN2, CCL2, CCL20, CXCL2, EGR1, IGF1R,IL-8, IL-11, IL6ST, IL13RA1, IFI16, COL4A1, COL4A2, FN1, FGA, LEPR and others are identified in early kidney transplant with AKI. Differentially expressed genes are associated with interstitial inflammation, synthesis of extracellular matrix, increased cell migration and effects of 7 infected cells had similarity and might act synergistically to increase disease severity. Cross talk between kidney damage in AKI or CKD and lung injury has been observed. AKI or CKD affects the acute lung injury through systemic pro-inflammatory cytokines and mediated through heme oxygenase 1, HMOX1 [55,56] or through modification of neutrophil and platelet functions [57].
Direct infection of SARS-CoV-2 kidney cells and inducing the damage could not be ruled out [28,33]. Infection with SARS-CoV alters expression of hundreds of genes in vitro. Similarly, altered expressions of thousands of genes have been described in CKD. It is unknown whether deregulated genes in these 2 different conditions were similar or belong to similar biological processes and pathways.

Hypothesis
Altered gene expressions due to infection and in chronic kidney disease could explain severity in COVID-19 with kidney defects

Altered expression of genes in SARS-CoV infected cells in vitro
Increased (up) and decreased (down) expression of host genes on infection with virus at different time points are downloaded from Gene Expression Omnibus (GEO) database, a public functional genomics data repository (https://www.ncbi.nlm.nih.gov/geo/) and catalogued in Enrichr at https://amp.pharm.mssm.edu/Enrichr/ [43,44]. We downloaded the data for SARS-CoV only.
Detailed result is shown in the Supplementary Tables S1A and S1B.

Altered expression of genes in chronic kidney disease
There are several reports for altered expression of genes in CKD in different tissues. We  Table 1.
Interacting partners of SARS-CoV-2 coded proteins are taken from published result [46,47] and shown in the Supplementary Table S4B.

Enrichment analysis
Enrichment analysis for the genes was carried out using online facility at Enrichr at https://amp.pharm.mssm.edu/Enrichr/ [43,44]. Enrichr is an integrative web-based software application for analysis of a gene-set comparing with various gene-set libraries. Given an input list of genes, it provides enrichment for different libraries like Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Gene Ontology (GO) terms for biological processes, DisGeNET, a discovery platform containing publicly available collections of genes and variants associated to human diseases.
We searched "Pubmed" (https://www.ncbi.nlm.nih.gov/pubmed/) for relevance of the genes associated with different biological processes and pathway. For comparison of different set of genes/proteins, we used an online facility at http://bioinformatics.psb.ugent.be/webtools/Venn/

Comparison of altered expression of genes in CKD and SARS-CoV infected cells in vitro
Kidney injury has been reported earlier for SARS-CoV infection [58]. SARS-CoV and closely related viruses SARS-CoV-2 have genomic sequence similarity (94.6% similar in amino acid sequence and 80% similar in nucleotide sequence) [59]. These two viruses also have structural similarities of proteins coded by them [60], some of the symptoms in infected patients by two viruses are similar. Comorbidity with kidney abnormalities [9,10,58], diabetes and cardiovascular disease [61] in COVID-19 and earlier SARS-CoV infected patients are also similar. However, SARS-CoV and SARS-CoV-2 differed in their infectivity and mortality in infected patients [62]. Thus, we assumed that altered gene expression in SARS-CoV would be similar. Both CKD and AKI are risk factors for severity of COVID-19 ( Table 1). Severity of AKI and repeated episodes of AKI are also associated with increased risk of CKD [34]. Thus we have used altered gene expression data in CKD in our analysis. Comparison of gene expression altered in SARS-CoV infected cells and deregulated genes in CKD in the same direction (increased and decreased), revealed that 2834unique genes were common (

Enrichment analysis with common gene deregulated in viral infected cells and CKD
We carried out enrichment analysis using Enrchedr for biological processes defined by Gene Ontology (GO), biological pathways as catalogued in KEEG pathways and gene-disease relations from DisGeNETas mentioned in material and methods. Result of such analysis is described below.

Biological Processes
More than 4000 biological processes (BPs) are associated with 2834 common deregulated genes; associations of 1019 BPs are significant (p≤0.05) without correction for multiple testing and 362 BPs are associated significantly (adjusted p≤ 0.05). The most significantly (adjusted p=1.42E -23 ) associated BP was regulation of transcription from RNA polymerase II promoter (GO:0006357).
Out of 2834 common genes, 359 genes belong to this BP. Many other transcription/transcription regulations related to BPs was also enriched. We grouped significantly associated BPs related to     Matsushita [66]).

Enrichment of terms in DisGeNET with common deregulated genes in SARS-CoV and CKD
DisGeNET contains publicly available collections of genes associated with disease and/or traits associated with disease. Gene-disease/trait relations are collected from expert-curated repositories, catalogues of Genome-Wide Association Studies, animal models and the published literature [67]. This data was accessed through https://amp.pharm.mssm.edu/Enrichr/ [43,44] .
Enrichment analysis revealed that 544 disease/trait was significantly (adjusted p≤0.05) associated with common deregulated genes in SARS-CoV and CKD. We have grouped the associated diseases (except cancers) related to (i) infectious diseases, (ii) kidney, (iii) heart, (iv) Defective renal functions in CKD increases the risk of CVD, mortality in CVD is 2-3 fold higher in patients with advanced stage of CKD [68]. CKD is relevant comorbidity in COPD patients; 7.1% of COPD patients have been diagnosed with CKD. CKD is a predictor for mortality independently from other cardiovascular comorbidities [69]. NAFLD might be a risk factor for the development and progression of CKD, sharing cardio-metabolic risk factors and possible common pathophysiological mechanisms [70]. Infection with hepatitis B and C results in chronic liver diseases and can lead to glomerular disease. Interconnection between chronic liver disease and CKD during after hepatitis B and C infections might be mediated through circulating immune complexes or complexes formed in situ [71]. SARS-CoV was observed in different tissues including lung, renal tubule, liver, pancreas and others but absent in heart [72]. infection. Even though CVD and liver disease in COVID-19 patients are risk for severity and mortality, it is not known whether SARS-CoV-2 infect directly to heart or liver [5].
Patients with autoimmune disease like rheumatoid arthritis (RA) are at a higher risk of infection with SARS-CoV-2 due to their immune-compromised state resulting from their underlying immune conditions. Immune-modulatory therapies such as biologics could be used for COVID-19. As of April 1, 2020, among COVID-19 patients originated from Europe, North America, South America, Asia, Africa, and Oceania, only 110 individuals with rheumatic disease have been identified [73]. In this study, 19 (17%) of 110 patients was identified with rheumatic disease [73]. So far no convincing evidences are available for the relationship of arthritis and osteoporosis with COVID-19. However, in a meta-analysis, significantly increased risk of incident CKD among patients with RA compared with individuals without RA was observed [74]. Increased circulating inflammatory cytokines representing as chronic inflammatory condition among aged individuals contributes to development of osteoporosis, CVD and CKD.
In patients with renal insufficiency, observed of bone and mineral disorders may accelerate aging and is a risk factor for cardiovascular death in these patients. Changes in mineral and bone metabolism in the early stages of CKD might accelerate aging, osteoporosis and CVD [75]. Thus association of common genes deregulated in CKD and SARS-CoV with arthritis and osteoporosis related disorders might represent relationship between CKD and these diseases. It remains to be found out whether arthritis and osteoporosis are also observed among COVID-19 patients.
A COVID-19 male patient of age 80 years with multiple comorbidities including severe anemia was reported. Treatment with antiviral drug and recombinant erythropoietin for 7days improved drastically the conditions. Erythropoietin modulates cytokines and exerts cytoprotective and antiapoptotic effects. Besides, erythropoietin removes intracellular iron, requires for viral enzymatic activity. This may provide an unfavorable condition for the growth of the virus [76]. This result shows that anemia may complicate the outcome of COVID-19. Eleven COVID-19 patients with Thalassemic patients did not show and additional severity [77]. CKD associated with anemia has poor outcome. Anemia in CKD results from a complex processes due to shortened erythrocyte survival, erythropoietin deficiency, defective iron homoeostasis and uremic-induced inhibitors [78].

Out of 2834 common genes deregulated in SARS-CoV infected cells in vitro and CKD, 1659
genes were associated with relevant BPs, KEGG pathways and disease terms for infections, kidney dysfunctions and other possible comorbid conditions like heart disease, diabetes, liver disease and others. Comparing the unique 569 host proteins that interact with proteins coded by SARS-CoV-2 and SARS-CoV (Supplementary Table S4B), we observed that 74 common deregulated genes in SARS-CoV infected cells and CKD also interact with viral proteins.

Discussion
Common genes are deregulated in same direction in SARS-CoV infected cells and CKD.
Various biological processes and pathways enriched with the common deregulated genes are relevant for infectious diseases in general as well as CKD. Deregulated genes are significantly associated with several diseases, especially related to lung, kidney, liver and heart. We further Role of infection in clinical course of kidney disease including excess morbidity in end-stage kidney disease has been observed and reviewed [63][64][65][66]. Role of cytokines in immune response and inflammation in kidney damage including AKI and CKD is well documented and has been reviewed [79]. Expression of 13 interleukin and interleukin receptors (IL11, IL17RA, IL17RC,   IL18, IL1A, IL1F10, IL1R1, IL1RAP, IL1RL2, IL20RB, IL34, IL6 and ILF3) [40]. Cytokine storm has also been observed in COVID-19 [80].  [82]. MERS-CoV infection alters DNA methylation modifying the expression of genes associated with antigen presentation [83]. Altered expression of several microRNA in acute respiratory infections including human coronavirus has been reported and reviewed [84,85]. Several long non-coding RNA was identified in viralinfected lungs from mouse. Subsets of long non-coding RNAs are likely to play important role in respiratory virus pathogenesis [86]. It remains to be found out whether similar mechanism (s) of gene regulation is operative in SARS-CoV-2 infection in COVID-19.

Conclusion
Molecular connection between COVID-19 and kidney disease/traits including AKI and CKD could be due to altered gene expression observed in SARS-infected cells and CKD. Patients with pre-existing renal abnormality like CKD when infected with SARS-CoV-2, pre-existing altered biological functions, localized or systemic, which are also deregulated due to infection would additively or synergistically altered. This might enhance the growth, survival and pathogenicity of the virus (Figure 2). This might be true for AKI. It remains to be found out whether CKD patients when infected with SARS-CoV-2 results in progression of CKD to ESRD. However, viral infection in COVID-19 might induce AKI [17]. It remains unknown whether AKI induced by viral infection would in long term induce CKD or not as has been observed for SARS-CoV infection to induce diabetes [87]. CKD has been prone to pneumonia [88]. However, evidences are lacking to show that CKD is susceptible to infection with SARS-CoV-2. Understanding the molecular relationship may not only help in management and treatment of kidney damage among COVID-19 patients but also help in treatment of COVID-19 patients.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Disclosure of potential conflicts of interest
The authors declare no conflict of interest.